Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
1
Short title Reduction of green leaf aldehydes 1
2
Corresponding Author 3
Kenji Matsui 4
Graduate School of Sciences and Technology for Innovation Yamaguchi University 5
Yamaguchi 753-8515 Japan 6
Telephone +81-83-933-5850 7
Fax +81-83-933-5820 8
Email matsuiyamaguchi-uacjp 9
10
Research Area 11
Biochemistry and Metabolism 12
13
One-sentence Summary 14
NADPH-dependent cinnamaldehyde and hexenal reductase affects the composition of 15
green leaf volatiles and is involved in indirect plant defenses 16
17
Funding 18
This work was supported partly by JSPS KAKENHI Grant Number 16H03283 (to K M) 19
The authors declare that they have no conflict of interest with the contents of this 20
article 21
22
23
Plant Physiology Preview Published on August 20 2018 as DOI101104pp1800632
Copyright 2018 by the American Society of Plant Biologists
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
2
Research Article 24
25
Identification of a hexenal reductase that modulates the composition of 26
green leaf volatiles 27
28
29
30
Toshiyuki Tanakaa Ayana Ikedab Kaori Shiojiric Rika Ozawad Kazumi Shikia 31
Naoko Nagai-Kunihiroa Kenya Fujitab Koichi Sugimotoa Katsuyuki T Yamatoe 32
Hideo Dohraf Toshiyuki Ohnishig Takao Koedukaab Kenji Matsuiab1 33
34
aDivision of Agricultural Sciences Graduate School of Sciences and Technology for 35
Innovation Yamaguchi University Yamaguchi 753-8515 Japan 36
bDepartment of Biological Chemistry Faculty of Agriculture Yamaguchi University 37
Yamaguchi 753-8515 Japan 38
cDepartment of Agriculture Ryukoku University Ooe Otsu Shiga 520-2194 Japan 39
dCenter for Ecological Research Kyoto University Hirano Otsu Shiga 520-2113 Japan 40
eFaculty of Biology-Oriented Science and Technology Kindai University Kinokawa 41
Wakayama 649-6493 Japan 42
fInstrumental Research Support Office Research Institute of Green Science and 43
Technology Shizuoka University Shizuoka 422-8529 Japan 44
gCollege of Agriculture Academic Institute Shizuoka University Shizuoka 422ndash8529 45
Japan 46
47
1Address correspondence to matsuiyamaguchi-uacjp 48
The author responsible for the distribution of materials to the findings presented in this 49
article in accordance with the policy described in the Instructions for Authors 50
(wwwplantphysiolorg) is Kenji Matsui (matsuiyamaguchi-uacjp) 51
52
Author Contributions 53
KM conceived this project designed experiments and wrote the article TT AI K 54
Shiki NNK KF and KSugimoto purified the enzyme and examined its properties 55
KTY and TK analyzed the data HD and TO determined amino acid sequences of 56 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
3
the enzyme TT KF and KM analyzed volatiles and examined phenotypes of 57
knockout mutants K Shioriji and RO performed bioassays with wasps 58
59
60
61
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
ABSTRACT 62
Green leaf volatiles (GLVs) including six-carbon (C6) aldehydes alcohols and esters 63
are formed when plant tissues are damaged GLVs play roles in direct plant defense at 64
wound sites indirect plant defense via the attraction of herbivore predators and 65
plant-plant communication GLV components provoke distinctive responses in their 66
target recipients and therefore control of GLV composition is important for plants to 67
appropriately manage stress responses Reduction of C6-aldehydes into C6-alcohols is a 68
key step in the control of GLV composition and is also important to avoid toxic buildup 69
of C6-aldehydes However the molecular mechanisms behind C6-aldehyde reduction 70
remain poorly understood In this study we purified an Arabidopsis thaliana 71
NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980 72
named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) CHR 73
T-DNA knockout mutant plants displayed a normal growth phenotype however we 74
observed significant suppression of C6-alcohol production following partial mechanical 75
wounding or herbivore infestation Our data also showed that the parasitic wasp Cotesia 76
vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants 77
compared to GLVs emitted from chr plants which corresponded with reduced 78
C6-alcohol levels in the mutant Moreover chr plants were more susceptible to 79
exogenous high-dose exposure to (Z)-3-hexenal as indicated by their markedly lowered 80
photosystem II activity Our study shows that reductases play significant roles in 81
changing GLV composition and are thus important in avoiding toxicity from volatile 82
carbonyls and in the attraction of herbivore predators 83
84
85
Key words Green leaf volatiles (Z)-3-Hexen-1-ol Hexenal reductase Carbonyl stress 86
Indirect defense 87
88
This article contains supplemental Figs S1-S9 and Tables S1-S2 89
90
91
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
INTRODUCTION 92
93
Plants form and emit a wide variety of volatile organic compounds Volatiles 94
from flowers attract pollinators and increase the fitness of plants by promoting efficient 95
reproduction and those from fruits attract seed-dispersing animals and help plants to 96
find new habitats (Dudareva et al 2013) Vegetative organs such as leaves stems and 97
roots also produce and emit volatiles and this process is generally induced by various 98
types of biotic and abiotic stresses as a defense response (Pierik et al 2014) 99
Green leaf volatiles (GLVs) are ubiquitously expressed with other groups of 100
volatile compounds such as terpenoids and amino acid derivatives GLVs are 101
derivatives of fatty acids and include six-carbon (C6) aldehydes alcohols and esters 102
(Fig 1 Matsui 2006 Scala et al 2013) In intact and healthy plant tissues GLV levels 103
are generally low but when tissues suffer stresses associated with disruption of cells 104
such as herbivore damage or attack by necrotrophic fungi GLV-forming pathways are 105
rapidly activated to yield large quantities of GLVs at damaged tissues (Matsui 2006 106
Scala et al 2013 Ameye et al 2017) GLVs at damage tissues participate in direct 107
plant defense by preventing the invasion of harmful microbes (Shiojiri et al 2006a 108
Kishimoto et al 2008) GLVs also function as infochemicals that are an indirect plant 109
defense via the attraction of natural predators of infesting herbivores (Kessler amp 110
Baldwin 2001 Shiojiri et al 2006a Shiojiri et al 2006b Joo et al 2018) GLVs have 111
also been associated with plant-plant communication (Sugimoto et al 2013) 112
Because the chemical properties of GLV components are distinct it is 113
assumed that each plays a distinctive role For example C6-aldehydes were shown to be 114
prominently involved in direct defense against the necrotrophic fungal pathogen 115
Botrytis cinerea whereas C6-alcohols and esters were less involved (Kishimoto et al 116
2008) The formyl groups in C6-aldehydes are potently reactive with nucleophiles such 117
as amines and alcohols and contribute to the suppression of pathogen growth When 118
carbon-carbon double bonds are conjugated to the formyl group as in (E)-2-hexenal 119
the αβ-unsaturated carbonyl moiety behaves as a more potent electrophile and is prone 120
to reactions with various biological molecules specifically reactions that eliminate their 121
intrinsic functions (Farmer and Muumlller 2013) Even though (Z)-3-hexenal is not a 122
reactive carbonyl species because the two double bonds are interrupted with one 123
methylene group bis-allylic hydrogen is prone to removal often resulting in 124 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
oxygenation to yield 4-hydroxy-(E)-2-hexenal or 4-oxo-(E)-2-hexenal (Pospiacutesil amp 125
Yamamoto 2017) These oxygenated hexenals are highly reactive carbonyl species and 126
(Z)-3-hexenal has specifically been identified as a potentially reactive compound 127
(Matsui et al 2012) C6-aldehydes were reported to be more effective bacteriostatic 128
chemicals than C6-alcohols and incorporation of a double bond either in the C2 or C3 129
position enhances their activities (Nakamura and Hatanaka 2002) albeit the formyl 130
group is not always a prerequisite for antimicrobial activity (Prost et al 2005) 131
GLVs are either attractive or repulsive to arthropods depending on their 132
composition and on the arthropod species (Wei amp Kang 2011 Scala et al 2013) 133
Females of the parasitic wasp Cotesia glomerata are attracted to (E)-2-hexenal and 134
(Z)-3-hexen-1-yl acetate but not to (Z)-3-hexen-1-ol (Shiojiri et al 2006) Moreover 135
(Z)-3-Hexen-1-yl acetate increased predation rates of a generalist natural predator 136
toward eggs of the herbivore Manduca quinquemaculata on Nicotiana attenuata plants 137
(Kessler and Baldwin 2001) Composition of GLVs elicited by herbivores also differ 138
between dawn and dusk as indicated by Joo et al (2018) who showed that GLV 139
aldehydes and alcohols were less abundant in comparison with GLV esters at dawn 140
whereas GLV ester levels were lower at dusk Higher predation rates on M sexta eggs 141
by Geocoris spp were observed with dawn GLV composition in nature suggesting that 142
the predator distinguished between GLV compositions (Joo et al 2018) 143
Concerning plant-plant communication (Z)-3-hexen-1-ol emitted from 144
Spodoptera litura-infested tomato leaves was shown to be absorbed by neighboring 145
tomato plants to form the defense compound (Z)-3-hexen-1-yl primeveroside (Sugimoto 146
et al 2013) It was also observed that the secretion of extrafloral nectar by lima beans 147
(Phaseolus lunatus) to attract predatory and parasitoid insects (ants and wasps) was 148
significantly induced after exposure of these plants to (Z)-3-hexen-1-yl acetate vapors 149
(Kost amp Heil 2006) These accumulating lines of evidence indicate that composition of 150
GLVs is crucial for plants to aptly cope with enemies and foes and that reduction of 151
C6-aldehydes to C6-alcohol is a crucial step in the control of GLV composition 152
Lipoxygenase (LOX) acts on linoleniclinoleic acids either in their free forms 153
or as acyl groups in glycerolipids to form corresponding fatty acidlipid hydroperoxides 154
which are subsequently cleaved by hydroperoxide lyase (HPL) to form C6-aldehydes 155
such as n-hexanal or (Z)-3-hexenal (Fig 1 Matsui 2006 Scala et al 2013 Mwenda amp 156
Matsui 2014) A portion of (Z)-3-hexenal is isomerized to (E)-2-hexenal in some plant 157 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
species (Kunishima et al 2016 Spyropoulou et al 2017) and a factor in oral 158
secretions of tobacco hawkmoth (Manduca sexta) catalyzes this isomerization in 159
Nicotiana attenuata resulting in higher attraction of the predator Geocoris spp 160
(Allmann amp Baldwin 2011) Other portions of C6-aldehydes are reduced to 161
corresponding C6-alcohols and portions of C6-alcohols are further converted into 162
corresponding acetates by acetyl-CoA transferase (DrsquoAuria et al 2007) It was assumed 163
that NAD(H)-dependent alcohol dehydrogenase (EC 1111) is involved in these 164
reduction steps (Hatanaka 1993) and this may be the case with completely disrupted 165
plant tissues Accordingly in completely homogenized leaves of Arabidopsis mutants 166
that were deficient in alcohol dehydrogenase (AtADH1 At1g77120) n-hexan-1-ol and 167
3-hexen-1-ol were formed at 62 and 51 of the levels found in parent wild-type 168
plants respectively (Bate et al 1998) In completely homogenized tomato tissues with 169
increased and suppressed expression levels of SlADH2 which is the tomato homolog of 170
AtADH1 n-hexan-1-ol and (Z)-3-hexen-1-ol levels roughly correlated with ADH 171
activities (Speirs et al 1998) In contrast in partially wounded leaf tissues of 172
Arabidopsis (Z)-3-hexenal was formed at disrupted tissues and diffused into the 173
neighboring intact tissues where it was reduced to (Z)-3-hexen-1-ol in an 174
NADPH-dependent manner (Matsui et al 2012) Hence an NADPH-dependent 175
C6-aldehyde reductase is likely accountable for the reduction of C6-aldehydes to 176
C6-alcohols Because plant leaves are commonly damaged only in part such as 177
following consumption by an herbivore such a NADPH-dependent reductase may be 178
ecophysiologically relevant but has not been identified yet In this study we purified 179
and identified a NADPH-dependent reductase preferring (Z)-3-hexenal which we 180
named CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) Involvement of 181
CHR in changing the composition of GLVs was verified using a T-DNA knockout 182
mutant disrupted in the corresponding CHR gene Morover chr mutant lines were 183
employed to assess the role of CHR in indirect defense of Arabidopsis via GLV 184
composition CHR involvement in detoxification of (Z)-3-hexenal was also examined 185
186
187
RESULTS 188
189
Purification of hexenal reductase 190 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
Following exposure to (Z)-3-hexenal vapors intact Arabidopsis leaves absorb 191
the compound into leaves reduce it to (Z)-3-hexen-1-ol and then re-emit it from the 192
leaves to the atmosphere (Matsui et al 2012) Therefore substantial enzyme activity 193
for converting (Z)-3-hexenal to (Z)-3-hexen-1-ol is already present in Arabidopsis 194
leaves under normal growth conditions even without stress treatments and tissue 195
disruptions to trigger GLV formation When crude extracts from Arabidopsis leaves 196
(No-0) were exposed to (Z)-3-hexenal exogenous NADPH was consumed (Fig 4) 197
Moreover the rate of NADH consumption was less than 20 of that found with 198
NADPH under the same reaction conditions Taken with our previous results (Matsui et 199
al 2012) this result indicated that Arabidopsis leaves carry a NADPH-dependent 200
(Z)-3-hexenal reductase 201
This reductase was purified from crude Arabidopsis leaf extracts using 202
ammonium sulfate fractionation followed by three consecutive chromatography steps 203
(Table I) During these steps reductase activity was identified as a single peak with a 204
substantial yield and SDS-PAGE analyses of fractions that were separated using a 205
HiTrap DEAE column in the third chromatography step indicated that a protein of 38 206
kDa correlated with the observed metabolic activity (Fig 2A) In the presence of 207
(Z)-3-hexenal the purified enzyme consumed NADPH most actively at pH 75 (Fig 208
2B) When a mixture of (Z)-3-hexenal and (E)-2-hexenal (154 and 46 microM respectively) 209
was used as a substrate in the absence of cofactor formation of the corresponding 210
alcohol was hardly detected (Fig 2C) Addition of NADH slightly facilitated reduction 211
of hexenals whereas NADPH resulted in more extensive formation of (Z)-3-hexen-1-ol 212
and (E)-2-hexen-1-ol (Fig 2D) However n-hexanal formation through reduction of the 213
carbon-carbon double bond in (Z)-3-hexenal and (E)-2-hexenal was barely increased 214
indicating that the enzyme has little double bond reductase activity 215
216
Identification of C6-aldehyde reductase gene 217
Internal amino acid sequences from the purified protein showed complete 218
identity with Arabidopsis CINNAMYL ALCOHOL DEHYDROGENASE 7 (AtCAD7 219
At4g37980 Supplemental Fig S1) The AtCAD7 gene is also known as 220
ELICITOR-ACTIVATED GENE 3 (ELI3 Kiedrowski et al 1992) and the protein 221
comprises 357 amino acids and has a molecular weight of 38245 Da close to that of the 222
purified enzyme estimated in SDS-PAGE analyses (Fig 2A) A MotifFinder search 223 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224
contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225
based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226
A recombinant protein encoded by the open reading frame of At4g37980 was 227
expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228
(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229
carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230
(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231
little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232
examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233
and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234
enzyme exhibited relatively high activities with aromatic aldehydes such as 235
2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236
sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237
biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238
2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239
aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240
Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241
n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242
to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243
enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244
HEXENAL REDUCTASE (CHR) 245
246
Involvement of CHR in GLV composition 247
To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248
insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249
S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250
differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251
Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252
impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253
Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254
for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255
selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
