Download pdf - Analysis of an Arabidopsis heat-sensitive mutant reveals that chlorophyll synthase is involved in reutilization of chlorophyllide during chlorophyll turnover

Analysis of an Arabidopsis heat-sensitive mutant reveals thatchlorophyll synthase is involved in reutilization ofchlorophyllide during chlorophyll turnover

Yao-Pin Lin1,2,3, Tsung-yuan Lee1, Ayumi Tanaka4 and Yee-yung Charng1,2,5,*1Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan,2Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei

115, Taiwan,3Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan,4Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan, and5Biotechnology Center, National Chung-Hsing University, Taichung 402, Taiwan

Received 20 May 2014; revised 27 June 2014; accepted 2 July 2014; published online 8 July 2014.

*For correspondence (e-mail [email protected]).

SUMMARY

Chlorophylls, the most abundant pigments in the photosynthetic apparatus, are constantly turned over as a

result of the degradation and replacement of the damage-prone reaction center D1 protein of photosystem

II. Results from isotope labeling experiments suggest that chlorophylls are recycled by reutilization of

chlorophyllide and phytol, but the underlying mechanism is unclear. In this study, by characterization of a

heat-sensitive Arabidopsis mutant we provide evidence of a salvage pathway for chlorophyllide a. A

missense mutation in CHLOROPHYLL SYNTHASE (CHLG) was identified and confirmed to be responsible for

a light-dependent, heat-induced cotyledon bleaching phenotype. Following heat treatment, mutant (chlg-1)

but not wild-type seedlings accumulated a substantial level of chlorophyllide a, which resulted in a surge of

phototoxic singlet oxygen. Immunoblot analysis suggested that the mutation destabilized the chlorophyll

synthase proteins and caused a conditional blockage of esterification of chlorophyllide a after heat stress.

Accumulation of chlorophyllide a after heat treatment occurred during recovery in the dark in the light-

grown but not the etiolated seedlings, suggesting that the accumulated chlorophyllides were not derived

from de novo biosynthesis but from de-esterification of the existing chlorophylls. Further analysis of the

triple mutant harboring the CHLG mutant allele and null mutations of CHLOROPHYLLASE1 (CLH1) and CLH2

indicated that the known chlorophyllases are not responsible for the accumulation of chlorophyllide a in

chlg-1. Taken together, our results show that chlorophyll synthase acts in a salvage pathway for chlorophyll

biosynthesis by re-esterifying the chlorophyllide a produced during chlorophyll turnover.

Keywords: chlorophyll turnover, chlorophyll synthase, chlorophyll biosynthesis, chlorophyllide, Arabidopsis

thaliana.

INTRODUCTION

Chlorophylls are the most abundant pigments used for

harvesting energy from visible light in plants, green algae

and cyanobacteria. The energy captured by chlorophylls is

then utilized for photosynthesis, a fundamental biochemi-

cal process for supporting life forms on Earth. This impor-

tant function means that chlorophyll (Chl) metabolism is a

topic that has been intensively studied. The major

pathways of anabolism and catabolism of Chls are well

understood and have been described in great detail in

several recent reviews (Tanaka et al., 2011; Tripathy and

Pattanayak, 2012; H€ortensteiner, 2013). In brief, Chls are

synthesized in plastids by coupling chlorophyllide (a chlo-

rin moiety synthesized from protoporphyrin after incorpo-

ration of Mg2+ by the tetrapyrrole pathway) with an

isoprenoid phytol tail derived from geranylgeranyldiphos-

phate (GGPP) formed by the methylerythritol phosphate

pathway (Kim et al., 2013). During leaf senescence, Chls

break down to non-fluorescent catabolites via the pheo-

phorbide a oxygenase pathway, which has catalytic com-

ponents in the plastid, cytosol and vacuole (H€ortensteiner,

© 2014 The AuthorsThe Plant Journal © 2014 John Wiley & Sons Ltd

14

The Plant Journal (2014) 80, 14–26 doi: 10.1111/tpj.12611

mailto:[email protected]

2013). Despite this understanding, a few critical processes,

such as Chl turnover at steady-state levels and in response

to environmental cues, remain enigmatic.

In the photosynthetic apparatus, Chls are bound to mul-

tisubunit protein complexes, i.e. photosystem I (PS I) and

photosystem II (PS II), which are involved in light harvest-

ing and conversion of photochemical energy (Nelson and

Yocum, 2006). A well-recognized side effect of oxygenic

photosynthesis is that it produces various radicals and

reactive oxygen species (ROS) that can damage PS II at all

light intensities (Aro et al., 2005). Environmental stress

factors, such as strong light, high and low temperature,

drought and salt, aggravate the problem (Aro et al., 1993;

Murata et al., 2007; Allakhverdiev et al., 2008). The reaction

center D1 protein in PS II is the target most vulnerable to

this photo damage, and repair of PS II requires the surgical

degradation of the damaged D1 protein and its replace-

ment by a newly synthesized D1 (Edelman and Mattoo,

2008; Komenda et al., 2012). Since the D1 protein is a Chl-

binding protein, the bound Chls must be released and

turned over during the repair process (Matile et al., 1999).

Indeed, constant turnover of Chl has been demonstrated in

plants, algae, and cyanobacteria (Riper et al., 1979; Raskin

et al., 1995; Feierabend and Dehne, 1996; Vavilin and

Vermaas, 2007; Beisel et al., 2010). In contrast to the well-

characterized turnover of the D1 protein, the mechanism

underlying Chl turnover is largely unknown (Komenda

et al., 2012; H€ortensteiner, 2013).

One probable fate of the Chls released from PS II has

been traced by experiments using an isotope-labeling tech-

nique in the cyanobacterium Synechocystis sp. PCC6803

(Vavilin and Vermaas, 2007). The study showed that a sub-

stantial part of the chlorophyllide derived from de-esterifi-

cation of Chl is recycled for the biosynthesis of new Chl

molecules, which are suggested to be used for repairing

PS II (Kopecna et al., 2012). These findings suggest the

existence of a salvage pathway involving de- and re-esteri-

fication of Chl, but the enzymes responsible for these

actions are unknown (H€ortensteiner, 2013).

