Rice 7-Hydroxymethyl Chlorophyll a Reductase Is Involved in the Promotion of Chlorophyll Degradation and Modulates Cell Death Signaling
Weilan Piao, Su-Hyun Han, Yasuhito Sakuraba, and Nam-Chon Paek
Abstract
The loss of green coloration via chlorophyll (Chl) degradation typically occurs during leaf senescence. To date, many Chl catabolic enzymes have been identified and shown to interact with light harvesting complex II to form a Chl degradation complex in senescing chloroplasts; this complex might metabolically channel phototoxic Chl catabolic intermediates to prevent oxidative damage to cells. The Chl catabolic enzyme 7-hydroxymethyl Chl a reductase (HCAR) converts 7-hydroxymethyl Chl a (7-HMC a) to Chl a. The rice (Oryza sativa) genome contains a single HCAR homolog (OsHCAR), but its exact role remains unknown. Here, we show that an oshcar knockout mutant exhibits persistent green leaves during both dark-induced and natural senescence, and accumulates 7-HMC a and pheophorbide a (Pheo a) in green leaf blades. Interestingly, both rice and Arabidopsis hcar mutants exhibit severe cell death at the vegetative stage; this cell death largely occurs in a light intensity-dependent manner. In addition, 7-HMC a treatment led to the generation of singlet oxygen (1O2) in Arabidopsis and rice protoplasts in the light. Under herbicide-induced oxidative stress conditions, leaf necrosis was more severe in hcar plants than in wild type, and HCAR-overexpressing plants were more tolerant to reactive oxygen species than wild type. Therefore, in addition to functioning in the conversion of 7-HMC a to Chl a in senescent leaves, HCAR may play a critical role in protecting plants from high light-induced damage by preventing the accumulation of 7-HMC a and Pheo a in developing and mature leaves at the vegetative stage.
INTRODUCTION
Leaf senescence is characterized by the gradual loss of green coloration, mainly due to chlorophyll (Chl) degradation. Since free Chl and its catabolic intermediates are highly phototoxic, they must be degraded rapidly and completely, along with photosynthetic proteins and other macromolecules. During this process, Chl is ultimately converted to non-phototoxic colorless catabolites, termed phyllobilins, through in the PAO/phyllobilin pathway (Hörtensteiner and Krautler, 2011); this reaction requires seven Chl catabolic enzymes (CCEs), which function in senescing chloroplasts (Hörtensteiner, 2013). Six CCE genes have been characterized in rice and/or Arabidopsis, including NON-YELLOW COLORING1 (NYC1, Horie et al., 2009; Kusaba et al., 2007) and NYC1-LIKE (NOL, Horie et al., 2009; Sato et al., 2009), along with genes encoding Chl b reductases, 7-hydroxymethyl Chl a reductase (HCAR, Meguro et al., 2011), pheophytinase (PPH, Schelbert et al., 2009), pheophorbide a oxygenase (PAO, Pružinská et al., 2003), and red Chl catabolite reductase (RCCR, Pružinská et al., 2007). Most mutants of these CCEs show a stay-green phenotype during natural senescence and/or artificially induced senescence, including dark- and phytohormone-induced leaf senescence, due to an impaired Chl degradation pathway (Horie et al., 2009; Hörtensteiner, 2009; Kusaba et al., 2007; Schelbert et al., 2009).
In addition to these CCEs, STAY-GREEN1 (SGR1, also termed NONYELLOWING1, NYE1) homologs have been isolated in various plant species, where they function as key positive regulators of Chl degradation; sgr mutants typically show a strong stay-green phenotype during both natural and dark-induced senescence (DIS) (Barry et al., 2008; Park et al., 2007; Ren et al., 2007; Sakuraba et al., 2015a). We previously showed that Arabidopsis SGR1/NYE1 physically interacts with the six CCEs and LHCII, forming a multi-protein complex that is likely important for rapid detoxification of Chl catabolic intermediates in senescing chloroplasts (Sakuraba et al., 2012; 2013).
