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Stress- and pathway-specific impacts of impaired jasmonoyl-isoleucine (JA-Ile) 1
catabolism on defense signaling and biotic stress resistance in Arabidopsis 2
3
Valentin Marquis1†, Ekaterina Smirnova1†, Laure Poirier1, Julie Zumsteg1, Fabian 4
Schweizer2, Philippe Reymond2 and Thierry Heitz1* 5
6 1 Institut de Biologie Moléculaire des Plantes (IBMP) du CNRS, Université de Strasbourg, Strasbourg, 7 France 8 2 Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland 9
10
† These authors contributed equally to the work 11
12 *Author for correspondence: 13 [email protected] 14 15 16 Short title: Pathway-specific impact of JA-Ile catabolism on induced defense 17 18
19
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 28, 2019. ; https://doi.org/10.1101/686709doi: bioRxiv preprint
2
ABSTRACT 20
21
Jasmonate (JA) synthesis and signaling are essential for plant defense upregulation upon 22
herbivore or microbial attacks. Stress-induced accumulation of jasmonoyl-isoleucine (JA-Ile), 23
the bioactive hormonal form triggering major transcriptional changes, is often dynamic and 24
transient, due to the existence of potent removal mechanisms. Two distinct but interconnected 25
JA-Ile turnover pathways have been described in Arabidopsis, either via cytochrome P450 26
(CYP94)-mediated oxidation, or through deconjugation by the amidohydrolases (AH) IAR3 27
and ILL6. Their impact was not well known because of gene redundancy and compensation 28
mechanisms when each pathway was partially impaired. Here we address the consequences of 29
fully blocking either or both pathways on JA homeostasis and defense signaling in three mutant 30
backgrounds: a double iar3 ill6 (2ah) mutant, a triple cyp94b1 b3 c1 mutant (3cyp), and a newly 31
generated quintuple (5ko) mutant deficient in all known JA-Ile-degrading activities. These lines 32
behaved very differently in response to either mechanical wounding, insect attack or fungal 33
infection, highlighting the stress-specific contributions and impacts of JA-Ile catabolic 34
pathways. Deconjugation and oxidative pathways contributed additively to JA-Ile removal 35
upon wounding, but their genetic impairement had opposite impacts on Spodoptera littoralis 36
larvae feeding: 2ah line was more resistant whereas 3cyp was more susceptible to insect attack. 37
In contrast, 2ah, 5ko but not 3cyp overaccumulated JA-Ile upon inoculation by Botrytis cinerea, 38
yet 3cyp was most resistant to the fungus. Despite of building-up unprecedented JA-Ile levels, 39
5ko displayed near WT levels of resistance in both bioassays. Molecular and metabolic analysis 40
indicated that restrained JA-Ile catabolism resulted in enhanced defense and resistance levels 41
only if genes encoding JAZ or JAM negative regulators were not simultaneously 42
overstimulated. Our data demonstrate that despite of acting on a shared hormonal substrate, AH 43
or/and CYP94 deficiency differentially impacts JA homeostasis, responses and tolerance to 44
related biotic stresses. 45
46
47
48
INTRODUCTION 49
50
Jasmonates (JAs) have been recognized for more than two decades as powerful regulators of 51
inducible defense responses protecting plants from damage inflicted by herbivorous insects or 52
microbial pathogens (Campos et al. 2014; Wasternack and Hause 2013). Even before the 53
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3
identification of jasmonoyl-isoleucine (JA-Ile) as the critical bioactive form in 2007 (Chini et 54
al. 2007; Thines et al. 2007), many derivatives of jasmonic acid (JA) were described, resulting 55
from hydroxylation, conjugation to sugars (Miersch et al. 2008), amino acids or amino 56
cyclopropane carboxylic acid, sulfation (Gidda et al. 2003), decarboxylation and many more 57
(Wasternack and Song 2017). The enzymes responsible for these modifications have not all 58
been identified and the function of these derivatives, if any, are generally unknown. The master 59
regulator jasmonoyl-(L)-isoleucine (JA-Ile) results from a specific conjugation reaction of 60
jasmonic acid (JA) by the enzyme JASMONATE RESISTANT 1 (JAR1). In the core 61
perception and signaling pathways, JA-Ile acts as a ligand promoting the assembly of the co-62
receptor CORONATINE INSENSITIVE 1 (COI1) with distinct JASMONATE ZIM-DOMAIN 63
PROTEIN (JAZ) that otherwise powerfully repress target transcription factors and responses. 64
After assembly, COI1-JAZ is recruited into the E3 ubiquitin ligase SCFCOI1 that tags JAZs for 65
proteolytic degradation, and provides the basis for JA-Ile-responsive gene de-repression (Chini 66
et al. 2007; Thines et al. 2007). Several hundreds to thousands of genes are under this control, 67
and depending on developmental stage, organ, nature of stimulus, and crosstalks with other 68
hormones, a wide array of JAZ-transcription factor combinations provide specificity to the 69
system with only one major hormonal ligand (Chini et al. 2016). Distinct JA-Ile-triggered 70
networks regulate leaf defenses against different types of aggressors and are integrated by 71
separate sets of transcription factors (TF). MYC2, a bHLH type TF, integrates (together with 72
MYC3 and MYC4) simultaneous JA/abscisic acid stimuli and defines a wound/insect-specific 73
branch reflected by induction of markers such as vegetative storage protein (VSP); 74
ERF1/ORA59 TFs integrate concomitant JA/ethylene signals into a microbe-specific branch 75
probed by plant defensin PDF1.2 (Pieterse et al. 2012; Wasternack and Hause 2013). 76
77
JA signaling, like any hormonal pathway, needs to be tightly controlled in time and space, 78
particularly because defense upregulation is costly and linked to overall growth inhibition. This 79
antagonism was long thought to be merely imposed by limited resources that need to be re-80
allocated from developmental to defense sinks (Havko et al. 2016), but recent advances have 81
shown that additional hardwire connections control growth-defense tradeoffs (Campos et al. 82
2014; Guo et al. 2018). For appropriate control of JA responses, plants rely on a number of 83
negative feedback mechanisms. In addition to JAZ repressor proteins, other negative regulators 84
were identified that repress JA responses at the promoter level, including JAV (Yan et al. 2018) 85
and the JAM subclass of bHLH (Sasaki-Sekimoto et al. 2013) proteins. Another way to 86
terminate jasmonate action is at the metabolic level by eliminating the active ligand JA-Ile. The 87
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4
characterization of JA-Ile catabolic pathways has shed light on hormone homeostasis regulation 88
and has also provided additional complexity in the JA metabolic grid. Two distinct enzymatic 89
pathways are known to modify or degrade the JA-Ile hormone. One consists in a two step 90
oxidation of the terminal (w) carbon of the JA moiety by cytochrome P450 monooxygenases 91
of the CYP94 family. In Arabidopsis, CYP94B3 and to a minor extent CYP94B1 generate 92
12OH-JA-Ile as a main product whereas CYP94C1 catalyzes the full oxidation of JA-Ile to 93
12COOH-JA-Ile (Figure 1) (Bruckhoff et al. 2016; Heitz et al. 2012; Kitaoka et al. 2011; Koo 94
et al. 2011, 2014). The first oxidation product retains reduced receptor-assembly capacity and 95
gene-inducing activity (Aubert et al. 2015; Koo et al. 2011; Poudel et al. 2019), but the second 96
product proved fully inactive (Aubert et al. 2015; Koo et al. 2014). The second pathway is 97
defined by deconjugating activity, first identified in Nicotiana attenuata (Woldemariam et al. 98
2012) and subsequently characterized in Arabidopsis as mediated by the amidohydrolases IAR3 99
and ILL6 (Figure 1) (Widemann et al. 2013). These enzymes act on JA-Ile and other JA-amino 100
acid conjugates but also on 12OH-JA-Ile generated by CYP94Bs, releasing JA and 12OH-JA 101
from these respective substrates (Widemann et al. 2013; Zhang et al. 2016). The interconnection 102
of the two catabolic pathways generates increased metabolic complexity because many of the 103
above-mentioned JAs derive in fact from JA-Ile catabolism (Heitz et al. 2016; Widemann et al. 104
2013). Both JA-Ile oxidases and amidohydrolases genes are induced by environmental cues and 105