Here, we report the serendipitous discovery of a Chl

turnover pathway by characterizing an Arabidopsis heat-

sensitive mutant carrying a missense mutation in a Chl

synthase gene. Our data show that the mutation led to

drastic accumulation of chlorophyllide a (Chlide a) with the

concomitant degradation of the D1 protein in Arabidopsis

seedlings in the dark after severe heat treatment. The heat-

induced accumulation of Chlide a was not due to a block-

age of the conversion of the de novo synthesized Chlide a

to Chl a, implying that de-esterification of Chl is the main

cause. We further proved that the well-known chlorophyl-

lase that is thought to de-esterify chlorophylls in vivo

(Jacob-Wilk et al., 1999; Tsuchiya et al., 1999; Harpaz-Saad

et al., 2007) is not required for the heat-induced formation

of Chlide a. Our results suggest that Chl synthase acts in a

salvage pathway for biosynthesis of Chl a by catalyzing the

re-esterification of the Chlide a produced during turnover

of Chl. This pathway probably maintains the homeostasis

of Chl a in the photosynthetic apparatus under stress as

well as normal conditions in plants.

RESULTS

Isolation and characterization of the Arabidopsis dlt4-1

mutant

Arabidopsis ethane methyl sulfonate-mutagenized mutants

with a defect in long-term acquired thermotolerance,

named dlt mutants, were isolated as described previously

(Wu et al., 2013) and in-depth studies were carried out on

the dlt4-1 allele (renamed chlg-1 later in this article). The

dlt4-1 seedlings had pale-green cotyledons and showed a

heat-sensitive phenotype (bleached cotyledons) following

thermotolerance assay (Figure 1a). Chlorophyll analysis

Figure 1. dlt4-1 is pale green and heat sensitive.

(a) Phenotypes of seedlings before and after LAT assay treatment (HS) with

3 days’ recovery. Seedlings of the wild type (WT) and mutants with the

same treatment were grown and treated on the same plate.

(b) Chlorophyll (Chl) contents and Chl a/b ratios of 5-day-old seedlings

under normal conditions. Fifty seedlings per line were collected for Chl

extraction.

(c) The heat sensitivity of seedlings in (a) was quantified and expressed as

the percentage of seedlings showing bleached cotyledons. The bars in (b)

and (c) indicate the mean � SD of three independent replicates with 50

seedlings each. Student’s t-test, mutant versus the wild type, *P < 0.05.

© 2014 The AuthorsThe Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 14–26

A salvage pathway for chlorophyllide a 15

showed that dlt4-1 seedlings had lower Chl content and a

higher Chl a/b ratio than the wild type (Figure 1b). Thermo-

tolerance analysis of the selfed F2 seedlings from a cross

between the wild type and the dlt4-1 mutant showed an

approximate 3:1 segregation ratio of normal:heat-sensitive

phenotype (Table S1 in Supporting Information), indicating

that dlt4-1 is a single recessive nuclear mutation. More-

over, the heat-sensitive phenotype was co-segregated with

the pale-green cotyledon phenotype.

To see whether the heat sensitivity of dlt4-1 was simply

due to the reduced Chl content, we examined the glk1 glk2

double mutant, which is reported to have a lower Chl con-

tent than the wild type in adult plants (Waters et al., 2009).

The GOLDEN2-LIKE (GLK) genes encode transcription fac-

tors that are required for chloroplast development (Rossini

et al., 2001). The Chl level in glk1 glk2 seedlings was lower

than that in the wild type and dlt4-1 (Figure 1b). However,

the double mutant plants did not show a heat-sensitive

phenotype like dlt4-1 (Figure 1a). By contrast, the heat-sen-

sitive mutant hsa32-1 (Charng et al., 2006) had a normal

Chl content (Figure 1a,b). These results suggest that the

decreased Chl level is not responsible for the thermotoler-

ance defect in dlt4-1 seedlings.

As well as showing defects in long-term acquired ther-

motolerance, dlt4-1 also showed significant defects in

basal thermotolerance, short-term acquired thermotoler-

ance and thermotolerance to moderately high temperature

(Figure S1a), suggesting that the dlt4-1 allele is detrimental

under a wide range of heat stress conditions. To see

whether these defects were due to a failure in heat shock

response, the levels of several heat shock proteins (HSPs)

in dlt4-1 seedlings were examined in response to heat

treatment. The protein levels of HSP101, HSP90, HSA32

and sHSP-C1 before and after treatment at 37°C for 1 h

were similar in the wild type and dlt4-1 (Figure S1b). This

result suggests that the heat sensitivity of dlt4-1 cannot be

ascribed to the HSP levels.

Cloning of dlt4-1 and complementation analysis associate

a missense mutation in the Chl synthase gene (CHLG)

with a mutant phenotype

In order to find out the identity of dlt4-1, the mutation site

responsible was located by map-based cloning. A G to A

transition was identified in exon 8 of the Chl synthase gene

(Figures 2a and S2), causing a replacement of glycine by

arginine at position 217 (Figure 2b). The Arabidopsis

genome contains a single copy of the Chl synthase gene,

previously cloned and named G4 (Gaubier et al., 1995;

Oster and R€udiger, 1997). G4 is more frequently annotated

as CHLG based on its homology with the chlG locus of

Synechocystis sp. PCC 6803 (Eckhardt et al., 2004). Trans-

formation of dlt4-1 with the recombinant CHLG genomic

DNA derived from wild-type Arabidopsis plants rescued

both the heat-sensitive phenotype and the pale-green

cotyledons of dlt4-1 (Table S2), indicating that it is a

mutant allele of CHLG. Therefore, henceforth, we will refer

to dlt4-1 as chlg-1. chlg-2, a null allele with T-DNA insertion

in exon 6 (Figure 2a), was obtained from the Arabidopsis

genome-wide mutagenesis project (Alonso et al., 2003). As

expected, the chlg-2 mutant is albino (Figure 3d) and does

not accumulate detectable Chl (Figure S3).