In addition to SGR1, all plants contain an additional SGR subgroup, SGR-LIKE (SGRL). The C-terminal sequence of SGRL differs considerably from that of SGR (Hörtensteiner, 2009; Sakuraba et al., 2015a). OsSGRL-overexpressing plants show a premature leaf yellowing phenotype (Rong et al., 2013). Similarly, overexpression of AtSGRL accelerates leaf yellowing under abiotic stresses, including high salinity and heat stress, at the vegetative stage (Sakuraba et al., 2014b), indicating that SGRL mainly contributes to Chl degradation during vegetative growth. Very recently, Shimoda et al. (2016) reported that SGR and SGRL homologs have Mg-dechelatase activity for Chl a and chlorophyllide a, respectively, representing the last of the seven CCEs in the PAO/phyllobilin pathway.
Cell death is a common process that occurs in response to pathogen attack or abiotic environmental stress. Cell death, often referred to as the “hypersensitive response (HR)”, has important functions in protecting plants from advancing pathogen infection (Greenberg et al., 1994). A few CCEs play an important role in inhibiting HR, because Chl catabolic intermediates act as strong photosensitizers in planta. Thus, some mutants of CCEs show an accelerated cell death phenotype. In Arabidopsis, accelerated cell death1 (acd1) and acd2 are null mutants of PAO and RCCR, respectively. Under normal photoperiodic growth conditions, acd1 and acd2 accumulate pheophorbide a (Pheo a) and RCC in their green leaves, respectively, causing excessive accumulation of reactive oxygen species (ROS). Consequently, acd1 and acd2 show a necrotic phenotype and growth retardation (Pružinská et al., 2003; 2007). Cell death symptoms were also observed in RNAi-mediated knockdown mutants of PAO and RCCR in rice (Tang et al., 2011). Interestingly, RCCR-overexpressing Arabidopsis plants exhibit increased tolerance to oxidative stress-induced cell death (Yao and Greenberg, 2006), indicating that some CCEs have the potential for controlling cell death mechanisms, possibly through the metabolic channeling of phototoxic Chl intermediates.
Arabidopsis HCAR, which catalyzes the reduction of 7-hydroxymethyl Chl a (7-HMC a) to Chl a, is a CCE that functions in senescing chloroplasts (Meguro et al., 2011). HCAR is a homolog of cyanobacterial divinyl reductases, which participate in Chl
biosynthesis (Ito et al., 2008). The Arabidopsis hcar (athcar) mutant shows a stay-green phenotype during DIS, accompanied by strong accumulation
of 7-HMC a and Pheo a (Meguro et al., 2011). Furthermore, HCAR physically interacts with other CCEs, such as SGR1/NYE1, NYC1,
NOL, RCCR, and LHCII, indicating that HCAR is a component of the Chl degradation complex
(Sakuraba et al., 2013). The rice genome encodes a homolog of HCAR (hereafter OsHCAR,
In this study, we performed a functional analysis of OsHCAR using a T-DNA insertion knockout mutant. The oshcar mutant showed a stay-green phenotype during DIS, along with strong accumulation of the Chl catabolic intermediates 7-HMC a and Pheo a, indicating that OsHCAR is a component of the Chl degradation complex in rice. In addition, both the oshcar and athcar mutants showed an accelerated cell death phenotype due to excessive accumulation of singlet oxygen (1O2). Furthermore, 7-HMC a-treated protoplasts produced large amounts of 1O2, indicating that rice and Arabidopsis HCAR play an important role in protecting pre-senescent leaf cells from cell death. We discuss the possible roles played by HCAR in Chl degradation and in protecting plants from cell death.
MATERIALS AND METHODS
Plant materials and growth conditions
The oshcar mutant and its parental wild-type japonica rice cultivar ‘Hwayoung’ were grown in a growth chamber under long days (LD, 14.5 h light, 30°C/9.5 h dark, 24°C, 300 μmol m−2 s−1) or in a paddy field under natural LD (>14-h light/day) in Suwon, Korea (37°N latitude). The T-DNA insertion oshcar mutant (stock number: PFG_2A-00576) was obtained from the Crop Biotech Institute at Kyung Hee University, Korea (Jeon et al., 2000). For DIS, detached or attached leaves of 3-week-old plants were incubated in complete darkness. The Arabidopsis thaliana (At) T-DNA insertion athcar mutant (SALK_018790C) and the AtHCAR-overexpressing (AtHCAR-OX) line were described previously (Sakuraba et al., 2013), and they were grown under LD (16 h light/day, 70 μmol m−2 s−1).