are co-regulated with the central regulon defined by JA pathway biosynthetic and signaling 106
genes. Their simultaneous enzymatic action shapes specific jasmonate patterns in different 107
tissues and stress conditions (Aubert et al. 2015; Heitz et al. 2012; Koo and Howe 2012; Koo 108
et al. 2014; Widemann et al. 2013, 2016). 109
110
Manipulating JA-Ile catabolic routes could be an attractive tool to engineer plants for enhanced 111
pathogen resistance or modulate other JA-dependent processes. Loss- or gain-of-function 112
mutant lines in CYP94 or AH genes have revealed profound impacts on JA metabolism but 113
contrasted consequences on JA responses. Arabidopsis lines ectopically overexpressing CYP94 114
or AH generally have reduced JA-Ile levels and a metabolic shift towards oxidized and/or 115
cleaved derivatives. As expected, increased turnover is correlated with attenuated JA responses, 116
for example increased sensitivity to Botrytis infection or to insect larvae feeding (Aubert et al. 117
2015; Koo et al. 2011). In comparison, analysis of single, double, or triple mutant lines has not 118
led to a unified conclusion as to how JA-Ile responses are impacted by gradual impairment of 119
JA-Ile catabolism. Deficiency in CYP94 or AH expression impacts JA homeostasis consistently 120
with their in vitro enzymatic activities. For example, cyp94b3 or b3c1 mutations lead to more 121
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5
JA-Ile upon wounding, concomitantly to reduced 12OH-JA-Ile and 12COOH-JA-Ile levels 122
(Heitz et al. 2012; Kitaoka et al. 2011; Koo et al. 2011). Double cyp94b3c1 and triple 123
cyp94b1b3c1 mutant plants exhibited slightly enhanced defense responses and tolerance to 124
fungal infection (Aubert et al. 2015). Strikingly, in another study, the double cyp94b1b3 and 125
triple cyp94b1b3c1 mutants exhibited deficient JA-Ile responses, questioning the current 126
signaling model (Poudel et al. 2016). On the other hand, AH-deficient lines have not yet been 127
tested for defense and resistance responses, so the contribution and impacts of this pathway are 128
unknown. 129
To overcome gene redundancy and compensation mechanisms between pathways that occur 130
frequently in JA metabolic mutants (Smirnova et al. 2017; Widemann et al. 2013), we 131
introduced in the present study new plant lines with higher order mutations (Figure 1): a double 132
iar3 ill6 mutant (thereafter called 2ah) impaired in the JA-Ile deconjugating pathway; and a 133
quintuple cyp94b1 cyp94b3 cyp94c1 iar3 ill6 (thereafter called 5ko) deficient in all 134
characterized enzymes turning over JA-Ile. We submitted these lines, along with the oxidation-135
deficient cyp94b1b3c1 mutant (thereafter called 3cyp), to parallel leaf stimulation by 136
mechanical wounding or B. cinerea infection, two environmental cues triggering strong JA 137
metabolism and signaling, but activating distinct transcriptional networks due to different cross-138
talks with other hormonal pathways (Pieterse et al. 2012). We determined the contribution of 139
each catabolic pathway on JA-Ile turnover for each stress and investigated the impact of 140
modified JA-Ile homeostasis on defense responses and tolerance to aggression by an herbivore 141
insect or by fungal infection (Figure 1). The data highlight new stress-specific impacts of fully 142
impairing either one or both JA-Ile catabolic pathways, where defense and aggressor resistance 143
intensities are more correlated (negatively) to the hyper-activation of negative feedback 144
signaling effectors rather than on actual JA-Ile levels. 145
146
147
RESULTS 148
149
Jasmonate profiling of higher order catabolic mutants reveals stimulus-specific impacts 150
on hormone homeostasis 151
152
Effective inactivation of the respective genes was verified in each mutant line by RT-qPCR 153
using RNA extracted from 1 h-wounded leaves (Supplemental Figure S1 and (Aubert et al. 154
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6
2015)). The four plant genotypes in the Col-0 ecotype (WT, 2ah, 3cyp, 5ko) were submitted 155
separately to mechanical wounding or to B. cinerea inoculation. Mechanical wounding 156
constitutes a synchronous and severe stimulus, and generates a massive jasmonate pulse where 157
compound-specific dynamics can be followed (Chung et al. 2008; Glauser et al. 2008; Heitz et 158
al. 2016). Therefore, a kinetic study was conducted with tissue collected at 1, 3 and 6 h post-159
wounding (hpw). Necrotic lesions inflicted by B. cinerea infection develop radially with fungal 160
hyphae continuously infecting new tissue, and 3 days post-inoculation (dpi) constitutes an 161
optimal time point for recording biochemical changes and for assessing antifungal resistance. 162
We quantified levels of JA, 12OH-JA, JA-Ile and its catabolites 12OH-JA-Ile and 12COOH-163
JA-Ile in the two biological responses. JA levels were only marginally affected by the mutations 164
(Supplemental Figure S2). Its oxidation product, 12OH-JA, is known to be partially formed via 165
conjugate intermediates (Figure 1) upon wounding (Widemann et al. 2013) and accordingly, 166
was less abundant in all mutants at 3 hpw but was only reduced in 2ah at 6 hpw (Supplemental 167
Figure S2). In contrast, 12OH-JA was less affected by mutations upon infection and was even 168
enhanced in 5ko, likely reflecting the predominant contribution of JAO enzymes directly 169
oxidizing JA in this material (Figure 1) (Smirnova et al. 2017). 170
171
Impacts on JA-Ile homeostasis upon wounding 172
JA-Ile showed the typical pattern (Chung et al. 2008; Glauser et al. 2008; Heitz et al. 2012; Koo 173
et al. 2011) in wounded WT leaves, peaking at 1 hpw and declined thereafter (Figure 2A). In 174
2ah and 3cyp lines, a significant overaccumulation of JA-Ile was recorded at 1 hpw and was 175
prolonged in 3cyp, extending data from mutants of lower order described previously (Heitz et 176
al. 2012; Widemann et al. 2013; Zhang et al. 2016). In the 5ko line that has both pathways 177
impaired, a huge JA-Ile accumulation was recorded that culminated between 3-6 hpw close to 178
40-50 nmol g FW-1. These data show that in wounded leaves, both AH and CYP94 pathways 179
contribute similarly to JA-Ile turnover and that their simultaneous inactivation synergistically 180
boosts hormone hyperaccumulation at later time points. Profiles of CYP94-generated JA-Ile 181
oxidation products were as expected: 12OH-JA-Ile levels were about double in 2ah compared 182
to WT, were suppressed in 3cyp and of note, were higher in 5ko than in 3cyp at 6 hpw (Figure 183
2A). 12COOH-JA-Ile was only detected from 3 hpw and evolved similarly to 12OH-JA-Ile in 184
2ah and 3cyp, but was barely detected in 5ko. 185
186
187
188
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7
Impacts on JA-Ile homeostasis upon Botrytis infection 189
The metabolic impacts were different in response to B. cinerea inoculation. JA-Ile levels were 190
similar to WT in 3cyp, confirming our previous data (Aubert et al. 2015) (Figure 2B). In 191
contrast, JA-Ile levels were strongly enhanced in 2ah but no further increase was observed in 192
5ko. This result indicates that the amidohydrolase pathway is essential for JA-Ile clearance upon 193
fungal infection and that blocking simultaneously the oxidative pathway does marginally 194
impact JA-Ile accumulation. 2ah also considerably enhanced 12OH-JA-Ile levels and 195
12COOH-JA-Ile, reinforcing initial data obtained with iar3 or ill6 single mutants (Widemann 196
et al. 2013). In 2ah, excess JA-Ile is likely oxidized into 12OH-JA-Ile that can no more be 197
deconjugated by IAR3 and ILL6 and part of this enlarged pool is further oxidized to 12COOH-198
JA-Ile by CYP94C1. Together, these experiments demonstrate stimulus-specific contributions 199
of AH and CYP94 catabolic pathways to JA-Ile turnover and accumulation of downstream 200
derivatives. 201
202
Impact of impaired catabolic pathways on defense and resistance responses is not 203
reflecting JA-Ile accumulation in mutant plants 204
205
As the mutations altered the steady-state levels of bioactive JA-Ile, the lines were examined for 206
induced expression of typical JA-regulated genes for each leaf stress model. In the case of 207