2
Research Article 24
25
Identification of a hexenal reductase that modulates the composition of 26
green leaf volatiles 27
28
29
30
Toshiyuki Tanakaa Ayana Ikedab Kaori Shiojiric Rika Ozawad Kazumi Shikia 31
Naoko Nagai-Kunihiroa Kenya Fujitab Koichi Sugimotoa Katsuyuki T Yamatoe 32
Hideo Dohraf Toshiyuki Ohnishig Takao Koedukaab Kenji Matsuiab1 33
34
aDivision of Agricultural Sciences Graduate School of Sciences and Technology for 35
Innovation Yamaguchi University Yamaguchi 753-8515 Japan 36
bDepartment of Biological Chemistry Faculty of Agriculture Yamaguchi University 37
Yamaguchi 753-8515 Japan 38
cDepartment of Agriculture Ryukoku University Ooe Otsu Shiga 520-2194 Japan 39
dCenter for Ecological Research Kyoto University Hirano Otsu Shiga 520-2113 Japan 40
eFaculty of Biology-Oriented Science and Technology Kindai University Kinokawa 41
Wakayama 649-6493 Japan 42
fInstrumental Research Support Office Research Institute of Green Science and 43
Technology Shizuoka University Shizuoka 422-8529 Japan 44
gCollege of Agriculture Academic Institute Shizuoka University Shizuoka 422ndash8529 45
Japan 46
47
1Address correspondence to matsuiyamaguchi-uacjp 48
The author responsible for the distribution of materials to the findings presented in this 49
article in accordance with the policy described in the Instructions for Authors 50
(wwwplantphysiolorg) is Kenji Matsui (matsuiyamaguchi-uacjp) 51
52
Author Contributions 53
KM conceived this project designed experiments and wrote the article TT AI K 54
Shiki NNK KF and KSugimoto purified the enzyme and examined its properties 55
KTY and TK analyzed the data HD and TO determined amino acid sequences of 56 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
3
the enzyme TT KF and KM analyzed volatiles and examined phenotypes of 57
knockout mutants K Shioriji and RO performed bioassays with wasps 58
59
60
61
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
ABSTRACT 62
Green leaf volatiles (GLVs) including six-carbon (C6) aldehydes alcohols and esters 63
are formed when plant tissues are damaged GLVs play roles in direct plant defense at 64
wound sites indirect plant defense via the attraction of herbivore predators and 65
plant-plant communication GLV components provoke distinctive responses in their 66
target recipients and therefore control of GLV composition is important for plants to 67
appropriately manage stress responses Reduction of C6-aldehydes into C6-alcohols is a 68
key step in the control of GLV composition and is also important to avoid toxic buildup 69
of C6-aldehydes However the molecular mechanisms behind C6-aldehyde reduction 70
remain poorly understood In this study we purified an Arabidopsis thaliana 71
NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980 72
named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) CHR 73
T-DNA knockout mutant plants displayed a normal growth phenotype however we 74
observed significant suppression of C6-alcohol production following partial mechanical 75
wounding or herbivore infestation Our data also showed that the parasitic wasp Cotesia 76
vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants 77
compared to GLVs emitted from chr plants which corresponded with reduced 78
C6-alcohol levels in the mutant Moreover chr plants were more susceptible to 79
exogenous high-dose exposure to (Z)-3-hexenal as indicated by their markedly lowered 80
photosystem II activity Our study shows that reductases play significant roles in 81
changing GLV composition and are thus important in avoiding toxicity from volatile 82
carbonyls and in the attraction of herbivore predators 83
84
85
Key words Green leaf volatiles (Z)-3-Hexen-1-ol Hexenal reductase Carbonyl stress 86
Indirect defense 87
88
This article contains supplemental Figs S1-S9 and Tables S1-S2 89
90
91
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
INTRODUCTION 92
93
Plants form and emit a wide variety of volatile organic compounds Volatiles 94
from flowers attract pollinators and increase the fitness of plants by promoting efficient 95
reproduction and those from fruits attract seed-dispersing animals and help plants to 96
find new habitats (Dudareva et al 2013) Vegetative organs such as leaves stems and 97
roots also produce and emit volatiles and this process is generally induced by various 98
types of biotic and abiotic stresses as a defense response (Pierik et al 2014) 99
Green leaf volatiles (GLVs) are ubiquitously expressed with other groups of 100
volatile compounds such as terpenoids and amino acid derivatives GLVs are 101
derivatives of fatty acids and include six-carbon (C6) aldehydes alcohols and esters 102
(Fig 1 Matsui 2006 Scala et al 2013) In intact and healthy plant tissues GLV levels 103
are generally low but when tissues suffer stresses associated with disruption of cells 104
such as herbivore damage or attack by necrotrophic fungi GLV-forming pathways are 105
rapidly activated to yield large quantities of GLVs at damaged tissues (Matsui 2006 106
Scala et al 2013 Ameye et al 2017) GLVs at damage tissues participate in direct 107
plant defense by preventing the invasion of harmful microbes (Shiojiri et al 2006a 108
Kishimoto et al 2008) GLVs also function as infochemicals that are an indirect plant 109
defense via the attraction of natural predators of infesting herbivores (Kessler amp 110
Baldwin 2001 Shiojiri et al 2006a Shiojiri et al 2006b Joo et al 2018) GLVs have 111
also been associated with plant-plant communication (Sugimoto et al 2013) 112
Because the chemical properties of GLV components are distinct it is 113
assumed that each plays a distinctive role For example C6-aldehydes were shown to be 114
prominently involved in direct defense against the necrotrophic fungal pathogen 115
Botrytis cinerea whereas C6-alcohols and esters were less involved (Kishimoto et al 116
2008) The formyl groups in C6-aldehydes are potently reactive with nucleophiles such 117
as amines and alcohols and contribute to the suppression of pathogen growth When 118
carbon-carbon double bonds are conjugated to the formyl group as in (E)-2-hexenal 119
the αβ-unsaturated carbonyl moiety behaves as a more potent electrophile and is prone 120
to reactions with various biological molecules specifically reactions that eliminate their 121
intrinsic functions (Farmer and Muumlller 2013) Even though (Z)-3-hexenal is not a 122
reactive carbonyl species because the two double bonds are interrupted with one 123
methylene group bis-allylic hydrogen is prone to removal often resulting in 124 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
oxygenation to yield 4-hydroxy-(E)-2-hexenal or 4-oxo-(E)-2-hexenal (Pospiacutesil amp 125
Yamamoto 2017) These oxygenated hexenals are highly reactive carbonyl species and 126
(Z)-3-hexenal has specifically been identified as a potentially reactive compound 127
(Matsui et al 2012) C6-aldehydes were reported to be more effective bacteriostatic 128
chemicals than C6-alcohols and incorporation of a double bond either in the C2 or C3 129
position enhances their activities (Nakamura and Hatanaka 2002) albeit the formyl 130
group is not always a prerequisite for antimicrobial activity (Prost et al 2005) 131
GLVs are either attractive or repulsive to arthropods depending on their 132
composition and on the arthropod species (Wei amp Kang 2011 Scala et al 2013) 133
Females of the parasitic wasp Cotesia glomerata are attracted to (E)-2-hexenal and 134
(Z)-3-hexen-1-yl acetate but not to (Z)-3-hexen-1-ol (Shiojiri et al 2006) Moreover 135
(Z)-3-Hexen-1-yl acetate increased predation rates of a generalist natural predator 136
toward eggs of the herbivore Manduca quinquemaculata on Nicotiana attenuata plants 137
(Kessler and Baldwin 2001) Composition of GLVs elicited by herbivores also differ 138
between dawn and dusk as indicated by Joo et al (2018) who showed that GLV 139
aldehydes and alcohols were less abundant in comparison with GLV esters at dawn 140
whereas GLV ester levels were lower at dusk Higher predation rates on M sexta eggs 141
by Geocoris spp were observed with dawn GLV composition in nature suggesting that 142
the predator distinguished between GLV compositions (Joo et al 2018) 143
Concerning plant-plant communication (Z)-3-hexen-1-ol emitted from 144
Spodoptera litura-infested tomato leaves was shown to be absorbed by neighboring 145
tomato plants to form the defense compound (Z)-3-hexen-1-yl primeveroside (Sugimoto 146
et al 2013) It was also observed that the secretion of extrafloral nectar by lima beans 147
(Phaseolus lunatus) to attract predatory and parasitoid insects (ants and wasps) was 148
significantly induced after exposure of these plants to (Z)-3-hexen-1-yl acetate vapors 149
(Kost amp Heil 2006) These accumulating lines of evidence indicate that composition of 150
GLVs is crucial for plants to aptly cope with enemies and foes and that reduction of 151
C6-aldehydes to C6-alcohol is a crucial step in the control of GLV composition 152
Lipoxygenase (LOX) acts on linoleniclinoleic acids either in their free forms 153
or as acyl groups in glycerolipids to form corresponding fatty acidlipid hydroperoxides 154
which are subsequently cleaved by hydroperoxide lyase (HPL) to form C6-aldehydes 155
such as n-hexanal or (Z)-3-hexenal (Fig 1 Matsui 2006 Scala et al 2013 Mwenda amp 156
Matsui 2014) A portion of (Z)-3-hexenal is isomerized to (E)-2-hexenal in some plant 157 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
species (Kunishima et al 2016 Spyropoulou et al 2017) and a factor in oral 158
secretions of tobacco hawkmoth (Manduca sexta) catalyzes this isomerization in 159
Nicotiana attenuata resulting in higher attraction of the predator Geocoris spp 160
(Allmann amp Baldwin 2011) Other portions of C6-aldehydes are reduced to 161
corresponding C6-alcohols and portions of C6-alcohols are further converted into 162
corresponding acetates by acetyl-CoA transferase (DrsquoAuria et al 2007) It was assumed 163
that NAD(H)-dependent alcohol dehydrogenase (EC 1111) is involved in these 164
reduction steps (Hatanaka 1993) and this may be the case with completely disrupted 165
plant tissues Accordingly in completely homogenized leaves of Arabidopsis mutants 166
that were deficient in alcohol dehydrogenase (AtADH1 At1g77120) n-hexan-1-ol and 167
3-hexen-1-ol were formed at 62 and 51 of the levels found in parent wild-type 168
plants respectively (Bate et al 1998) In completely homogenized tomato tissues with 169
increased and suppressed expression levels of SlADH2 which is the tomato homolog of 170
AtADH1 n-hexan-1-ol and (Z)-3-hexen-1-ol levels roughly correlated with ADH 171
activities (Speirs et al 1998) In contrast in partially wounded leaf tissues of 172
Arabidopsis (Z)-3-hexenal was formed at disrupted tissues and diffused into the 173
neighboring intact tissues where it was reduced to (Z)-3-hexen-1-ol in an 174
NADPH-dependent manner (Matsui et al 2012) Hence an NADPH-dependent 175
C6-aldehyde reductase is likely accountable for the reduction of C6-aldehydes to 176
C6-alcohols Because plant leaves are commonly damaged only in part such as 177
following consumption by an herbivore such a NADPH-dependent reductase may be 178
ecophysiologically relevant but has not been identified yet In this study we purified 179
and identified a NADPH-dependent reductase preferring (Z)-3-hexenal which we 180
named CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) Involvement of 181
CHR in changing the composition of GLVs was verified using a T-DNA knockout 182
mutant disrupted in the corresponding CHR gene Morover chr mutant lines were 183
employed to assess the role of CHR in indirect defense of Arabidopsis via GLV 184
composition CHR involvement in detoxification of (Z)-3-hexenal was also examined 185
186
187
RESULTS 188
189
Purification of hexenal reductase 190 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
Following exposure to (Z)-3-hexenal vapors intact Arabidopsis leaves absorb 191
the compound into leaves reduce it to (Z)-3-hexen-1-ol and then re-emit it from the 192
leaves to the atmosphere (Matsui et al 2012) Therefore substantial enzyme activity 193
for converting (Z)-3-hexenal to (Z)-3-hexen-1-ol is already present in Arabidopsis 194
leaves under normal growth conditions even without stress treatments and tissue 195
disruptions to trigger GLV formation When crude extracts from Arabidopsis leaves 196
(No-0) were exposed to (Z)-3-hexenal exogenous NADPH was consumed (Fig 4) 197
Moreover the rate of NADH consumption was less than 20 of that found with 198
NADPH under the same reaction conditions Taken with our previous results (Matsui et 199
al 2012) this result indicated that Arabidopsis leaves carry a NADPH-dependent 200
(Z)-3-hexenal reductase 201
This reductase was purified from crude Arabidopsis leaf extracts using 202
ammonium sulfate fractionation followed by three consecutive chromatography steps 203
(Table I) During these steps reductase activity was identified as a single peak with a 204
substantial yield and SDS-PAGE analyses of fractions that were separated using a 205
HiTrap DEAE column in the third chromatography step indicated that a protein of 38 206
kDa correlated with the observed metabolic activity (Fig 2A) In the presence of 207
(Z)-3-hexenal the purified enzyme consumed NADPH most actively at pH 75 (Fig 208
2B) When a mixture of (Z)-3-hexenal and (E)-2-hexenal (154 and 46 microM respectively) 209
was used as a substrate in the absence of cofactor formation of the corresponding 210
alcohol was hardly detected (Fig 2C) Addition of NADH slightly facilitated reduction 211
of hexenals whereas NADPH resulted in more extensive formation of (Z)-3-hexen-1-ol 212
and (E)-2-hexen-1-ol (Fig 2D) However n-hexanal formation through reduction of the 213
carbon-carbon double bond in (Z)-3-hexenal and (E)-2-hexenal was barely increased 214
indicating that the enzyme has little double bond reductase activity 215
216
Identification of C6-aldehyde reductase gene 217
Internal amino acid sequences from the purified protein showed complete 218
identity with Arabidopsis CINNAMYL ALCOHOL DEHYDROGENASE 7 (AtCAD7 219
At4g37980 Supplemental Fig S1) The AtCAD7 gene is also known as 220
ELICITOR-ACTIVATED GENE 3 (ELI3 Kiedrowski et al 1992) and the protein 221
comprises 357 amino acids and has a molecular weight of 38245 Da close to that of the 222
purified enzyme estimated in SDS-PAGE analyses (Fig 2A) A MotifFinder search 223 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224
contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225
based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226
A recombinant protein encoded by the open reading frame of At4g37980 was 227
expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228
(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229
carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230
(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231
little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232
examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233
and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234
enzyme exhibited relatively high activities with aromatic aldehydes such as 235
2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236
sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237
biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238
2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239
aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240
Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241
n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242
to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243
enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244
HEXENAL REDUCTASE (CHR) 245
246
Involvement of CHR in GLV composition 247
To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248
insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249
S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250
differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251
Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252
impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253
Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254
for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255
selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
3
the enzyme TT KF and KM analyzed volatiles and examined phenotypes of 57
knockout mutants K Shioriji and RO performed bioassays with wasps 58
59
60
61
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
ABSTRACT 62
Green leaf volatiles (GLVs) including six-carbon (C6) aldehydes alcohols and esters 63
are formed when plant tissues are damaged GLVs play roles in direct plant defense at 64
wound sites indirect plant defense via the attraction of herbivore predators and 65
plant-plant communication GLV components provoke distinctive responses in their 66
target recipients and therefore control of GLV composition is important for plants to 67
appropriately manage stress responses Reduction of C6-aldehydes into C6-alcohols is a 68
key step in the control of GLV composition and is also important to avoid toxic buildup 69
of C6-aldehydes However the molecular mechanisms behind C6-aldehyde reduction 70