Chl synthase protein is dramatically reduced in chlg-1

Glycine-217 of Arabidopsis Chl synthase is located within

the predicted fourth transmembrane helix and is highly

conserved among the land plant proteins (Figure 2b). To

investigate the effect of the amino acid replacement, a rab-

bit antiserum was raised against a synthetic peptide

derived from the N-terminal sequence of predicted mature

Arabidopsis Chl synthase for immunoblot analysis (Fig-

ure 2b). The antiserum recognizes a single major band

around 29 kDa in the crude extract of the wild type but not

Figure 2. Point mutation in dlt4-1 (chlg-1) resulted in an amino acid change,

which was located at one of the putative transmembrane motifs in chloro-

phyll (Chl) synthase.

(a) Schematic genomic DNA structure of the CHLG gene. Exons are pre-

sented as black boxes. Mutated sites in chlg-1 and chlg-2 are indicated by

black and white triangles, respectively.

(b) Protein structure of Arabidopsis CHLG. The start and end amino acids

are numbered. The upper inset shows the peptide sequence for raising anti-

Chl synthase antibody. The chloroplast transit peptide (light gray box) and

the putative transmembrane helices (dark gray box) were predicted by the

ChloroP (Emanuelsson et al., 2007) and HMMTOP (Tusnady and Simon,

2001) programs, respectively. Alignment of the fourth transmembrane helix

of CHLG orthologs is shown, and identical residues are marked by a black

background. The arrow indicates the mutation site in chlg-1. The star indi-

cates the conserved proline residue mutated in rice ygl1. At, Arabidopsis

thaliana; Os, Oryza sativa; Ps, Picea sitchensis; Pp, Physcomitrella patens;

Cr, Chlamydomonas reinhardtii; Cya, Cyanothece sp. PCC 7425.


16 Yao-Pin Lin et al.

chlg-2 seedlings (Figure 3a). The detected protein is

authentic Chl synthase because it is not present in the

knock-out mutant and its size is close to the calculated

molecular mass of Chl synthase without the predicted tran-

sit peptide. Interestingly, the level of Chl synthase in chlg-1

is dramatically reduced to about 10% of that in the wild

type (Figure 3a). There is no significant difference at the

transcript level (Figure 3b), indicating that chlg-1 does not

cause instability of the transcripts. The reduced level of Chl

synthase protein is correlated with reduced Chl content in

chlg-1. However, with about 10% of the wild-type level of

Chl synthase protein, the mutant accumulated about 60%

of total Chl compared with the wild type (Figure 1b),

suggesting that the mutant enzyme is catalytically active

under normal conditions. Besides Chl synthase, Lhcb1, a

light-harvesting Chl a/b-binding protein, is reduced to

about 60% of the wild-type level in chlg-1 (Figure 3a),

correlating well with the total Chl content. There was no

obvious difference in abundance in other proteins in PS I

and PS II, such as PsaA, D1 and CP47, between the wild

type and chlg-1 (Figure 3a).

Furthermore, blue native polyacrylamide gel electropho-

resis (PAGE) and immunoblot analyses were performed to

examine whether the mutated Chl synthase and its reduc-

tion in protein level affect thylakoid membrane complexes

in chlg-1. For this purpose, we used 4-week-old plants to

obtain the thylakoid more easily. Figure 3(c) shows that

the patterns of thylakoid membrane pigmented complexes

on blue native gel looked similar to previously published

results and the identities of these complexes were

Figure 3. The level of chlorophyll (Chl) synthase protein is much lower in chlg-1 than in the wild type.

(a) Immunoblot analysis of total proteins of 5-day-old seedlings. The band intensity was normalized to tubulin, with that of the wild type (WT) assigned as 1,

and indicated below each band.

(b) Quantitative RT-PCR analysis of WT and chlg-1 transcripts. Relative transcript levels (%) of CHLG were normalized to that of ACTIN2 with the WT assigned as

100. The bar indicates the mean � SD of a representative experiment with three replicates. No significance was observed in Student’s t-test with chlg-1 versus

WT, P > 0.05.

(c) Thylakoid proteins equal to 10 lg chlorophyll/lane were analyzed by blue native gel and immunoblotting. The representative shown is from four biological

repeats. PSI, PSII, photosystems I and II; LHCII, light-harvesting complex II.

(d) Phenotypes after 3 days’ recovery of 5-day-old seedlings with or without heat treatment (HS) at 40°C for 1 h. Since the growth of chlg-2 was much slower

than that of the wild type and chlg-1, 15-day-old instead of 5-day-old chlg-2 seedlings were used in this assay.



assigned based on this similarity (J€arvi et al., 2011).

Immunoblots showed that Chl synthase has a wide

distribution in thylakoid membrane complexes in the wild

type (Figure 3c), providing information lacking in the pro-

tein co-migration database for Arabidopsis thylakoids

(Takabayashi et al., 2013). In general, the patterns of the

pigmented complexes were largely unaltered in the mutant

despite a drastic reduction in the level of Chl synthase.

Similarly, the distribution of Lhcb1 and D1 proteins

detected by immunoblot was not substantially affected.

To address whether the heat-sensitive phenotype of

chlg-1 was simply a consequence of a reduction in Chl

synthase protein, we examined the response of the null

mutant chlg-2 to heat treatment. Despite being albino,

chlg-2 grew on sucrose-containing medium and survived

after heat stress treatment at a non-permissive tempera-

ture for chlg-1 (Figure 3d), indicating that the reduction in

Chl synthase protein is not a direct cause of the heat-

sensitive phenotype of chlg-1.