Plant transformation
To produce Arabidopsis lines ectopically expressing OsHCAR, pMDC43 harboring 35S:OsHCAR was transformed into Agrobacterium tumefaciens stain GV3101. Agrobacterium-mediated transformation of the athcar mutant was performed using the floral-dip method (Zhang et al., 2006). Transformants were confirmed by genomic PCR using OsHCAR-specific primers (
Chl quantification
To measure total Chl concentrations, pigments were extracted from leaf tissues with 80% ice-cold acetone. Chl concentrations were determined by spectrophotometry, as described previously (Porra et al., 1989).
SDS-PAGE and immunoblot analysis
Total protein extracts were prepared from leaf tissues, using the middle section of the third leaf in the main culm of each 2-month-old rice plant grown under LD. Leaf tissues were ground in liquid nitrogen, and 10 mg aliquots were homogenized with 100 μl of sample buffer (50 mM Tris, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS, and 6% 2-mercaptoethanol). The homogenates were centrifuged at 10,000 × g for 3 min, and the supernatants were denatured at 80°C for 5 min. A 4 μl aliquot of each sample was subjected to 12% (w/v) SDS-PAGE, followed by electroblotting onto a Hybond-p membrane (GE Healthcare). Antibodies against photosystem proteins Lhca1, Lhca2, Lhcb1, Lhcb2, Lhcb4, Lhcb5, CP43, and PsaA (Agrisera, Sweden) were used for immunoblot analysis, and RbcL was visualized by Coomassie Brilliant Blue (CBB) staining. The level of each protein was examined using the ECL system with WESTSAVE (AbFRONTIER, Korea) according to the manufacturer’s protocol.
Measurement of Fv/Fm ratios
The Fv/Fm ratios of wild type and oshcar plants grown in the paddy field were measured using an OS-30p instrument (OptiSciences, USA), as previously described (Sakuraba et al., 2015b). The middle section of each flag leaf was adapted in the dark for 5 min to complete oxidation of QA. After dark treatment, the Fv/Fm ratio was measured in the paddy field. For both experiments, more than three experimental replicates per plant were conducted.
RNA isolation and quantitative reverse-transcription PCR (qPCR) analysis
For the RT reactions, total RNA was extracted from rice leaf blades and other tissues
using an RNA Extraction Kit (Macrogen, Korea). First-strand cDNA was prepared with
2 μg total RNA using M-MLV reverse transcriptase and oligo(dT)15 primer (Promega) in a total volume of 25 μl and diluted with 75 μl water. For quantitative
reverse-transcription PCR (qPCR), a 20 ml mixture was prepared including first-strand
cDNA equivalent to 2 μl total RNA, 10 μl 2× GoTaq master mix (Promega), 6 μl distilled
water, and gene-specific forward and reverse primers (
HPLC analysis
Leaves were weighed and pulverized in acetone using a Shake Master grinding apparatus (BioMedical Science), and the extracts were centrifuged for 15 min at 22,000 × g. The pigments were separated on a Symmetry C8 column (150 × 4.6 mm; Waters) as described previously (Zapata et al., 2000). The elution profiles were monitored by measuring the absorbance at 653 nm (SPD-M10A; Shimadzu), and pigments were identified based on their retention times and spectral patterns. Pigment quantification was performed based on the areas of the peaks.
