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BREAKTHROUGH REPORT 1
2
3
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Arabidopsis STAY-GREEN, Mendel’s Green Cotyledon Gene, Encodes 5
Magnesium-Dechelatase 6
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Yousuke Shimoda1, Hisashi Ito1,2*, Ayumi Tanaka1,2 9
10 1 Institute of Low Temperature Science, Hokkaido University, N19 W8, Kita-ku, Sapporo, 060-0819, Japan 11 2 CREST, Japan Science and Technology Agency, N19 W8, Kita-ku, Sapporo, 060-0819, Japan 12
Corresponding author: Hisashi Ito, ito98@lowtem.hokudai.ac.jp 13
14
Short Title: Identification of Mg-dechelatase. 15
One-Sentence Summary: Arabidopsis STAY-GREEN is a functional Mg-dechelatase that extracts Mg from free 16
chlorophyll and from chlorophyll in complexes, thus acting in chlorophyll degradation and photosystem 17
degradation. 18
19
The author responsible for distribution of materials integral to the findings presented in this article in accordance 20
with the policy described in the Instructions for Authors (www.plantcell.org) is: Hisashi Ito 21
(ito98@lowtem.hokudai.ac.jp). 22
23
ABSTRACT 24
Pheophytin a is an essential component of oxygenic photosynthetic organisms, because the primary charge 25
separation between chlorophyll a and pheophytin a is the first step in the conversion of light energy. In addition, 26
conversion of chlorophyll a to pheophytin a is the first step of chlorophyll degradation. Pheophytin is synthesized 27
by extracting magnesium (Mg) from chlorophyll; the enzyme Mg-dechelatase catalyzes this reaction. In this study, 28
we report that Mendel’s green cotyledon gene, STAY-GREEN (SGR), encodes Mg-dechelatase. The Arabidopsis 29
thaliana genome has three SGR genes, STAY-GREEN1 (SGR1), STAY-GREEN2 (SGR2), and STAY-GREEN LIKE 30
(SGRL). Recombinant SGR1/2 extracted Mg from chlorophyll a but had very low or no activity against 31
chlorophyllide a; in contrast, SGRL had higher dechelating activity against chlorophyllide a compared to 32
chlorophyll a. All SGRs could not extract Mg from chlorophyll b. Enzymatic experiments using the photosystem 33
and light-harvesting complexes showed that SGR extracts Mg not only from free chlorophyll but also from 34
chlorophyll in the chlorophyll-protein complexes. Furthermore, most of the chlorophyll and chlorophyll-binding 35
Plant Cell Advance Publication. Published on September 7, 2016, doi:10.1105/tpc.16.00428
©2016 American Society of Plant Biologists. All Rights Reserved
2
proteins disappeared when SGR was transiently expressed by a chemical induction system. Thus, SGR is not only 36
involved in chlorophyll degradation but also contributes to photosystem degradation. 37
38
INTRODUCTION 39
Chlorophyll and its derivatives play essential roles in photosynthesis, where chlorophyll harvests light 40
energy and transfers it to the reaction center. Most chlorophyll molecules in photosynthesis are involved 41
in this process. Green plants have two different chlorophyll species, chlorophyll a and b, which harvest 42
light energy; the biosynthetic pathway for these chlorophylls has been studied extensively (Tanaka and 43
Tanaka, 2007). Chlorophyll a is synthesized from 5-aminolevulinic acid through multiple steps. At the 44
last step of chlorophyll synthesis, a portion of chlorophyll a is converted to chlorophyll b by 45
chlorophyllide a oxygenase via 7-hydroxymethyl chlorophyll a. Chlorophyll b is reconverted to 46
chlorophyll a by chlorophyll b reductase (CBR) and 7-hydroxymethyl chlorophyll a reductase (HCAR) 47
(Meguro et al., 2011). Arabidopsis thaliana has two isozymes of CBR, NON-YELLOW COLORING 1 48
(NYC1) and NYC1-LIKE (NOL) (Kusaba et al., 2007; Horie et al., 2009). This pathway, known as the 49
chlorophyll cycle, interconverts chlorophyll a and chlorophyll b (Figure 1). All the enzymes responsible 50
for chlorophyll synthesis and for the chlorophyll cycle have been identified and the chlorophyll 51
metabolic pathway has been determined. 52
Another important function of chlorophyll is to drive electron transfer and pheophytin a plays a 53
crucial role in this function. In the reaction center of photosystem II (PSII), the primary charge 54
separation between P680 (chlorophyll a; PSII primary donor) and pheophytin a occurs; this is the first 55
step in the conversion of light to chemical energy in photosynthesis (Holzwarth et al., 2006). Pheophytin 56
a is synthesized by extracting magnesium (Mg) from chlorophyll a. The enzyme responsible for this 57
reaction has been tentatively called Mg-dechelatase, although it is still not evident whether other 58
enzymes catalyze Mg-dechelation, or whether it occurs spontaneously under acidic conditions. 59
Mg-dechelation is an important process in the formation of PSII, because PSII assembly starts with the 60
3
formation of the D1/D2 complex of which pheophytin a is an indispensable component (Nickelsen and 61
Rengstl, 2013). 62
Mg-dechelatase also has a physiological function during senescence. A recent study showed that the 63
first step of chlorophyll degradation is the conversion of chlorophyll a to pheophytin a (Christ and 64
Hörtensteiner, 2013). Pheophytin a is then converted to pheophorbide a by pheophytin pheophorbide 65
hydrolase (pheophytinase; PPH); pheophorbide a is then oxidatively ring-opened to the red chlorophyll 66
catabolite by pheophorbide a oxygenase (PaO); this is followed by the reduction to fluorescent 67
chlorophyll catabolite by red chlorophyll catabolite reductase (RCCR) (Rodoni et al., 1997; Schelbert et 68
al., 2009). Interestingly, chlorophyll b cannot directly enter into this degradation pathway but must be 69
converted to chlorophyll a before degradation; this is due to the substrate specificity of the latter 70
degradation enzymes (Hortensteiner, 2006). The degradation of chlorophyll is a key part of nitrogen 71
recycling and also important in avoiding cellular damage. If chlorophyll degradation is not properly 72
regulated, severe photodamage occurs and cell death is induced (Pruzinska et al., 2003; Hirashima et al., 73
2009; Hortensteiner and Krautler, 2011). Among chlorophyll degradation enzymes, Mg-dechelatase is 74
especially important for regulation because it catalyzes the step in which chlorophyll is committed to 75
degradation. 76
As Mg-dechelatase has indispensable functions in the formation of PSII and the degradation of 77
chlorophyll, many attempts have been made to identify it; however, all these efforts failed. This has been 78
partly due to the difficulty of detecting dechelation activity in vitro using chlorophyll(ide) as a substrate 79
(Hortensteiner and Krautler, 2011). Instead of chlorophyll, an artificial substrate chlorophyllin (a 80
semi-synthetic derivative of chlorophyll), has been widely used to measure Mg-dechelatase activity. 81
However, this might lead to a failure in identifying Mg-dechelatase because the real substrate of 82
Mg-dechelatase is chlorophyll a. 83
Most of the mutants of chlorophyll degradation enzymes, such as PPH (Schelbert et al., 2009), PaO 84
4
(Pruzinska et al., 2003), and CBR (Kusaba et al., 2007; Horie et al., 2009), exhibit a stay-green 85
phenotype. It is therefore reasonable to assume that the mutation of Mg-dechelatase would also cause a 86
strong stay-green phenotype because it catalyzes the first step of the chlorophyll degradation pathway. 87
Mendel studied the mechanisms of inheritance using seven pea mutants, including a green cotyledon 88
mutant. Recently, Mendel’s green cotyledon gene was shown to encode the STAY-GREEN (SGR) 89
protein. The SGR mutation induces a stay-green phenotype not only in Mendel’s green cotyledon 90
(Armstead et al., 2007; Sato et al., 2007), but also in many other plants (Park et al., 2007; Ren et al., 91
2007). Many studies have been carried out to elucidate the function of SGR and a hypothesis for SGR 92
function was proposed based on protein–protein interaction experiments. Sakuraba et al. (Sakuraba et al., 93
2012) found that SGR physically interacted with the light-harvesting complex of PSII (LHCII), and also 94
with six chlorophyll degradation enzymes including HCAR, NOL, NYC1, PaO, PPH, and RCCR; they 95
proposed a complex of SGR with LHCII and chlorophyll degradation enzymes that allows the metabolic 96
channeling of chlorophyll degradation intermediates. However, the question remained whether SGR can 97
simultaneously bind six proteins and whether SGR has other functions. 98
We speculated that the SGR gene could encode Mg-dechelatase because all the sgr mutants showed 99
strong stay-green phenotypes. To examine this possibility, we carried out in vitro and in vivo 100
experiments. When SGR was transiently expressed in Arabidopsis, chlorophyll was degraded and this 101
was accompanied by the accumulation of a small amount of pheophytin a. Recombinant SGR proteins 102
prepared using a wheat germ protein expression system converted chlorophyll a to pheophytin a, but 103
SGR had no activity against chlorophyll b. When we incubated SGR with chlorophyll-protein 104
complexes isolated with a sucrose density gradient, chlorophyll a was efficiently converted to 105
pheophytin a. Based on these experiments, we concluded that Mendel’s green cotyledon gene (SGR) 106
encodes Mg-dechelatase. We discuss the enzymatic properties of SGR in relation to the degradation of 107
photosystems. 108
5
109
RESULTS 110
Mg-Dechelating Activity of Recombinant SGR 111
The Arabidopsis thaliana genome contains three SGR genes, STAY-GREEN1 (SGR1; AT4G22920), 112
STAY-GREEN2 (SGR2; AT4G11910), and STAY-GREEN LIKE (SGRL; AT1G44000) (Sakuraba et al., 113