Heat sensitivity of chlg-1 coincides with heat-induced

accumulation of Chlide a, degradation of Chl synthase and

D1 proteins, and a surge in ROS

Since Chl synthase is responsible for the esterification of

Chlide a and Chlide b (Oster and R€udiger, 1997), the

reduced level of Chl synthase in chlg-1 may lead to accu-

mulation of its substrates Chlide a and/or Chlide b, which

are phototoxic compounds, after heat treatment. If this is

the case, the heat-sensitive phenotype of chlg-1 should be

light dependent. Indeed, the bleaching of chlg-1 cotyledons

was prevented when the plants were recovered in the dark

for 2 days immediately after heat treatment, but not in

those plants exposed to a light/dark cycle (Figure 4). To

determine whether Chlide a/b accumulated in chlg-1, high-

performance liquid chromatography (HPLC) analysis was

performed. In the wild type there was no obvious variation

in Chlide a accumulation before or after heat treatment at

40°C for 1 h. In chlg-1, the Chlide a level was at least two-

fold higher than that of the wild type under non-stress con-

ditions, and was drastically increased at 6 h after heat

treatment, reaching a peak level of 18 nmol g�1 fresh

weight (Figure 5a,b). A substantial amount of Chlide a

remained detectable (about 50% of the peak level) in chlg-1

even 48 h after heat treatment (Figure 5b). Accumulation

of Chlide a in chlg-1 was reduced at lower temperatures.

No substantial increase in Chlide a was observed when the

heat treatment was conducted at 35°C, and a moderate

increase was observed at 37°C (Figure 5c). No cotyledon

bleaching was found in the mutant after heat treatment at

35 or 37°C for 1 h (Figure 5d). Unlike Chlide a, Chlide b

could not be detected in either the wild type or the mutant

before or after heat treatment at 40°C for 1 h. We also

examined the effect of heat treatment on the levels of Chl

synthase, D1, PsaA, CP47 and Lhcb1 by immunoblotting.

Heat treatment at 40°C for 1 h reduced Chl synthase and

D1 protein by about 40–50% in both the wild type and

chlg-1, while PsaA, CP47 and Lhcb1 were less affected (Fig-

ure 5e). These results suggest that the heat treatment

induced degradation of Chl synthase and the D1 proteins.

Chlorophyllide a is a photosensitizer that induces the

production of singlet oxygen (Kim et al., 2013). To see

whether the heat-induced over-accumulation of Chlide a

increased production of singlet oxygen in chlg-1, the fluo-

rescence probe reagent singlet oxygen sensor green

(SOSG) was used for histochemical staining. In the pres-

ence of singlet oxygen, SOSG emits green fluorescence,

with excitation and emission maxima at 504 and 525 nm,

respectively (Rag�as et al., 2009). Emission of green fluores-

cence was observed in chlg-1 cotyledons after heat treat-

ment at 40°C for 1 h with 4-h recovery at 22°C in light, but

not in the wild type subjected to the same treatment (Fig-

ure 6). The fluorescence signal in chlg-1 was diminished

when recovery took place in the dark (Figure S4). No sub-

stantial difference was observed between the wild type and

chlg-1 treated at 35 or 37°C. Consistently, the autofluores-

cence of chlorophylls was reduced substantially only in

chlg-1 treated at 40°C and recovered in light. These results

suggest that over-accumulation of Chlide a induced the

production of singlet oxygen in the presence of light in the

mutant. Both Chlide a and the accumulation of singlet oxy-

gen showed good correlation with the bleaching phenotype

in chlg-1 at 40°C but not at lower temperatures, strongly

suggesting that the light-dependent heat sensitivity of chlg-

1 is due to an over-accumulation of phototoxic Chlide a.

Chlorophyllide a in chlg-1 is derived from de-esterification

of Chl a, which is independent of chlorophyllases CLH1

and CLH2

Chlorophyllide a can either be generated from protochloro-

phyllide (Pchlide) catalyzed by Pchlide oxidoreductase

Figure 4. The heat sensitivity of chlg-1 is light dependent.

(a) chlg-1 is bleached after heat treatment with recovery in the light/dark

cycle.

(b) chlg-1 is not bleached after heat treatment with recovery in the dark. The

schematics show the conditions for heat treatment. L/D, 16-h light/8-h dark

cycle; D, 24-h dark. Seedlings with the same treatment were grown and

treated on the same plate.



(POR) (Warren et al., 2009) or from de-esterification of Chl

a catalyzed by chlorophyllase (Harpaz-Saad et al., 2007).

Therefore, it was of interest to know via which route Chlide

a was generated in chlg-1 after heat stress. We considered

the de novo biosynthesis of Chlide a unlikely to be the

source because it was accumulated in seedlings recovered

in the dark after heat treatment (Figure 5) and the conver-

sion of Pchlide to Chlide a is light dependent (Warren

et al., 2009). We further examined the response to heat

treatment in etiolated seedlings in which Chlide a can only

be generated by POR. Without heat shock treatment, no

significant difference was observed between the wild type

and chlg-1 in levels of Pchlide and Chlide a before and

after exposure to light, respectively (Figure 7b). As

expected, Chl a was produced at a lower level in chlg-1

than in the wild type due to a reduced level of CHLG pro-

tein (Figure 7b,c). Heat treatment at 40°C for 1 h decreased

the level of Chlide a in both the wild type and the mutant

after 24 h of recovery in light, and no difference was

observed between the wild type and chlg-1 (Figure 7b).

Immunoblot analysis showed no substantial difference in

POR level between the wild type and chlg-1 before or after

heat treatment (Figure 7c). No cotyledon bleaching was

observed in the heat-treated etiolated chlg-1 seedlings

(Figure 7d). These results suggest that Chlide a accumu-

lated in the light-grown chlg-1 seedlings is not derived

from de novo biosynthesis but from de-esterification of Chl

a catalyzed by chlorophyllase.