wounding, transcripts of MYC2, an early-responsive transcription factor (TF) gene controlling 208
late targets, accumulated similarly at 1 hpw, but declined less than WT in mutants at 3 hpw 209
(3cyp and 5ko) and 6 hpw (2ah and 5ko) (Figure 3A). Two VSP genes were examined as late 210
markers of the anti-insect branch of the JA defense pathway (Pieterse et al. 2012; Wasternack 211
and Hause 2013). In WT, both VSP1 and VSP2 expression peaked at 3 hpw before declining to 212
low levels at 6 hpw (Figure 3A). Expression was globally enhanced in mutant lines compared 213
to WT, but with distinct patterns. VSP1 was strongly enhanced in 2ah at all time points, while 214
in 3cyp and 5ko, gain in transcript levels was only observed at 6 hpw when WT signal faded. 215
For VSP2, only 2ah displayed enhanced expression at all 3 time points. This indicates that 216
impaired JA-Ile catabolism results in persistent defense marker expression, but this is not 217
commensurate with JA-Ile accumulation, because 5ko displays lower defense than 2ah. To 218
determine the physiological impacts of such altered hormone and defense profiles, we used an 219
insect feeding assay as a biological readout of signaling. When larvae of Spodoptera littoralis 220
were placed on leaves of the four genotypes for 8 to 9 days, contrasted results were recorded: 221
larvae fed on 2ah plants were consistently lighter than those feeding on WT, indicating stronger 222
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8
anti-insect resistance (Figure 3B). This opposed to 3cyp, that sustained higher insect 223
development, confirming impaired resistance reported previously by Poudel et al. (2016). 224
Finally, similarly to fungal infection, 5ko displayed WT level of resistance to herbivory. These 225
conclusions were drawn from 3 successive trials (Supplemental Figure S3) with large insect 226
populations. 227
A similar analysis was conducted for the response to B. cinerea infection. The antimicrobial 228
branch of JA-Ile-dependent defense signaling was probed by monitoring ORA59 TF and 229
PDF1.2 marker expression. Surprisingly, both genes exhibited reduced expression in 2ah and 230
5ko at 3 dpi, in contrast to 3cyp that maintained WT levels (Figure 4A). Anti-fungal resistance 231
assay indicated that only 3cyp displayed smaller lesions, in agreement with our previous report 232
(Aubert et al. 2015), while 3cyp and to a lesser extent 2ah supported reduced fungal biomass 233
as estimated from B. cinerea cutinase signal (Figure 4B). Content in camalexin, a major 234
antimicrobial phytoalexin in Arabidopsis, was determined but no significant differences were 235
found between genotypes. Together, the data show that impairing either one or both JA-Ile 236
catabolic pathways has distinct and stress-specific consequences on JA-mediated defense and 237
resistance responses. 238
239
Impaired JA-Ile catabolism results in gain in resistance only when negative feedback 240
effectors are not overinduced 241
242
Upon parallel investigation of the two leaf defense models, increased JA-Ile accumulation due 243
to impaired catabolism did not correlate systematically with stronger defense or resistance 244
phenotypes. Conversely, enhanced resistance can occur without elevated hormone levels 245
(Figures 2, 3 and 4). These observations suggest that other players may be at work to limit or 246
counteract overinduction of defense responses under deficient JA-Ile turnover. Obvious 247
candidates for such negative regulation are genes encoding JAZ repressors (Browse 2009) or 248
JAM bHLH factors competing with MYC transcription factors (Sasaki-Sekimoto et al. 2013), 249
both classes of genes being JA-Ile-responsive. We selected JAZ genes that were previously 250
described as highly induced and sensitive to catabolic pathway manipulation (Aubert et al. 251
2015; Heitz et al. 2012; Widemann et al. 2013). As shown in Figure 5A, JAZ8, JAZ10, JAM1 252
and JAM2 expression was maximal in all lines at 1 hpw with only slight enhancement in some 253
mutants, but their transcripts were clearly more persistent in 3cyp and 5ko lines at 3 and 6 hpw, 254
particularly for JAZ, compared to 2ah and WT. This result establishes in 3cyp and 5 ko a 255
correlation between hyperaccumulation of JA-Ile (Figure 2), the overinduction of JAZ and JAM 256
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9
genes (Figure 5), near-WT defense induction and WT (5ko) or deficient (3cyp) levels of insect 257
resistance (Figure 3) compared to WT. In contrast, despite of enhanced JA-Ile levels upon 258
wounding, 2ah does not display persistent expression of these repressors (Figure 5, 3 and 6 259
hpw), and this correlates with more robust defense and reduced insect herbivory (Figure 3B). 260
In response to B. cinerea infection, JAZ1, JAZ8 and JAM1 transcripts were overinduced in 2ah 261
and 5ko (JAM2 only in 5ko) (Figure 5B), that overaccumulate JA-Ile, correlating with reduced 262
defense (Figure 4A) and WT antifungal resistance (Figure 4B). On the contrary, these genes 263
were not overinduced in 3cyp, the only genotype exhibiting significantly increased antifungal 264
resistance (Figure 4B). Therefore, the comparative analysis showed that elevated defense and 265
resistance under deficient JA-Ile turnover is induced only if negative effectors are themselves 266
not over-responding to JA-Ile accumulation. 267
To determine if JA-Ile catabolism mutations affected also basal gene expression in leaves 268
before stress, we analyzed transcript levels of the same target genes in untouched leaves in a 269
separate set of plants. Although higher fluctuations were recorded within a given genetic 270
background than for stimulated tissues, two trends emerged: MYC2 and ORA59 TFs transcripts 271
were significantly upregulated in 3cyp and 5ko lines, but levels of their respective targets VSP 272
and PDF1.2 were equal to or lower than WT in these lines (Supplemental Figure S4). 273
Strikingly, all 5 analyzed genes encoding JAZ and JAM negative regulators were expressed at 274
slightly but significantly higher levels in mutant (mostly 3cyp and 5ko) compared to WT. This 275
result indicates that severe or even total block in JA-Ile catabolism does not increase basal 276
expression of typical defense marker genes in Arabidopsis leaves. 277
278
DISCUSSION 279
280
Hormonal compounds must be timely and spatially controlled to ensure coordinated 281
physiological responses. Catabolic pathways have been characterized for all major plant 282
receptor-binding hormonal ligands and are integral components of hormone homeostasis and 283
action. Since their relatively recent discovery, JA-Ile catabolic pathways have been the focus 284
of intense biochemical and physiological studies that disclosed their relationship with JA 285
signaling. As a common theme in hormone catabolism (Kawai et al. 2014; Mizutani and Ohta 286
2010), an oxidative inactivation pathway was characterized, with CYP94 enzymes defining a 287
two-step JA-Ile oxidation process. In contrast to most hormones for which conjugation 288
corresponds to inactivation or generation of storage forms (Piotrowska and Bajguz 2011), JA 289
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10
requires conjugation to the amino acid isoleucine as the critical activation step. Consistently, 290
deconjugation by the amidohydrolases IAR3 and ILL6 was characterized in Arabidopsis as a 291
second JA-Ile removal pathway, acting in a JAR1 reverse reaction. 292
Understanding the functions of JA-Ile catabolism potentially offers avenues to tailor increased 293
or on-demand defense signaling in diverse situations, and was addressed repetitively by loss-294
of-function studies in the two catabolic pathways (Aubert et al. 2015; Heitz et al. 2012; Kitaoka 295
et al. 2011; Koo et al. 2011, 2014; Luo et al. 2016; Poudel et al. 2016; Woldemariam et al. 296
2012). In the present work, we conducted a comprehensive analysis of selected genotypes that 297
allowed to evaluate the respective impact of each biochemical pathway on alterations in JA 298
metabolism, defense regulation and resistance to biotic stress. The data clarify some former 299
unexpected results and fill significant knowledge gaps by establishing novel pathway- and 300
stress-specific features. It is remarkable that all mutant lines, including the newly generated 5ko 301