remain poorly understood In this study we purified an Arabidopsis thaliana 71
NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980 72
named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) CHR 73
T-DNA knockout mutant plants displayed a normal growth phenotype however we 74
observed significant suppression of C6-alcohol production following partial mechanical 75
wounding or herbivore infestation Our data also showed that the parasitic wasp Cotesia 76
vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants 77
compared to GLVs emitted from chr plants which corresponded with reduced 78
C6-alcohol levels in the mutant Moreover chr plants were more susceptible to 79
exogenous high-dose exposure to (Z)-3-hexenal as indicated by their markedly lowered 80
photosystem II activity Our study shows that reductases play significant roles in 81
changing GLV composition and are thus important in avoiding toxicity from volatile 82
carbonyls and in the attraction of herbivore predators 83
84
85
Key words Green leaf volatiles (Z)-3-Hexen-1-ol Hexenal reductase Carbonyl stress 86
Indirect defense 87
88
This article contains supplemental Figs S1-S9 and Tables S1-S2 89
90
91
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
INTRODUCTION 92
93
Plants form and emit a wide variety of volatile organic compounds Volatiles 94
from flowers attract pollinators and increase the fitness of plants by promoting efficient 95
reproduction and those from fruits attract seed-dispersing animals and help plants to 96
find new habitats (Dudareva et al 2013) Vegetative organs such as leaves stems and 97
roots also produce and emit volatiles and this process is generally induced by various 98
types of biotic and abiotic stresses as a defense response (Pierik et al 2014) 99
Green leaf volatiles (GLVs) are ubiquitously expressed with other groups of 100
volatile compounds such as terpenoids and amino acid derivatives GLVs are 101
derivatives of fatty acids and include six-carbon (C6) aldehydes alcohols and esters 102
(Fig 1 Matsui 2006 Scala et al 2013) In intact and healthy plant tissues GLV levels 103
are generally low but when tissues suffer stresses associated with disruption of cells 104
such as herbivore damage or attack by necrotrophic fungi GLV-forming pathways are 105
rapidly activated to yield large quantities of GLVs at damaged tissues (Matsui 2006 106
Scala et al 2013 Ameye et al 2017) GLVs at damage tissues participate in direct 107
plant defense by preventing the invasion of harmful microbes (Shiojiri et al 2006a 108
Kishimoto et al 2008) GLVs also function as infochemicals that are an indirect plant 109
defense via the attraction of natural predators of infesting herbivores (Kessler amp 110
Baldwin 2001 Shiojiri et al 2006a Shiojiri et al 2006b Joo et al 2018) GLVs have 111
also been associated with plant-plant communication (Sugimoto et al 2013) 112
Because the chemical properties of GLV components are distinct it is 113
assumed that each plays a distinctive role For example C6-aldehydes were shown to be 114
prominently involved in direct defense against the necrotrophic fungal pathogen 115
Botrytis cinerea whereas C6-alcohols and esters were less involved (Kishimoto et al 116
2008) The formyl groups in C6-aldehydes are potently reactive with nucleophiles such 117
as amines and alcohols and contribute to the suppression of pathogen growth When 118
carbon-carbon double bonds are conjugated to the formyl group as in (E)-2-hexenal 119
the αβ-unsaturated carbonyl moiety behaves as a more potent electrophile and is prone 120
to reactions with various biological molecules specifically reactions that eliminate their 121
intrinsic functions (Farmer and Muumlller 2013) Even though (Z)-3-hexenal is not a 122
reactive carbonyl species because the two double bonds are interrupted with one 123
methylene group bis-allylic hydrogen is prone to removal often resulting in 124 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
oxygenation to yield 4-hydroxy-(E)-2-hexenal or 4-oxo-(E)-2-hexenal (Pospiacutesil amp 125
Yamamoto 2017) These oxygenated hexenals are highly reactive carbonyl species and 126
(Z)-3-hexenal has specifically been identified as a potentially reactive compound 127
(Matsui et al 2012) C6-aldehydes were reported to be more effective bacteriostatic 128
chemicals than C6-alcohols and incorporation of a double bond either in the C2 or C3 129
position enhances their activities (Nakamura and Hatanaka 2002) albeit the formyl 130
group is not always a prerequisite for antimicrobial activity (Prost et al 2005) 131
GLVs are either attractive or repulsive to arthropods depending on their 132
composition and on the arthropod species (Wei amp Kang 2011 Scala et al 2013) 133
Females of the parasitic wasp Cotesia glomerata are attracted to (E)-2-hexenal and 134
(Z)-3-hexen-1-yl acetate but not to (Z)-3-hexen-1-ol (Shiojiri et al 2006) Moreover 135
(Z)-3-Hexen-1-yl acetate increased predation rates of a generalist natural predator 136
toward eggs of the herbivore Manduca quinquemaculata on Nicotiana attenuata plants 137
(Kessler and Baldwin 2001) Composition of GLVs elicited by herbivores also differ 138
between dawn and dusk as indicated by Joo et al (2018) who showed that GLV 139
aldehydes and alcohols were less abundant in comparison with GLV esters at dawn 140
whereas GLV ester levels were lower at dusk Higher predation rates on M sexta eggs 141
by Geocoris spp were observed with dawn GLV composition in nature suggesting that 142
the predator distinguished between GLV compositions (Joo et al 2018) 143
Concerning plant-plant communication (Z)-3-hexen-1-ol emitted from 144
Spodoptera litura-infested tomato leaves was shown to be absorbed by neighboring 145
tomato plants to form the defense compound (Z)-3-hexen-1-yl primeveroside (Sugimoto 146
et al 2013) It was also observed that the secretion of extrafloral nectar by lima beans 147
(Phaseolus lunatus) to attract predatory and parasitoid insects (ants and wasps) was 148
significantly induced after exposure of these plants to (Z)-3-hexen-1-yl acetate vapors 149
(Kost amp Heil 2006) These accumulating lines of evidence indicate that composition of 150
GLVs is crucial for plants to aptly cope with enemies and foes and that reduction of 151
C6-aldehydes to C6-alcohol is a crucial step in the control of GLV composition 152
Lipoxygenase (LOX) acts on linoleniclinoleic acids either in their free forms 153
or as acyl groups in glycerolipids to form corresponding fatty acidlipid hydroperoxides 154
which are subsequently cleaved by hydroperoxide lyase (HPL) to form C6-aldehydes 155
such as n-hexanal or (Z)-3-hexenal (Fig 1 Matsui 2006 Scala et al 2013 Mwenda amp 156
Matsui 2014) A portion of (Z)-3-hexenal is isomerized to (E)-2-hexenal in some plant 157 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
species (Kunishima et al 2016 Spyropoulou et al 2017) and a factor in oral 158
secretions of tobacco hawkmoth (Manduca sexta) catalyzes this isomerization in 159
Nicotiana attenuata resulting in higher attraction of the predator Geocoris spp 160
(Allmann amp Baldwin 2011) Other portions of C6-aldehydes are reduced to 161
corresponding C6-alcohols and portions of C6-alcohols are further converted into 162
corresponding acetates by acetyl-CoA transferase (DrsquoAuria et al 2007) It was assumed 163
that NAD(H)-dependent alcohol dehydrogenase (EC 1111) is involved in these 164
reduction steps (Hatanaka 1993) and this may be the case with completely disrupted 165
plant tissues Accordingly in completely homogenized leaves of Arabidopsis mutants 166
that were deficient in alcohol dehydrogenase (AtADH1 At1g77120) n-hexan-1-ol and 167
3-hexen-1-ol were formed at 62 and 51 of the levels found in parent wild-type 168
plants respectively (Bate et al 1998) In completely homogenized tomato tissues with 169
increased and suppressed expression levels of SlADH2 which is the tomato homolog of 170
AtADH1 n-hexan-1-ol and (Z)-3-hexen-1-ol levels roughly correlated with ADH 171
activities (Speirs et al 1998) In contrast in partially wounded leaf tissues of 172
Arabidopsis (Z)-3-hexenal was formed at disrupted tissues and diffused into the 173
neighboring intact tissues where it was reduced to (Z)-3-hexen-1-ol in an 174
NADPH-dependent manner (Matsui et al 2012) Hence an NADPH-dependent 175
C6-aldehyde reductase is likely accountable for the reduction of C6-aldehydes to 176
C6-alcohols Because plant leaves are commonly damaged only in part such as 177
following consumption by an herbivore such a NADPH-dependent reductase may be 178
ecophysiologically relevant but has not been identified yet In this study we purified 179
and identified a NADPH-dependent reductase preferring (Z)-3-hexenal which we 180
named CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) Involvement of 181
CHR in changing the composition of GLVs was verified using a T-DNA knockout 182
mutant disrupted in the corresponding CHR gene Morover chr mutant lines were 183
employed to assess the role of CHR in indirect defense of Arabidopsis via GLV 184
composition CHR involvement in detoxification of (Z)-3-hexenal was also examined 185
186
187
RESULTS 188
189
Purification of hexenal reductase 190 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
Following exposure to (Z)-3-hexenal vapors intact Arabidopsis leaves absorb 191
the compound into leaves reduce it to (Z)-3-hexen-1-ol and then re-emit it from the 192
leaves to the atmosphere (Matsui et al 2012) Therefore substantial enzyme activity 193
for converting (Z)-3-hexenal to (Z)-3-hexen-1-ol is already present in Arabidopsis 194
leaves under normal growth conditions even without stress treatments and tissue 195
disruptions to trigger GLV formation When crude extracts from Arabidopsis leaves 196
(No-0) were exposed to (Z)-3-hexenal exogenous NADPH was consumed (Fig 4) 197
Moreover the rate of NADH consumption was less than 20 of that found with 198
NADPH under the same reaction conditions Taken with our previous results (Matsui et 199
al 2012) this result indicated that Arabidopsis leaves carry a NADPH-dependent 200
(Z)-3-hexenal reductase 201
This reductase was purified from crude Arabidopsis leaf extracts using 202
ammonium sulfate fractionation followed by three consecutive chromatography steps 203
(Table I) During these steps reductase activity was identified as a single peak with a 204
substantial yield and SDS-PAGE analyses of fractions that were separated using a 205
HiTrap DEAE column in the third chromatography step indicated that a protein of 38 206
kDa correlated with the observed metabolic activity (Fig 2A) In the presence of 207
(Z)-3-hexenal the purified enzyme consumed NADPH most actively at pH 75 (Fig 208
2B) When a mixture of (Z)-3-hexenal and (E)-2-hexenal (154 and 46 microM respectively) 209
was used as a substrate in the absence of cofactor formation of the corresponding 210
alcohol was hardly detected (Fig 2C) Addition of NADH slightly facilitated reduction 211
of hexenals whereas NADPH resulted in more extensive formation of (Z)-3-hexen-1-ol 212
and (E)-2-hexen-1-ol (Fig 2D) However n-hexanal formation through reduction of the 213
carbon-carbon double bond in (Z)-3-hexenal and (E)-2-hexenal was barely increased 214
indicating that the enzyme has little double bond reductase activity 215
216
Identification of C6-aldehyde reductase gene 217
Internal amino acid sequences from the purified protein showed complete 218
identity with Arabidopsis CINNAMYL ALCOHOL DEHYDROGENASE 7 (AtCAD7 219
At4g37980 Supplemental Fig S1) The AtCAD7 gene is also known as 220
ELICITOR-ACTIVATED GENE 3 (ELI3 Kiedrowski et al 1992) and the protein 221
comprises 357 amino acids and has a molecular weight of 38245 Da close to that of the 222
purified enzyme estimated in SDS-PAGE analyses (Fig 2A) A MotifFinder search 223 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224
contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225
based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226
A recombinant protein encoded by the open reading frame of At4g37980 was 227
expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228
(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229
carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230
(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231
little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232
examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233
and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234
enzyme exhibited relatively high activities with aromatic aldehydes such as 235
2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236
sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237
biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238
2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239
aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240
Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241
n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242
to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243
enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244
HEXENAL REDUCTASE (CHR) 245
246
Involvement of CHR in GLV composition 247
To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248
insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249
S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250
differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251
Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252
impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253
Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254
for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255
selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
ABSTRACT 62
Green leaf volatiles (GLVs) including six-carbon (C6) aldehydes alcohols and esters 63
are formed when plant tissues are damaged GLVs play roles in direct plant defense at 64
wound sites indirect plant defense via the attraction of herbivore predators and 65
plant-plant communication GLV components provoke distinctive responses in their 66
target recipients and therefore control of GLV composition is important for plants to 67
appropriately manage stress responses Reduction of C6-aldehydes into C6-alcohols is a 68
key step in the control of GLV composition and is also important to avoid toxic buildup 69
of C6-aldehydes However the molecular mechanisms behind C6-aldehyde reduction 70
remain poorly understood In this study we purified an Arabidopsis thaliana 71
NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980 72
named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) CHR 73
T-DNA knockout mutant plants displayed a normal growth phenotype however we 74
observed significant suppression of C6-alcohol production following partial mechanical 75
wounding or herbivore infestation Our data also showed that the parasitic wasp Cotesia 76
vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants 77
compared to GLVs emitted from chr plants which corresponded with reduced 78
C6-alcohol levels in the mutant Moreover chr plants were more susceptible to 79
exogenous high-dose exposure to (Z)-3-hexenal as indicated by their markedly lowered 80
photosystem II activity Our study shows that reductases play significant roles in 81
changing GLV composition and are thus important in avoiding toxicity from volatile 82
carbonyls and in the attraction of herbivore predators 83
84
85
Key words Green leaf volatiles (Z)-3-Hexen-1-ol Hexenal reductase Carbonyl stress 86
Indirect defense 87
88
This article contains supplemental Figs S1-S9 and Tables S1-S2 89
90
91
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
INTRODUCTION 92
93
Plants form and emit a wide variety of volatile organic compounds Volatiles 94
from flowers attract pollinators and increase the fitness of plants by promoting efficient 95
reproduction and those from fruits attract seed-dispersing animals and help plants to 96
find new habitats (Dudareva et al 2013) Vegetative organs such as leaves stems and 97
roots also produce and emit volatiles and this process is generally induced by various 98
types of biotic and abiotic stresses as a defense response (Pierik et al 2014) 99
Green leaf volatiles (GLVs) are ubiquitously expressed with other groups of 100
volatile compounds such as terpenoids and amino acid derivatives GLVs are 101
derivatives of fatty acids and include six-carbon (C6) aldehydes alcohols and esters 102
(Fig 1 Matsui 2006 Scala et al 2013) In intact and healthy plant tissues GLV levels 103
are generally low but when tissues suffer stresses associated with disruption of cells 104
such as herbivore damage or attack by necrotrophic fungi GLV-forming pathways are 105
rapidly activated to yield large quantities of GLVs at damaged tissues (Matsui 2006 106
Scala et al 2013 Ameye et al 2017) GLVs at damage tissues participate in direct 107
plant defense by preventing the invasion of harmful microbes (Shiojiri et al 2006a 108
Kishimoto et al 2008) GLVs also function as infochemicals that are an indirect plant 109
defense via the attraction of natural predators of infesting herbivores (Kessler amp 110
Baldwin 2001 Shiojiri et al 2006a Shiojiri et al 2006b Joo et al 2018) GLVs have 111