2014). First, we used recombinant mature SGR proteins expressed in Escherichia coli for enzymatic 114
experiments, but we did not observe any Mg-dechelating activity. Next, we examined the 115
Mg-dechelating activity of mature SGR proteins prepared by a wheat germ protein expression system 116
(Supplemental Figure 1). Recombinant SGR1 had high dechelating activity against chlorophyll a but 117
very low activity against chlorophyllide a (Figure 2A, Supplemental Data Set 1). Substrates and 118
products were identified by their absorption spectra (Supplemental Figure 2) and by their HPLC 119
retention time (Shimoda et al., 2012).The substrate specificity of SGR2 was almost the same as that of 120
SGR1, which is consistent with the high amino acid sequence similarity between SGR1 and SGR2 121
(Supplemental Figure 3). In contrast, SGRL had much higher activity against chlorophyllide a than 122
against chlorophyll a (Figure 2B). None of the three SGRs (SGR1, SGR2, and SGRL) extracted Mg 123
from chlorophyll b. These results suggest that SGR has Mg-dechelating activity and that substrate 124
specificity is different between SGR1/2 and SGRL. 125
To confirm that in vitro Mg dechelation is an enzymatic reaction catalyzed by SGR, the following 126
experiments were carried out by using recombinant SGRL protein because it has the highest activity 127
among three SGRs. Mg-dechelating activity was completely lost by heating at 95ºC (Figure 3). Purified 128
SGRL, showing a single or a major band on SDS-PAGE, had Mg-dechelating activity (Supplemental 129
Figure 4) suggesting that SGR has Mg-dechelating activity without any other factors. A time course 130
study showed that the amount of the products (pheophytin a or pheophorbide a) increased depending on 131
the incubation time and the product never increased without SGRL proteins in the reaction mixture 132
6
(Figure 4A). Increasing concentrations of chlorophyll a and chlorophyllide a substrates were 133
accompanied by enhanced conversion to their respective products (Figure 4B). The non-linearity 134
observed using chlorophyll a as a substrate differs from the almost linear increase in product formation 135
obtained with chlorophyllide a (Figure 4B, left panel); this could arise from a number of factors and a 136
more detailed analysis is required. The amount of the product depended on the concentration of SGRL 137
(Figure 4C). All these results strongly indicate that release of Mg from chlorophylls occurs 138
enzymatically by SGR. 139
140
Mg-Dechelating Activity of SGR in Cells 141
Recombinant SGR showed Mg-dechelating activity in vitro; however, it is not evident whether SGR 142
functions as a Mg-dechelatase in cells. To answer this question, we transiently expressed SGR1 with a 143
chemical induction system containing dexamethasone (DEX), and we examined the accumulation of 144
pheophytin a, a product of Mg-dechelatase (Figure 5A). SGR1 expression increased the level of 145
pheophytin a. Although the increase in pheophytin a suggested the occurrence of Mg-dechelation by 146
SGR1, the absolute level of pheophytin a was very low (Figure 5B). One possible reason for this is that 147
synthesized pheophytin a is immediately degraded by the next enzyme, PPH. In order to examine this 148
possibility, SGR1 was transiently induced in pph background and the pigments were analyzed (Figure 149
5B). Pheophytin a accumulated more in the pph background than in the WT by DEX treatment (Figure 150
5C), indicating that SGR could function as a Mg-dechelatase in cells. 151
For further confirmation of SGR function, we introduced mature SGR1 into the cyanobacterium 152
Synechococcus elongatus PCC7942 (hereafter Synechococcus) (Supplemental figure 5A). The 153
Synechococcus genome has no SGR, or any homologous gene, indicating that Synechococcus has no 154
SGR system for Mg-dechelation. If SGR requires other protein components, it would not be expected to 155
function as a Mg-dechelatase in Synechococcus cells. Our immunoblot analysis showed that SGR1 was 156
7
successfully expressed in Synechococcus (Supplemental figure 5B). Chlorophyll content was low 157
(Figure 6A) and pheophytin a and pheophorbide a accumulated in large amounts (Figure 6B) in 158
Synechococcus expressing SGR1, indicating that SGR1 functions as Mg-dechelatase in Synechococcus 159
cells. Interestingly, the level of pheophorbide a was comparable to that of pheophytin a, which was quite 160
different from the results obtained with the Arabidopsis leaves in which pheophorbide a was not 161
detected (Figure 5B). Pheophorbide a might be synthesized from pheophytin a by an unknown PPH-like 162
enzyme in Synechococcus cells. Based on these experiments, we finally concluded that SGR encodes a 163
Mg-dechelatase and that no other protein is required for the dechelating activity of SGR. 164
165
Expression of SGR in Arabidopsis 166
To elucidate the impact of SGR on chlorophyll metabolism and the relationship between SGR and other 167
chlorophyll metabolic enzymes, we constitutively overexpressed the cDNA of the SGR1 gene in 168
wild-type (WT) Arabidopsis plants and mutants, such as ch1-1 (mutant of chlorophyllide a oxygenase), 169
and the cbr and pph mutants. These transgenic plants exhibited low chlorophyll content and retarded 170
growth (Figure 7). We assumed that the plants would not grow when SGR1 was expressed in large 171
amounts and that only the mutants with low expression levels of SGR would survive. Low chlorophyll 172
content was also observed when SGR2 or SGRL was constitutively overexpressed (Supplemental Figure 173
6, Supplemental Figure 7). 174
Based on these severe phenotypes, we concluded that constitutive overexpression is not appropriate 175
for the study of SGR function. Instead, we transiently expressed SGR1 in fully greened leaves using a 176
DEX induction system (Figure 8). Three independent transgenic lines transiently overexpressing SGR1 177
in a WT background (line numbers 3, 19, and 34) are shown in Figure 8A. After 24 h of DEX treatment, 178
approximately half of the chlorophyll was degraded in the WT background (Figure 8C). Chlorophyll 179
degradation was also observed in the pph mutant background; however, 70% of chlorophyll still 180
8
remained after 24 h of DEX treatment. Interestingly, the level of chlorophyll b was not significantly 181
changed by DEX treatment in a cbr mutant background, although chlorophyll a was extensively 182
degraded. This is consistent with experiments demonstrating that SGR did not extract Mg from 183
chlorophyll b (Figure 2A). Reduction of chlorophyll content was also observed when SGR2 or SGRL 184
was transiently induced by DEX induction system (Supplemental Figure 6, Supplemental Figure 7). 185
Next, we used excised leaves from either a WT or ch1-1 background to examine the effect of SGR1 186
expression on chloroplast proteins (Figure 9). After DEX treatment, chlorophyll levels decreased to 20% 187
of the initial level in both WT and ch1-1 backgrounds (Figure 9B). The rate of chlorophyll degradation 188
was slightly faster in the ch1-1 background than in the WT background. Upon DEX treatment, a 189
reduction in chlorophyll content was accompanied by a decrease in chlorophyll-binding proteins of both 190
photosystems and LHC (Figure 9D). Degradation of PSI (CP1) and Lhca1 was slightly faster than that 191
of PSII (CP43, CP47, D1, D2) and Lhcb1. This was confirmed by a low-temperature fluorescence 192
spectrum (Supplemental Figure 8) in which PSI fluorescence (approximately 735 nm) decreased rapidly, 193
compared to PSII fluorescence (688 nm and 695 nm). In contrast, the levels of the cytochrome b6f 194
complex, a thylakoid membrane protein, and ribulose-1,5-bisphosphate carboxylase/oxygenase, a 195
soluble protein, were not significantly affected by DEX treatment. These results indicate that SGR 196
regulates the first step of photosystem degradation by dechelating Mg from chlorophyll molecules. An 197
experiment examining electrolyte leakage confirmed that degradation of chlorophyll and 198
chlorophyll-binding proteins was not caused by cell death in these plants (Supplemental Figure 9). 199
200
Chlorophyll a in the Pigment-Protein Complex is a Substrate of SGR 201
Most of the chlorophyll was degraded within 24 h of SGR1 expression. This suggests that SGR is able to 202
release Mg not only from free chlorophyll but also from chlorophyll existing in photosystems because 203
all of the chlorophyll binds to proteins in the chloroplast. To examine this possibility, we incubated 204
9
recombinant SGR with PSI or LHCII purified by sucrose density gradient centrifugation. Pheophytin a 205
accumulated after incubation with both substrates (Figure 10). The level of chlorophyll b was unchanged 206
following incubation with SGR1 or SGRL, indicating that chlorophyll b is not a substrate of these 207
proteins. SGR1 and SGRL had high catalytic activity against chlorophyll a in both PSI and LHCII. 208
These observations suggest that SGR directly attacked the pigment-protein complexes and converted 209
chlorophyll a to pheophytin a. 210
211
DISCUSSION 212
SGR Encodes Mg-Dechelatase 213
The main enzymes of the chlorophyll degradation pathway have been identified previously, with the 214
exception of Mg-dechelatase (Hortensteiner and Krautler, 2011). It has long been debated whether 215
Mg-dechelation is brought about by an enzyme (Costa et al., 2002) or small substance (Suzuki et al., 216
2005), or whether it takes place spontaneously in a low pH environment (Christ and Hörtensteiner, 217
2013), as Mg-dechelatase has not been identified despite great efforts. In this study, we demonstrated 218