defective in all known JA-Ile-catabolizing enzymes, were undistinguishable from WT for 302
growth under standard conditions. This comes along with the observation that CYP94 and 303
IAR3/ILL6 deficiency does not increase basal JA-Ile accumulation or elevate defense gene 304
expression in resting plants. We conclude that abolition of JA-Ile catabolism does neither turn 305
on constitutive JA responses nor provoke growth inhibition. 306
307
Wounding/insect stress and infection by a necrotroph both induce strong JA metabolism and 308
signaling (Campos et al. 2014), but due to different interactions with other hormonal networks 309
(Pieterse et al. 2012), the transcriptome and defensive outcome display only partial overlap. 310
Previous reports showed that single mutations in either the oxidative or cleavage pathway were 311
sufficient to overaccumulate JA-Ile upon leaf wounding (Heitz et al. 2012; Kitaoka et al. 2011; 312
Koo et al. 2011). This feature was extended in 3cyp or 2ah lines fully impaired in one or the 313
other pathway, but here we demonstrate that their simultaneous deficiency raises JA-Ile to 314
remarkably high levels, indicating that upon massive and rapid JA-Ile biosynthesis triggered by 315
mechanical wounding, no additional enzymatic pathway can efficiently turnover and limit 316
accumulation of JA-Ile. In response to B. cinerea infection, a comparable JA-Ile 317
overaccumulation was recorded in 2ah and 5ko lines, but in marked contrast 3cyp infected 318
leaves accumulated WT levels of JA-Ile, suggesting that in this background, the activity of the 319
AH pathway is sufficient to prevent abnormal hormone build-up. The CYP94 oxidation 320
pathway is however active in infected WT plants, because oxidized derivatives are readily 321
detected, albeit less abundantly than upon wounding. A preponderance of the AH pathway upon 322
infection is supported by the strongly enhanced JA-Ile levels in 2ah and 5ko lines. This indicates 323
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11
that CYP94 cannot substitute for AH deficiency and that IAR3/ILL6 enzymes are essential for 324
proper JA-Ile homeostasis upon necrotroph attack. 325
326
We next investigated the signaling output of perturbed JA-Ile homeostasis in the two leaf 327
defense models and found that transcript levels of canonical regulatory and defense markers 328
and resistance phenotypes were overall poorly correlated with changes in JA-Ile levels in 329
mutant lines. Upon wounding, 3cyp was closest to WT in term of VSP induction, contrasting 330
with the deficient defense performance of this genotype. Both AH-deficient lines gained a more 331
persistent VSP1/2 expression, more pronounced for VSP2 in 5ko. This behavior was 332
accompanied by a lower weight gain in S. littoralis larvae in 2ah, but not in 5ko, despite of its 333
massive JA-Ile content. The situation with antimicrobial resistance also revealed a strong 334
distortion of the usually linear relationship between hormone, defense and resistance levels, as 335
the high JA-Ile-accumulating lines 2ah and 5ko exhibited depressed defense signaling and WT 336
B. cinerea resistance. In contrast, 3cyp, while maintaining WT JA-Ile levels, was able to better 337
defend against the fungus, as reported earlier (Aubert et al. 2015). In this case, the normal 338
behavior of the ORA59-PDF1.2 module or camalexin levels do not seem to account for the 339
better tolerance. Nevertheless, this result shows that more JA-dependent resistance can arise 340
without an obvious over-accumulation of JA-Ile. 341
342
The JA-Ile regulatory network is complex and sets in motion cascades of positive and negative 343
transcriptional regulators (Hickman et al. 2017). We hypothesized that known elements 344
defining negative feedback loops may counteract some of the effects of chronic JA-Ile 345
overaccumulation to restrain defense induction. This notion was introduced earlier for some 346
JAZ genes in single or double mutants of one pathway, but its relevance was difficult to assess 347
in single biological situations analyzed separately (Aubert et al. 2015; Heitz et al. 2012; Koo et 348
al. 2011). Here we analyzed expression of JAZ and JAM, a second type of negative regulator 349
inhibiting transcription of JA-Ile- and MYC-regulated target genes (Liu et al. 2019; Sasaki-350
Sekimoto et al. 2013) in the different pathway/genotype/stress combinations. We found a 351
seemingly robust correlation between the overinduction of several JAZ and JAM genes and the 352
absence of enhanced expression of defense response under impaired JA-Ile turnover (Figure 6). 353
We selected JAZ genes that were previously found to be sensitive to altered JA-Ile homeostasis 354
(Aubert et al. 2015; Heitz et al. 2012). JAZ8, JAZ10, JAM1 and JAM2 persisted longer than in 355
WT in 3cyp and 5ko after wounding, but for unknown reasons behaved like WT in 2ah, 356
correlating with sustained VSP expression and better tolerance to feeding by S. littoralis larvae 357
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in this latter line. In the case of B. cinerea infection, a similar logic was respected, but in 358
different genotypes. Both 2ah and 5ko displayed significantly higher expression of either JAZ 359
or JAM or both genes, but this was not observed in 3cyp. This correlated well with the former 360
lines showing low defense and WT necrotic lesion sizes, and 3cyp maintaining WT defense and 361
being more resistant to infection. It should be noted that in 3cyp, ORA59-PDF1.2 as well as 362
camalexin accumulation behaved like in WT, so other defense determinants must account for 363
the observed phenotypes. 364
Together these results demonstrate that the two JA-Ile catabolic pathways contribute 365
differentially to JA-Ile removal upon two distinct leaf stresses, and further variations may be 366
expected in other organs or under other environmental constraints. 367
368
The impact of rewiring genetically JA-Ile catabolic capacity may vary between plant species 369
and needs further investigations. For example, the oxidative or deconjugation pathway were 370
silenced transiently (CYP94) or stably (JIH) in Nicotiana attenuata, and in two studies, clearly 371
enhanced JA-Ile-dependent defenses and insect resistance were described (Luo et al. 2016; 372
Woldemariam et al. 2012). We showed that it is possible to genetically extend the half-life and 373
amplitude of JA-Ile burst in Arabidopsis, but this has contrasted consequences on signaling. 374
The precise mechanism underlying control of signaling when JA-Ile catabolism is impaired 375
remains unknown. Stress-specific “sensing” of excess JA-Ile on JAZ or JAM promoters may be 376
mediated by the different interactions engaged between JA signaling and other hormonal 377
pathways, such as the synergy with ethylene upon necrotroph attack, or with abscisic acid upon 378
wounding or herbivory (Pieterse et al., 2012). The observation that blocking either catabolic 379
pathway acting on the same substrate has distinct signaling and physiological outcomes 380
suggests that signaling consequences of JA metabolism are not mediated solely through the 381
control of JA-Ile levels. Modification of abundance of enzymatic products may also contribute 382
to alter the signaling process(s). A recent paper reported that in addition to JA-Ile, 12OH-JA-383
Ile was an active jasmonate signaling through COI1 and contributing to wound responses 384
(Poudel et al. 2019). Such an interpretation would be consistent with our description of 385
metabolic and defensive features of 2ah in wound/insect responses, but not with 3cyp in the 386
Botrytis assay. In addition, one must keep in mind that IAR3 also accepts auxin conjugates as 387
substrates, which may also be at the basis of hormonal cross-talk and alter the signaling output 388
(Widemann et al. 2013; Zhang et al. 2016). The only cases where restrained catabolism ends-389
up in enhanced tolerance to fungal or insect attack is when JAZ and/or JAM repressor transcripts 390
are not overstimulated to reinforce negative feedback loops. The factors determining when 391
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13
negative regulators take control remain unknown but their elucidation will be of major 392