Reactive oxygen species detection
For singlet oxygen (1O2) detection, Singlet Oxygen Sensor Green (SOSG; Invitrogen) reagent was used, as previously described (Han et al., 2012). Leaves of 2-week-old plants were treated with 50 mM SOSG in 10 mM sodium phosphate buffer (pH 7.5). After 30 min incubation, fluorescence emission following excitation at 480 nm was imaged using a laser scanning confocal microscope (LSM510, Carl Zeiss-LSM510). The red autofluorescence from Chl was also detected following excitation at 543 nm. Detection of hydrogen peroxide (H2O2) and superoxide (O2−) was carried out as previously described (Li et al., 2010), with minor modifications. Hydrogen peroxide (H2O2) and superoxide (O2−) were detected using 3,3-diaminobendizine (DAB) and nitroblue tetrazolium chloride (NBT), respectively. Leaves of 2-month-old plants grown in a paddy field were sampled and incubated in 0.1% DAB (Sigma) or 0.05% NBT (Duchefa) in 50 mM sodium phosphate buffer (pH 7.5) at room temperature overnight with gentle shaking. Chlorophyll was completely removed by incubation in 90% ethanol at 80°C.
Yeast two-hybrid analysis
The full-length cDNAs of rice HCAR, SGR, NYC1, NOL, PPH, PAO, and RCCR in entry vectors were inserted into destination vectors pDEST32 (bait) and pDEST22
(prey) (Invitrogen). Yeast strain MaV203 was used for cotransformation of bait and
prey clones, and β-galactosidase activity was measured via a liquid assay using chlorophenol
red-β-
Trypan blue staining
Trypan blue staining was performed as described by Koch and Slusarenko (1990) with minor modifications. Arabidopsis leaves and rice leaf discs exposed to herbicide-induced oxidative stress were incubated overnight in lactophenol-trypan blue solution (10 ml lactic acid, 10 ml glycerol, 10 g phenol, and 10 mg trypan blue dissolved in 10 ml distilled water). The stained leaves were boiled for 1 min and decolorized in 60% glycerol solution.
Oxidative stress assay
For MV treatment, one-month-old rice plants were sprayed with 50 μM methyl viologen dichloride (MV, Sigma), and three-week-old Arabidopsis plants were sprayed with 10 μM MV. The plants were incubated under continuous light conditions for the indicated times.
Protoplast isolation and light treatment
Rice leaf protoplasts were isolated from 15- to 20-d-old plants as described previously (Liang et al., 2003). Arabidopsis protoplasts were isolated from 3-week-old rosette leaves as described previously (Wu et al., 2009). The protoplasts were resuspended with protoplast incubation solution (500 mM manntiol, 20 mM KCl, 4 mM MES [pH5.8]), and incubated in the dark (control), under moderate light (200 μmol m−2 s−1), or under high light (500 μmol m−2 s−1), without or with 10 μM Chl b (Sigma-Aldrich), Pheo a (Sigma-Aldrich), and 7-HMC a, at 22°C under fluorescent light. 1O2 production in the protoplasts was visualized using singlet oxygen sensor green (SOSG, Invitrogen), and green fluorescence was observed under a Confocal Laser Scanning Microscope II (LSM710, Carl Zeiss).