that SGR encodes Mg-dechelatase. 219
There are no reports that discuss the metal dechelation mechanism. However, ferrochelatase 220
catalyzes the reverse reaction of metal dechelation. According to the study of ferrochelatase, metal 221
chelation consists of metal binding to the ferrochelatase, deprotonation from two -NH and insertion of 222
Fe into protoporphyrin IX (Wang et al., 2009). Glutamate, tyrosine and histidine residues play a central 223
role in these processes. Interestingly, these amino acid residues are conserved in SGRs. It is possible to 224
speculate that Mg is dechelated by the reverse reaction of ferrochelatase i.e. two protonations followed 225
by Mg-dechelation. Amino acid substitution experiments will uncover the dechelation mechanism of 226
SGR. 227
The sgr mutants have been extensively studied since Mendel, and exhibit a strong stay-green 228
10
phenotype without exception (Sato et al., 2007). This stay-green phenotype is consistent with our 229
conclusion that SGR encodes Mg-dechelatase because it catalyzes the committed step of chlorophyll 230
degradation. Sakuraba et al.(Sakuraba et al., 2012) proposed that SGR binds six chlorophyll degradation 231
enzymes and forms a large complex (SGR-chlorophyll catabolic enzymes-LHCII complex) that enables 232
efficient metabolic trafficking. The ch1-1 mutant lacks LHCII because chlorophyll b is not synthesized. 233
However, SGR efficiently catalyzes the dechelating reaction in the ch1-1 mutant as in the WT 234
background. These observations suggest that LHCII is not required for SGR function. In addition, if 235
SGR binds many proteins (i.e., six chlorophyll degradation enzymes), it might be difficult for SGR to 236
have access to the substrate of the chlorophyll-protein complexes. Another question is whether it is 237
possible for SGR to simultaneously bind six proteins from a structural viewpoint. The hypothesis of the 238
SGR-chlorophyll catabolic enzymes-LHCII complex should be re-examined. However, complexes 239
consisting of two proteins (SGR-PPH, SGR-HCAR, and SGR-LHCII) should be considered because 240
LHCII is a substrate of SGR and because SGR must accept chlorophyll a from HCAR and transfer 241
pheophytin a to PPH. 242
243
Substrate Specificity and Physiological Functions of SGRs 244
Initially, we examined the dechelating activity of recombinant SGR expressed in E. coli; however, we 245
did not observe any activity that was consistent with previous reports (Hortensteiner, 2009). Then, we 246
used the recombinant SGR prepared with a wheat germ protein expression system instead of E. coli; 247
high dechelating activity was observed, indicating that the activity of SGR largely depends on 248
protein-producing systems. Enzymatic experiments with recombinant SGR showed interesting substrate 249
specificity among different SGRs; SGR1/2 extracted Mg from chlorophyll a but showed very low or no 250
activity against chlorophyllide a. Considering the expression of SGR1/2 predominantly during 251
senescence (Sakuraba et al., 2014) and its substrate specificity, SGR1/2 might be involved in chlorophyll 252
11
degradation during senescence. This hypothesis is consistent with the strong stay-green phenotype of the 253
sgr1/sgr2 double mutant (Wu et al., 2016). In contrast, the SGRL protein is expressed during greening 254
(Sakuraba et al., 2014). Interestingly, SGRL showed higher activity against chlorophyllide a than against 255
chlorophyll a. The conversion of chlorophyllide a to pheophorbide a by SGRL might not be an 256
experimental artifact but might have a physiological function. Chidgey et al. (Chidgey et al., 2014) 257
proposed that chlorophyllide a is a component of the machinery involved in the formation of 258
photosystems. Lin et al. reported that chlorophyllide a, which is derived from chlorophyll a, is reused 259
for chlorophyll synthesis (Lin et al., 2014). The level of chlorophyllide a might partly be regulated by 260
SGRL. Conversion of chlorophyllide a to pheophorbide a by SGRL suggests a new chlorophyll 261
degradation pathway via chlorophyllide a (chlorophyllide pathway). 262
SGR1, SGR2, and SGRL could not extract Mg from chlorophyll b. This is consistent with the 263
chemical experimental results that chlorophyll b is much more stable in acidic conditions compared to 264
chlorophyll a (Saga and Tamiaki, 2012). The question remains as to whether SGR could not extract Mg 265
from chlorophyll b due to the stabilization of Mg in chlorophyll b by the effect of 7-formyl group, or 266
whether SGR evolved to fit to chlorophyll a. 267
268
Regulation of Photosystem Dynamics by Chlorophyll Metabolic Enzymes 269
Two hypotheses exist for the degradation of photosystems. One is that some proteases are responsible 270
for the first step of this process; chlorophyll degradation enzymes immediately degrade the resulting free 271
chlorophylls. The other hypothesis is that chlorophyll degradation enzymes catalyze the first step of 272
photosystem degradation and the resulting apoproteins are degraded by proteases. The present SGR 273
study supports the latter hypothesis. When we transiently induced SGR in fully greened leaves, 274
chlorophyll levels decreased. This suggests that SGR extracts Mg from chlorophyll embedded in 275
chlorophyll-protein complexes because all the chlorophyll molecules exist as chlorophyll-protein 276
12
complexes. This idea was supported by in vitro experiments; chlorophyll a was converted to pheophytin 277
a when isolated chlorophyll-protein complexes were incubated with SGR (Figure 10). Interestingly, 278
chlorophyll-binding proteins also disappeared along with a decrease in chlorophyll upon induction of 279
SGR (Figure 9D), suggesting that chlorophyll-depleted apoproteins are immediately degraded in 280
thylakoid membranes. If protein degradation occurred before chlorophyll degradation, a large amount of 281
free chlorophyll would accumulate and the stay-green phenotype would not be observed in sgr mutants, 282
because free chlorophyll rapidly induces bleaching. These phenomena are similar to those of CBR 283
(Horie et al., 2009); CBR converts chlorophyll b in LHCII to 7-hydroxymethyl chlorophyll a; this is the 284
first step of chlorophyll b degradation. Chlorophyll b and LHCII are never degraded in the cbr mutant, 285
although chlorophyll a is degraded as in WT plants. These in vitro and in vivo experiments with SGR 286
and CBR strongly suggest that two enzymes, Mg-dechelatase (SGR) and CBR, primarily regulate the 287
degradation of photosystems (PSI, PSII, LHCI and LHCII) in green plants. 288
Another possible role of Mg-dechelatase is to supply pheophytin a for the formation of PSII. A 289
supply of pheophytin a might also be required for the PSII repair cycle. Presently, we have no 290
experimental evidence to support the involvement of SGR in these processes; even if the pheophytin a 291
required for these processes is low, we cannot exclude the possibility that the pheophytin a required for 292
the formation and repair cycle of PSII is generated spontaneously. However, the formation and repair 293
cycle of PSII must be strictly regulated depending on the developmental stage and environmental 294
conditions. It might be difficult to supply enough pheophytin a needed for these processes simply 295
through spontaneous generation. Therefore, it is reasonable to assume that SGR or some other 296
Mg-dechelatase participate in the formation and repair cycle of the PSII. Further study is required to 297
understand the involvement of SGR (Mg-dechelatase) in these processes. 298
299
MATERIALS 300
13
Plant Materials and Growth Conditions 301
Arabidopsis thaliana wild-type (ecotype Columbia) and mutant (ch1-1 (chlorophyllide a oxygenase) 302
(Yamasato et al., 2005), cbr (nyc1 and nol) (Horie et al., 2009) and pph (Hu et al., 2015)) plants were 303
used in this study. We grew plants on soil or half-strength Murashige and Skoog (MS) medium 304
containing 1% (w/v) sucrose, 0.8% (w/v) agar, and 0.05% 2-(N-morpholino)ethanesulfonic acid (MES) 305
buffer (pH 5.8) under 14 h light/10 h dark conditions (70 µmol photons m-2 s-1, white light, fluorescent 306
bulbs) at 24°C. 307
We cultivated Synechococcus elongatus PCC7942 in BG-11 medium with shaking at 40–50 rpm under 308
continuous light (20–30 µmol photons m-2 s-1) at 24°C; we used the logarithmically growing cells for 309
pigment and immunoblot analysis. 310
311
Arabidopsis Transformation 312
We used a polymerase chain reaction (PCR) assay (KOD-Plus-; Toyobo) to prepare Arabidopsis SGR1, 313
SGR2 and SGRL cDNA with a C-terminal FLAG-tag, using the primers listed in Supplemental Table 1, 314
and cloned it into the pGreenII vector (Hellens et al., 2000) under the control of the 35S promoter from 315
the cauliflower mosaic virus using the SalI and NotI sites. To chemically induce expression of SGR in 316
Arabidopsis, we expressed SGR under the control of the pOp6 promoter and the synthetic transcription 317
factor, LhGR (Craft et al., 2005; Wielopolska et al., 2005). We subcloned SGR cDNA with a C-terminal 318
FLAG-tag into a Gateway pENTR 4 Dual Selection Vector (Invitrogen) using an In-Fusion cloning 319
system (Clontech Laboratories) and then introduced it into a binary vector, pOpON, using the Gateway 320
recombination system. We constructed the pOpON vector from pOpOff2 by removing the antisense 321
fragments with the KpnI and XbaI restriction enzymes. We introduced the glufosinate-resistant gene into 322
the ClaI site. We transferred these constructs into the WT plants, and into the ch1-1, cbr, and pph 323
mutants. 324
14
325
Synechococcus Transformation 326