importance to maximize the potential output of JA-regulated defenses. This provides the ground 393
for future work at the protein, promoter and chromatin levels to determine how inhibitory 394
mechanisms could be at work. 395
396
397
METHODS 398
399
Plant growth and treatments 400
401
Arabidopsis thaliana genotypes used were in the Col0 ecotype and were grown under a 12 h 402
light/12 h dark photoperiod in a growth chamber. The individual T-DNA insertion lines were 403
obtained from the Nottingham Arabidopsis Stock Center (NASC). The 3cyp line was obtained 404
by crossing the alleles cyp94b1-1 (SALK_129672), cyp94b3-1 (CS302217) and cyp94c1-1 405
(SALK_55455) (Aubert et al. 2015). The 2ah line was obtained after crossing the lines iar3-5 406
(SALK_069047) and ill6-2 (SALK_024894C). The quintuple 5ko line was obtained by crossing 407
the 3cyp line with a double iar3-5 ill6-1 (GK412-E11). 408
B. cinerea inoculation and resistance scoring were as described in (Aubert et al. 2015). For 409
mechanical wounding experiments, 5 or 6 fully expanded leaves were wounded three times 410
across mid-vein with a hemostat. At increasing time points following damage, leaf samples 411
were quickly harvested and flash-frozen in liquid nitrogen before storing at -80°C until analysis. 412
Insect performance assay 413
Four-week-old Arabidopsis plants were challenged with freshly hatched Spodoptera littoralis 414
larvae (eggs obtained from Syngenta, Stein AG, Switzerland). Five larvae were placed on each 415
of 11 pots, each containing 2 plants. Plants were placed in a transparent plastic box and kept in 416
a growth chamber during the experiment (10 h light/14 h dark, 100 µmol m-2 s-1 of light, 20-417
22°C and 65% relative humidity). After 8-9 days of feeding, larvae were weighed on a precision 418
balance (Mettler-Toledo, Greifensee, Switzerland). The experiment was performed 419
successively three times (different sampling dates). 420
421
RT-qPCR gene expression assays 422
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14
Total RNA was extracted from plant leaves with TRI reagent (Molecular Research Center). 423
One microgram of RNA was reverse transcribed using the SuperScript IV reverse transcription 424
system (Thermo Fisher Scientific). Real-time PCR was performed on 10 ng of cDNA as 425
described in Berr et al. (2010) using a LightCycler 480 II instrument (Roche Applied Science). 426
The housekeeping genes EXP (At4g26410) and TIP41 (At4g34270) were used as internal 427
references for qPCR on cDNA derived from infected/wounded or non-stimulated leaves. 428
Measurement of fungal biomass was performed as described in (Smirnova et al. 2017) except 429
that ACT2 (At3g18780) and UBQ10 (At4g05320) were used as reference genes. Gene-specific 430
primer sequences used for qPCR are listed in Supplemental Table 1. 431
432
Jasmonate and camalexin profiling 433
434
Jasmonates were identified and quantified by ultra high performance liquid chromatography 435
coupled to tandem mass spectrometry (UHPLC-MS/MS). About 50-100 mg frozen plant 436
material was extracted with 10 volumes (10 µL per mg) of ice-cold extraction solution 437
(MeOH:water:acetic acid 70:29:0.5, v/v/v) containing 9,10-dihydro-JA and 9,10-dihydro-JA-438
Ile as internal standards for workup recovery. Grinding was performed with a glass-bead 439
Precellys tissue homogenizer (Bertin Instruments, France) in 2 mL screw-capped tubes. After 440
30 min incubation at 4°C on a rotating wheel, homogenates were cleared by centrifugation 441
before concentration of supernatants under a stream of N2 and overnight conservation at -20°C. 442
After a second centrifugation step, extracts were submitted to quantitative LC-MS/MS analysis 443
on an EvoQ Elite LC-TQ (Bruker) equiped with an electrospray ionisation source (ESI) and 444
coupled to a Dionex UltiMate 3000 UHPLC system (Thermo). Five µL plant extract were 445
injected. Chromatographic separation was achieved using an Acquity UPLC HSS T3 column 446
(100 x 2.1 mm, 1.8 µm; Waters) and pre-column. The mobile phase consisted of (A) water and 447
(B) methanol, both containing 0.1 % formic acid. The run started by 2 min of 95 % A, then 448
a linear gradient was applied to reach 100 % B at 10 min, followed by isocratic run using B 449
during 3 min. Return to initial conditions was achieved in 1 min, with a total run time of 15 450
min. The column was operated at 35 °C with a flow-rate of 0.30 mL min-1. Nitrogen was used 451
as the drying and nebulizing gas. The nebulizer gas flow was set to 35 L h-1, and the desolvation 452
gas flow to 30 L h-1. The interface temperature was set to 350 °C and the source temperature to 453
300 °C. The capillary voltage was set to 3.5 kV, the ionization was in positive or negative mode. 454
Low mass and high mass resolution were 2 for the first mass analyzer and 2 for the second. 455
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15
Data acquisition was performed with the MS Workstation 8 for the mass spectrometry and the 456
liquid chromatography was piloted with Bruker Compass Hystar 4.1 SR1 software. The data 457
analysis was performed with the MS Data Review software. Absolute quantifications were 458
achieved by comparison of sample signal with dose-response curves established with pure 459
compounds and recovery correction based on internal standard signal. The transitions were, in 460
negative mode: JA 209.3>59.3; JA-Ile 322.3>130.2; 12OH-JA-Ile 338.3>130.2; 12COOH-JA-461
Ile 352.2>130.1; 12OH-JA 225.2>59.3; in positive mode: camalexin 201.0>59.3. 462
463
Statistical analysis 464
All statistical analysis was performed using InfoStat 2015d (http:// www.infostat.com.ar). 465
Comparisons of sample means were performed by one-way analysis of variance (P % 0.05 or 466
P % 0.01) and Tukey’s post-hoc multiple comparisons tests (P < 0.05 or P < 0.01), and 467
significant differences of means were determined. 468
469
LEGENDS TO FIGURES 470
Figure 1. Positions of the impaired enzymatic steps in JA-Ile catabolic mutants and 471
simplified view of analyzed signaling and defense responses 472
Left box: Necrotroph fungus infection or mechanical wounding/insect attack trigger jasmonic 473
acid (JA) biosynthesis. Some JA is conjugated to isoleucine by JAR1 enzyme to form bioactive 474
jasmonoyl-isoleucine (JA-Ile). JA-Ile is turned over by a two-step oxidation to 12OH-JA-Ile 475
and 12COOH-JA-Ile by the cytochrome P450 enzymes CYP94B3/B1 and CYP94C1, or by 476
conjugate cleavage mediated by the amidohydrolases (AH) IAR3 and ILL6. These AH also 477
cleave 12OH-JA-Ile to release 12OH-JA. Red crosses indicate impaired JA-Ile catabolic steps 478
in higher order mutants utilized in this study: oxidation-deficient (triple cyp94b1 b3 c1 mutant, 479
3cyp), deconjugation-deficient (double iar3 ill6, 2ah) or deficient for both pathways (quintuple 480
mutant, 5ko). Right box: in absence of JA-Ile, JAZ repressors block transcription of target 481
genes. Upon biosynthesis, JA-Ile promotes the assembly of the COI1-JAZ co-receptor which 482
is recruited into the SCFCOI1 E3 ubiquitin ligase directing proteolytic degradation of JAZ 483
repressors. Transcription factors (TFs) like MYC2 or ORA59 become activated in association 484
with mediator 25 subunit (MED25) and allow the transcription of numerous stimulus-specific 485
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16
defense responses. The anti-insect branch was probed by the MYC2-VSP genes, and the 486
antimicrobial branch by the ORA59 and PDF1.2 genes. Among JA-Ile targets are also genes 487
encoding JAZ repressors or JAM proteins that compete with MYC2 and ORA59, to attenuate 488
signaling. In mutant plants impaired for JA-Ile catabolism, overinduction of this negative 489
feedback loop may occur and prevent hyper-stimulation of JA-Ile-dependent defenses and 490
associated enhanced resistance. 491
Figure 2. JA-Ile and oxidized catabolites accumulation in 2ah, 3cyp and 5ko mutants after 492
mechanical wounding or Botrytis cinerea Infection. 493
Six-week-old plants were wounded 3 times across leaf mid-vein (A) or drop-inoculated on two 494
sites per leaf with a suspension containing 2.5x106 fungal spores mL-1 (B). Treated leaves were 495