also been associated with plant-plant communication (Sugimoto et al 2013) 112
Because the chemical properties of GLV components are distinct it is 113
assumed that each plays a distinctive role For example C6-aldehydes were shown to be 114
prominently involved in direct defense against the necrotrophic fungal pathogen 115
Botrytis cinerea whereas C6-alcohols and esters were less involved (Kishimoto et al 116
2008) The formyl groups in C6-aldehydes are potently reactive with nucleophiles such 117
as amines and alcohols and contribute to the suppression of pathogen growth When 118
carbon-carbon double bonds are conjugated to the formyl group as in (E)-2-hexenal 119
the αβ-unsaturated carbonyl moiety behaves as a more potent electrophile and is prone 120
to reactions with various biological molecules specifically reactions that eliminate their 121
intrinsic functions (Farmer and Muumlller 2013) Even though (Z)-3-hexenal is not a 122
reactive carbonyl species because the two double bonds are interrupted with one 123
methylene group bis-allylic hydrogen is prone to removal often resulting in 124 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
oxygenation to yield 4-hydroxy-(E)-2-hexenal or 4-oxo-(E)-2-hexenal (Pospiacutesil amp 125
Yamamoto 2017) These oxygenated hexenals are highly reactive carbonyl species and 126
(Z)-3-hexenal has specifically been identified as a potentially reactive compound 127
(Matsui et al 2012) C6-aldehydes were reported to be more effective bacteriostatic 128
chemicals than C6-alcohols and incorporation of a double bond either in the C2 or C3 129
position enhances their activities (Nakamura and Hatanaka 2002) albeit the formyl 130
group is not always a prerequisite for antimicrobial activity (Prost et al 2005) 131
GLVs are either attractive or repulsive to arthropods depending on their 132
composition and on the arthropod species (Wei amp Kang 2011 Scala et al 2013) 133
Females of the parasitic wasp Cotesia glomerata are attracted to (E)-2-hexenal and 134
(Z)-3-hexen-1-yl acetate but not to (Z)-3-hexen-1-ol (Shiojiri et al 2006) Moreover 135
(Z)-3-Hexen-1-yl acetate increased predation rates of a generalist natural predator 136
toward eggs of the herbivore Manduca quinquemaculata on Nicotiana attenuata plants 137
(Kessler and Baldwin 2001) Composition of GLVs elicited by herbivores also differ 138
between dawn and dusk as indicated by Joo et al (2018) who showed that GLV 139
aldehydes and alcohols were less abundant in comparison with GLV esters at dawn 140
whereas GLV ester levels were lower at dusk Higher predation rates on M sexta eggs 141
by Geocoris spp were observed with dawn GLV composition in nature suggesting that 142
the predator distinguished between GLV compositions (Joo et al 2018) 143
Concerning plant-plant communication (Z)-3-hexen-1-ol emitted from 144
Spodoptera litura-infested tomato leaves was shown to be absorbed by neighboring 145
tomato plants to form the defense compound (Z)-3-hexen-1-yl primeveroside (Sugimoto 146
et al 2013) It was also observed that the secretion of extrafloral nectar by lima beans 147
(Phaseolus lunatus) to attract predatory and parasitoid insects (ants and wasps) was 148
significantly induced after exposure of these plants to (Z)-3-hexen-1-yl acetate vapors 149
(Kost amp Heil 2006) These accumulating lines of evidence indicate that composition of 150
GLVs is crucial for plants to aptly cope with enemies and foes and that reduction of 151
C6-aldehydes to C6-alcohol is a crucial step in the control of GLV composition 152
Lipoxygenase (LOX) acts on linoleniclinoleic acids either in their free forms 153
or as acyl groups in glycerolipids to form corresponding fatty acidlipid hydroperoxides 154
which are subsequently cleaved by hydroperoxide lyase (HPL) to form C6-aldehydes 155
such as n-hexanal or (Z)-3-hexenal (Fig 1 Matsui 2006 Scala et al 2013 Mwenda amp 156
Matsui 2014) A portion of (Z)-3-hexenal is isomerized to (E)-2-hexenal in some plant 157 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
species (Kunishima et al 2016 Spyropoulou et al 2017) and a factor in oral 158
secretions of tobacco hawkmoth (Manduca sexta) catalyzes this isomerization in 159
Nicotiana attenuata resulting in higher attraction of the predator Geocoris spp 160
(Allmann amp Baldwin 2011) Other portions of C6-aldehydes are reduced to 161
corresponding C6-alcohols and portions of C6-alcohols are further converted into 162
corresponding acetates by acetyl-CoA transferase (DrsquoAuria et al 2007) It was assumed 163
that NAD(H)-dependent alcohol dehydrogenase (EC 1111) is involved in these 164
reduction steps (Hatanaka 1993) and this may be the case with completely disrupted 165
plant tissues Accordingly in completely homogenized leaves of Arabidopsis mutants 166
that were deficient in alcohol dehydrogenase (AtADH1 At1g77120) n-hexan-1-ol and 167
3-hexen-1-ol were formed at 62 and 51 of the levels found in parent wild-type 168
plants respectively (Bate et al 1998) In completely homogenized tomato tissues with 169
increased and suppressed expression levels of SlADH2 which is the tomato homolog of 170
AtADH1 n-hexan-1-ol and (Z)-3-hexen-1-ol levels roughly correlated with ADH 171
activities (Speirs et al 1998) In contrast in partially wounded leaf tissues of 172
Arabidopsis (Z)-3-hexenal was formed at disrupted tissues and diffused into the 173
neighboring intact tissues where it was reduced to (Z)-3-hexen-1-ol in an 174
NADPH-dependent manner (Matsui et al 2012) Hence an NADPH-dependent 175
C6-aldehyde reductase is likely accountable for the reduction of C6-aldehydes to 176
C6-alcohols Because plant leaves are commonly damaged only in part such as 177
following consumption by an herbivore such a NADPH-dependent reductase may be 178
ecophysiologically relevant but has not been identified yet In this study we purified 179
and identified a NADPH-dependent reductase preferring (Z)-3-hexenal which we 180
named CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) Involvement of 181
CHR in changing the composition of GLVs was verified using a T-DNA knockout 182
mutant disrupted in the corresponding CHR gene Morover chr mutant lines were 183
employed to assess the role of CHR in indirect defense of Arabidopsis via GLV 184
composition CHR involvement in detoxification of (Z)-3-hexenal was also examined 185
186
187
RESULTS 188
189
Purification of hexenal reductase 190 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
Following exposure to (Z)-3-hexenal vapors intact Arabidopsis leaves absorb 191
the compound into leaves reduce it to (Z)-3-hexen-1-ol and then re-emit it from the 192
leaves to the atmosphere (Matsui et al 2012) Therefore substantial enzyme activity 193
for converting (Z)-3-hexenal to (Z)-3-hexen-1-ol is already present in Arabidopsis 194
leaves under normal growth conditions even without stress treatments and tissue 195
disruptions to trigger GLV formation When crude extracts from Arabidopsis leaves 196
(No-0) were exposed to (Z)-3-hexenal exogenous NADPH was consumed (Fig 4) 197
Moreover the rate of NADH consumption was less than 20 of that found with 198
NADPH under the same reaction conditions Taken with our previous results (Matsui et 199
al 2012) this result indicated that Arabidopsis leaves carry a NADPH-dependent 200
(Z)-3-hexenal reductase 201
This reductase was purified from crude Arabidopsis leaf extracts using 202
ammonium sulfate fractionation followed by three consecutive chromatography steps 203
(Table I) During these steps reductase activity was identified as a single peak with a 204
substantial yield and SDS-PAGE analyses of fractions that were separated using a 205
HiTrap DEAE column in the third chromatography step indicated that a protein of 38 206
kDa correlated with the observed metabolic activity (Fig 2A) In the presence of 207
(Z)-3-hexenal the purified enzyme consumed NADPH most actively at pH 75 (Fig 208
2B) When a mixture of (Z)-3-hexenal and (E)-2-hexenal (154 and 46 microM respectively) 209
was used as a substrate in the absence of cofactor formation of the corresponding 210
alcohol was hardly detected (Fig 2C) Addition of NADH slightly facilitated reduction 211
of hexenals whereas NADPH resulted in more extensive formation of (Z)-3-hexen-1-ol 212
and (E)-2-hexen-1-ol (Fig 2D) However n-hexanal formation through reduction of the 213
carbon-carbon double bond in (Z)-3-hexenal and (E)-2-hexenal was barely increased 214
indicating that the enzyme has little double bond reductase activity 215
216
Identification of C6-aldehyde reductase gene 217
Internal amino acid sequences from the purified protein showed complete 218
identity with Arabidopsis CINNAMYL ALCOHOL DEHYDROGENASE 7 (AtCAD7 219
At4g37980 Supplemental Fig S1) The AtCAD7 gene is also known as 220
ELICITOR-ACTIVATED GENE 3 (ELI3 Kiedrowski et al 1992) and the protein 221
comprises 357 amino acids and has a molecular weight of 38245 Da close to that of the 222
purified enzyme estimated in SDS-PAGE analyses (Fig 2A) A MotifFinder search 223 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224
contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225
based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226
A recombinant protein encoded by the open reading frame of At4g37980 was 227
expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228
(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229
carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230
(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231
little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232
examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233
and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234
enzyme exhibited relatively high activities with aromatic aldehydes such as 235
2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236
sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237
biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238
2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239
aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240
Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241
n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242
to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243
enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244
HEXENAL REDUCTASE (CHR) 245
246
Involvement of CHR in GLV composition 247
To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248
insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249
S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250
differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251
Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252
impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253
Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254
for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255
selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
INTRODUCTION 92
93
Plants form and emit a wide variety of volatile organic compounds Volatiles 94
from flowers attract pollinators and increase the fitness of plants by promoting efficient 95
reproduction and those from fruits attract seed-dispersing animals and help plants to 96
find new habitats (Dudareva et al 2013) Vegetative organs such as leaves stems and 97
roots also produce and emit volatiles and this process is generally induced by various 98
types of biotic and abiotic stresses as a defense response (Pierik et al 2014) 99
Green leaf volatiles (GLVs) are ubiquitously expressed with other groups of 100
volatile compounds such as terpenoids and amino acid derivatives GLVs are 101
derivatives of fatty acids and include six-carbon (C6) aldehydes alcohols and esters 102
(Fig 1 Matsui 2006 Scala et al 2013) In intact and healthy plant tissues GLV levels 103
are generally low but when tissues suffer stresses associated with disruption of cells 104
such as herbivore damage or attack by necrotrophic fungi GLV-forming pathways are 105
rapidly activated to yield large quantities of GLVs at damaged tissues (Matsui 2006 106
Scala et al 2013 Ameye et al 2017) GLVs at damage tissues participate in direct 107
plant defense by preventing the invasion of harmful microbes (Shiojiri et al 2006a 108
Kishimoto et al 2008) GLVs also function as infochemicals that are an indirect plant 109
defense via the attraction of natural predators of infesting herbivores (Kessler amp 110
Baldwin 2001 Shiojiri et al 2006a Shiojiri et al 2006b Joo et al 2018) GLVs have 111
also been associated with plant-plant communication (Sugimoto et al 2013) 112
Because the chemical properties of GLV components are distinct it is 113
assumed that each plays a distinctive role For example C6-aldehydes were shown to be 114
prominently involved in direct defense against the necrotrophic fungal pathogen 115
Botrytis cinerea whereas C6-alcohols and esters were less involved (Kishimoto et al 116
2008) The formyl groups in C6-aldehydes are potently reactive with nucleophiles such 117
as amines and alcohols and contribute to the suppression of pathogen growth When 118
carbon-carbon double bonds are conjugated to the formyl group as in (E)-2-hexenal 119
the αβ-unsaturated carbonyl moiety behaves as a more potent electrophile and is prone 120
to reactions with various biological molecules specifically reactions that eliminate their 121
intrinsic functions (Farmer and Muumlller 2013) Even though (Z)-3-hexenal is not a 122
reactive carbonyl species because the two double bonds are interrupted with one 123
methylene group bis-allylic hydrogen is prone to removal often resulting in 124 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
oxygenation to yield 4-hydroxy-(E)-2-hexenal or 4-oxo-(E)-2-hexenal (Pospiacutesil amp 125
Yamamoto 2017) These oxygenated hexenals are highly reactive carbonyl species and 126
(Z)-3-hexenal has specifically been identified as a potentially reactive compound 127
(Matsui et al 2012) C6-aldehydes were reported to be more effective bacteriostatic 128
chemicals than C6-alcohols and incorporation of a double bond either in the C2 or C3 129
position enhances their activities (Nakamura and Hatanaka 2002) albeit the formyl 130
group is not always a prerequisite for antimicrobial activity (Prost et al 2005) 131
GLVs are either attractive or repulsive to arthropods depending on their 132
composition and on the arthropod species (Wei amp Kang 2011 Scala et al 2013) 133
Females of the parasitic wasp Cotesia glomerata are attracted to (E)-2-hexenal and 134
(Z)-3-hexen-1-yl acetate but not to (Z)-3-hexen-1-ol (Shiojiri et al 2006) Moreover 135
(Z)-3-Hexen-1-yl acetate increased predation rates of a generalist natural predator 136
toward eggs of the herbivore Manduca quinquemaculata on Nicotiana attenuata plants 137
(Kessler and Baldwin 2001) Composition of GLVs elicited by herbivores also differ 138
between dawn and dusk as indicated by Joo et al (2018) who showed that GLV 139
aldehydes and alcohols were less abundant in comparison with GLV esters at dawn 140
whereas GLV ester levels were lower at dusk Higher predation rates on M sexta eggs 141
by Geocoris spp were observed with dawn GLV composition in nature suggesting that 142
the predator distinguished between GLV compositions (Joo et al 2018) 143
Concerning plant-plant communication (Z)-3-hexen-1-ol emitted from 144
Spodoptera litura-infested tomato leaves was shown to be absorbed by neighboring 145
tomato plants to form the defense compound (Z)-3-hexen-1-yl primeveroside (Sugimoto 146
et al 2013) It was also observed that the secretion of extrafloral nectar by lima beans 147
(Phaseolus lunatus) to attract predatory and parasitoid insects (ants and wasps) was 148
significantly induced after exposure of these plants to (Z)-3-hexen-1-yl acetate vapors 149
(Kost amp Heil 2006) These accumulating lines of evidence indicate that composition of 150
GLVs is crucial for plants to aptly cope with enemies and foes and that reduction of 151
C6-aldehydes to C6-alcohol is a crucial step in the control of GLV composition 152
Lipoxygenase (LOX) acts on linoleniclinoleic acids either in their free forms 153
or as acyl groups in glycerolipids to form corresponding fatty acidlipid hydroperoxides 154
which are subsequently cleaved by hydroperoxide lyase (HPL) to form C6-aldehydes 155
such as n-hexanal or (Z)-3-hexenal (Fig 1 Matsui 2006 Scala et al 2013 Mwenda amp 156
Matsui 2014) A portion of (Z)-3-hexenal is isomerized to (E)-2-hexenal in some plant 157 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
species (Kunishima et al 2016 Spyropoulou et al 2017) and a factor in oral 158
secretions of tobacco hawkmoth (Manduca sexta) catalyzes this isomerization in 159
Nicotiana attenuata resulting in higher attraction of the predator Geocoris spp 160
(Allmann amp Baldwin 2011) Other portions of C6-aldehydes are reduced to 161
corresponding C6-alcohols and portions of C6-alcohols are further converted into 162
corresponding acetates by acetyl-CoA transferase (DrsquoAuria et al 2007) It was assumed 163
that NAD(H)-dependent alcohol dehydrogenase (EC 1111) is involved in these 164
reduction steps (Hatanaka 1993) and this may be the case with completely disrupted 165
plant tissues Accordingly in completely homogenized leaves of Arabidopsis mutants 166