RESULTS
Rice hcar mutants exhibit a stay-green phenotype during leaf senescence
The Oryza sativa genome contains only a single HCAR homolog (OsHCAR; LOC_Os04g25400), and the amino acid sequence of OsHCAR is highly similar to those
of HCAR homologs in other plant species (
Like most CCE mutants in Arabidopsis, athcar mutants show a stay-green phenotype during DIS (Meguro et al., 2011; Sakuraba et al., 2013). Thus, we examined the phenotype of the oshcar mutant during DIS. At the vegetative stage, the leaf color of oshcar was almost the same as that of wild type. However, the entire plant showed a stay-green
phenotype after 14 days of dark incubation (14 DDI; Fig. 1C). We also confirmed that detached leaf segments of oshcar also showed stay-green phenotype after 6 DDI (Fig. 1D). The stay-green phenotype was observed in the OsHCAR/oshcar heterozygous progenies that were segregated from the T-DNA heterozygous (OsHCAR/oshcar) plants, similar to the oshcar homozygous progenies (
We then examined the senescence phenotype of oshcar during natural senescence. To this end, we grew the plants in a paddy field under
natural LD conditions (~14 h light/day at 37° N latitude, Suwon, Korea). During grain
filling, oshcar exhibited delayed senescence (
To examine whether OsHCAR could recover the defects in the athcar mutant, we developed transgenic plants overexpressing OsHCAR in the athcar background (35S:OsHCAR/athcar). We evaluated the expression of OsHCAR in three independent transgenic lines by RT-PCR (
Rice HCAR is enzymatically equivalent to Arabidopsis HCAR
HCAR is a CCE that converts 7-HMC a to Chl a (Fig. 2A). In Arabidopsis, 7-HMC a accumulates in the hcar mutant, although it is detected only under DIS conditions (Meguro et al., 2011). To examine whether 7-HMC a accumulates in the oshcar mutant, we performed HPLC analysis. Chl and its intermediates were extracted from
green leaves (0 DDI) and dark-treated leaves (5 DDI) from wild type and oshcar plants. The substrate of HCAR, 7-HMC a, was undetectable in wild-type leaves, but it accumulated to levels high enough to
detect in hcar leaves before and after dark treatment (Figs. 2B and 2C), which also occurs in the athcar mutant (
We previously used yeast two-hybrid and co-immunoprecipitation assays to show that in Arabidopsis, HCAR physically interacts with other CCEs (SGR1/NYE1, NYC1, NOL, PPH, PAO, and RCCR), directly or indirectly, at LHCII (Sakuraba et al., 2013). Thus, it is highly likely that HCAR also interacts with other CCEs in rice. To investigate this notion, we performed yeast two-hybrid assays to examine the pairwise interactions between HCAR and six CCEs (SGR, NYC1, NOL, PPH, PAO, and RCCR). HCAR interacted with five CCEs, whereas PPH failed to interact with HCAR (Fig. 3). Interestingly, HCAR interacted with itself, suggesting that it may form dimers that interact with other CCEs at LHCII and induce Chl degradation.
The expression pattern of OsHCAR differs from that of AtHCAR during leaf senescence
To investigate how HCAR transcription is regulated in rice, we used RT-qPCR to examine the expression levels
of HCAR in different tissues, including root, leaf sheath, leaf blade, tiller, tiller base,
and internode tissue. HCAR mRNA was highly abundant in leaf sheath, leaf blade, and internode tissue, which
contain Chls (
Cell death occurs in hcar leaves during vegetative growth
In both Arabidopsis and rice, knockout and/or knockdown mutants of PAO and RCCR show an accelerated cell death phenotype, even under normal growth conditions, due to excess accumulation of phototoxic Chl intermediates (Pružinská et al., 2003, 2007; Tang et al., 2011). Thus, it is possible that HCAR is involved in regulating cell death signaling by modulating the metabolic processes underlying Chl degradation.
At 80 d after seeding (DAS) in the paddy field, the oshcar mutant exhibited accelerated cell death, especially in the tip sectors of older leaves
(Figs. 5A–5C). In addition, under long-day (LD) conditions in growth chambers, the oshcar mutant showed accelerated cell death symptoms under high light, while cell death
was barely detectable under low light (
The rice hcar mutant is susceptible to oxidative stress-induced cell death
It has previously been reported that the atrccr mutant, also known as acd2, is highly susceptible, whereas AtRCCR-OX plants are more tolerant to the treatment of methyl viologen, which induces oxidative
stress by blocking the electron transport during photosynthesis in the chloroplast
(Yao and Greenberg, 2006). To investigate whether the oshcar mutant also shows increased susceptibility to oxidative stress, we treated mutant
and wild-type seedlings with 50 μM methyl viologen (MV). As expected, after 6 d of
MV treatment, oshcar seedlings wilted much more quickly than WT (Figs. 6A and 6B). This leaf necrosis phenotype was also confirmed in oshcar heterozygous lines, while WT segregates did not show the phenotype, similar to Hwayoung
WT (
Arabidopsis HCAR is involved in cell death signaling
Whereas the oshcar mutant showed accelerated cell death symptoms in both the paddy field and growth chamber (Figs. 5 and 6), this phenotype has not been reported for the athcar mutant, as previous studies of this mutant have mainly focused on its senescence phenotype during DIS (Meguro et al., 2011; Sakuraba et al., 2013). We therefore examined whether cell death occurs in athcar under normal conditions. Like oshcar, the 4-week-old athcar plants showed an accelerated cell death phenotype when grown under normal light (100 μmol m−2 s−1) conditions (Fig. 7A), which was confirmed by trypan blue staining (Fig. 7B). Under high light (500 μmol m−2 s−1) conditions, the cell death phenotype was more severe in athcar than in wild type and was observed even in 3-week-old plants (Figs. 7A and 7C). Furthermore, athcar plants were clearly smaller than wild-type plants (Fig. 7A), probably because severe cell death inhibits vegetative growth.