We transformed Synechococcus with a GeneArt Synechococcus Protein Expression Kit (Invitrogen). We 327
used PCR (KOD- Plus-) to amplify the coding region of SGR1 lacking transit peptide using the primers 328
listed in Supplemental Table 1 and cloned it into the pSyn6 vector (Invitrogen) using an In-Fusion 329
system. We followed the manufacturer’s protocol to transform Synechococcus. 330
331
DEX Treatment 332
We grew plants on soil for 3.5–4.5 weeks under 14 h light/10 h dark conditions at 24°C. We placed 333
excised third and fourth rosette leaves on wet filter paper containing 3 mM MES (pH 5.8). For DEX 334
treatment, we prepared DEX as a 20 mM stock in dimethyl sulfoxide. We sprayed the plants with DEX 335
(10 µM) supplemented with 0.015% Silwet L-77; plants were then incubated under continuous light 336
(70 µmol photons m-2 s-1) for 24–30 h at 24°C. When whole plants were treated with DEX, they were 337
grown on MS medium for 2 weeks under 14 h light/10 h dark conditions and sprayed with 10 µM DEX 338
supplemented with 0.015% Silwet L-77 and then incubated under continuous light (70 µmol photons m-2 339
s-1) for 24 h at 24°C. The mock treatment consisted of a Silwet L-77 solution containing 0.05% dimethyl 340
sulfoxide. 341
342
Mg-Dechelatase Assay 343
We obtained chlorophyllide a from chlorophyll a by hydrolysis with recombinant chlorophyllase 344
(Tsuchiya et al., 1999; Shimoda et al., 2012); we isolated the LHCII trimer and photosystem I (PSI) 345
particles and purified them with sucrose gradient centrifugation (Shimoda et al., 2012). We synthesized 346
recombinant SGR and green fluorescent protein (GFP) with an in vitro transcription/translation system 347
(TNT SP6 High-Yield Wheat Germ Protein Expression System; Promega). We removed transit peptides 348
15
and introduced a FLAG-tag at the C-terminus of the SGR1, SGR2, and SGRL proteins (SGR1-FLAG, 349
SGR2-FLAG, and SGRL-FLAG). We amplified the DNA fragments using the primers listed in 350
Supplemental Table 1, and cloned them into the pF3A WG (BYDV) Flexi vector (Promega). We purified 351
plasmid DNA with the PureYield Plasmid Miniprep System (Promega). After expression of the 352
recombinant proteins according to the manufacturer’s protocol, we added one part mixture to three parts 353
buffer in a 50 µl reaction buffer to a final concentration of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 354
and 0.05% polysorbate 20. We dissolved pigments in 80% acetone, and we added 0.8 µl of the acetone 355
solution (375 µM substrate stock) to the reaction buffer. We used chlorophyll a, chlorophyllide a, and 356
chlorophyll b (300 pmol) for the analyses (6 µM final concentration for standard assay). When we used 357
PSI particles or LHCII as the substrate for the Mg-dechelatase assay, we diluted these translation 358
solutions twice in 50 µl of reaction buffer to a final concentration of 50 mM Tris-HCl (pH 7.5), 100 mM 359
NaCl, and 0.05% polysorbate 20; we added 1 µl of solution containing PSI particles or LHCII to the 360
reaction buffer. For every reaction, we used 200 pmol of chlorophyll a in PSI particle or LHCII. We 361
incubated the mixtures at 25°C in the dark for 60 min. In the case of SGRL incubated with chlorophyll a 362
and chlorophyllide a, the incubation time was 15 min. We added nine volumes of acetone after the 363
reaction. After centrifugation at 21,600g for 15 min at 4°C, we analyzed the pigments with 364
high-performance liquid chromatography (HPLC). All reported chlorophyll quantities are the mean 365
values of three independent samples. 366
367
Pigment Analysis 368
We ground the leaves in pure acetone stored at -30°C , using a Shake Master homogenizer (Biomedical 369
Science) cooled in liquid nitrogen (Hu et al., 2013). We harvested Synechococcus cells by centrifugation 370
at 4°C. We disrupted the cells in pure methanol stored at 4°C, with a Shake Master homogenizer cooled 371
in liquid nitrogen. We separated the pigments on a Symmetry C8 column (150 × 4.6 mm; Waters). We 372
16
analyzed the pigments extracted from the plants using the solvent (methanol/acetonitrile/acetone = 1:2:1 373
(v/v)) at the flow rate of 1.0 ml/min. We analyzed the pigments extracted from the reaction mixture 374
according to Zapata et al. (Zapata et al., 2000). We monitored the elution profiles with a diode array 375
detector (SPD-M10AVP; Shimadzu) and a fluorescence detector monitoring 680 nm fluorescence with 376
410 nm excitation (RF-20A; Shimadzu). 377
378
Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) and Immunoblot 379
Analysis 380
To extract all the proteins from the leaf tissue, we ground the leaf tissue in liquid nitrogen and 381
homogenized it in 20 volumes (v/w) of protein extraction buffer containing 50 mM Tris-HCl (pH 8.0), 382
12% (w/v) sucrose, 2% (w/v) lithium lauryl sulfate, and 1.5% (w/v) dithiothreitol. We denatured the 383
samples at 90°C for 2 min; then, we mixed the samples with an equal volume of urea lysis buffer 384
containing 10 mM Tris-HCl (pH 8.0), 10% (w/v) sucrose, 2% (w/v) SDS, 1 mM 385
ethylenediaminetetraacetic acid, 4 mM dithiothreitol, a small amount of bromophenol blue, and 10 M 386
urea; finally, we centrifuged the samples at 21,600g for 5 min at 25°C. We harvested the Synechococcus 387
cells by centrifugation at 4 °C. We resuspended the pellets in the aforementioned protein extraction 388
buffer. We disrupted the resuspended cells (300 µl) by vigorous shaking with glass beads (200 mg, 389
0.1 mm in diameter; M&S Instruments) using a Shake Master homogenizer for 5 min at 4°C. We 390
denatured the samples at 90°C for 2 min and then centrifuged them at 21,600g for 5 min at 25°C. We 391
determined protein concentrations using a Bradford Ultra Kit (Expedeon) with bovine serum albumin 392
(Sigma-Aldrich) as the protein standard. We subjected proteins to SDS-PAGE with a polyacrylamide gel 393
(14%) containing 4 M urea. After electrophoresis, we transferred the proteins to polyvinylidene 394
difluoride membranes. We normalized samples by their fresh weight for Arabidopsis and the volume of 395
reaction mixture for recombinant proteins. We analyzed 5 μg of Synechococcus protein. We stained 396
17
proteins with a Quick-CBB kit (Wako Chemicals). We purchased antibodies against the D1 (Arabidopsis 397
D1 protein, C-terminal, AS05084, Lot1207), D2 (Arabidopsis D2 protein, AS06146100), CP47 398
(Arabidopsis CP47, AS04038), Lhca1 (Arabidopsis Lhca1 protein, AS01005, Lot0512), Lhcb1 399
(Arabidopsis Lhcb1 protein, AS01004, Lot1501), and cytochrome b6f (Arabidopsis Cytb6 protein 400
N-terminal, AS03034, Lot0612) complex proteins from Agrisera. We purchased monoclonal antibodies 401
against FLAG-tag from Sigma-Aldrich (F1804, LotSLBK1346V). We purchased monoclonal antibodies 402
against GFP from Roche (11814460001, Lot12600500). We prepared the anti-CP1 (PsaA/PsaB) and 403
anti-CP43 antibodies as previously described (Tanaka et al., 1991). We raised anti-SGR1 antibodies 404
against peptides corresponding to residues GPLWEAVSPDGHKTETLPE of the Arabidopsis SGR1 405
protein. 406
407
RNA isolation and quantitative real-time PCR 408
We extracted total RNA from leaf tissues using the RNeasy Mini Kit (Qiagen) according to the 409
manufacturer’s instructions. We synthesized the cDNA using the PrimeScriptRT reagent kit with gDNA 410
eraser (TaKaRa). We performed quantitative real-time PCR using gene-specific primers as listed in 411
Supplemental Table 1, the iQ SYBR Green Supermix (Bio-Rad) and a MyiQ2 Two-Color Real-Time 412
PCR Detection System (Bio-Rad). We obtained the data using the iQ5 Optical System software 413
(Bio-Rad). 414
415
Purification of recombinant SGRL-FLAG 416
We synthesized recombinant SGRL with a FLAG-tag (SGRL-FLAG) using wheat germ expression 417
system. We diluted translation solutions containing expressed SGRL-FLAG five times in 0.5 ml of 418
buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl and were then incubated with FLAG 419
antibody-linked magnetic beads (Wako Chemicals) using a rotator for 15 min at 20°C. We washed the 420
18
beads four times with buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.05% 421
polysorbate 20. We eluted SGRL-FLAG by incubation with buffer containing 50 mM Tris-HCl (pH 7.5), 422
100 mM NaCl, 0.05% polysorbate 20, and 1 mg/ml DYKDDDDK peptide for 15 min at 20°C. We 423
detected purified SGRL-FLAG using gel electrophoresis followed by silver staining. 424
425
Spectroscopy of Low-Temperature Chlorophyll Fluorescence 426
We used a fluorescence spectrometer to measure the fluorescence spectra of leaves emitted at 77 K 427
(F-2500; Hitachi). The excitation wavelength was 440 nm. We normalized fluorescence intensities to an 428
emission intensity of 690 nm. 429
430
Electrolyte Leakage 431
We measured electrolyte leakage in excised leaves before or after DEX treatment as previously 432
described (Shimoda et al., 2012). We performed more than five replicates for each assay. 433
434
Accession Numbers 435
Sequence data from this article can be found in the EMBL/GenBank data libraries under accession 436
numbers SGR1 (AT4G22920), SGR2 (AT4G11910), and SGRL (AT1G44000). 437
438
Supplemental Data 439
Supplemental Figure 1. Expression of recombinant proteins. 440
Supplemental Figure 2. In-line absorption spectra of pheophytin a and pheophorbide a. 441
Supplemental Figure 3. Mg-dechelating activity and substrate specificity of recombinant SGR2. 442
Supplemental Figure 4. Mg-dechelating activity of purified SGRL. 443
Supplemental Figure 5. SGR1 expression in Synechococcus. 444
19
Supplemental Figure 6. SGR2 overexpression and induction in Arabidopsis. 445