In Arabidopsis, CHLOROPHYLLASE1 (CLH1) and CLH2

code for chlorophyllases that de-esterify Chls (Tsuchiya

et al., 1999). To determine whether CLH1 and CLH2 are

responsible for the heat-induced accumulation of Chlide a

in chlg-1, we generated a triple mutant chlg-1 clh1-1 clh2-2

for epistatic analysis. The triple mutant was obtained by

crossing chlg-1 and the clh1-1 clh2-2 double mutant and

confirmed by genotyping (Figure S5). In clh1-1 clh2-2,

levels of Chlide a were slightly lower than those in the wild

type before and after heat treatment (Figure 8a). This slight

Figure 5. Heat sensitivity of chlg-1 coincides with accumulation of chlorophyllide (Chlide) a and degradation of chlorophyll (Chl) synthase (CHLG) and D1 pro-

teins.

(a) High-performance liquid chromatography analysis of Chlide in 5-day-old seedlings after heat treatment at 40°C for 1 h and followed by 2 h recovery in the

dark (H). N, non-heated control. The position of the Chlide a peak is indicated by an arrow. WT, wild type.

(b) The Chlide a level before (pre) and after heat treatment at 40°C for 1 h and followed by different recovery times in the dark.

(c) The Chlide a level after heat treatment at different temperatures for 1 h and followed by 2 h recovery (2 h) in the dark. Total Chl was extracted from 50 seed-

lings per line. The bar in (b) and (c) indicates the mean � SD of representative experiments from four independent biological repeats with three replicates each.

(d) Phenotypes of seedlings after heat treatments at different temperatures for 1 h and followed by 3 days’ recovery in a light/dark cycle. Seedlings of the WT

and chlg-1 with the same treatment were grown and treated on the same plate.

(e) Immunoblot analysis of total proteins from 5-day-old seedlings after heat treatment at 40°C for 1 h (H). N, non-heated control. The band intensity was nor-

malized to tubulin, with that of the WT assigned as 1, and indicated below each band.



difference was probably due to the action of chlorophyl-

lase during pigment extraction (Hu et al., 2013). However,

in chlg-1 clh1-1 clh2-2, the Chlide a level was dramatically

increased as it was in chlg-1 after heat stress (Figure 8a).

Over-accumulation of Chlide a in the triple mutant also

coincided with its light-dependent heat-sensitive pheno-

type (Figure 8b). These results indicate that CLH1 and

CLH2 are not responsible for the heat-induced accumula-

tion of Chlide a in chlg-1.

DISCUSSION

A missense mutation in chlg-1 results in the replacement

of a conserved glycine-217 residue by arginine, a bulkier

and positively charged residue, at the predicted fourth

transmembrane helix in Arabidopsis Chl synthase (Fig-

ure 2b). Based on the reduction of the mutant protein in

chlg-1 under normal and heat stress conditions (Fig-

ures 3a, 5e and 7d), we propose that this mutation destabi-

lizes the protein and prevents esterification of Chlide a,

especially after severe heat stress, leading to the accumu-

lation of phototoxic Chlide a. Previously, a rice yellow-

green leaf mutant, ygl1, was shown to have a reduction in

Chl synthase activity (Wu et al., 2007). The ygl1 allele is

caused by a missense mutation in the Chl synthase gene

that leads to the replacement of a conserved Pro residue

by Ser near the Gly residue reported here (Figure 2b). A

significant reduction in total Chl and a higher Chl a versus

Chl b ratio is reported for ygl1, which is similar to the find-

ings reported here for chlg-1 (Figure 1b). Consistently, the

ygl1 mutant also accumulates Chlide to a slightly higher

level under normal conditions, as does Arabidopsis chlg-1

(Figure 5b). It is not known whether heat stress can further

increase the accumulation of Chlide in the rice mutant. It is

notable that reducing the level of Chl synthase in trans-

genic tobacco plants using an antisense approach also

resulted in a reduction in total Chl content and a higher

Chl a/b ratio, but interestingly did not result in the accumu-

lation of Chlide (Shalygo et al., 2009). It remains to be seen

whether the accumulation of Chlide is associated with the

mutated Chl synthases or with the exact level of the

enzyme activity. The higher Chl a/b ratio in three different

plants with deficient Chl synthase suggests a common

effect on Chl b conversion by limiting the biosynthesis of

Chl. A relationship between reduction in Chl content and

increased Chl a/b ratio has been reported in wheat and bar-

ley mutants (Falbel and Staehelin, 1994). The mechanism

underlying this relationship is not clear. Nevertheless, a

hypothesis of differential affinity to Chl a in the apopro-

teins of the chlorophyll–protein complexes has been pro-

posed, explaining how the availability of Chl a affects the

Chl a/b ratio (Shimada et al., 1990).

Over-accumulation of Chlide a in chlg-1 turns out to be

an important clue for understanding how Chl turns over at

steady state. In our system, severe heat stress treatment

results in accelerated degradation of the D1 protein (Fig-

ure 5e), which is consistent with a previous report (Maru-

tani et al., 2012). The concomitant accumulation of Chlide

a in chlg-1 should result from the degradation of Chl a

released from D1 instead of de novo biosynthesis from

Pchlide (Figures 5 and 7). The Chl a released from degrada-

tion of 50% of the D1 protein should account for about 1%

of total Chl a. This estimation is based on the assumption

that the ratio of (Chl a + b)/PS II reaction center protein in

Arabidopsis thylakoid membrane is about 400 (Malkin

et al., 1981; Melis, 1984), six Chl a molecules bound to

each D1–D2 complex in PS II (Zouni et al., 2001) are

released upon D1 degradation, and the Chl a/b ratio is

about three in the wild type. The percentage of Chl a

bound to the D1–D2 complex should be 1.5-fold higher in

chlg-1 than in the wild type because, in the mutant, the Chl

a level was reduced by 40% (Figure 1b) and the D1 level by

less than 10% (Figure 5e). If the Chl a released is all de-

esterified, it would result in accumulation of Chlide a

equivalent to about 1.5% of total Chl a in chlg-1. This esti-

mation is smaller but close to the actual number obtained.