We then examined whether the athcar mutant is also highly susceptible to MV-induced oxidative stress. At 3 d after MV
treatment, 4-week-old athcar plants displayed more severe cell death symptoms than wild type, whereas AtHCAR-OX plants were more tolerant to this treatment than wild type (Figs. 7D and 7E). The athcar mutant was also highly susceptible to oxyfluorfen-induced oxidative stress (
We then subjected 35S:OsHCAR/athcar plants to MV-induced oxidative stress, finding that they were more tolerant of this
stress than wild type (
The production of singlet oxygen in hcar mutant protoplasts is caused by the accumulation of 7-HMC a and Pheo a
To analyze the role of HCAR in the cell death response in more detail, we monitored
the production of singlet oxygen in protoplasts from wild-type and oshcar plants in rice. In the dark, neither wild-type nor oshcar protoplasts produced 1O2 (Fig. 8A). After 2 h of low-light treatment, however, we detected green fluorescence corresponding
to 1O2 accumulation in wild-type protoplasts (Fig. 8B). Furthermore, we detected a much stronger fluorescence in oshcar protoplasts that were incubated for a longer period of time under low light (Fig. 8B). However, green fluorescence was barely detected in wild-type protoplasts under
high light (Figs. 8B and 8C), indicating that oshcar protoplasts produce 1O2 in a light-dependent manner. To examine whether the accumulation of 7-HMC a leads to 1O2 production in oshcar protoplasts, we purified 7-HMC a from Chl b (
High levels of Pheo a accumulate in athcar plants during dark incubation (Meguro et al., 2011). We therefore investigated whether Pheo a also accumulates in the oshcar mutant. Before dark incubation, Pheo a was barely detected in wild-type and oshcar leaves. After 4 DDI, however, high levels of Pheo a accumulated in hcar leaves (Figs. 9A and 9B). We also investigated whether the levels of PAO mRNA and/or PAO protein are related to Pheo a accumulation in oshcar leaves. By RT-qPCR analysis, we found that the mRNA level of PAO in oshcar leaves was not significantly different to that of wild-type leaves (Fig. 9C), like the expression patterns of other CCE genes, such as SGR1, NYC1, NOL, PPH, and RCCR (
DISCUSSION
Rice HCAR is a functional homolog of Arabidopsis HCAR involved in Chl breakdown
AtHCAR is a key enzyme in the PAO/phyllobilin pathway for Chl degradation; it catalyzes
the conversion of 7-HMC a to Chl a in vivo and in vitro, and the athcar mutant shows a stay-green phenotype during DIS (Meguro et al., 2011). In this study, we found that, like the athcar mutant, the oshcar mutant exhibits persistent leaf greenness much longer than wild type during DIS and
natural senescence (Fig. 1;
In Arabidopsis, HCAR interacts with other CCEs, such as SGR1/NYE1, NYC1, NOL, and RCCR, during leaf senescence (Sakuraba et al., 2013). Similarly, in rice, OsHCAR also interacted with SGR, NYC1, and NOL in yeast two-hybrid assays (Fig. 3). Interestingly, we also found that OsHCAR interacted with itself (Fig. 3), suggesting that OsHCAR can form homodimers or -trimers. The importance of the HCAR-HCAR interaction remains unclear. Very recently, Wang and Liu (2016) determined the crystal structure of AtHCAR and found that it has the potential to form trimers, which is likely important for its interaction with LHCII. Similarly, we previously showed that Arabidopsis SGR1/NYE1 forms a homodimer and/or hetero-dimer with other SGR homologs in Arabidopsis, i.e., SGR2 and SGR-LIKE (SGRL), possibly to help control the rate of Chl degradation (Sakuraba et al., 2014b; 2014c). In this respect, HCAR dimer (or trimer) formation is probably important for enhancing its functions, e.g., its interaction capacity with LHCII and other CCEs, similar to SGR/Mg-dechelatase proteins.