Supplemental Figure 7. SGRL overexpression and induction in Arabidopsis. 446
Supplemental Figure 8. Low-temperature fluorescence spectroscopy of SGR1-induced leaves. 447
Supplemental Figure 9. Electrolyte leakage of SGR1-induced leaves. 448
Supplemental Table 1. Primers used in this study. 449
Supplemental Dataset 1. Raw data of HPLC analysis of pigments. 450
451
452
Acknowledgements 453
We thank Dr. A. Takabayashi, Y. Akiyama, and K. Matsuda for their useful comments on this study. The 454
Ministry of Education, Culture, Sports, Science, and Technology, Japan, supported this work with a 455
Grant-in-Aid for Scientific Research no. 15H04381 to A.T. We thank CSIRO, Max-Planck-Gesellschaft 456
zur Forderung der Wissenschaften c.V. (MPG) and Dr. Ian Moore of the University of Oxford for 457
providing the pOpOff vector. 458
459
AUTHOR CONTRIBUTIONS 460
Y.S., H.I., and A.T designed the research. Y.S. performed the research. Y.S., H.I., and A.T analyzed the461
data. A.T. wrote the article. 462
463
Figure Legends 464
Figure 1. Chlorophyll metabolic pathway in land plants. 465
Mg-dechelatase was identified in this study. 466
CAO, chlorophyllide a oxygenase; CBR, chlorophyll b reductase; CS, chlorophyll synthase; HCAR, 467
7-hydroxymethyl chlorophyll a reductase; PPH, pheophytin pheophorbide hydrolase; POR,468
20
NADPH:protochlorophyllide oxidoreductase. 469
470
Figure 2. Mg-dechelating activity and substrate specificity of recombinant SGR1 and SGRL. 471
(A) Pigment analysis after incubation of chlorophyll derivatives with SGR. Chlorophyll a and 472
chlorophyllide a were incubated with recombinant GFP, SGR1 with a FLAG-tag (SGR1-FLAG) for 60 473
min or with recombinant SGRL with a FLAG-tag (SGRL-FLAG) for 15 min. Recombinant proteins 474
were prepared with a wheat germ protein expression system and diluted 3-fold with the reaction buffer 475
without purification. GFP was used as a negative control because it has a similar molecular weight as 476
SGR. Chlorophyll b was incubated with recombinant GFP, SGR1-FLAG and SGRL-FLAG for 60 min. 477
The concentration of substrates was 6 µM. After incubation, pigments were analyzed using 478
high-performance liquid chromatography. Pigments were detected at 410 nm for chlorophyll a 479
derivatives or 435 nm for chlorophyll b derivatives. 480
(B) An increase in chlorophyll derivatives by SGR activity. The levels of pheophytin a and 481
pheophorbide a were determined after incubation of recombinant GFP and SGR1 with a FLAG-tag 482
(SGR1-FLAG, SGRL-FLAG) with 6 µM of chlorophyll a and chlorophyllide a (n = 3 ± SD). The 483
incubation times of SGR1-FLAG and SGRL-FLAG were 60 and 15 min, respectively. Recombinant 484
proteins were prepared with a wheat germ protein expression system and diluted with the reaction buffer 485
without purification. GFP was used as a negative control because it has similar molecular weight as 486
SGR. 487
488
Figure 3. Mg-dechelating activity of heat-denatured SGRL. 489
Recombinant GFP and SGRL-FLAG were denatured by heat treatment for 5 min at 95ºC . Chlorophyll a 490
and chlorophyllide a were incubated with non-denatured or denatured recombinant GFP and 491
SGRL-FLAG for 60 min at 25ºC. Recombinant proteins were prepared by a wheat germ protein 492
21
expression system and diluted 3-fold with the reaction buffer without purification. GFP was used as a 493
negative control because it has similar molecular weight as SGR. The concentration of substrates was 6 494
µM. After incubation, pigments were analyzed using high-performance liquid chromatography. 495
Pigments were detected at 410 nm. 496
497
Figure 4. Biochemical analysis of SGRL. 498
(A) Time-dependent formation of Mg-free chlorophyll derivatives by SGRL-FLAG. Chlorophyll a or 499
chlorophyllide a were incubated with recombinant GFP (open circles) and SGRL-FLAG (closed circles) 500
for up to 60 min or 10 min at 25ºC, respectively. Recombinant proteins were prepared by a wheat germ 501
protein expression system and diluted 3-fold with the reaction buffer without purification. GFP was used 502
as a negative control because it has similar molecular weight as SGR. The concentration of substrates 503
was 6 µM. After incubation, the level of pheophytin a and pheophorbide a were determined using 504
high-performance liquid chromatography (n=3±SD). 505
(B) Kinetic analysis of Mg-dechelating of SGRL-FLAG. Various concentration of chlorophyll a or 506
chlorophyllide a were incubated with recombinant GFP and SGRL-FLAG for 30 min or 5 min at 25ºC , 507
respectively. Recombinant proteins were prepared by a wheat germ protein expression system and 508
diluted 3-fold with the reaction buffer without purification. GFP was used as a negative control because 509
it has similar molecular weight as SGR. After incubation, the level of pheophytin a and pheophorbide a 510
were determined using high-performance liquid chromatography (n=3±SD). The inset shows 511
Lineweaver-Burk plot of kinetic data of Mg-dechelating of SGRL-FLAG. 512
(C) SGRL-FLAG concentration-dependent formation of Mg-free chlorophyll derivatives. Chlorophyll a 513
or chlorophyllide a were incubated with various concentrations of recombinant GFP (open circles) and 514
SGRL-FLAG (closed circles) for 30 min or 5 min at 25ºC , respectively. Translation solutions containing 515
expressed GFP and SGRL-FLAG were diluted three, six, or twelve times in 50 µl of reaction buffer. 516
22
GFP was used as a negative control because it has similar molecular weight as SGR. The concentration 517
of substrates was 6 µM. After incubation, the level of pheophytin a and pheophorbide a were determined 518
using high-performance liquid chromatography (n=3±SD). 519
520
Figure 5. SGR1 functions as a Mg-dechelatase in cells. 521
(A) SGR1 accumulation in the transformants. Inducible SGR1 with a FLAG-tag (SGR1-FLAG) was 522
introduced into WT (pOpON:SGR1- FLAG /WT #19) and pph (pOpON:SGR1-FLAG/pph) plants. 523
SGR1- FLAG was induced by DEX application in the transformants. After DEX or mock treatment for 524
24 h, Proteins were extracted from the plants and SGR1 was detected by immunoblotting analysis using 525
an anti-FLAG antibody. 526
(B) Pigment analysis after SGR1 induction. After DEX or mock treatment for 24 h, pigments were 527
extracted from the plants and analyzed using high-performance liquid chromatography. Fluorescence 528
intensity was monitored (410 nm excitation, 680 nm fluorescence). 529
(C) Pheophytin a contents in the transformants. After DEX or mock treatment for 24 h, pigments were 530
extracted from the plants and the amount of pheophytin a was determined (n = 4 ± SD). 531
532
Figure 6. SGR1 functions in Synechococcus. 533
(A) Chlorophyll a contents of Synechococcus. Chlorophyll a content of Synechococcus harboring the 534
pSyn6 vector (pSyn6-empty) or SGR1 cloned into the pSyn6 vector (pSyn6-SGR1) were determined 535
(n = 3 ± SD). Pigment content is shown based on OD750. 536
(B) Derivatives of chlorophyll a in Synechococcus. Pheophytin a and pheophorbide a contents of WT 537
and transformed Synechococcus were determined (n = 3 ± SD). 538
539
Figure 7. SGR1 overexpression in Arabidopsis. 540
23
(A) Visual phenotype of the transformants. SGR1 with a FLAG-tag (SGR1-FLAG) was overexpressed 541
in WT plants and in the ch1-1, cbr, and pph mutants. Scale bar: 1 cm. 542
(B) SGR1 accumulation in the transformants. Proteins were extracted from the plants and SGR1 was 543
detected by immunoblotting analysis using an anti-FLAG antibody. 544
(C) Chlorophyll content of the transformants. Chlorophyll was extracted from the plants and the amount 545
of chlorophyll a and b was determined (n = 3 ± SD). 546
547
Figure 8. SGR1 induction in Arabidopsis. 548
(A) Visual phenotype of the transformants. SGR1 with a FLAG-tag (SGR1-FLAG) was induced by 549
DEX application for 24 h in WT plants and in the ch1-1, cbr, and pph mutants grown for 2 weeks. Three 550
independent transformants in a WT background are shown. Scale bar: 1 cm. 551
(B) SGR1 accumulation in the transformants. Proteins were extracted from the plants and SGR1 was 552
detected by immunoblotting analysis using an anti-FLAG antibody. 553
(C) Chlorophyll contents of the transformants. Chlorophyll was extracted from the plants and the 554
amount of chlorophyll a and b was determined (n = 4 ± SD). 555
556
Figure 9. Degradation of chlorophyll and chlorophyll-binding protein by the induction of SGR1. 557
(A) Color changes of leaves. Inducible SGR1 with a FLAG-tag was introduced into WT 558
(pOpON:SGR1-FLAG/WT #19) or ch1-1 (pOpON:SGR1-FLAG/ch1-1) plants. DEX or mock-treated 559
excised leaves were observed for up to 30 h. Scale bar: 0.5 cm. 560
(B) Chlorophyll contents of leaves. Chlorophyll contents of pOpON:SGR1-FLAG/WT #19 and 561
pOpON:SGR1-FLAG/ch1-1 were determined before and after DEX or mock treatment for up to 30 h 562
(n = 4 ± SD). Comparisons were made to a 0 h control. 563
(C) SGR1 accumulation in leaves. Proteins were extracted from leaves and SGR1 was detected using 564
24
immunoblotting analysis with an anti-FLAG antibody. 565
(D) Chloroplast protein content in leaves. Proteins were extracted from the pOpON:SGR1-FLAG/WT 566
#19 and pOpON:SGR1-FLAG/ch1-1 excised leaves before and after DEX or mock treatment for up to 567
30 h. The large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcL) was detected using 568
Coomassie Brilliant Blue staining. 569
570
Figure 10. Magnesium extraction from chlorophyll in the chlorophyll-protein complex by SGR. 571
(A) Pigment analysis after incubation of PSI with SGR. PSI was isolated from Arabidopsis and 572
