Mol. Cells 2017; 40(10): 773-786
Published online October 17, 2017
https://doi.org/10.14348/molcells.2017.0127
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: sakuraba0425@gmail.com (YS); ncpaek@snu.ac.kr (NCP)
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
Keywords 7-hydroxymethyl chlorophyll a reductase (HCAR), cell death, chlorophyll, chlorophyll catabolic enzyme, rice
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
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;
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).
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,
Arabidopsis HCAR, which catalyzes the reduction of 7-hydroxymethyl Chl
In this study, we performed a functional analysis of
The
To produce Arabidopsis lines ectopically expressing
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).
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.
The
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 (
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 ×
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.
The full-length cDNAs of rice
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.
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.
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
The
Like most CCE mutants in Arabidopsis,
We then examined the senescence phenotype of
To examine whether
HCAR is a CCE that converts 7-HMC
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.
To investigate how
In both Arabidopsis and rice, knockout and/or knockdown mutants of
At 80 d after seeding (DAS) in the paddy field, the
It has previously been reported that the
Whereas the
We then examined whether the
We then subjected
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
High levels of Pheo
AtHCAR is a key enzyme in the PAO/phyllobilin pathway for Chl degradation; it catalyzes the conversion of 7-HMC
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
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
In this study, we found that the
It is also possible that excessive accumulation of 7-HMC
Mol. Cells 2017; 40(10): 773-786
Published online October 31, 2017 https://doi.org/10.14348/molcells.2017.0127
Copyright © The Korean Society for Molecular and Cellular Biology.
Weilan Piao1, Su-Hyun Han1, Yasuhito Sakuraba1,2,*, and Nam-Chon Paek1,*
1Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea
Correspondence to:*Correspondence: sakuraba0425@gmail.com (YS); ncpaek@snu.ac.kr (NCP)
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
Keywords: 7-hydroxymethyl chlorophyll a reductase (HCAR), cell death, chlorophyll, chlorophyll catabolic enzyme, rice
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
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;
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).
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,
Arabidopsis HCAR, which catalyzes the reduction of 7-hydroxymethyl Chl
In this study, we performed a functional analysis of
The
To produce Arabidopsis lines ectopically expressing
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).
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.
The
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 (
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 ×
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.
The full-length cDNAs of rice
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.
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.
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
The
Like most CCE mutants in Arabidopsis,
We then examined the senescence phenotype of
To examine whether
HCAR is a CCE that converts 7-HMC
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.
To investigate how
In both Arabidopsis and rice, knockout and/or knockdown mutants of
At 80 d after seeding (DAS) in the paddy field, the
It has previously been reported that the
Whereas the
We then examined whether the
We then subjected
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
High levels of Pheo
AtHCAR is a key enzyme in the PAO/phyllobilin pathway for Chl degradation; it catalyzes the conversion of 7-HMC
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
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
In this study, we found that the
It is also possible that excessive accumulation of 7-HMC
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