harvested at 1, 3 and 6 hours post wounding (hpw) or 3 days post inoculation (dpi) on WT 496
(black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green bars) plants. Jasmonates were 497
extracted and quantified by LC–MS/MS. JA-Ile, 12OH-JA-Ile and 12COOH-JA-Ile levels were 498
expressed in nmol g-1 fresh weight (FW). Histograms represent the mean ± SEM of three 499
(wounding) or four (B. cinerea) biological replicates. Columns labeled with different letters 500
indicate a significant difference between genotypes at each time point as determined by one-501
way ANOVA with Tukey post-hoc test, P < 0.05. 502
Figure 3. Expression profiles of JA-Ile-dependent genes in response to mechanical 503
wounding and impact on insect feeding in JA-Ile catabolic mutants. 504
(A) Expression profiles of jasmonate-dependent MYC2, VSP1 and VSP2 marker genes in 505
response to wounding in WT (black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green 506
bars) mutants. Relative expression of each target gene at 1, 3 or 6 hours post wounding (hpw) 507
is represented. Gene expression was determined by real-time PCR using gene-specific primers 508
and normalized using EXP and TIP41 reference genes. Transcript quantification was performed 509
on three biological replicates analyzed in duplicate. Histograms represent mean expression ± 510
SEM. Columns labeled with different letters indicate a significant difference between 511
genotypes at each time point as determined by one-way ANOVA with Tukey posthoc test, P < 512
0.05. 513
(B) Insect feeding assay. S. littoralis larvae were placed on leaves of 4-week-old plants. After 514
8-9 days, larval weight was determined. Histograms represent mean ± SEM from 3 independent 515
trials (presented in Supplemental Figure S3). The number of total larvae on each genotype is 516
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17
indicated within the bars. Different letters indicate significant differences at P < 0.05 (linear 517
mixed model). 518
Figure 4. Expression profiles of JA-Ile-dependent defense genes and resistance levels in 519
response to B. cinerea infection in 2ah, 3cyp and 5ko mutants. 520
(A) Expression profiles of jasmonate-regulated ORA59 and PDF1.2 genes in response to 521
infection by B. cinerea in WT (black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green 522
bars) mutants. Relative expression of each target gene at 3 day post infection (dpi) is 523
represented. Gene expression was determined by real-time PCR using gene-specific primers 524
and normalized using EXP and TIP41 reference genes. Transcript quantification was performed 525
on three biological replicates analyzed in duplicate. Histograms represent mean expression ± 526
SEM. Columns labeled with different letters indicate a significant difference between 527
genotypes at each time point as determined by one-way ANOVA with Tukey posthoc test, P < 528
0.05. 529
(B) Disease resistance assessment. Two sites per leaf were inoculated across the main vein with 530
5 µL of spore suspension containing 2.5x106 spores mL-1. Left panel: representative leaves of 531
each genotype were detached and photographed at 3 dpi. Middle panel: histograms represent 532
the mean lesion diameters ± SEM of about 100 lesion sites from 10 to 15 plants for each 533
genotype. Right panel: evaluation of fungal growth by real-time qPCR with B. cinerea cutinase-534
specific primers on genomic DNA extracted from 3-day- infected leaves. Quantification was 535
performed on three biological replicates analyzed in duplicate. Columns labeled with different 536
letters indicate a significant difference as determined by one-way ANOVA with Tukey post-537
hoc test (P < 0.01). 538
Figure 5. Expression profiles of JA-Ile dependant negative feedback genes in response to 539
wounding and infection in 2ah, 3cyp and 5ko mutants. 540
Expression profiles of jasmonate-dependent genes in response to wounding (A, JAZ8, JAZ10, 541
JAM1 and JAM2) or infection by Botrytis cinerea (B, JAZ1, JAZ8, JAM1 and JAM2) in WT 542
(black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green bars) mutants. Relative 543
expression of each target gene at 1, 3 or 6 hours post wounding (hpw) and 3 day post infection 544
(dpi) is represented. Gene expression was determined by real-time PCR using gene-specific 545
primers and normalized using EXP and TIP41 reference genes. Transcript quantification was 546
performed on three biological replicates analyzed in duplicate. Histograms represent mean 547
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18
expression ± SEM. Columns labeled with different letters indicate a significant difference 548
between genotypes at each time point as determined by one-way ANOVA with Tukey posthoc 549
test, P < 0.05. 550
Figure 6. Relationships between impaired JA-Ile catabolism, JA-Ile levels, 551
defense/repressor gene induction, and resistance to attackers. 552
The data show distinct circuitry for the two leaf responses analyzed. The signs -, 0, or + indicate 553
lower, equal, or increased response respectively, in stimulated mutant genotypes compared to 554
WT. The two catabolic pathways have differential contributions to JA-Ile turnover in response 555
to wounding or infection. JA-Ile accumulation is enhanced to different extents, except in 556
infected 3cyp, and this has variable consequences on defense amplitude and aggressor 557
resistance. The trend emerging is that impaired hormone catabolism does, with or without JA-558
Ile overaccumulation, result in better defense/resistance, only if negative effectors like JAZ or 559
JAM are themselves not overinduced. 560
561
ACKNOWLEDGMENTS 562
J. Browse (WSU, Pullman, USA) for providing a segregating population of iar3-5 ill6-2 line. 563
564
FUNDING 565
This work was supported by basic funding of CNRS, grant ANR-12-BSV8-005 (Jasmonox) from the 566 Agence Nationale de la Recherche to ES, and IdEx-2014-208e Interdisciplinary grant to LP from 567 Université de Strasbourg and CNRS. VM is recipient of a predoctoral fellowship from the Université de 568 Strasbourg and the Ministère de l’Enseignement Supérieur et de la Recherche. This research was 569 supported by a grant from the Swiss National Science Foundation (31003A_169278 to P.R.). 570 571
572
REFERENCES 573
574
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 28, 2019. ; https://doi.org/10.1101/686709doi: bioRxiv preprint
19
Aubert Y, Widemann E, Miesch L, Pinot F, Heitz T (2015) CYP94-mediated jasmonoyl-575
isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses 576
to Botrytis cinerea infection. J Exp Bot 66:3879–3892 577
Browse J (2009) Jasmonate passes muster: a receptor and targets for the defense hormone. Annu 578
Rev Plant Biol 60:183–205 579
Bruckhoff V, Haroth S, Feussner K, Konig S, Brodhun F, Feussner I (2016) Functional 580
Characterization of CYP94-Genes and Identification of a Novel Jasmonate Catabolite in 581
Flowers. PLoS One 11:e0159875 582
Campos ML, Kang JH, Howe GA (2014) Jasmonate-triggered plant immunity. J Chem Ecol 583
40:657–675 584
Chini A, Fonseca S, Fernandez G, Adie B, Chico JM, Lorenzo O, Garcia-Casado G, Lopez-585
Vidriero I, Lozano FM, Ponce MR, Micol JL, Solano R (2007) The JAZ family of 586
repressors is the missing link in jasmonate signalling. Nature 448:666–671 587
Chini A, Gimenez-Ibanez S, Goossens A, Solano R (2016) Redundancy and specificity in 588
jasmonate signalling. Curr Opin Plant Biol 33:147–156 589
Chung HS, Koo AJ, Gao X, Jayanty S, Thines B, Jones AD, Howe GA (2008) Regulation and 590
function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and 591
herbivory. Plant Physiol 146:952–964 592
Gidda SK, Miersch O, Levitin A, Schmidt J, Wasternack C, Varin L (2003) Biochemical and 593
molecular characterization of a hydroxyjasmonate sulfotransferase from Arabidopsis 594
thaliana. J Biol Chem 278:17895–17900 595
Glauser G, Grata E, Dubugnon L, Rudaz S, Farmer EE, Wolfender JL (2008) Spatial and 596
temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response 597
to wounding. J Biol Chem 283:16400–16407 598
Guo Q, Major IT, Howe GA (2018) Resolution of growth-defense conflict: mechanistic insights 599
from jasmonate signaling. Curr Opin Plant Biol 44:72–81 600
Havko NE, Major IT, Jewell JB, Attaran E, Browse J, Howe GA (2016) Control of Carbon 601