incubated with recombinant GFP, SGR1 with a FLAG-tag (SGR1-FLAG) and SGRL with a FLAG-tag 573
(SGRL-FLAG) for 60 min. Recombinant proteins were prepared by a wheat germ protein expression 574
system and diluted with the same volume of reaction buffer without purification. GFP was used as a 575
negative control because it has similar molecular weight as SGR. After incubation, pigments were 576
analyzed using high-performance liquid chromatography. Pigments were detected at 410 nm. 577
(B) Pigment analysis after incubation of LHCII with SGR. LHCII was isolated from Arabidopsis and 578
incubated with recombinant GFP, SGR1-FLAG and SGRL-FLAG for 60 min. Recombinant proteins 579
were prepared with a wheat germ protein expression system and diluted with same volume of the 580
reaction buffer without purification. GFP was used as a negative control because it has a similar 581
molecular weight as SGR. After incubation, pigments were analyzed using high-performance liquid 582
chromatography. Pigments were detected at 410 nm. 583
584
585
References 586
Armstead, I., Donnison, I., Aubry, S., Harper, J., Hörtensteiner, S., James, C., Mani, J., Moffet, M., Ougham, 587
H., Roberts, L., Thomas, A., Weeden, N., Thomas, H., and King, I. (2007). Cross-Species Identification 588
25
of Mendel's I Locus. Science 315, 73. 589
Chidgey, J.W., Linhartová, M., Komenda, J., Jackson, P.J., Dickman, M.J., Canniffe, D.P., Koník, P., Pilný, 590
J., Hunter, C.N., and Sobotka, R. (2014). A Cyanobacterial Chlorophyll Synthase-HliD Complex 591
Associates with the Ycf39 Protein and the YidC/Alb3 Insertase. Plant Cell 26, 1267-1279. 592
Christ, B., and Hörtensteiner, S. (2013). Mechanism and Significance of Chlorophyll Breakdown. J. Plant 593
Growth Regul. 33, 4-20. 594
Costa, M.L., Civello, P.M., Chaves, A.R., and Martínez, G.A. (2002). Characterization of Mg-dechelatase 595
activity obtained from Fragaria × ananassa fruit. Plant Physiol. Biochem. 40, 111-118. 596
Craft, J., Samalova, M., Baroux, C., Townley, H., Martinez, A., Jepson, I., Tsiantis, M., and Moore, I. (2005). 597
New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis. 598
Plant J. 41, 899-918. 599
Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S., and Mullineaux, P.M. (2000). pGreen: a versatile and 600
flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42, 601
819-832. 602
Hirashima, M., Tanaka, R., and Tanaka, A. (2009). Light-independent cell death induced by accumulation of 603
pheophorbide a in Arabidopsis thaliana. Plant Cell Physiol. 50, 719-729. 604
Holzwarth, A.R., Müller, M.G., Reus, M., Nowaczyk, M., Sander, J., and Rögner, M. (2006). Kinetics and 605
mechanism of electron transfer in intact photosystem II and in the isolated reaction center: 606
Pheophytin is the primary electron acceptor. Proc. Natl. Acad. Sci. U. S. A. 103, 6895-6900. 607
Horie, Y., Ito, H., Kusaba, M., Tanaka, R., and Tanaka, A. (2009). Participation of chlorophyll b reductase in 608
the initial step of the degradation of light-harvesting chlorophyll a/b-protein complexes in 609
Arabidopsis. J. Biol. Chem. 284, 17449-17456. 610
Hortensteiner, S. (2006). Chlorophyll degradation during senescence. Annu. Rev. Plant Biol. 57, 55-77. 611
Hortensteiner, S. (2009). Stay-green regulates chlorophyll and chlorophyll-binding protein degradation 612
26
during senescence. Trends Plant Sci. 14, 155-162. 613
Hortensteiner, S., and Krautler, B. (2011). Chlorophyll breakdown in higher plants. Biochim. Biophys. Acta 614
1807, 977-988. 615
Hu, X., Tanaka, A., and Tanaka, R. (2013). Simple extraction methods that prevent the artifactual conversion 616
of chlorophyll to chlorophyllide during pigment isolation from leaf samples. Plant Methods 9, 19. 617
Hu, X., Makita, S., Schelbert, S., Sano, S., Ochiai, M., Tsuchiya, T., Hasegawa, S.F., Hortensteiner, S., 618
Tanaka, A., and Tanaka, R. (2015). Reexamination of Chlorophyllase Function Implies Its 619
Involvement in Defense against Chewing Herbivores. Plant Physiol. 167, 660-670. 620
Kusaba, M., Ito, H., Morita, R., Iida, S., Sato, Y., Fujimoto, M., Kawasaki, S., Tanaka, R., Hirochika, H., 621
Nishimura, M., and Tanaka, A. (2007). Rice NON-YELLOW COLORING1 is involved in 622
light-harvesting complex II and grana degradation during leaf senescence. Plant Cell 19, 1362-1375. 623
Lin, Y.-P., Lee, T.-y., Tanaka, A., and Charng, Y.-y. (2014). Analysis of an Arabidopsis heat-sensitive mutant 624
reveals that chlorophyll synthase is involved in reutilization of chlorophyllide during chlorophyll 625
turnover. Plant J. 80, 14-26. 626
Meguro, M., Ito, H., Takabayashi, A., Tanaka, R., and Tanaka, A. (2011). Identification of the 627
7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis. Plant Cell 23, 628
3442-3453. 629
Nickelsen, J., and Rengstl, B. (2013). Photosystem II Assembly: From Cyanobacteria to Plants. Annu. Rev. 630
Plant Biol. 64, 609-635. 631
Park, S.Y., Yu, J.W., Park, J.S., Li, J., Yoo, S.C., Lee, N.Y., Lee, S.K., Jeong, S.W., Seo, H.S., Koh, H.J., Jeon, 632
J.S., Park, Y.I., and Paek, N.C. (2007). The senescence-induced staygreen protein regulates 633
chlorophyll degradation. Plant Cell 19, 1649-1664. 634
Pruzinska, A., Tanner, G., Anders, I., Roca, M., and Hortensteiner, S. (2003). Chlorophyll breakdown: 635
pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 636
27
gene. Proc. Natl. Acad. Sci. U. S. A. 100, 15259-15264. 637
Ren, G., An, K., Liao, Y., Zhou, X., Cao, Y., Zhao, H., Ge, X., and Kuai, B. (2007). Identification of a Novel 638
Chloroplast Protein AtNYE1 Regulating Chlorophyll Degradation during Leaf Senescence in 639
Arabidopsis. Plant Physiol. 144, 1429-1441. 640
Rodoni, S., Muhlecker, W., Anderl, M., Krautler, B., Moser, D., Thomas, H., Matile, P., and Hortensteiner, S. 641
(1997). Chlorophyll Breakdown in Senescent Chloroplasts (Cleavage of Pheophorbide a in Two 642
Enzymic Steps). Plant Physiol. 115, 669-676. 643
Saga, Y., and Tamiaki, H. (2012). Demetalation of Chlorophyll Pigments. Chemistry & Biodiversity 9, 644
1659-1683. 645
Sakuraba, Y., Park, S.-Y., Kim, Y.-S., Wang, S.-H., Yoo, S.-C., Hörtensteiner, S., and Paek, N.-C. (2014). 646
Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf 647
Senescence. Mol. Plant 7, 1288-1302. 648
Sakuraba, Y., Schelbert, S., Park, S.-Y., Han, S.-H., Lee, B.-D., Andrès, C.B., Kessler, F., Hörtensteiner, S., 649
and Paek, N.-C. (2012). Stay-green and chlorophyll catabolic enzymes interact at light-harvesting 650
complex II for chlorophyll detoxification during leaf senescence in Arabidopsis. Plant Cell 24, 507-518. 651
Sato, Y., Morita, R., Nishimura, M., Yamaguchi, H., and Kusaba, M. (2007). Mendel's green cotyledon gene 652
encodes a positive regulator of the chlorophyll-degrading pathway. Proc. Natl. Acad. Sci. U. S. A. 104, 653
14169-14174. 654
Schelbert, S., Aubry, S., Burla, B., Agne, B., Kessler, F., Krupinska, K., and Hortensteiner, S. (2009). 655
Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf 656
senescence in Arabidopsis. Plant Cell 21, 767-785. 657
Shimoda, Y., Ito, H., and Tanaka, A. (2012). Conversion of chlorophyll b to chlorophyll a precedes magnesium 658
dechelation for protection against necrosis in Arabidopsis. Plant J. 72, 501-511. 659
Suzuki, T., Kunieda, T., Murai, F., Morioka, S., and Shioi, Y. (2005). Mg-dechelation activity in radish 660
28
cotyledons with artificial and native substrates, Mg-chlorophyllin a and chlorophyllide a. Plant 661
Physiol. Biochem. 43, 459-464. 662
Tanaka, A., Yamamoto, Y., and Tsuji, H. (1991). Formation of chlorophyll-protein complexes during greening 663
2. Redistribution of chlorophyll among apoproteins. Plant Cell Physiol. 32, 195-204. 664
Tanaka, R., and Tanaka, A. (2007). Tetrapyrrole Biosynthesis in Higher Plants. Annu. Rev. Plant Biol. 58, 665
321-346. 666
Tsuchiya, T., Ohta, H., Okawa, K., Iwamatsu, A., Shimada, H., Masuda, T., and Takamiya, K. (1999). Cloning 667
of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the 668
induction by methyl jasmonate. Proc. Natl. Acad. Sci. U. S. A. 96, 15362-15367. 669
Wang, Y., Shen, Y., and Ryde, U. (2009). QM/MM study of the insertion of metal ion into protoporphyrin IX by 670
ferrochelatase. J. Inorg. Biochem. 103, 1680-1686. 671
Wielopolska, A., Townley, H., Moore, I., Waterhouse, P., and Helliwell, C. (2005). A high-throughput 672
inducible RNAi vector for plants. Plant Biotechnol. J. 3, 583-590. 673
Wu, S., Li, Z., Yang, L., Xie, Z., Chen, J., Zhang, W., Liu, T., Gao, S., Gao, J., Zhu, Y., Xin, J., Ren, G., and 674
Kuai, B. (2016). NON-YELLOWING2 (NYE2), a Close Paralog of NYE1, Plays a Positive Role in 675
Chlorophyll Degradation in Arabidopsis. Mol. Plant 9, 624-627. 676
Yamasato, A., Nagata, N., Tanaka, R., and Tanaka, A. (2005). The N-terminal domain of chlorophyllide a 677
oxygenase confers protein instability in response to chlorophyll b accumulation in Arabidopsis. Plant 678
Cell 17, 1585-1597. 679
Zapata, M., Rodríguez, F., and Garrido, J.L. (2000). Separation of chlorophylls and carotenoids from marine 680
phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing 681
mobile phases. Mar. Ecol. Prog. Ser. 195, 29-45. 682
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Fig. 1
NN
NN
CH3
CH3
CH3H3C
H3C
CH2
Mg
OCOOCH3
O
OH
NN
NN
CH3
CH3
CH3H3C
H3C
CH2
Mg
OCOOCH3
O
O C20H39
NNH
HNN
CH3
CH3
CH3H3C
H3C
CH2
OCOOCH3
O
O C20H39
NN
NN
CHO
CH3
CH3H3C
H3C
CH2
Mg
OCOOCH3
O
O C20H39
Figure 1. Chlorophyll metabolic pathway in land plants.
Mg-dechelatase was identified in this study.
CAO, chlorophyllide a oxygenase; CBR, chlorophyll b reductase; CS, chlorophyll synthase;
HCAR, 7-hydroxymethyl chlorophyll a reductase; PPH, pheophytin pheophorbide hydrolase;
POR, NADPH:protochlorophyllide oxidoreductase.
