Arabidopsis STAY-GREEN, Mendel’s Green Cotyledon Gene,
Encodes Magnesium-Dechelatase
Yousuke Shimoda,
a
Hisashi Ito,
a,b,1
and Ayumi Tanaka
a,b
a
Institute of Low Temperature Science, Hokkaido University, Kita-ku, Sapporo 060-0819, Japan
b
CREST, Japan Science and Technology Agency, Kita-ku, Sapporo 060-0819, Japan
Pheophytin a is an essential component of oxygenic photosynthetic organisms because the primary charge separation
between chlorophyll a and pheophytin a is the first step in the conversion of light energy. In addition, conversion of chlorophyll
a to pheophytin a is the first step of chlorophyll degradation. Pheophytin is synthesized by extracting magnesium (Mg) from
chlorophyll; the enzyme Mg-dechelatase catalyzes this reaction. In this study, we report that Mendel’s green cotyledon gene,
STAY-GREEN (SGR), encodes Mg-dechelatase. The Arabidopsis thaliana genome has three SGR genes, SGR1, SGR2, and
STAY-GREEN LIKE (SGRL). Recombinant SGR1/2 extracted Mg from chlorophyll a but had very low or no activity against
chlorophyllide a; by contrast, SGRL had higher dechelating activity against chlorophyllide a compared with chlorophyll a. All
SGRs could not extract Mg from chlorophyll b. Enzymatic experiments using the photosystem and light-harvesting complexes
showed that SGR extracts Mg not only from free chlorophyll but also from chlorophyll in the chlorophyll-protein complexes.
Furthermore, most of the chlorophyll and chlorophyll binding proteins disappeared when SGR was transiently expressed by
a chemical induction system. Thus, SGR is not only involved in chlorophyll degradation but also contributes to photosystem
degradation.
INTRODUCTION
Chlorophyll and its derivatives play essential roles in photosynthesis,
where chlorophyll ha rvests light energy and transfers it to the reaction
center. Most chlorophyll molecules in photosynthesis are involved in
this process. Green plants have two different chlorophyll species,
chlorophyll a and b, which harvest light energy; the biosynthetic
pathway for these chlorophylls has been studied extensively (Tanaka
and Tanaka,2007). Chlorophyll ais synthesized from 5-aminolevulinic
acid through multiple steps. At the last step of chlorophyll synthesis,
a portion of chlorophyll a is converted to chlorophyll b by chloro-
phyllide a oxygenase via 7-hydroxymethyl chlorophyll a. Chlorophyll
b is reconverted to chlorophyll a by chlorophyll b reductase (CBR) and
7-hydroxymethyl chlorophyll a reductase (HCAR) (Meguro et al.,
2011). Arabidopsis thaliana has two isozymes of CBR, NON-YELLOW
COLORING1 (NYC1) and NYC1-LIKE (NOL) (Kusaba et al., 2007;
Horie et al., 2009). This pathway, known as the chlorophyll cycle,
interconverts chlorophyll a and chlorophyll b (Figure 1). All the en-
zymes responsible for chlorophyll synthesis and for the chlorophyll
cycle have been identified, and the chlorophyll metabolic pathway
has been determined.
Another important function of chlorophyll is to drive electron
transfer, and pheophytin a plays a crucial role in this function. In the
reaction center of PSII, the primary charge separation between
P680 (chlorophyll a; PSII primary donor) and pheophytin a occurs;
this is the first step in the conversion of light to chemical energy in
photosynthesis (Holzwarth et al., 2006). Pheophytin a is synthe-
sized by extracting magnesium (Mg) from chlorophyll a. The en-
zyme responsible for this reaction has been tentatively called
Mg-dechelatase, although it is still not evident whether other
enzymes catalyze Mg-dechelation or whether it occurs sponta-
neously under acidic conditions. Mg-dechelation is an important
process in the formation of PSII because PSII assembly starts with
the formation of the D1/D2 complex of which pheophytin a is an
indispensable component (Nickelsen and Rengstl, 2013).