Assimilation and Partitioning by Jasmonate: An Accounting of Growth-Defense 602
Tradeoffs. Plants (Basel) 5 603
Heitz T, Smirnova E, Widemann E, Aubert Y, Pinot F, Menard R (2016) The Rise and Fall of 604
Jasmonate Biological Activities. Subcell Biochem 86:405–426 605
Heitz T, Widemann E, Lugan R, Miesch L, Ullmann P, Desaubry L, Holder E, Grausem B, 606
Kandel S, Miesch M, Werck-Reichhart D, Pinot F (2012) Cytochromes P450 CYP94C1 607
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 28, 2019. ; https://doi.org/10.1101/686709doi: bioRxiv preprint
20
and CYP94B3 catalyze two successive oxidation steps of plant hormone Jasmonoyl-608
isoleucine for catabolic turnover. J Biol Chem 287:6296–6306 609
Hickman R, Van Verk MC, Van Dijken AJH, Mendes MP, Vroegop-Vos IA, Caarls L, 610
Steenbergen M, Van der Nagel I, Wesselink GJ, Jironkin A, Talbot A, Rhodes J, De Vries 611
M, Schuurink RC, Denby K, Pieterse CMJ, Van Wees SCM (2017) Architecture and 612
Dynamics of the Jasmonic Acid Gene Regulatory Network. Plant Cell 29:2086–2105 613
Kawai Y, Ono E, Mizutani M (2014) Evolution and diversity of the 2-oxoglutarate-dependent 614
dioxygenase superfamily in plants. Plant J 78:328–343 615
Kitaoka N, Matsubara T, Sato M, Takahashi K, Wakuta S, Kawaide H, Matsui H, Nabeta K, 616
Matsuura H (2011) Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-617
hydroxylase, a key enzyme in the oxidative catabolism of jasmonate. Plant Cell Physiol 618
52:1757–1765 619
Koo AJ, Cooke TF, Howe GA (2011) Cytochrome P450 CYP94B3 mediates catabolism and 620
inactivation of the plant hormone jasmonoyl-L-isoleucine. Proc Natl Acad Sci U S A 621
108:9298–9303 622
Koo AJ, Howe GA (2012) Catabolism and deactivation of the lipid-derived hormone 623
jasmonoyl-isoleucine. Front Plant Sci 3:19 624
Koo AJ, Thireault C, Zemelis S, Poudel AN, Zhang T, Kitaoka N, Brandizzi F, Matsuura H, 625
Howe GA (2014) Endoplasmic reticulum-associated inactivation of the hormone 626
jasmonoyl-L-isoleucine by multiple members of the cytochrome P450 94 family in 627
Arabidopsis. J Biol Chem 289:29728–29738 628
Liu Y, Du M, Deng L, Shen J, Fang M, Chen Q, Lu Y, Wang Q, Li C, Zhai Q (2019) MYC2 629
Regulates the Termination of Jasmonate Signaling via an Autoregulatory Negative 630
Feedback Loop. Plant Cell 31:106–127 631
Luo J, Wei K, Wang S, Zhao W, Ma C, Hettenhausen C, Wu J, Cao G, Sun G, Baldwin IT, Wu 632
J, Wang L (2016) COI1-Regulated Hydroxylation of Jasmonoyl-L-isoleucine Impairs 633
Nicotiana attenuata's Resistance to the Generalist Herbivore Spodoptera litura. J Agric 634
Food Chem 64:2822–2831 635
Miersch O, Neumerkel J, Dippe M, Stenzel I, Wasternack C (2008) Hydroxylated jasmonates 636
are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-637
off in jasmonate signaling. New Phytol 177:114–127 638
Mizutani M, Ohta D (2010) Diversification of P450 genes during land plant evolution. Annu 639
Rev Plant Biol 61:291–315 640
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 28, 2019. ; https://doi.org/10.1101/686709doi: bioRxiv preprint
21
Pieterse CM, Van der Does D, Zamioudis C, Leon-Reyes A, Van Wees SC (2012) Hormonal 641
modulation of plant immunity. Annu Rev Cell Dev Biol 28:489–521 642
Piotrowska A, Bajguz A (2011) Conjugates of abscisic acid, brassinosteroids, ethylene, 643
gibberellins, and jasmonates. Phytochemistry 72:2097–2112 644
Poudel AN, Holtsclaw RE, Kimberlin A, Sen S, Zeng S, Joshi T, Lei Z, Sumner LW, Singh 645
K, Matsuura H, Koo AJ (2019) 12-Hydroxy-jasmonoyl-L-isoleucine is an active 646
jasmonate that signals through CORONATINE INSENSITIVE 1 and contributes to the 647
wound response in Arabidopsis. Plant Cell Physiol pii: pcz109. doi:10.1093/pcp/pcz109 648
Poudel AN, Zhang T, Kwasniewski M, Nakabayashi R, Saito K, Koo AJ (2016) Mutations in 649
jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound 650
responses in Arabidopsis thaliana. Biochim Biophys Acta 1861:1396–1408 651
Sasaki-Sekimoto Y, Jikumaru Y, Obayashi T, Saito H, Masuda S, Kamiya Y, Ohta H, Shirasu 652
K (2013) Basic helix-loop-helix transcription factors JASMONATE-ASSOCIATED 653
MYC2-LIKE1 (JAM1), JAM2, and JAM3 are negative regulators of jasmonate responses 654
in Arabidopsis. Plant Physiol 163:291–304 655
Smirnova E, Marquis V, Poirier L, Aubert Y, Zumsteg J, Menard R, Miesch L, Heitz T (2017) 656
Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and 657
Resistance Responses against Botrytis cinerea Infection. Mol Plant 10:1159–1173 658
Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K, He SY, Howe GA, 659
Browse J (2007) JAZ repressor proteins are targets of the SCF(COI1) complex during 660
jasmonate signalling. Nature 448:661–665 661
Wasternack C, Hause B (2013) Jasmonates: biosynthesis, perception, signal transduction and 662
action in plant stress response, growth and development. An update to the 2007 review in 663
Annals of Botany. Ann Bot 111:1021–1058 664
Wasternack C, Song S (2017) Jasmonates: biosynthesis, metabolism, and signaling by proteins 665
activating and repressing transcription. J Exp Bot 68:1303–1321 666
Widemann E, Miesch L, Lugan R, Holder E, Heinrich C, Aubert Y, Miesch M, Pinot F, Heitz 667
T (2013) The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine 668
hormone turnover and generate 12-hydroxyjasmonic acid upon wounding in Arabidopsis 669
leaves. J Biol Chem 288:31701–31714 670
Widemann E, Smirnova E, Aubert Y, Miesch L, Heitz T (2016) Dynamics of Jasmonate 671
Metabolism upon Flowering and across Leaf Stress Responses in Arabidopsis thaliana. 672
Plants (Basel) 5 673
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 28, 2019. ; https://doi.org/10.1101/686709doi: bioRxiv preprint
22
Woldemariam MG, Onkokesung N, Baldwin IT, Galis I (2012) Jasmonoyl-L-isoleucine 674
hydrolase 1 (JIH1) regulates jasmonoyl-L-isoleucine levels and attenuates plant defenses 675
against herbivores. Plant J 72:758–767 676
Yan C, Fan M, Yang M, Zhao J, Zhang W, Su Y, Xiao L, Deng H, Xie D (2018) Injury Activates 677
Ca(2+)/Calmodulin-Dependent Phosphorylation of JAV1-JAZ8-WRKY51 Complex for 678
Jasmonate Biosynthesis. Mol Cell 70:136–149.e7 679
Zhang T, Poudel AN, Jewell JB, Kitaoka N, Staswick P, Matsuura H, Koo AJ (2016) Hormone 680
crosstalk in wound stress response: wound-inducible amidohydrolases can 681
simultaneously regulate jasmonate and auxin homeostasis in Arabidopsis thaliana. J Exp 682
Bot 67:2107–2120 683
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O
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Figure 1. Positions of the impaired enzymatic steps in JA-‐Ile catabolic mutants and simplified view of analyzedsignaling and defense responsesLeft box: Necrotroph fungus infection or mechanical wounding/insect attack trigger jasmonic acid (JA) biosynthesis.Some JA is conjugated to isoleucine by JAR1 enzyme to form bioactive jasmonoyl-‐isoleucine (JA-‐Ile). JA-‐Ile is turnedover by a two-‐step oxidation to 12OH-‐JA-‐Ile and 12COOH-‐JA-‐Ile by the cytochrome P450 enzymes CYP94B3/B1 andCYP94C1, or by conjugate cleavage mediated by the amidohydrolases (AH) IAR3 and ILL6. These AH also cleave12OH-‐JA-‐Ile to release 12OH-‐JA. Red crosses indicate impaired JA-‐Ile catabolic steps in higher order mutants utilizedin this study: oxidation-‐deficient (triple cyp94b1 b3 c1 mutant, 3cyp), deconjugation-‐deficient (double iar3 ill6, 2ah)or deficient for both pathways (quintuple mutant, 5ko). Right box: in absence of JA-‐Ile, JAZ repressors blocktranscription of target genes. Upon biosynthesis, JA-‐Ile promotes the assembly of the COI1-‐JAZ co-‐receptor which isrecruited into the SCFCOI1 E3 ubiquitin ligase directing proteolytic degradation of JAZ repressors. Transcriptionfactors (TFs) like MYC2 or ORA59 become activated in association with mediator 25 subunit (MED25) and allow thetranscription of numerous stimulus-‐specific defense responses. The anti-‐insect branch was probed by the MYC2-‐VSP genes, and the antimicrobial branch by the ORA59 and PDF1.2 genes. Among JA-‐Ile targets are also genesencoding JAZ repressors or JAM proteins that compete with MYC2 and ORA59, to attenuate signaling. In mutantplants impaired for JA-‐Ile catabolism, overinduction of this negative feedback loop may occur and prevent hyper-‐stimulation of JA-‐Ile-‐dependent defenses and associated enhanced resistance.