Fig. 2
B
A Chlorophyll a Chlorophyllide a Chlorophyll b
Pheo a Pheo a
Pheide a Pheide a
Mg-
fre
e c
hlo
rop
hyl
l de
riva
tive
s
(µM
/ 6
0m
in)
Mg-
fre
e c
hlo
rop
hyl
l de
riva
tive
s
(µM
/ 1
5m
in)
Figure 2. Mg-dechelating activity and substrate specificity of recombinant SGR1 and SGRL.
(A) Pigment analysis after incubation of chlorophyll derivatives with SGR. Chlorophyll a and
chlorophyllide a were incubated with recombinant GFP, SGR1 with a FLAG-tag (SGR1-
FLAG) for 60 min or with recombinant SGRL with a FLAG-tag (SGRL-FLAG) for 15 min.
Recombinant proteins were prepared with a wheat germ protein expression system and diluted
3-fold with the reaction buffer without purification. GFP was used as a negative control
because it has a similar molecular weight as SGR. Chlorophyll b was incubated with
recombinant GFP, SGR1-FLAG and SGRL-FLAG for 60 min. The concentration of substrates
was 6 µM. After incubation, pigments were analyzed using high-performance liquid
chromatography. Pigments were detected at 410 nm for chlorophyll a derivatives or 435 nm for
chlorophyll b derivatives.
(B) An increase in chlorophyll derivatives by SGR activity. The levels of pheophytin a and
pheophorbide a were determined after incubation of recombinant GFP and SGR1 with a
FLAG-tag (SGR1-FLAG, SGRL-FLAG) with 6 µM of chlorophyll a and chlorophyllide a (n =
3 ± SD). The incubation times of SGR1-FLAG and SGRL-FLAG were 60 and 15 min,
respectively. Recombinant proteins were prepared with a wheat germ protein expression system
and diluted with the reaction buffer without purification. GFP was used as a negative control
because it has similar molecular weight as SGR.
Fig. 3
Chlorophyll a Chlorophyllide a
Figure 3. Mg-dechelating activity of heat-denatured SGRL.
Recombinant GFP and SGRL-FLAG were denatured by heat treatment for 5 min at 95ºC.
Chlorophyll a and chlorophyllide a were incubated with non-denatured or denatured
recombinant GFP and SGRL-FLAG for 60 min at 25ºC. Recombinant proteins were
prepared by a wheat germ protein expression system and diluted 3-fold with the reaction
buffer without purification. GFP was used as a negative control because it has similar
molecular weight as SGR. The concentration of substrates was 6 µM. After incubation,
pigments were analyzed using high-performance liquid chromatography. Pigments were
detected at 410 nm.
Fig. 4
A
B
C
Ph
eo
ph
orb
ide
a (
µM
)
Incubation time (min)
Ph
eo
ph
ytin
a (
µM
)
0 µM 3 µM 6 µM
12 µM
0 1/12 1/6 1/3 0 1/12 1/6 1/3
Incubation time (min)
Dilution ratio (v/v)
0 µM 3 µM 6 µM
12 µM
Ph
eo
ph
ytin
a (
µM
) P
he
op
hyt
in a
(µ
M)
Dilution ratio (v/v)
Ph
eo
ph
orb
ide
a (
µM
) P
he
op
ho
rbid
e a
(µ
M)
(S) Chlorophyll a (S) Chlorophyllide a
(S) Chlorophyll a (S) Chlorophyllide a
(S) Chlorophyll a (S) Chlorophyllide a
1/P
he
o a
(µ
M)
1/Chl a (µM)
1/P
he
ide
a (
µM
)
1/Chlide a (µM)
Figure 4. Biochemical analysis of SGRL.
(A) Time-dependent formation of Mg-free chlorophyll derivatives by SGRL-FLAG.
Chlorophyll a or chlorophyllide a were incubated with recombinant GFP (open circles)
and SGRL-FLAG (closed circles) for up to 60 min or 10 min at 25ºC, respectively.
Recombinant proteins were prepared by a wheat germ protein expression system and
diluted 3-fold with the reaction buffer without purification. GFP was used as a negative
control because it has similar molecular weight as SGR. The concentration of substrates
was 6 µM. After incubation, the level of pheophytin a and pheophorbide a were
determined using high-performance liquid chromatography (n=3±SD). (B) Kinetic analysis of Mg-dechelating of SGRL-FLAG. Various concentration of
chlorophyll a or chlorophyllide a were incubated with recombinant GFP and SGRL-
FLAG for 30 min or 5 min at 25ºC , respectively. Recombinant proteins were prepared
by a wheat germ protein expression system and diluted 3-fold with the reaction buffer
without purification. GFP was used as a negative control because it has similar molecular
weight as SGR. After incubation, the level of pheophytin a and pheophorbide a were
determined using high-performance liquid chromatography (n=3±SD). The inset shows
Lineweaver-Burk plot of kinetic data of Mg-dechelating of SGRL-FLAG. (C) SGRL-FLAG concentration-dependent formation of Mg-free chlorophyll derivatives.
Chlorophyll a or chlorophyllide a were incubated with various concentrations of
recombinant GFP (open circles) and SGRL-FLAG (closed circles) for 30 min or 5 min at
25ºC , respectively. Translation solutions containing expressed GFP and SGRL-FLAG
were diluted three, six, or twelve times in 50 µl of reaction buffer. GFP was used as a
negative control because it has similar molecular weight as SGR. The concentration of
substrates was 6 µM. After incubation, the level of pheophytin a and pheophorbide a
were determined using high-performance liquid chromatography (n=3±SD).
Fig. 5
A
B WT pph
pOpON:SGR1-FLAG /WT #19
pOpON:SGR1-FLAG /pph
C
Figure 5. SGR1 functions as a Mg-dechelatase
in cells.
(A) SGR1 accumulation in the transformants.
Inducible SGR1 with a FLAG-tag (SGR1-
FLAG) was introduced into WT (pOpON:SGR1-
FLAG /WT #19) and pph (pOpON:SGR1-
FLAG/pph) plants. SGR1- FLAG was induced
by DEX application in the transformants. After
DEX or mock treatment for 24 h, Proteins were
extracted from the plants and SGR1 was
detected by immunoblotting analysis using an
anti-FLAG antibody.
(B) Pigment analysis after SGR1 induction.
After DEX or mock treatment for 24 h, pigments
were extracted from the plants and analyzed
using high-performance liquid chromatography.
Fluorescence intensity was monitored (410 nm
excitation, 680 nm fluorescence).
(C) Pheophytin a contents in the transformants.
After DEX or mock treatment for 24 h, pigments
were extracted from the plants and the amount
of pheophytin a was determined (n = 4 ± SD).
Fig. 6
A B
Figure 6. SGR1 functions in Synechococcus.
(A) Chlorophyll a contents of Synechococcus. Chlorophyll a content of Synechococcus
harboring the pSyn6 vector (pSyn6-empty) or SGR1 cloned into the pSyn6 vector (pSyn6-
SGR1) were determined (n = 3 ± SD). Pigment content is shown based on OD750.
(B) Derivatives of chlorophyll a in Synechococcus. Pheophytin a and pheophorbide a contents
of WT and transformed Synechococcus were determined (n = 3 ± SD).
Figure 7. SGR1 overexpression in Arabidopsis.
(A) Visual phenotype of the transformants. SGR1 with a FLAG-tag (SGR1-FLAG) was
overexpressed in WT plants and in the ch1-1, cbr, and pph mutants. Scale bar: 1 cm.
(B) SGR1 accumulation in the transformants. Proteins were extracted from the plants and
SGR1 was detected by immunoblotting analysis using an anti-FLAG antibody.
(C) Chlorophyll content of the transformants. Chlorophyll was extracted from the plants and
the amount of chlorophyll a and b was determined (n = 3 ± SD).
Figure 8. SGR1 induction in Arabidopsis.
(A) Visual phenotype of the transformants. SGR1 with a FLAG-tag (SGR1-FLAG) was
induced by DEX application for 24 h in WT plants and in the ch1-1, cbr, and pph mutants
grown for 2 weeks. Three independent transformants in a WT background are shown. Scale
bar: 1 cm. (B) SGR1 accumulation in the transformants. Proteins were extracted from the plants and
SGR1 was detected by immunoblotting analysis using an anti-FLAG antibody. (C) Chlorophyll contents of the transformants. Chlorophyll was extracted from the plants and
the amount of chlorophyll a and b was determined (n = 4 ± SD).
Figure 9. Degradation of chlorophyll and chlorophyll-binding protein by the induction of
SGR1.
(A) Color changes of leaves. Inducible SGR1 with a FLAG-tag was introduced into WT
(pOpON:SGR1-FLAG/WT #19) or ch1-1 (pOpON:SGR1-FLAG/ch1-1) plants. DEX or mock-
treated excised leaves were observed for up to 30 h. Scale bar: 0.5 cm.
(B) Chlorophyll contents of leaves. Chlorophyll contents of pOpON:SGR1-FLAG/WT #19 and
pOpON:SGR1-FLAG/ch1-1 were determined before and after DEX or mock treatment for up to
30 h (n = 4 ± SD). Comparisons were made to a 0 h control.
(C) SGR1 accumulation in leaves. Proteins were extracted from leaves and SGR1 was detected
using immunoblotting analysis with an anti-FLAG antibody.
(D) Chloroplast protein content in leaves. Proteins were extracted from the pOpON:SGR1-
FLAG/WT #19 and pOpON:SGR1-FLAG/ch1-1 excised leaves before and after DEX or mock
treatment for up to 30 h. The large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase
(RbcL) was detected using Coomassie Brilliant Blue staining.
Fig. 10
A B PSI LHCII
Figure 10. Magnesium extraction from chlorophyll in the chlorophyll-protein complex by SGR.
(A) Pigment analysis after incubation of PSI with SGR. PSI was isolated from Arabidopsis and
incubated with recombinant GFP, SGR1 with a FLAG-tag (SGR1-FLAG) and SGRL with a
FLAG-tag (SGRL-FLAG) for 60 min. Recombinant proteins were prepared by a wheat germ
protein expression system and diluted with the same volume of reaction buffer without
purification. GFP was used as a negative control because it has similar molecular weight as
SGR. After incubation, pigments were analyzed using high-performance liquid chromatography.