Chlorophyllide a was accumulated up to 18 nmol g�1 fresh

weight in chlg-1 (Figure 5b), which is equivalent to about

Figure 6. The singlet oxygen level is greatly increased in chlg-1 after heat

treatment. Cotyledons of a 5-day-old seedling cut after 1 h of treatment at

the indicated temperatures were incubated in 10 lM of singlet oxygen sen-

sor green (SOSG) solution at 22°C under light for 4 h. Fluorescence images

were taken under a microscope equipped with a digital camera. The repre-

sentative shown is from four independent replicates.



1.9% of Chl a in the mutant (Figure 1b). This calculation

suggests that the Chlide a accumulated in chlg-1 mainly

comes from the de-esterification of Chl a released from the

degraded D1 complex. This notion is in good agreement

with isotope-labeling studies in Synechocystis, which sug-

gest that most chlorophyll is de-esterified upon dissocia-

tion and repair of damaged PS II (Vavilin and Vermaas,

2007).

The accumulation of Chlide a in chlg-1 is probably due

to the sudden increase in Chl turnover after heat shock,

which could apparently not be processed efficiently in the

mutant with reduced Chl synthase function. These findings

support a previously proposed pathway of steady-state Chl

turnover involving de- and re-esterification of Chl (Vavilin

and Vermaas, 2007). Interestingly, no accumulation of

Chlide b was observed in chlg-1 after heat stress (Fig-

ure 5a). This observation is consistent with previous find-

ings in labeling experiments that Chl turnover mainly

affects Chl a (Beisel, Jahnke, Hofmann, K€oppchen, Schurr

and Matsubara 2010, Feierabend and Dehne, 1996). The

level of Chlide a under non-stress conditions in chlg-1 is

higher than that in the wild type (Figure 5b), suggesting

that Chl synthase is also involved in Chl turnover under

normal conditions. The slight to moderate increase in

Chlide a under normal or milder heat stress (such as 37°Cfor 1 h) may not reach the threshold level that causes a

detrimental phototoxic effect in the mutant. This notion is

supported by a good correlation in the levels of Chlide a

and singlet oxygen in the mutant (Figures 5b and 6).

The involvement of Chl synthase in the reutilization of

Chlide a generated from turnover of Chl points to the exis-

tence of a salvage pathway in degradation and biosynthe-

sis of Chl a. We propose a working model in which the

salvage pathway for Chlide a is integrated with the major

pathways of Chl metabolism known to date (Figure 9). The

biosynthesis and salvage pathways for the phytol moiety

in Chl (Ischebeck et al., 2006; Kim et al., 2013) are not

shown for simplicity. Since Chlide a can also be converted

to Chlide b catalyzed by Chlide a oxidase (CAO) (Oster

et al., 2000), the salvaged Chlide a may be used in the bio-

synthesis of Chl b via a pathway known as the Chl cycle

(Figure 9), which is an important mechanism regulating

Figure 7. Accumulation of chlorophyllide (Chlide) a and the heat sensitive phenotype were not observed in greening etiolated seedlings of chlg-1 after heat

treatment.

(a) Schematic of heat treatment and sampling time of (b) and (d).

(b) Contents of protochlorophyllide (Pchlide), Chlide a and chlorophyll (Chl) a in etiolated seedlings without (N) or with (H) the heat treatment. Bar indicates the

mean � SD of representative experiments with three replicates. Student’s t-test, mutant versus the wild type, *P < 0.05. WT, wild type.

(c) Immunoblot analysis with total proteins from 5-day-old etiolated seedlings after heat treatment at 40°C for 1 h. pre, before heat treatment (HS). 0 to 48 h,

recovery time in light after heat treatment. POR, protochlorophyllide oxidoreductase; CHLG, Chl synthase.

(d) Phenotypes of etiolated seedlings before and after heat treatment and recovery in the light for 48 h. Seedlings of WT and chlg-1 with the same treatment

were grown on the same plate.



the construction and destruction of the peripheral light-

harvesting complexes (Tanaka and Tanaka, 2011). Whether

the salvage pathway and the Chl cycle are spatially

connected remains to be determined. One common ques-

tion in these two processes is how Chl a is converted to

Chlide a. Chlorophyllase (chlorophyll chlorophyllidohydro-

lase, EC 3.1.1.14) is the only enzyme known to de-esterify

Chl and form chlorophyllide in vitro (Takamiya et al.,

2000). However, given its extra-chloroplastic location, the

in vivo function of chlorophyllase remains controversial

(H€ortensteiner, 2013). Studies on Arabidopsis CLH1 in

response to necrotrophic pathogens suggest that chloro-

phyllase is involved in plant damage control (Kariola et al.,

2005). In addition, wheat chlorophyllase was shown to be

very stable at elevated temperatures (Arkus et al., 2005).

Based on these previous reports, we speculated that chlo-

rophyllase might act as a damage control enzyme in

response to severe heat stress that perturbs membrane

integrity. However, our results do not support this notion.

Genetic data show that chlorophyllases CLH1 and CLH2 are

not responsible for the accumulation of Chlide a in heat-

treated chlg-1 (Figure 8), suggesting the existence of an

alternative enzyme hydrolyzing Chl. Of note, chlorophyl-

lase has recently been implicated in the breakdown of Chl

during pigment extraction, and producing Chlide as an arti-

fact (Hu et al., 2013). The strong accumulation of Chlide a

in the presence and absence of the functional CLH genes

in chlg-1 excludes the possibility of Chlide a accumulation

in the mutant being artifactual (Figure 8a).

Recently, genetic evidence convincingly showed that

pheophytin pheophorbide hydrolase (or pheophytinase,

PPH), not chlorophyllase as has long been postulated, is

responsible for the breakdown of Chl during leaf senes-

cence in Arabidopsis (Schenk et al., 2007; Schelbert et al.,

2009). These findings indicate that removal of the phytol

chain from pheophytin a, not Chl a, is the first committed

step in Chl catabolism via the PAO pathway (Figure 9).