Mg-dechelatase also has a physiological function during se-
nescence. A recent study showed that the first step of chlorophyll
degradation is the conversion of chlorophyll a to pheophytin a (Christ
and Hörtensteiner, 2013). Pheophytin a is then converted to pheo-
phorbide a by pheophytin pheophorbide hydrolase (pheophytinase;
PPH); pheophorbide a is then oxidatively ring-opened to the red
chlorophyll catabolite by pheophorbide a oxygenase (PaO). This is
followed by the reduction to fluorescent chlorophyll catabolite by red
chlorophyll catabolite reductase (RCCR) (Rodoni et al., 1997;
Schelbert et al., 2009). Interestingly, chlorophyll b cannot directly
enter into this degradation pathway but must be converted to
chlorophyll a before degradation; this is due to the substrate spec-
ificity of the latter degradation enzymes (Hörtensteiner, 2006). The
degradation of chlorophyll is a key part of nitrogen recycling and
is important in avoiding cellular damage. If chlorophyll degradation is
not properly regulated, severe photodamage occurs and cell death is
induced (Pruzinská et al., 2003; Hirashima et al., 2009; Hörtensteiner
and Kräutler, 2011). Among chlorophyll degradation enzymes,
Mg-dechelatase is especially important for regulation because it
catalyzes the step in which chlorophyll is committed to degradation.
As Mg-dechelatase has indispensable functions in the forma-
tion of PSII and the degradation of chlorophyll, many attempts
have been made to identify it; however, all these efforts failed. This
1
Address correspondence to ito98@lowtem.hokudai.ac.jp.
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructio ns for Autho rs (www. plantc el l.org ) is: Hisas hi Ito (ito98@
lowtem.hokudai.ac.jp).
www.plantcell.org/cgi/doi/10.1105/tpc.16.00428
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has been partly due to the difficulty of detecting dechelation
activity in vitro using chlorophyll(ide) as a substrate (Hörtensteiner
and Kräutler, 2011). Instead of chlorophyll, an artificial substrate
chlorophyllin (a semisynthetic derivative of chlorophyll) has been
widely used to measure Mg-dechelatase activity. However, this
might lead to a failure in identifying Mg-dechelatase because the
real substrate of Mg-dechelatase is chlorophyll a.
Most of the mutants of chlorophyll degradation enzymes, such as
PPH (Schelbert et al., 2009), PaO (Pruzinská et al., 2003), and CBR
(Kusaba et al., 2007; Horie et al., 2009), exhibit a stay-green phe-
notype. It is therefore reasonable to assume that the mutation of
Mg-dechelatase would also cause a strong stay-green phenotype
because it catalyzes the first step of the chlorophyll degradation
pathway. Mendel studied the mechanisms of inheritance using seven
pea (Pisum sativum) mutants, including a green cotyledon mutant.
Recently, Mendel’s green cotyledon gene was shown to encode the
STAY-GREEN (SGR) protein. The SGR mutation induces a stay-
green phenotype not only in Mendel’s green cotyledon (Armstead
et al., 2007; Sato et al., 2007), but also in many other plants (Park et al.,
2007; Ren et al., 2007). Many studies have been performed to elu-
cidate the function of SGR and a hypothesis for SGR function was
proposed based on protein-protein interaction experiments.
Sakuraba et al. (2012) found that SGR physically interacted with the
light-harvesting complex of PSII (LHCII) and also with six chlorophyll
degradation enzymes including HCAR, NOL, NYC1, PaO, PPH, and
RCCR; they proposed a complex of SGR with LHCII and chlorophyll
degradation enzymes that allows the metabolic channeling of
chlorophyll degradation intermediates. However, the question
remained whether SGR can simultaneously bind six proteins and
whether SGR has other functions.
We speculated that the SGR genecould encode Mg-dechelatase
because all the sgr mutants showed strong stay-green phenotypes.
To examine this possibility, we performed in vitro and in vivo ex-
periments. When SGR was transiently expressed in Arabidopsis,
chlorophyll was degraded and this was accompanied by the ac-
cumulation of a small amount of pheophytin a. Recombinant SGR
proteins prepared using a wheat germ protein expression system
converted chlorophyll a to pheophytin a, but SGR had no activity
against chlorophyll b. When we incubated SGR with chlorophyll-
protein complexes isolated with a sucrose density gradient, chlo-
rophyll a was efficiently converted to pheophytin a. Based on these
experiments, we concluded that Mendel’sgreencotyledongene
(SGR) encodes Mg-dechelatase. We discuss the enzymatic
properties of SGR in relation to the degradation of photosystems.