JA JA-Ile
12OH-JA 12OH-JA-Ile
12COOH-JA-Ile
SCFCOI1 signalling
JAO2JAO3JAO4
Stimulus
JAR1
CYP94B3CYP94B1
3cyp
CYP94C1
IAR3/ILL6
IAR3/ILL6
5ko
5ko2ah
5ko2ah
3cyp5ko
JA-Ile
TFJAZ
COI1COI1
Transcription
JAZ
JAM
JAZ
JAMJAZ
Defense cascadeNegative feedback
PDF1.2 VSP1 VSP2
Antimicrobial
Repression
JAZ
JAZ
Anti-insect
Physiological responses
MED25TF
Jasmonatemetabolism Resistance
?
Infection Wounding Herbivory
ORA59 MYC2
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JA-‐Ile
12OH-‐JA-‐Ile
12CO
OH-‐JA-‐Ile
Figure 2. JA-‐Ile and oxidized catabolites accumulation in 2ah, 3cyp and 5ko mutants after mechanicalwounding or Botrytis cinerea Infection.Six-‐week-‐old plants were wounded 3 times across leaf mid-‐vein (A) or drop-‐inoculated on two sites perleaf with a suspension containing 2.5x106 fungal spores mL-‐1 (B). Treated leaves were harvested at 1, 3and 6 hours post wounding (hpw) or 3 days post inoculation (dpi) on WT (black bars), 2ah (yellow bars),3cyp (blue bars) and 5ko (green bars) plants. Jasmonates were extracted and quantified by LC–MS/MS.JA-‐Ile, 12OH-‐JA-‐Ile and 12COOH-‐JA-‐Ile levels were expressed in nmol g-‐1 fresh weight (FW). Histogramsrepresent the mean ± SEM of three (wounding) or four (B. cinerea) biological replicates. Columns labeledwith different letters indicate a significant difference between genotypes at each time point asdetermined by one-‐way ANOVA with Tukey post-‐hoc test, P < 0.05.
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Figure 3. Expression profiles of JA-‐Ile-‐dependent genes in response to mechanical wounding and impact oninsect feeding in JA-‐Ile catabolic mutants.(A) Expression profiles of jasmonate-‐dependent MYC2, VSP1 and VSP2marker genes in response to wounding in WT (black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green bars) mutants. Relative expression of each target gene at 1, 3 or 6 hours post wounding (hpw) is represented. Gene expression was determined by real-‐time PCR using gene-‐specific primers and normalized using EXP and TIP41 reference genes. Transcript quantification was performed on three biological replicates analyzed in duplicate. Histograms represent mean expression ± SEM. Columns labeled with different letters indicate a significant difference between genotypes at each time point as determined by one-‐way ANOVA with Tukey posthoc test, P < 0.05.(B) Insect feeding assay. S. littoralis larvae were placed on leaves of 4-‐week-‐old plants. After 8-‐9 days, larval weight was determined. Histograms represent mean ± SEM from 3 independent trials (presented in Supplemental Figure S3). The number of total larvae on each genotype is indicated within the bars. Different letters indicate significant differences at P < 0.05 (linear mixed model).
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Figure 4. Expression profiles of JA-‐Ile-‐dependent defense genes and resistance levels in response to B.cinerea infection in 2ah, 3cyp and 5komutants.(A) Expression profiles of jasmonate-‐regulated ORA59 and PDF1.2 genes in response to infection by B.cinerea in WT (black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green bars) mutants. Relativeexpression of each target gene at 3 day post infection (dpi) is represented. Gene expression wasdetermined by real-‐time PCR using gene-‐specific primers and normalized using EXP and TIP41 referencegenes. Transcript quantification was performed on three biological replicates analyzed in duplicate.Histograms represent mean expression ± SEM. Columns labeled with different letters indicate a significantdifference between genotypes at each time point as determined by one-‐way ANOVA with Tukey posthoctest, P < 0.05.(B) Disease resistance assessment. Two sites per leaf were inoculated across the main vein with 5 µL ofspore suspension containing 2.5x106 spores mL-‐1. Left panel: representative leaves of each genotype weredetached and photographed at 3 dpi. Middle panel: histograms represent the mean lesion diameters ±SEM of about 100 lesion sites from 10 to 15 plants for each genotype. Right panel: evaluation of fungalgrowth by real-‐time qPCR with B. cinerea cutinase-‐specific primers on genomic DNA extracted from 3-‐day-‐infected leaves. Quantification was performed on three biological replicates analyzed in duplicate.Columns labeled with different letters indicate a significant difference as determined by one-‐way ANOVAwith Tukey post-‐hoc test (P < 0.01).
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3 dpi3 dpi1 hpw 3 hpw 6 hpw 1 hpw 3 hpw 6 hpw
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A B
Figure 5. Expression profiles of JA-‐Ile dependant negative feedback genes in response to wounding andinfection in 2ah, 3cyp and 5komutants.Expression profiles of jasmonate-‐dependent genes in response to wounding (A, JAZ8, JAZ10, JAM1 andJAM2) or infection by Botrytis cinerea (B, JAZ1, JAZ8, JAM1 and JAM2) in WT (black bars), 2ah (yellowbars), 3cyp (blue bars) and 5ko (green bars) mutants. Relative expression of each target gene at 1, 3 or 6hours post wounding (hpw) and 3 day post infection (dpi) is represented. Gene expression wasdetermined by real-‐time PCR using gene-‐specific primers and normalized using EXP and TIP41 referencegenes. Transcript quantification was performed on three biological replicates analyzed in duplicate.Histograms represent mean expression ± SEM. Columns labeled with different letters indicate asignificant difference between genotypes at each time point as determined by one-‐way ANOVA withTukey posthoc test, P < 0.05.
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Figure 6. Relationships between impaired JA-‐Ile catabolism, JA-‐Ile levels, defense/repressorgene induction, and resistance to attackers.The data show distinct circuitry for the two leaf responses analyzed. The signs -‐, 0, or +indicate lower, equal, or increased response respectively, in stimulated mutant genotypescompared to WT. The two catabolic pathways have differential contributions to JA-‐Ileturnover in response to wounding or infection. JA-‐Ile accumulation is enhanced to differentextents, except in infected 3cyp, and this has variable consequences on defense amplitudeand aggressor resistance. The trend emerging is that impaired hormone catabolism does,with or without JA-‐Ile overaccumulation, result in better defense/resistance, only if negativeeffectors like JAZ or JAM are themselves not overinduced.
Mutant&genotype
Impaired&pathway& JA5Ile&levels&
Defense&transcript&induction&
Repressor&transcript&induction
Resistance&phenotype
3cyp oxidation + 0 ++ *+*2ah deconjugation + ++ 0 +5ko both +++ + ++ 0
Mutant&genotype
Impaired&pathway JA5Ile&levels&
Defense&transcript&induction&
Repressor&transcript&induction
Resistance&phenotype
3cyp oxidation 0 0 0 +2ah deconjugation ++ * + 0/+5ko both ++ * + 0
B.#cinerea&infection
Wound/Insect
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