that were deficient in alcohol dehydrogenase (AtADH1 At1g77120) n-hexan-1-ol and 167
3-hexen-1-ol were formed at 62 and 51 of the levels found in parent wild-type 168
plants respectively (Bate et al 1998) In completely homogenized tomato tissues with 169
increased and suppressed expression levels of SlADH2 which is the tomato homolog of 170
AtADH1 n-hexan-1-ol and (Z)-3-hexen-1-ol levels roughly correlated with ADH 171
activities (Speirs et al 1998) In contrast in partially wounded leaf tissues of 172
Arabidopsis (Z)-3-hexenal was formed at disrupted tissues and diffused into the 173
neighboring intact tissues where it was reduced to (Z)-3-hexen-1-ol in an 174
NADPH-dependent manner (Matsui et al 2012) Hence an NADPH-dependent 175
C6-aldehyde reductase is likely accountable for the reduction of C6-aldehydes to 176
C6-alcohols Because plant leaves are commonly damaged only in part such as 177
following consumption by an herbivore such a NADPH-dependent reductase may be 178
ecophysiologically relevant but has not been identified yet In this study we purified 179
and identified a NADPH-dependent reductase preferring (Z)-3-hexenal which we 180
named CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) Involvement of 181
CHR in changing the composition of GLVs was verified using a T-DNA knockout 182
mutant disrupted in the corresponding CHR gene Morover chr mutant lines were 183
employed to assess the role of CHR in indirect defense of Arabidopsis via GLV 184
composition CHR involvement in detoxification of (Z)-3-hexenal was also examined 185
186
187
RESULTS 188
189
Purification of hexenal reductase 190 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
Following exposure to (Z)-3-hexenal vapors intact Arabidopsis leaves absorb 191
the compound into leaves reduce it to (Z)-3-hexen-1-ol and then re-emit it from the 192
leaves to the atmosphere (Matsui et al 2012) Therefore substantial enzyme activity 193
for converting (Z)-3-hexenal to (Z)-3-hexen-1-ol is already present in Arabidopsis 194
leaves under normal growth conditions even without stress treatments and tissue 195
disruptions to trigger GLV formation When crude extracts from Arabidopsis leaves 196
(No-0) were exposed to (Z)-3-hexenal exogenous NADPH was consumed (Fig 4) 197
Moreover the rate of NADH consumption was less than 20 of that found with 198
NADPH under the same reaction conditions Taken with our previous results (Matsui et 199
al 2012) this result indicated that Arabidopsis leaves carry a NADPH-dependent 200
(Z)-3-hexenal reductase 201
This reductase was purified from crude Arabidopsis leaf extracts using 202
ammonium sulfate fractionation followed by three consecutive chromatography steps 203
(Table I) During these steps reductase activity was identified as a single peak with a 204
substantial yield and SDS-PAGE analyses of fractions that were separated using a 205
HiTrap DEAE column in the third chromatography step indicated that a protein of 38 206
kDa correlated with the observed metabolic activity (Fig 2A) In the presence of 207
(Z)-3-hexenal the purified enzyme consumed NADPH most actively at pH 75 (Fig 208
2B) When a mixture of (Z)-3-hexenal and (E)-2-hexenal (154 and 46 microM respectively) 209
was used as a substrate in the absence of cofactor formation of the corresponding 210
alcohol was hardly detected (Fig 2C) Addition of NADH slightly facilitated reduction 211
of hexenals whereas NADPH resulted in more extensive formation of (Z)-3-hexen-1-ol 212
and (E)-2-hexen-1-ol (Fig 2D) However n-hexanal formation through reduction of the 213
carbon-carbon double bond in (Z)-3-hexenal and (E)-2-hexenal was barely increased 214
indicating that the enzyme has little double bond reductase activity 215
216
Identification of C6-aldehyde reductase gene 217
Internal amino acid sequences from the purified protein showed complete 218
identity with Arabidopsis CINNAMYL ALCOHOL DEHYDROGENASE 7 (AtCAD7 219
At4g37980 Supplemental Fig S1) The AtCAD7 gene is also known as 220
ELICITOR-ACTIVATED GENE 3 (ELI3 Kiedrowski et al 1992) and the protein 221
comprises 357 amino acids and has a molecular weight of 38245 Da close to that of the 222
purified enzyme estimated in SDS-PAGE analyses (Fig 2A) A MotifFinder search 223 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224
contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225
based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226
A recombinant protein encoded by the open reading frame of At4g37980 was 227
expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228
(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229
carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230
(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231
little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232
examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233
and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234
enzyme exhibited relatively high activities with aromatic aldehydes such as 235
2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236
sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237
biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238
2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239
aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240
Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241
n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242
to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243
enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244
HEXENAL REDUCTASE (CHR) 245
246
Involvement of CHR in GLV composition 247
To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248
insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249
S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250
differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251
Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252
impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253
Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254
for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255
selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
oxygenation to yield 4-hydroxy-(E)-2-hexenal or 4-oxo-(E)-2-hexenal (Pospiacutesil amp 125
Yamamoto 2017) These oxygenated hexenals are highly reactive carbonyl species and 126
(Z)-3-hexenal has specifically been identified as a potentially reactive compound 127
(Matsui et al 2012) C6-aldehydes were reported to be more effective bacteriostatic 128
chemicals than C6-alcohols and incorporation of a double bond either in the C2 or C3 129
position enhances their activities (Nakamura and Hatanaka 2002) albeit the formyl 130
group is not always a prerequisite for antimicrobial activity (Prost et al 2005) 131
GLVs are either attractive or repulsive to arthropods depending on their 132
composition and on the arthropod species (Wei amp Kang 2011 Scala et al 2013) 133
Females of the parasitic wasp Cotesia glomerata are attracted to (E)-2-hexenal and 134
(Z)-3-hexen-1-yl acetate but not to (Z)-3-hexen-1-ol (Shiojiri et al 2006) Moreover 135
(Z)-3-Hexen-1-yl acetate increased predation rates of a generalist natural predator 136
toward eggs of the herbivore Manduca quinquemaculata on Nicotiana attenuata plants 137
(Kessler and Baldwin 2001) Composition of GLVs elicited by herbivores also differ 138
between dawn and dusk as indicated by Joo et al (2018) who showed that GLV 139
aldehydes and alcohols were less abundant in comparison with GLV esters at dawn 140
whereas GLV ester levels were lower at dusk Higher predation rates on M sexta eggs 141
by Geocoris spp were observed with dawn GLV composition in nature suggesting that 142
the predator distinguished between GLV compositions (Joo et al 2018) 143
Concerning plant-plant communication (Z)-3-hexen-1-ol emitted from 144
Spodoptera litura-infested tomato leaves was shown to be absorbed by neighboring 145
tomato plants to form the defense compound (Z)-3-hexen-1-yl primeveroside (Sugimoto 146
et al 2013) It was also observed that the secretion of extrafloral nectar by lima beans 147
(Phaseolus lunatus) to attract predatory and parasitoid insects (ants and wasps) was 148
significantly induced after exposure of these plants to (Z)-3-hexen-1-yl acetate vapors 149
(Kost amp Heil 2006) These accumulating lines of evidence indicate that composition of 150
GLVs is crucial for plants to aptly cope with enemies and foes and that reduction of 151
C6-aldehydes to C6-alcohol is a crucial step in the control of GLV composition 152
Lipoxygenase (LOX) acts on linoleniclinoleic acids either in their free forms 153
or as acyl groups in glycerolipids to form corresponding fatty acidlipid hydroperoxides 154
which are subsequently cleaved by hydroperoxide lyase (HPL) to form C6-aldehydes 155
such as n-hexanal or (Z)-3-hexenal (Fig 1 Matsui 2006 Scala et al 2013 Mwenda amp 156
Matsui 2014) A portion of (Z)-3-hexenal is isomerized to (E)-2-hexenal in some plant 157 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
species (Kunishima et al 2016 Spyropoulou et al 2017) and a factor in oral 158
secretions of tobacco hawkmoth (Manduca sexta) catalyzes this isomerization in 159
Nicotiana attenuata resulting in higher attraction of the predator Geocoris spp 160
(Allmann amp Baldwin 2011) Other portions of C6-aldehydes are reduced to 161
corresponding C6-alcohols and portions of C6-alcohols are further converted into 162
corresponding acetates by acetyl-CoA transferase (DrsquoAuria et al 2007) It was assumed 163
that NAD(H)-dependent alcohol dehydrogenase (EC 1111) is involved in these 164
reduction steps (Hatanaka 1993) and this may be the case with completely disrupted 165
plant tissues Accordingly in completely homogenized leaves of Arabidopsis mutants 166
that were deficient in alcohol dehydrogenase (AtADH1 At1g77120) n-hexan-1-ol and 167
3-hexen-1-ol were formed at 62 and 51 of the levels found in parent wild-type 168
plants respectively (Bate et al 1998) In completely homogenized tomato tissues with 169
increased and suppressed expression levels of SlADH2 which is the tomato homolog of 170
AtADH1 n-hexan-1-ol and (Z)-3-hexen-1-ol levels roughly correlated with ADH 171
activities (Speirs et al 1998) In contrast in partially wounded leaf tissues of 172
Arabidopsis (Z)-3-hexenal was formed at disrupted tissues and diffused into the 173
neighboring intact tissues where it was reduced to (Z)-3-hexen-1-ol in an 174
NADPH-dependent manner (Matsui et al 2012) Hence an NADPH-dependent 175
C6-aldehyde reductase is likely accountable for the reduction of C6-aldehydes to 176
C6-alcohols Because plant leaves are commonly damaged only in part such as 177
following consumption by an herbivore such a NADPH-dependent reductase may be 178
ecophysiologically relevant but has not been identified yet In this study we purified 179
and identified a NADPH-dependent reductase preferring (Z)-3-hexenal which we 180
named CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) Involvement of 181
CHR in changing the composition of GLVs was verified using a T-DNA knockout 182
mutant disrupted in the corresponding CHR gene Morover chr mutant lines were 183
employed to assess the role of CHR in indirect defense of Arabidopsis via GLV 184
composition CHR involvement in detoxification of (Z)-3-hexenal was also examined 185
186
187
RESULTS 188
189
Purification of hexenal reductase 190 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
Following exposure to (Z)-3-hexenal vapors intact Arabidopsis leaves absorb 191
the compound into leaves reduce it to (Z)-3-hexen-1-ol and then re-emit it from the 192
leaves to the atmosphere (Matsui et al 2012) Therefore substantial enzyme activity 193
for converting (Z)-3-hexenal to (Z)-3-hexen-1-ol is already present in Arabidopsis 194
leaves under normal growth conditions even without stress treatments and tissue 195
disruptions to trigger GLV formation When crude extracts from Arabidopsis leaves 196
(No-0) were exposed to (Z)-3-hexenal exogenous NADPH was consumed (Fig 4) 197
Moreover the rate of NADH consumption was less than 20 of that found with 198
NADPH under the same reaction conditions Taken with our previous results (Matsui et 199
al 2012) this result indicated that Arabidopsis leaves carry a NADPH-dependent 200
(Z)-3-hexenal reductase 201
This reductase was purified from crude Arabidopsis leaf extracts using 202
ammonium sulfate fractionation followed by three consecutive chromatography steps 203
(Table I) During these steps reductase activity was identified as a single peak with a 204
substantial yield and SDS-PAGE analyses of fractions that were separated using a 205
HiTrap DEAE column in the third chromatography step indicated that a protein of 38 206
kDa correlated with the observed metabolic activity (Fig 2A) In the presence of 207
(Z)-3-hexenal the purified enzyme consumed NADPH most actively at pH 75 (Fig 208
2B) When a mixture of (Z)-3-hexenal and (E)-2-hexenal (154 and 46 microM respectively) 209
was used as a substrate in the absence of cofactor formation of the corresponding 210
alcohol was hardly detected (Fig 2C) Addition of NADH slightly facilitated reduction 211
of hexenals whereas NADPH resulted in more extensive formation of (Z)-3-hexen-1-ol 212
and (E)-2-hexen-1-ol (Fig 2D) However n-hexanal formation through reduction of the 213
carbon-carbon double bond in (Z)-3-hexenal and (E)-2-hexenal was barely increased 214
indicating that the enzyme has little double bond reductase activity 215
216
Identification of C6-aldehyde reductase gene 217
Internal amino acid sequences from the purified protein showed complete 218
identity with Arabidopsis CINNAMYL ALCOHOL DEHYDROGENASE 7 (AtCAD7 219
At4g37980 Supplemental Fig S1) The AtCAD7 gene is also known as 220
ELICITOR-ACTIVATED GENE 3 (ELI3 Kiedrowski et al 1992) and the protein 221
comprises 357 amino acids and has a molecular weight of 38245 Da close to that of the 222
purified enzyme estimated in SDS-PAGE analyses (Fig 2A) A MotifFinder search 223 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224
contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225
based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226
A recombinant protein encoded by the open reading frame of At4g37980 was 227
expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228
(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229
carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230
(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231
little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232
examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233
and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234
enzyme exhibited relatively high activities with aromatic aldehydes such as 235
2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236
sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237
biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238
2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239
aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240
Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241
n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242
to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243
enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244
HEXENAL REDUCTASE (CHR) 245
246
Involvement of CHR in GLV composition 247
To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248
insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249
S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250
differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251
Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252
impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253
Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254
for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255
selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
species (Kunishima et al 2016 Spyropoulou et al 2017) and a factor in oral 158
secretions of tobacco hawkmoth (Manduca sexta) catalyzes this isomerization in 159
Nicotiana attenuata resulting in higher attraction of the predator Geocoris spp 160
(Allmann amp Baldwin 2011) Other portions of C6-aldehydes are reduced to 161
corresponding C6-alcohols and portions of C6-alcohols are further converted into 162
corresponding acetates by acetyl-CoA transferase (DrsquoAuria et al 2007) It was assumed 163
that NAD(H)-dependent alcohol dehydrogenase (EC 1111) is involved in these 164
reduction steps (Hatanaka 1993) and this may be the case with completely disrupted 165
plant tissues Accordingly in completely homogenized leaves of Arabidopsis mutants 166
that were deficient in alcohol dehydrogenase (AtADH1 At1g77120) n-hexan-1-ol and 167
3-hexen-1-ol were formed at 62 and 51 of the levels found in parent wild-type 168
plants respectively (Bate et al 1998) In completely homogenized tomato tissues with 169
increased and suppressed expression levels of SlADH2 which is the tomato homolog of 170
AtADH1 n-hexan-1-ol and (Z)-3-hexen-1-ol levels roughly correlated with ADH 171
activities (Speirs et al 1998) In contrast in partially wounded leaf tissues of 172
Arabidopsis (Z)-3-hexenal was formed at disrupted tissues and diffused into the 173
neighboring intact tissues where it was reduced to (Z)-3-hexen-1-ol in an 174