RESULTS
Mg-Dechelating Activity of Recombinant SGR
The Arabidopsis genome contains three SGR genes, SGR1
(AT4G22920), SGR2 (AT4G11910), and STAY-GREEN LIKE
(SGRL; AT1G44000) (Sakuraba et al., 2014). First, we used
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.
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recombinant mature SGR proteins expressed in Escherichia coli
for enzymatic experiments, butwedidnotobserveany
Mg-dechelating activity. Next, we examined the Mg-dechelating
activity of mature SGR proteins prepared by a wheat germ protein
expression system (Supplemental Figure 1). Recombinant SGR1
had high dechelating activity against chlorophyll a but very low
activity against chlorophyllide a (Figure 2A; Supplemental Data Set
1). Substrates and products were identified by their absorption
spectra (Supplemental Figure 2) and by their HPLC retention time
(Shimoda et al., 2012).The substrate specificity of SGR2 was
almost the same as that of SGR1, which is consistent with the
high amino acid sequence similarity b etween SGR1 and SGR2
(Supplemental Figure 3). In contrast, SGRL had much hi gher
activity against chlorophyllide a than against chlorophyll a (Figure
2B). None of the three SGRs (SGR1, SGR2, and SGRL) extracted
Mg from chlorophyll b. These results suggest that SGR has
Mg-dechelating activity and that substrate specificity is differ ent
between SGR1/2 and SGRL.
To confirm that in vitro Mg dechelation is an enzymatic reaction
catalyzed by SGR, the following experiments were performed
using recombinant SGRL protein because it has the highest ac-
tivity among three SGRs. Mg-dechelating activity was completely
lost by heating at 95°C (Figure 3). Purified SGRL, sho wing
a single or a major band on SDS-PAGE, had Mg-dechelating
activity (Supplemental Figure 4), suggesting that SGR has
Mg-dechelating activity without any other factors. A time-course
study showed that the amount of the products (pheophytin a or
pheophorbide a) increased depending on the incubation time and
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 pro teins 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 mM. After incubation, pigments were analyzed using HPLC. 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 and SGRL-FLAG) with 6 mM of chlorophyll a and chlorophyllide a (n =36
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 puri fication. GFP was used as a negative control because it has sim ilar molecular weight as SGR.
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the product never increased without SGRL proteins in the re-
action mi xture (Figure 4A). Increasing concentrations of
chlorophyll a and chlorophyllide a substrates were accompa-
nied by enhanced conversion to their respective products
(Figure 4B). The nonlinearity observ ed using chlo rophyll a as
a substrate differs from the almost linear increase in product
formation obtained with chlorophyllide a (Figure 4B, left panel);
this could arise from a number of factors and a more detailed
analysis is required. The amount of the product depended on
the concentrati on of SGRL (Figure 4C). All these r esults
strongly indicate that release of Mg from c hlorophylls occurs
enzymatically by SGR.
Mg-Dechelating Activity of SGR in Cells
Recombinant SGR showed Mg-dechelating activity in vitro;
however, it is not evident whether SGR functions as a Mg-
dechelatase in cells. To answer this question, we transiently
expressed SGR1 with a chemical induction system containing
dexamethasone (DEX), and we examined the accumulation of
pheophytin a, a product of Mg-dec helatase (Figure 5A). SGR1
expression increased the level of pheophytin a.Although
the increase in pheophytin a suggested the occurrence of
Mg-dechelation by SGR1, the absolute level of pheophytin
a wasverylow(Figure5B).Onepossiblereasonforthisisthat
synthesized pheophytin a is immediately degraded by the next
enzyme, PPH. In order to examine this pos sibility, S GR1 was
transiently induced in pph background and the pigments were
analyzed (Figure 5B). Pheophyt in a accumulate d more in the
pph background than in the wild type by DEX treatment (Figure
5C), indicating that SGR could function as a Mg-dech elatase
in cells.
For further confirmation of SGR function, we introduced mature
SGR1 into the cyano bacterium Synechococcus elongatus
PCC7942 (hereafter Synechococcus) (Supplemental Figure 5A).
