arabidopsis stay-green, mendel's green cotyledon … · 5 arabidopsis stay-green, mendel’s...

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BREAKTHROUGH REPORT 1

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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, [email protected] 13

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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

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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

([email protected]). 22

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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

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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

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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

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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

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(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

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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

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(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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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



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).

A

B

C

Fig. 7

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).

A

B

C

Fig. 8

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).

Fig. 9

A B

C

D

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.

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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.


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.


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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.


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.


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.


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.


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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zapata,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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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

This information is current as of August 29, 2018

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