Given the similarity between the structure of pheophytin

and Chl, which differ only by one magnesium ion, it is

tempting to speculate that the enzyme catalyzing the

de-esterification of Chl a in the salvage pathway may share

Figure 8. Chlorophyllases CLH1 and CLH2 are not required for the accumu-

lation of chlorophyllide (Chlide) a in chlg-1.

(a) The Chlide a contents of 5-day-old seedlings after heat treatment at 40°Cfor 1 h followed by 2 h recovery in the dark (H). N, non-heated control. The

bar indicates the mean � SD of representative experiments with three repli-

cates. Student’s t-test, *P < 0.05. WT, wild type.

(b) Phenotyping of seedlings with or without heat treatment and followed

by 2 days’ recovery in the indicated light/dark (L/D) cycle.

Figure 9. A working model of chlorophyll (Chl) metabolism including the Chl a salvage pathway revealed in this study. POR, protochlorophyllide oxidoreduc-

tase; CHLG, Chl synthase; CAO, chloropyllide a oxygenase; CBR, Chl b reductase; HCAR, hydroxymethyl Chl a reductase; MCS, metal chelating substance; PPH,

pheophytinase; PAO, pheophorbide a oxygenase NCCs, non-fluorescent catabolites; LHC, light-harvesting complex; PSII, photosystem II. The enzyme responsi-

ble for de-esterification of Chl a is unknown and is marked with a question mark.



sequence homology with PPH. Two PPH homologs,

AT5G19850 and AT4G36530, have been identified by

phylogenetic analysis (Schelbert et al., 2009). Products of

these genes are present in the chloroplast proteome

(Ferro et al., 2010). Unlike PPH, which is predominantly

expressed in senescent leaves, transcripts of AT5G19850

and AT4G36530 are abundantly present in green tissues

of all ages (Winter et al., 2007); this is an expression

pattern more likely to be associated with steady-state

Chl turnover. This information makes AT5G19850 and

AT4G36530 attractive candidates for future genetic manip-

ulation.

In conclusion, our results show that Chl synthase is

involved in reutilization of Chlide a in a salvage pathway of

Chl a de- and re-esterification. These findings raise several

further questions about the role of the salvage pathway in

PS II repair, particularly the role of the de-esterification of

Chl a. It has been proposed that the phytol chain of Chl a

may hinder the degradation of D1 protein by FtsH protease

(Nixon et al., 2010). Answers to these questions await the

identification of the involved Chl hydrolyzing enzyme(s)

and further genetic manipulation. The chlg-1 mutant

isolated in this study will be very useful for this future

endeavor. It will also be interesting to know how Chl

synthase participates in the delivery of chlorophylls to

different pigment–protein complexes in higher plants. This

probably involves direct interactions of Chl synthase with

different protein complexes, as suggested by the wide

distribution of Chl synthase in Arabidopsis thylakoids

(Figure 3c). Recently, pull-down experiments using tagged

Chl synthase in Synechocystis PCC 6803 retrieved Chl syn-

thase-interacting proteins, including high-light-inducible

protein HliD, Ycf39 (a putative assembly factor for PS II)

and YidC insertase (Chidgey et al., 2014). A similar tech-

nique may be applicable in Arabidopsis by introducing

tagged Chl synthase into the chlg-2 mutant for pull-down

experiments. Identification of the components in different

Chl synthase-interacting complexes may help us to under-

stand how different Chl metabolic pathways are channeled

and regulated.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

The ethane methyl sulfonate-mutagenized mutant chlg-1/dlt4-1 inthe Col-0 background was isolated by a forward genetic approachas described in a previous study (Wu et al., 2013). The Chl syn-thase knockout mutant chlg-2 (Salk_112733) was obtained fromthe Arabidopsis Biological Resource Center (http://abrc.osu.edu/).The double mutants glk1 glk2 and clh1-1 clh 2-2 were gifts fromDr Jane A. Langdale, University of Oxford (Waters et al., 2009)and Dr Stefan H€ortensteiner, University of Zurich (Schenk et al.,2007), respectively. The triple mutant chlg-1 clh1-1 clh 2-2 wasgenerated by crossing chlg-1 and clh1-1 clh 2-2, followed by geno-typing using gene-specific primers (sequences listed in Figure S4).

Seeds were sterilized, sown on a plate containing 0.8% agar withhalf-strength Murashige–Skoog medium and 1% sucrose, andimbibed for 2 days at 4°C in the dark. After imbibition, plates wereincubated at 22°C in a culture room with a 16-h light(100 lmol m�2 sec�1)/8-h dark cycle.

Heat treatments

For the thermotolerance assays, 3- to 5-day-old seedlings weretreated as described in Liu et al. (2011). For Chl metabolismassays under heat stress, 5-day-old seedlings grown on agar med-ium plates were treated in a water bath at 35, 37 or 40°C for 1 h inthe dark.

Positional cloning of chlg-1 and complementation testing

Map-based cloning was applied to the F2 population from thecross between chlg-1 (in the Col-0 background) and the wild type(in the Ler background) with the help of molecular markers (http://www.arabidopsis.org/browse/Cereon/index.jsp) as described inJander (2006). After rough mapping, the mutated site in chlg-1was located to chromosome 3 within the region between themarkers ciw4 and CER470385. Further fine mapping narroweddown the locus to the region flanked by the markers CER470044and CER45226, and the genes within this region were examinedby sequencing.

For the complementation test of chlg-1, genomic DNA ofArabidopsis CHLG was amplified by PCR with primer set 50-GTTGCCACGTGTCTCTCAAC-30 and 50-GTTTGTGGATACATGTTTAGAATCAATAC-30. The 4109-bp PCR fragment, including 1015 bpupstream of the transcriptional start site, was cloned into the vec-tor pCR8/GW/TOPO (Invitrogen, http://www.invitrogen.com/),sequenced to verify no mutation, and subcloned into the binaryvector pBGW,0. Transformation and selection of transgenic plantswere performed as described previously (Charng et al., 2007).