Pigments were detected at 410 nm. (B) Pigment analysis after incubation of LHCII with SGR. LHCII was isolated from
Arabidopsis and incubated with recombinant GFP, SGR1-FLAG and SGRL-FLAG for 60 min.
Recombinant proteins were prepared with a wheat germ protein expression system and diluted
with same volume of the reaction buffer without purification. GFP was used as a negative
control because it has a similar molecular weight as SGR. After incubation, pigments were
analyzed using high-performance liquid chromatography. Pigments were detected at 410 nm.
Parsed CitationsArmstead, I., Donnison, I., Aubry, S., Harper, J., Hörtensteiner, S., James, C., Mani, J., Moffet, M., Ougham, H., Roberts, L., Thomas,A., Weeden, N., Thomas, H., and King, I. (2007). Cross-Species Identification of Mendel's I Locus. Science 315, 73.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chidgey, J.W., Linhartová, M., Komenda, J., Jackson, P.J., Dickman, M.J., Canniffe, D.P., Koník, P., Pilný, J., Hunter, C.N., andSobotka, R. (2014). A Cyanobacterial Chlorophyll Synthase-HliD Complex Associates with the Ycf39 Protein and the YidC/Alb3Insertase. Plant Cell 26, 1267-1279.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Christ, B., and Hörtensteiner, S. (2013). Mechanism and Significance of Chlorophyll Breakdown. J. Plant Growth Regul. 33, 4-20.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Costa, M.L., Civello, P.M., Chaves, A.R., and Martínez, G.A. (2002). Characterization of Mg-dechelatase activity obtained fromFragaria × ananassa fruit. Plant Physiol. Biochem. 40, 111-118.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Craft, J., Samalova, M., Baroux, C., Townley, H., Martinez, A., Jepson, I., Tsiantis, M., and Moore, I. (2005). New pOp/LhG4 vectorsfor stringent glucocorticoid-dependent transgene expression in Arabidopsis. Plant J. 41, 899-918.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hellens, R.P., Edwards, E.A., Leyland, N.R., Bean, S., and Mullineaux, P.M. (2000). pGreen: a versatile and flexible binary Ti vectorfor Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42, 819-832.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hirashima, M., Tanaka, R., and Tanaka, A. (2009). Light-independent cell death induced by accumulation of pheophorbide a inArabidopsis thaliana. Plant Cell Physiol. 50, 719-729.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Holzwarth, A.R., Müller, M.G., Reus, M., Nowaczyk, M., Sander, J., and Rögner, M. (2006). Kinetics and mechanism of electrontransfer in intact photosystem II and in the isolated reaction center: Pheophytin is the primary electron acceptor. Proc. Natl. Acad.Sci. U. S. A. 103, 6895-6900.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Horie, Y., Ito, H., Kusaba, M., Tanaka, R., and Tanaka, A. (2009). Participation of chlorophyll b reductase in the initial step of thedegradation of light-harvesting chlorophyll a/b-protein complexes in Arabidopsis. J. Biol. Chem. 284, 17449-17456.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hortensteiner, S. (2006). Chlorophyll degradation during senescence. Annu. Rev. Plant Biol. 57, 55-77.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hortensteiner, S. (2009). Stay-green regulates chlorophyll and chlorophyll-binding protein degradation during senescence. TrendsPlant Sci. 14, 155-162.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hortensteiner, S., and Krautler, B. (2011). Chlorophyll breakdown in higher plants. Biochim. Biophys. Acta 1807, 977-988.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hu, X., Tanaka, A., and Tanaka, R. (2013). Simple extraction methods that prevent the artifactual conversion of chlorophyll tochlorophyllide during pigment isolation from leaf samples. Plant Methods 9, 19.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hu, X., Makita, S., Schelbert, S., Sano, S., Ochiai, M., Tsuchiya, T., Hasegawa, S.F., Hortensteiner, S., Tanaka, A., and Tanaka, R.
(2015). Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores. Plant Physiol.167, 660-670.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kusaba, M., Ito, H., Morita, R., Iida, S., Sato, Y., Fujimoto, M., Kawasaki, S., Tanaka, R., Hirochika, H., Nishimura, M., and Tanaka, A.(2007). Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence.Plant Cell 19, 1362-1375.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lin, Y.-P., Lee, T.-y., Tanaka, A., and Charng, Y.-y. (2014). Analysis of an Arabidopsis heat-sensitive mutant reveals that chlorophyllsynthase is involved in reutilization of chlorophyllide during chlorophyll turnover. Plant J. 80, 14-26.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Meguro, M., Ito, H., Takabayashi, A., Tanaka, R., and Tanaka, A. (2011). Identification of the 7-hydroxymethyl chlorophyll a reductaseof the chlorophyll cycle in Arabidopsis. Plant Cell 23, 3442-3453.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nickelsen, J., and Rengstl, B. (2013). Photosystem II Assembly: From Cyanobacteria to Plants. Annu. Rev. Plant Biol. 64, 609-635.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Park, S.Y., Yu, J.W., Park, J.S., Li, J., Yoo, S.C., Lee, N.Y., Lee, S.K., Jeong, S.W., Seo, H.S., Koh, H.J., Jeon, J.S., Park, Y.I., andPaek, N.C. (2007). The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19, 1649-1664.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Pruzinska, A., Tanner, G., Anders, I., Roca, M., and Hortensteiner, S. (2003). Chlorophyll breakdown: pheophorbide a oxygenase isa Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene. Proc. Natl. Acad. Sci. U. S. A. 100, 15259-15264.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ren, G., An, K., Liao, Y., Zhou, X., Cao, Y., Zhao, H., Ge, X., and Kuai, B. (2007). Identification of a Novel Chloroplast Protein AtNYE1Regulating Chlorophyll Degradation during Leaf Senescence in Arabidopsis. Plant Physiol. 144, 1429-1441.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Rodoni, S., Muhlecker, W., Anderl, M., Krautler, B., Moser, D., Thomas, H., Matile, P., and Hortensteiner, S. (1997). ChlorophyllBreakdown in Senescent Chloroplasts (Cleavage of Pheophorbide a in Two Enzymic Steps). Plant Physiol. 115, 669-676.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Saga, Y., and Tamiaki, H. (2012). Demetalation of Chlorophyll Pigments. Chemistry & Biodiversity 9, 1659-1683.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sakuraba, Y., Park, S.-Y., Kim, Y.-S., Wang, S.-H., Yoo, S.-C., Hörtensteiner, S., and Paek, N.-C. (2014). Arabidopsis STAY-GREEN2Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence. Mol. Plant 7, 1288-1302.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sakuraba, Y., Schelbert, S., Park, S.-Y., Han, S.-H., Lee, B.-D., Andrès, C.B., Kessler, F., Hörtensteiner, S., and Paek, N.-C. (2012).Stay-green and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leafsenescence in Arabidopsis. Plant Cell 24, 507-518.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sato, Y., Morita, R., Nishimura, M., Yamaguchi, H., and Kusaba, M. (2007). Mendel's green cotyledon gene encodes a positiveregulator of the chlorophyll-degrading pathway. Proc. Natl. Acad. Sci. U. S. A. 104, 14169-14174.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schelbert, S., Aubry, S., Burla, B., Agne, B., Kessler, F., Krupinska, K., and Hortensteiner, S. (2009). Pheophytin pheophorbidehydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell 21, 767-785.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Shimoda, Y., Ito, H., and Tanaka, A. (2012). Conversion of chlorophyll b to chlorophyll a precedes magnesium dechelation forprotection against necrosis in Arabidopsis. Plant J. 72, 501-511.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Suzuki, T., Kunieda, T., Murai, F., Morioka, S., and Shioi, Y. (2005). Mg-dechelation activity in radish cotyledons with artificial andnative substrates, Mg-chlorophyllin a and chlorophyllide a. Plant Physiol. Biochem. 43, 459-464.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tanaka, A., Yamamoto, Y., and Tsuji, H. (1991). Formation of chlorophyll-protein complexes during greening 2. Redistribution ofchlorophyll among apoproteins. Plant Cell Physiol. 32, 195-204.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tanaka, R., and Tanaka, A. (2007). Tetrapyrrole Biosynthesis in Higher Plants. Annu. Rev. Plant Biol. 58, 321-346.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tsuchiya, T., Ohta, H., Okawa, K., Iwamatsu, A., Shimada, H., Masuda, T., and Takamiya, K. (1999). Cloning of chlorophyllase, the keyenzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate. Proc. Natl. Acad. Sci. U. S. A.96, 15362-15367.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang, Y., Shen, Y., and Ryde, U. (2009). QM/MM study of the insertion of metal ion into protoporphyrin IX by ferrochelatase. J.Inorg. Biochem. 103, 1680-1686.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wielopolska, A., Townley, H., Moore, I., Waterhouse, P., and Helliwell, C. (2005). A high-throughput inducible RNAi vector for plants.Plant Biotechnol. J. 3, 583-590.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wu, S., Li, Z., Yang, L., Xie, Z., Chen, J., Zhang, W., Liu, T., Gao, S., Gao, J., Zhu, Y., Xin, J., Ren, G., and Kuai, B. (2016). NON-YELLOWING2 (NYE2), a Close Paralog of NYE1, Plays a Positive Role in Chlorophyll Degradation in Arabidopsis. Mol. Plant 9, 624-627.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yamasato, A., Nagata, N., Tanaka, R., and Tanaka, A. (2005). The N-terminal domain of chlorophyllide a oxygenase confers proteininstability in response to chlorophyll b accumulation in Arabidopsis. Plant Cell 17, 1585-1597.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zapata, M., Rodríguez, F., and Garrido, J.L. (2000). Separation of chlorophylls and carotenoids from marine phytoplankton: a newHPLC method using a reversed phase C8 column and pyridine-containing mobile phases. Mar. Ecol. Prog. Ser. 195, 29-45.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
DOI 10.1105/tpc.16.00428; originally published online September 7, 2016;Plant Cell
Yousuke Shimoda, Hisashi Ito and Ayumi TanakaArabidopsis STAY-GREEN, Mendel's Green Cotyledon Gene, Encodes Magnesium-Dechelatase
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