Although the physiological role of OsHCAR is almost equivalent to that of AtHCAR,
we found that the mRNA expression pattern of OsHCAR considerably differs from that of AtHCAR. Similar to AtNOL (Sakuraba et al., 2013), AtHCAR expression decreased during DIS, while the expression of OsHCAR and OsNOL increased under both DIS and natural senescence (Fig. 4). These results strongly suggest that the requirement for HCAR and NOL activity somehow
differs in Arabidopsis and rice, especially in chloroplasts at the senescence phase.
Indeed, the phenotypes of Arabidopsis and rice nol null mutants are quite different during leaf senescence: the rice nol plants display a stay-green phenotype (Sato et al., 2009), whereas the Arabidopsis nol plants undergo normal yellowing (Horie et al., 2009). Similarly, the oshcar mutant showed a strong stay-green phenotype during natural senescence in the paddy
field (
HCAR plays an important role in preventing cell death signaling in rice and Arabidopsis
Under high-light conditions, Chl and its intermediates produce high levels of singlet oxygen, because their excited forms react with triplet oxygen, leading to the production of the highly reactive oxygen species 1O2. Protoporphyrin IX (Proto IX) and protochlorophyllide (Pchlide) are strong photosensitizers in the Chl biosynthesis pathway (Jung et al., 2008; Nagata et al., 2005; op den Camp et al., 2003), whereas Pheo a and RCC are strong photosensitizers in the Chl degradation pathway: Arabidopsis pao and rccr mutants, also known as acd1 and acd2, respectively, show severe cell death symptoms, accompanied by the production of singlet oxygen (Pružinská et al., 2007). However, it was previously unclear whether other Chl intermediates can also have photo-toxic effects.
In this study, we found that the oshcar mutant suffered from cell death symptoms in mature leaves under normal growth conditions
in both the paddy field and growth chamber. We also found that oshcar mutant is also susceptible to herbicide-induced oxidative stress conditions (Fig. 5). and similar cell death symptoms in the Arabidopsis hcar mutant was also observed (Fig. 6). It has been known that MV blocks the electron transport during photosynthesis in
the chloroplasts, leading to the production of 1O2. Produced 1O2 indirectly promotes the degradation of Chls, as well as photosystem apparatus, and
further 1O2 is produced by the accumulation of 7-HMC a and Pheo a in hcar mutants, similar to rccr mutant (Yao and Greenberg, 2006). Furthermore, both rice and Arabidopsis protoplasts incubated with purified 7-HMC
a produced large amounts of singlet oxygen (Fig. 8;
It is also possible that excessive accumulation of 7-HMC a changes the pigment composition in Chl-containing photosystem proteins in non-senescent
green leaves. Indeed, in transgenic Arabidopsis plants overexpressing chlorophyllide
a oxygenase (CAO-OX), high levels of Chl b accumulate in leaves, leading to changes in Chl a/b ratios in Chl-binding photosystem proteins, including LHCII and the subunits of core
complexes (Sakuraba et al., 2009; Yamasato et al., 2005). Furthermore, CAO-OX plants exhibit cell death symptoms under high-light conditions, because their
altered Chl composition ultimately changes their energy transfer capacity (Sakuraba et al., 2010), indicating that maintaining the proper balance of Chl composition in photosystem
complexes is important for preventing cell death due to excessive light damage. Similarly,
it is highly likely that the excessive 7-HMC a in the hcar mutants enters into the binding sites of Chls instead of photosystem proteins, leading
to severe cell death symptoms (Figs. 5
Supplementary data
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