Chlorophyll extraction and pigment identification

Chlorophyll extraction was performed following the method out-lined in Hu et al. (2013) with minor modifications. Briefly, about 50whole seedlings at the indicated stages were collected, flash fro-zen in liquid nitrogen and ground. Chlorophylls were extractedwith 100% acetone pre-cooled at �20°C (1 ml per 100 mg freshweight). The total Chl content and Chl a/b ratio were calculated bythe formula given in Arnon (1949). Standard Chlide a and b solu-tions were prepared from the hydrolyzation of Chl a and b (Sigma,http://www.sigmaaldrich.com/), respectively, by recombinant chlo-rophyllase, AtCLH1, prepared as previously described (Tsuchiyaet al., 1999). Chlorophyllides in the Chl extract were separatedusing an ultra-performance liquid chromatography system (UPLC;Waters, http://www.waters.com/) and a BEH C18 reverse-phasecolumn with a mobile phase containing (i) methanol:acetonitrile:0.25 M pyridine at 45:35:20 and (ii) 100% acetone as described inGarrido et al. (2003). The Pchlide content was calculated by itsabsorption spectra with a fluorescence spectrophotometer (Bio-tech Synergy MX; http://www.biotek.com/) following a previouslydescribed method (Hukmani and Tripathy, 1992).

Extraction of RNA and quantitative RT-PCR

Total RNA extraction and cDNA preparation of 5-day-old seedlingswere performed as described in Liu and Charng (2013). Expressionof the genes CHLG and ACTIN2 was analyzed by quantitative (q)RT-PCR with the primer sets 50-GACCCCAGAGGATGTTGCTA30 + 50-GGCTCATTAATTGCGTCGAT-30 and 50-GGCAAGTCATCACGATTGG-30 + 50- CAGCTTCCATTCCCACAAAC-30, respectively.



http://www.arabidopsis.org/browse/Cereon/index.jsp

http://www.arabidopsis.org/browse/Cereon/index.jsp

http://www.invitrogen.com/

http://www.sigmaaldrich.com/

http://www.waters.com/

http://www.biotek.com/

Protein extraction and immunoblotting

Total protein extraction and immunoblot analysis were performedas described previously (Liu et al., 2011). Rabbit polyclonal antise-rum against Arabidopsis Chl synthase was produced by using asynthetic peptide (N0-ETDTDKVKSQTPDKAPAGGC) as the immu-nogen. Peptide synthesis, immunization of animals and antibodypurification were performed by LTK Biotechnology (http://www.ltk.com.tw/). Antibodies against HSP101, HSP90, HSA32,sHSP-C1 and tubulin were as described in the work of Liu et al.(2011). Antibodies against POR (AS05 067), PsaA (AS06 172), PsbA(D1, AS05 084), PsbB (CP47 AS04 038 and Lhcb1 (AS09 522) wereobtained from Agrisera (http://www.agrisera.com/).

Thylakoid preparation and blue native PAGE

Preparation of thylakoid from the leaves of 4-week-old plants wasperformed as previously described (Chu and Li, 2011). The Invitro-gen NativePAGE system was employed for running blue nativePAGE. Briefly, the isolated thylakoid was resuspended in samplebuffer (BN2008; Invitrogen) with 1% n-dodecyl-D-maltopyranoside(DDM; Sigma) and centrifuged at 17 000 g for 30 min at 4°C.After adding 0.1% G-250 running dye (BN2008; Invitrogen), thesupernatant was loaded onto the gel (4–16% Bis-Tris gel,BN2112BX10; Invitrogen) in a volume equal to 10 lg Chl per lane.Immunoblotting following blue native PAGE was performed asdescribed above.

Singlet oxygen detection

After the indicated treatments, cotyledons of 5-day-old seedlingwere immediately cut and mounted on slides and 10 lM aque-ous solution of SOSG reagent (Invitrogen) was added. Afterincubation at 22°C in a transparent humid-box in light(100 lmol m�2 sec�1) for 4 h, images were captured by a Z1microscope (Zeiss, http://www.zeiss.com/) with a 488/525 nmexcitation/emission filter set.

Accession numbers

Sequence data presented in this article can be found in TheArabidopsis Information Resource (http://www.arabidopsis.org/)or NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) websites under thefollowing accession numbers: orthologs of Chl synthase in Arabid-opsis thaliana, At3 g51820; Oryza sativa, NP_001055272; Piceasitchensis, ACN41022; Physcomitrella patens, XP_001752661;Chlamydomonas reinhardtii, XP_001701588; Cyanothece, YP_002484523.

ACKNOWLEDGEMENTS

This research was supported by grants from the National ScienceCouncil of Taiwan (grants 100-2311-B-001-007 and 101-2311-B-001-010) to YYC. We thank the Small Molecule Metabolomics CoreFacility, Institute of Plant and Microbial Biology and the ScientificInstrument Center, Academia Sinica for technical assistance inUPLC operation. The DNA sequencing service was provided bythe Institute of Biomedical Sciences, Academia Sinica. We thankDrs Chiung-Chih Chu and Hsou-min Li for the instruction of chlo-roplast isolation and Ms Miranda Loney for English editing. Wealso thank the Institute of Plant and Microbial Biology for lendingus the fluorescence spectrophotometer.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online ver-sion of this article.Figure S1. Thermotolerance assays of dlt4-1 (chlg-1) under differ-ent heat stress regimes.Figure S2. Map-based cloning of dlt4-1 (chlg-1).Figure S3. Chromatography analysis of chlorophyll contents in thewild type and chlg-2.Figure S4. Surge of singlet oxygen level in chlg-1 after heat treat-ment is light dependent.Figure S5. Genotyping of triple mutant chlg-1 clh1-1 clh2-2.

Table S1. Phenotype segregation of F2 seedlings from the crossbetween the wild-type and dlt4-1 mutant.Table S2. Complementation test in the T2 seedlings of chlg-1transformed with the wild-type CHLG gene.

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