The Synechococcus genome has no SGR, or any homologous
gene, indicating that Synechococcus has no SGR system for
Mg-dechelation. If SGR requires other protein components, it
would not be expected to function as a Mg-dechelatase in Syn-
echococcus cells. Our immunoblot analysis showed that SGR1
was successfully expressed in Synechococcus (Supplemental
Figure 5B). Chlorophyll content was low (Figure 6A) and pheo-
phytin a and pheophorbide a accumulated in large amounts
(Figure 6B) in Synechococcus expressing SGR1, indicating that
SGR1 functions as Mg-dechelatase in Synechococcus cells. In-
terestingly, the level of pheophorbide a was comparable to that of
pheophytin a, which was quite different from the results obtained
with the Arabidopsis leaves in which pheophorbide a was not
detected (Figure 5B). Pheophorbide a
might be synthesized from
pheophytin a by an unknown PPH-like enzyme in Synechococcus
cells. Based on these experiments, we finally concluded that SGR
encodes a Mg-dechelatase and that no other protein is required
for the dechelating activity of SGR.
Expression of SGR in Arabidopsis
To elucidate the impact of SGR on chlorophyll metabolism and the
relationship between SGR and other chlorophyll metabolic
enzymes, we constitutively overexpressed the cDNA of the SGR1
gene in wild-type Arabidopsis plants and mutants, such as ch1-1
(mutant of chlorophyllide a oxygenase) and the cbr and pph
mutants. These transgenic plants exhibited low chlorophyll
content and retarded growth (Figure 7). We assumed that the
plants would not grow when SGR1 was expressed in large
amounts and that only the mutants with low expression levels of
SGR would survive. Low chlorophyll content was also observed
when SGR2 or SGRL was c onstitutively o verexpressed
(Supplemental Figures 6 and 7).
Based on these severe phenotypes, we concluded that
constitutive overexpre ssion is not appropriate for the study of
SGR function. Instead, we transiently expressed SGR1 in fully
greened leaves using a DEX in duction system (Figu re 8). Three
independent transgenic lines trans ientl y overexpressing SGR1
in a wild-type background (line nu mbers 3, 19, and 34) are shown
in Figure 8A. After 24 h of DEX trea tment, approximately half of
the chlorophyll was degraded in the wild-type background
(Figure 8C). Chlorophyll degradation was also observed in the
pph mutant backgro und; however, 70% of chlorophyll still
Figure 3. Mg-Dechelating Activity of Heat-Denatured SGRL.
Recombinant GFP and SGRL-FLAG were denatured by heat treatment for
5 min at 95°C. Chlorophyll a and chlorophyllide a were incubated with
nondenatured or denatured recombinant GFP and SGRL-FLAG for 60 min
at 25°C. 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 mM. After
incubation, pigments were analyzed using HPLC. Pigments were detected
at 410 nm.
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remained after 24 h of DEX treatment. Interestingl y, the level of
chlorophyll b was not significantly changed by DEX treatment in
a cbr mutant background, although chlorophyll a was exten-
sively degraded. This is consistent with experiments demon-
strating that SGR did not extract Mg from chlorophyll b (Figure
2A). Reduction of chlo rophyll conte nt was also observed when
SGR2 or SGRL wa s t ransiently induced by DEX inducti on system
(Supplemental Figures 6 and 7).
Next, we used excised leaves from either a wild-type or ch1-1
background to examine the effect of SGR1 expression on chlo-
roplast proteins (Figure 9). After DEX treatment, chlorophyll levels
decreased to 20% of the initial level in both wild-type and ch1-1
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 25°C, 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 mM. After incubation, the level of pheophytin a and pheophorbide a was determined using HPLC
(n =36
SD).
(B) Kinetic analysis of Mg-dechelating of SGRL-FLAG. Various concentrations of chlorophyll a or chlorophyllide a were incubated with recombinant GFP and
SGRL-FLAG for 30 or 5 min at 25°C, 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 HPLC (n =36
SD). The inset shows Lineweaver-Burk plot of kinetic data of Mg-dechelating of
SGRL-FLAG.
(C) SGRL-FLAG concentration-dependent formation o f Mg-free chlorophyll derivatives. Chlorophyll a or chlorophyllide a were incubated with various con-
centrationsof recombinant GFP (open circles) and SGRL-FLAG (closed circles) for 30 or 5 min at 25°C,respectively.Translation solutions containing expressed
GFP and SGRL-FLAG were diluted 3, 6, or 12 times in 50 mL of reaction buffer. GFP was used as a negative control because it has similar molecular weight as
SGR. The concentration of substrates was 6 mM. After incubation, the levels of pheophytin a and pheophorbide a were determined using HPLC (n =36SD).
Identification of Mg-Dechelata se 2151
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