NADPH-dependent manner (Matsui et al 2012) Hence an NADPH-dependent 175
C6-aldehyde reductase is likely accountable for the reduction of C6-aldehydes to 176
C6-alcohols Because plant leaves are commonly damaged only in part such as 177
following consumption by an herbivore such a NADPH-dependent reductase may be 178
ecophysiologically relevant but has not been identified yet In this study we purified 179
and identified a NADPH-dependent reductase preferring (Z)-3-hexenal which we 180
named CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) Involvement of 181
CHR in changing the composition of GLVs was verified using a T-DNA knockout 182
mutant disrupted in the corresponding CHR gene Morover chr mutant lines were 183
employed to assess the role of CHR in indirect defense of Arabidopsis via GLV 184
composition CHR involvement in detoxification of (Z)-3-hexenal was also examined 185
186
187
RESULTS 188
189
Purification of hexenal reductase 190 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
Following exposure to (Z)-3-hexenal vapors intact Arabidopsis leaves absorb 191
the compound into leaves reduce it to (Z)-3-hexen-1-ol and then re-emit it from the 192
leaves to the atmosphere (Matsui et al 2012) Therefore substantial enzyme activity 193
for converting (Z)-3-hexenal to (Z)-3-hexen-1-ol is already present in Arabidopsis 194
leaves under normal growth conditions even without stress treatments and tissue 195
disruptions to trigger GLV formation When crude extracts from Arabidopsis leaves 196
(No-0) were exposed to (Z)-3-hexenal exogenous NADPH was consumed (Fig 4) 197
Moreover the rate of NADH consumption was less than 20 of that found with 198
NADPH under the same reaction conditions Taken with our previous results (Matsui et 199
al 2012) this result indicated that Arabidopsis leaves carry a NADPH-dependent 200
(Z)-3-hexenal reductase 201
This reductase was purified from crude Arabidopsis leaf extracts using 202
ammonium sulfate fractionation followed by three consecutive chromatography steps 203
(Table I) During these steps reductase activity was identified as a single peak with a 204
substantial yield and SDS-PAGE analyses of fractions that were separated using a 205
HiTrap DEAE column in the third chromatography step indicated that a protein of 38 206
kDa correlated with the observed metabolic activity (Fig 2A) In the presence of 207
(Z)-3-hexenal the purified enzyme consumed NADPH most actively at pH 75 (Fig 208
2B) When a mixture of (Z)-3-hexenal and (E)-2-hexenal (154 and 46 microM respectively) 209
was used as a substrate in the absence of cofactor formation of the corresponding 210
alcohol was hardly detected (Fig 2C) Addition of NADH slightly facilitated reduction 211
of hexenals whereas NADPH resulted in more extensive formation of (Z)-3-hexen-1-ol 212
and (E)-2-hexen-1-ol (Fig 2D) However n-hexanal formation through reduction of the 213
carbon-carbon double bond in (Z)-3-hexenal and (E)-2-hexenal was barely increased 214
indicating that the enzyme has little double bond reductase activity 215
216
Identification of C6-aldehyde reductase gene 217
Internal amino acid sequences from the purified protein showed complete 218
identity with Arabidopsis CINNAMYL ALCOHOL DEHYDROGENASE 7 (AtCAD7 219
At4g37980 Supplemental Fig S1) The AtCAD7 gene is also known as 220
ELICITOR-ACTIVATED GENE 3 (ELI3 Kiedrowski et al 1992) and the protein 221
comprises 357 amino acids and has a molecular weight of 38245 Da close to that of the 222
purified enzyme estimated in SDS-PAGE analyses (Fig 2A) A MotifFinder search 223 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224
contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225
based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226
A recombinant protein encoded by the open reading frame of At4g37980 was 227
expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228
(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229
carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230
(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231
little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232
examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233
and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234
enzyme exhibited relatively high activities with aromatic aldehydes such as 235
2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236
sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237
biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238
2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239
aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240
Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241
n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242
to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243
enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244
HEXENAL REDUCTASE (CHR) 245
246
Involvement of CHR in GLV composition 247
To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248
insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249
S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250
differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251
Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252
impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253
Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254
for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255
selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
8
Following exposure to (Z)-3-hexenal vapors intact Arabidopsis leaves absorb 191
the compound into leaves reduce it to (Z)-3-hexen-1-ol and then re-emit it from the 192
leaves to the atmosphere (Matsui et al 2012) Therefore substantial enzyme activity 193
for converting (Z)-3-hexenal to (Z)-3-hexen-1-ol is already present in Arabidopsis 194
leaves under normal growth conditions even without stress treatments and tissue 195
disruptions to trigger GLV formation When crude extracts from Arabidopsis leaves 196
(No-0) were exposed to (Z)-3-hexenal exogenous NADPH was consumed (Fig 4) 197
Moreover the rate of NADH consumption was less than 20 of that found with 198
NADPH under the same reaction conditions Taken with our previous results (Matsui et 199
al 2012) this result indicated that Arabidopsis leaves carry a NADPH-dependent 200
(Z)-3-hexenal reductase 201
This reductase was purified from crude Arabidopsis leaf extracts using 202
ammonium sulfate fractionation followed by three consecutive chromatography steps 203
(Table I) During these steps reductase activity was identified as a single peak with a 204
substantial yield and SDS-PAGE analyses of fractions that were separated using a 205
HiTrap DEAE column in the third chromatography step indicated that a protein of 38 206
kDa correlated with the observed metabolic activity (Fig 2A) In the presence of 207
(Z)-3-hexenal the purified enzyme consumed NADPH most actively at pH 75 (Fig 208
2B) When a mixture of (Z)-3-hexenal and (E)-2-hexenal (154 and 46 microM respectively) 209
was used as a substrate in the absence of cofactor formation of the corresponding 210
alcohol was hardly detected (Fig 2C) Addition of NADH slightly facilitated reduction 211
of hexenals whereas NADPH resulted in more extensive formation of (Z)-3-hexen-1-ol 212
and (E)-2-hexen-1-ol (Fig 2D) However n-hexanal formation through reduction of the 213
carbon-carbon double bond in (Z)-3-hexenal and (E)-2-hexenal was barely increased 214
indicating that the enzyme has little double bond reductase activity 215
216
Identification of C6-aldehyde reductase gene 217
Internal amino acid sequences from the purified protein showed complete 218
identity with Arabidopsis CINNAMYL ALCOHOL DEHYDROGENASE 7 (AtCAD7 219
At4g37980 Supplemental Fig S1) The AtCAD7 gene is also known as 220
ELICITOR-ACTIVATED GENE 3 (ELI3 Kiedrowski et al 1992) and the protein 221
comprises 357 amino acids and has a molecular weight of 38245 Da close to that of the 222
purified enzyme estimated in SDS-PAGE analyses (Fig 2A) A MotifFinder search 223 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224
contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225
based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226
A recombinant protein encoded by the open reading frame of At4g37980 was 227
expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228
(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229
carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230
(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231
little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232
examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233
and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234
enzyme exhibited relatively high activities with aromatic aldehydes such as 235
2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236
sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237
biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238
2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239
aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240
Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241
n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242
to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243
enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244
HEXENAL REDUCTASE (CHR) 245
246
Involvement of CHR in GLV composition 247
To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248
insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249
S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250
differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251
Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252
impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253
Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254
for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255
selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224
contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225
based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226
A recombinant protein encoded by the open reading frame of At4g37980 was 227
expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228
(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229
carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230
(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231
little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232
examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233
and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234
enzyme exhibited relatively high activities with aromatic aldehydes such as 235
2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236
sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237
biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238
2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239
aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240
Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241
n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242
to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243
enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244
HEXENAL REDUCTASE (CHR) 245
246
Involvement of CHR in GLV composition 247
To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248
insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249
S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250
differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251
Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252
impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253
Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254
for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255
selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257
homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258
that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259
extracts from chr hpl and chr HPL plants however were not significantly different 260
from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261
Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262
emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263
these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264
significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265
of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266
mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267
depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268
by the presence of functional CHR Because total amounts of GLVs differed between 269
these two genotypes we investigated whether their abilities to form C6-aldehydes 270
through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271
HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272
step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273
NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274
1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275
and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276
levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277
Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278
were slightly higher than that for No-0 leaves suggesting that the ability to form 279
(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281
wounding of chr HPL leaves 282
283
CHR is involved in indirect defenses 284
We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285
emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286
diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287
females preferred P xylostella-damaged plants over intact plants but did not show any 288
preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290
xylostella-infested plants and intact plants did not differ significantly Examinations of 291
wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292
HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293
showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294
HPL plants (Supplemental Fig S7) 295
Quantities of volatiles emitted at 24 h after the onset of P xylostella 296
infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297
volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298
conditions employed here with the selected ion monitoring mode of GC-MS In these 299
analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300
glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301
genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302
whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303
HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304
305
Involvement of CHR in detoxification of hexenal 306
The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307
indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308
(Z)-3-hexenal vapor at 133 or 267 nmol cm-3
FvFm remained relatively unchanged 309
FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3
310
(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311
(chr hpl) treated with 133 nmol cm-3
(Z)-3-hexenal maintained normal FvFm values 312
however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313
(Z)-3-hexenal Following treatment with 400 nmol cm-3
(Z)-3-hexenal FvFm of the 314
mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315
FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316
hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317
symptoms after treatment with 133 nmol cm-3
(Z)-3-hexenal (Fig 7B) However 318
treatment with 267 nmol cm-3
(Z)-3-hexenal caused the leaves of mutant plants to wilt 319
slightly and their curl at their margins and 400 nmol cm-3
(Z)-3-hexenal treatment 320
caused obvious damage in both genotypes with more serious effects seen in the mutant 321
plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
12
323
324
DISCUSSION 325
In this study we confirmed that At4g37980 which had been previously designated 326
CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327
aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328
Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329
lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330
knockout mutant plants with active HPL formed significantly lower concentrations of 331
(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332
leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333
knockout mutant plants with active HPL These results indicate that the enzyme 334
encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335
that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336
the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337
CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338
dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339
[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340
Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341
against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342
caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343
were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344
AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345
floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346
corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347
contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348
corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349
observations this study indicates that CHR has limited involvement in lignin 350
biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351
(Z)-3-hexen-1-ol during GLV-bursts 352
BLASTP searches were performed against NCBI Protein Reference 353
Sequences and those with BLAST scores of more than 250 and with biochemically 354
confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
13
sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356
the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357
Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358
AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359
et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360
In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361
confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362
CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363
including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364
roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365
dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366
(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367
as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368
alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369
which were thought to be involved in the lignin biosynthesis were also located within 370
the clade however their in vivo functions have not been completely confirmed and for 371
example AtCAD8 knockout mutants showed no differences in lignin contents compared 372
with that in corresponding wild-type plants even though recombinant AtCAD8 had 373
slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374
2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375
involved in monolignol synthesis whereas those encoding oxidoreductases of the 376
specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377
take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378
metabolites In further studies TBLASTN searches were performed using CHR as a 379
query with the whole genome sequences of six representative plant species (Populus 380
trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381
Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382
scores of more than 250 also gave a tree with two clades comprising ORSM family and 383
true CAD family members (Supplementary Fig S8) These observations imply that 384
ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385
and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386
the present genes 387
Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
14
thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389
biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390
Accordingly several studies examined the effects of mutations or genetic manipulation 391
of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392
tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393
complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394
from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395
was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396
involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397
that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398
reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399
of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400
plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401
increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402
freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403
did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404
results suggest that NADH-dependent ADH is at least partly involved in the reduction 405
of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406
previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407
wounding of leaves and diffuse into neighboring intact tissues where reduction 408
proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409
This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410
C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411
C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412
performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413
NADPH-dependent CHR respectively Further studies are expected to extend these 414
findings into other plant species 415
GLVs are employed by some herbivores to find food and by some predators 416
and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417
vary depending on each component of GLVs For example C glomerata is a parasitoid 418
of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419
but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420
of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
15
predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422
attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423
was favored under certain conditions (Yang et al 2016) In this context it is assumed 424
that adjustment of GLV composition is beneficial for plants allowing management of 425
both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426
plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427
production in response to infestations of P xylostella larvae was lower in knockout 428
mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429
from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430
vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431
Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432
C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433
formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434
required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435
a role in this Arabidopsis plant-herbivore-carnivore system 436
We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437
Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438
reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439
reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440
exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441
significant malfunctions of photosynthesis likely due to their inability to reduce 442
(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443
phase in this study specifically 267 and 400 nmol cm-3
at which a difference in 444
susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445
However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446
tissues can reach up to ca 15 micromol cm-3
(Matsui et al 2012 Mochizuki et al 2016) 447
therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448
concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449
of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450
least under the assay conditions employed in this study Nevertheless ecophysiological 451
significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452
studies These may include direct determination of (Z)-3-hexenal concentration in intact 453
leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
16
amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455
phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456
harmful effects on leaf tissues 457
458
459
EXPERIMENTAL PROCEDURES 460
Plant materials 461
Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462
T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463
the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464
wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465
volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466
(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467
CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468
population using genomic PCR analyses with the primers listed in Supplementary Table 469
S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470
plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471
grown in a chamber at 22degC under fluorescent lights (60 micromol m-2
s-1
) with a 14-h 472
light10-h dark photoperiod 473
474
475
Enzyme assays 476
Hexenal reductase activity was monitored according to the oxidation of NADPH by 477
continuously following decreases in absorption at 340 nm using a photometer 478
(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479
using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480
concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481
Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482
assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483
the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484
absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485
recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486
10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
17
specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488
and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489
Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490
mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491
activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492
constructed and analyzed using Hyper 32 (version 100) 493
Enzyme activities were also evaluated using GC-MS analyses of the products 494
Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495
with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496
20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497
volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498
incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499
CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500
(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501
fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502
to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503
GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504
m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505
column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506
min-1
to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507
of 1 mL min-1
The glass insert was an SPME Sleeve (Supelco) and splitless injections 508
were performed with a sampling time of 1 min To remove all compounds from the 509
matrix the fiber was held in the injection port for 10 min Injector and interface 510
temperatures were 200C and 230C respectively The mass detector was operated in 511
electron impact mode with an ionization energy of 70 eV Compounds were identified 512
using retention indexes and MS profiles of corresponding authentic specimens and 513
quantitative analyses were performed with standard curves that were generated for each 514
compound using aqueous suspensions containing Tween 20 Standard solutions were 515
mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516
solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517
above and calibration curves were constructed for each compound 518
519
Purification of hexenal reductase 520 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
18
Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521
were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522
in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523
phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524
Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525
cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526
Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527
extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528
dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529
(pH 80) and 4 mM dithiothreitol 530
After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531
rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532
650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533
ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534
buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535
sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536
desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537
equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538
Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539
potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540
columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541
10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542
dithiothreitol 543
Fractions with substantial activity were combined concentrated using an 544
Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545
column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546
Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547
washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548
NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549
SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550
(see Fig 1A) 551
After purification the protein band of 38 kDa excised from the SDS-PAGE 552
gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
19
using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554
performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555
NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556
nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557
Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558
Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559
linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1
560
Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561
(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562
nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563
Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564
were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565
spectra were generated by collision-induced dissociation in the linear ion trap To 566
identify the protein MS and MSMS data were converted to a MGF file using 567
NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568
then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569
with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570
taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571
oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572
Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573
574
cDNA cloning 575
Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576
Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577
degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578
USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579
(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580
5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581
5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582
sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583
T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584
then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585
resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
20
were grown in Luria broth supplemented with kanamycin (50 microg mL-1
) and 587
chloramphenicol (30 microg mL-1
) at 37degC until the optical density at 600 nm reached 06ndash588
08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589
was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590
Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591
resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592
045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1
lysozyme After 593
incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594
with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595
then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596
applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597
25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598
After washing the column in the same buffer the recombinant Arabidopsis CHR 599
enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600
601
Volatiles from leaves 602
Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603
cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604
mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605
avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606
wounded using forceps without injuring the mid vein The jar was then tightly closed 607
and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608
headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609
using GC-MS under the conditions used in enzyme assays Molecular ion 610
chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611
and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612
quantification Calibration curves were constructed from GC-MS analyses under the 613
same conditions by placing known amounts of authentic compounds [suspended in 614
01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615
formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616
homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617
and pestle To determine volatiles in intact leaves buffer was replaced with solution 618
containing 1 g mL-1
CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
21
transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620
twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621
tert-butyl ether containing 1 microg mL-1
of nonanyl acetate GC-MS analyses were then 622
performed as described above 623
To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624
had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625
diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626
30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627
described above but in the selected ion monitoring (SIM) mode with mz 6910 628
[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]
+ 629
630
Flight responses of C vestalis 631
P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632
and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633
with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634
vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635
parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637
for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638
mm diameter and 130 mm length containing honey and were kept in a 639
climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640
darkness Females were never kept for more than 10 d At least 1 h before the start of 641
each experiment oviposition-inexperienced female wasps were transferred to another 642
climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643
continuous light 644
Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645
plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646
times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647
plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648
(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649
xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650
were removed and the cleaned plants were used as infested plants The other group of 651
plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
the two groups of plants and the first landing by each wasp on a plant was recorded as 653
its choice of the two groups Upon landing on a plant wasps were immediately removed 654
from the cage using an insect aspirator If the wasp did not land on any of the plants 655
within 30 min it was recorded as no-choice The trial was repeated for four times and 656
plants were replaced for each trial Preferences of female C vestalis for volatiles 657
emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658
for 24 h were also assessed 659
660
Susceptibility to (Z)-3-hexenal vapor 661
Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662
fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663
(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664
were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665
duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2
666
s-1
Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667
(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668
(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669
The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670
light (60 micromol m-2
s-1
) For each plant fluorescence measurements were conducted at 671
the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672
after the onset of treatment The plants were dark-acclimated for at least 15 min before 673
PAM analysis 674
675
Phylogenetic analysis 676
Amino acid sequences of selected proteins were initially aligned using the multiple 677
sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678
alignments were inspected and manually edited to remove gaps and unconserved 679
regions using the alignment editing program AliView 121 (Larsson 2014) 680
Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681
326 (Huelsenbeck and Ronquist 2001) under the WAG model 682
683
ACCESSION NUMBERS 684
Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
accession numbers NP_195511 686
687
688
689
690
691
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
SUPPLEMENTAL DATA 692
693
The following supplemental materials are available 694
695
Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696
697
Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698
protein with a His-tag following isolation from E coli 699
700
Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701
leaves 702
703
Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704
(Z)-3-hexenal n-heptanal and cinnamaldehyde 705
706
Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707
mutant plants 708
709
Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710
leaves 711
712
Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713
714
Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715
716
Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717
718
Supplemental Table S1 Primer sequences used for genotyping 719
720
Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721
8 722
723
Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
In this research we used the supercomputer of ACCMS Kyoto University Japan for 725
phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726
State University for English proofreading 727
728
FIGURE LEGENDS 729
Figure 1 The Green leaf volatile synthetic pathway in plants 730
Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731
yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732
to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733
lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734
enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735
reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736
(Z)-3-hexen-1-yl acetate 737
738
Figure 2 Purification and properties of hexenal reductase 739
A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740
volumes of fractions were loaded onto each lane and proteins were stained with 741
Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742
11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743
with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744
partially purified enzyme C GC-MS chromatograms of products formed from 745
(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746
quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747
(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748
activity are indicated by nd not detected 749
750
Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751
Data are presented as averages plusmn SE (n = 4) 752
753
Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Arabidopsis leaves 755
Activities were determined in four biological replicates (n = 4) and are presented as 756
averages plusmn SE Different letters indicate significant differences as identified using two 757
way ANOVA and Tukey post-hoc tests P lt 005 758
759
Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760
leaves 761
A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762
of each genotype) and partially wounded leaves (upper chromatogram of each 763
genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764
black and red respectively The inset shows a representative image of mutant (chr hpl) 765
plant after partial wounding B Quantities of green leaf volatiles from intact and 766
partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767
Different letters represent significant differences as identified using two way ANOVA 768
and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769
770
Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771
P xylostella infestation 772
A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773
Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774
above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775
(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776
001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777
subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778
(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779
plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780
headspaces of glass vials containing three plants were collected after a 24-h infestation 781
period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782
as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783
(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784
785
Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786
and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788
circles) plants were exposed to 0 133 267 or 400-nmol cm-3
(Z)-3-hexenal vapor in a 789
closed glass jars and photosystem II activity was estimated using the chlorophyll 790
fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791
identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792
significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793
mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794
400-nmol cm-3
(Z)-3-hexenal vapor 795
796
Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797
oxidoreductases involved in specialized metabolism (ORSMs) 798
Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799
as a query Proteins with BLAST scores of more than 250 were chosen and those 800
studied at least at the protein level were selected to construct the tree (Supplementary 801
Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802
mutant expression studies for the gene indicated by blue stars The scale bar represents 803
009 amino acid substitutions per site 804
805
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806
Purification step Total protein Total activity Specific
activity
Purification Yield
mg nkat nkatmg -Fold
Crude extract 1707 1062 166 1 100
Ammonium
sulfate
1634 592 260 058 557
Butyl-Toyopearl 837 900 108 173 847
Hydroxyapatite 028 212 662 119 199
HiTrap DEAE 0031 394 788 209 037
807
808
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809
cinnamaldehyde 810
Substrate Km (microM) Vmax (nkat mg-1
)
(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280
n-Heptanal 840 plusmn 3451 286 plusmn 5253
Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030
811
812
813
814
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
References 815
Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816
rapid isomerization of green leaf volatiles Science 329 1075-1078 817
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818
Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819
Phytol doi101111nph14671 820
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821
M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822
in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823
biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825
differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826
dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828
purification and substrate specificity Arch Biochem Biophys 216 605-615 829
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830
Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832
Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833
dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834
Biol 80 289-297 835
DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836
of a BAHD acyltransferase responsible for producing the green leaf volatile 837
(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839
(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840
downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841
Physiol 140 1484-1493 842
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843
thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844
817-838 845
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846
(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848
1529-1544 849
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850
metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852
throughput Nucl Acids Res 32 1792-1797 853
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854
for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855
Planta 225 23-39 856
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857
signaling Ann Rev Plant Biol 64 429-450 858
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859
34 1201-1218 860
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861
trees Bioinformatics 17 754-755 862
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863
of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865
aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866
characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868
Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869
robust indirect defence in nature Funct Ecol 32 136-149 870
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871
7 improvements in performance and usability Mol Biol Evol 30 722-780 872
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873
emissions in nature Science 291 2141-2144 874
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875
Rapid activation of a novel plant defense gene is strictrly dependent on the 876
Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878
B Kang C Lewis N G (2004) Functional reclassification of the putative 879
cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
Sci USA 101 1455-1460 881
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882
activities of C6-aldehydes are important constitutents for defense responses in 883
Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885
neighbouring plants J Ecol 94 619-628 886
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887
(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888
roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889
DOI101038srep08258 890
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891
Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892
leaf aldehyde in plants J Biol Chem 191 14023-14033 893
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894
non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895
9-18 896
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897
data sets Bioinformatics 30 3276-3278 898
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899
metabolism Curr Opin Plant Biol 9 274-280 900
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901
metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902
distinct ecophysiological requirements PLoS ONE 7 e36433 903
doi101371journalpone0036433 904
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905
Bot 51 659-668 906
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907
production of green leaf volatiles by lipase-dependent and independent pathway 908
Plant Biotechnol 31 445-452 909
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910
antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911
Agric Food Chem 50 7639-7644 912
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
community perspective Plant Cell Environ 37 1845-1853 914
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915
products Biochim Biophys Acta 1861 457-466 916
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917
Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918
Evaluation of the antimicrobial activities of plant oxylipins supports their 919
involvement in defense against pathogen Plant Physiol 139 1902-1913 920
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921
Completion of the seven-step pathway from tabersonine to the anticancer drug 922
precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923
6224-6229 924
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925
of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926
Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928
(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929
responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931
volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932
Mol Sci 14 17781-17811 933
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934
Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935
volatile biosynthesis in plants an approach for improving plant resistance against 936
both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938
(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939
searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940
plutellae J Chem Ecol 32 969-979 941
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942
L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943
dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944
mutants Plant Physiol 132 848-860 945
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947
involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948
2059-2076 949
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950
manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951
balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953
Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954
of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955
DOI103389fpls201701342 956
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957
(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958
localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959
336-341 960
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961
R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962
(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963
neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964
USA 111 7144-7149 965
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966
molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967
2725-2729 968
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969
F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970
Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971
saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972
1018-1039 973
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974
dehydrogenase Expression during fruit ripening and under hypoxic conditions 975
FEBS Lett 123 39-42 976
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977
Behav 6 369-371 978
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981
(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982
cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983
984
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
985
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983
Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved