STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis

During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel's green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC.     

Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence

Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII)-bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Map-based cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence.     

Participation of Chlorophyll b Reductase in the Initial Step of the Degradation of Light-harvesting Chlorophyll a/b-Protein Complexes in Arabidopsis

The light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) is the most abundant membrane protein in green plants, and its degradation is a crucial process for the acclimation to high light conditions and for the recovery of nitrogen (N) and carbon (C) during senescence. However, the molecular mechanism of LHCII degradation is largely unknown. Here, we report that chlorophyll b reductase, which catalyzes the first step of chlorophyll b degradation, plays a central role in LHCII degradation. When the genes for chlorophyll b reductases NOL and NYC1 were disrupted in Arabidopsis thaliana, chlorophyll b and LHCII were not degraded during senescence, whereas other pigment complexes completely disappeared. When purified trimeric LHCII was incubated with recombinant chlorophyll b reductase (NOL), expressed in Escherichia coli, the chlorophyll b in LHCII was converted to 7-hydroxymethyl chlorophyll a. Accompanying this conversion, chlorophylls were released from LHCII apoproteins until all the chlorophyll molecules in LHCII dissociated from the complexes. Chlorophyll-depleted LHCII apoproteins did not dissociate into monomeric forms but remained in the trimeric form. Based on these results, we propose the novel hypothesis that chlorophyll b reductase catalyzes the initial step of LHCII degradation, and that trimeric LHCII is a substrate of LHCII degradation.     

Molecular cloning and characterization of a chlorophyll degradation regulatory gene from bamboo

Leaf senescence constituted the final stage of leaf development and it is always accompanied by the leaf yellowing. The non-yellowing gene (NYE1), initially identified from Arabidopsis in our laboratory, is a key regulatory gene responsible for chlorophyll degradation during senescence. In this study, an orthologue of AtNYE1 was isolated from the bamboo (Bambusa emeiensis cv. Viridiflavus) and tentatively named BeNYE1. The full length sequence of 1 386 bp contains an open reading frame of 801 bp. The protein encoded by BeNYE1 consists of 266 amino acids. Sequence analysis revealed that BeNYE1 had high similarity with other NYE/SGR proteins from various monocotyledon species. BeNYE1 was strongly induced by natural senescence and dark-induced senescence in bamboo. Driven by a 1.5 kb upstream fragment of AtNYE1, BeNYE1 could rescue the stay-green phenotype of nye1-1. The constitutive over-expression of BeNYE1 could accelerate the chlorophyll degradation. These results indicated that BeNYE1 might play an important role in the regulation of chlorophyll degradation during leaf senescence in bamboo.     

Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice

Yellowing, which is related to the degradation of chlorophyll and chlorophyll–protein complexes, is a notable phenomenon during leaf senescence. NON-YELLOW COLORING1 ( NYC1 ) in rice encodes a membrane-localized short-chain dehydrogenase/reductase (SDR) that is thought to represent a chlorophyll  b reductase necessary for catalyzing the first step of chlorophyll  b degradation. Analysis of the nyc1 mutant, which shows the stay-green phenotype, revealed that chlorophyll  b degradation is required for the degradation of light-harvesting complex II and thylakoid grana in leaf senescence. Phylogenetic analysis further revealed the existence of NYC1-LIKE (NOL) as the most closely related protein to NYC1. In the present paper, the nol mutant in rice was also found to show a stay-green phenotype very similar to that of the nyc1 mutant, i.e. the degradation of chlorophyll  b was severely inhibited and light-harvesting complex II was selectively retained during senescence, resulting in the retention of thylakoid grana even at a late stage of senescence. The nyc1 nol double mutant did not show prominent enhancement of inhibition of chlorophyll degradation. NOL was localized on the stromal side of the thylakoid membrane despite the lack of a transmembrane domain. Immunoprecipitation analysis revealed that NOL and NYC1 interact physically in vitro . These observations suggest that NOL and NYC1 are co-localized in the thylakoid membrane and act in the form of a complex as a chlorophyll  b reductase in rice.     

Arabidopsis STAYGREEN-LIKE (SGRL) promotes abiotic stress-induced leaf yellowing during vegetative growth

During leaf senescence in Arabidopsis, STAYGREEN 1 (SGR1) and SGR2 regulate chlorophyll degradation positively and negatively, respectively. SGR-LIKE (SGRL) is also expressed in pre-senescing leaves, but its function remains largely unknown. Here we show that under abiotic stress, Arabidopsis plants overexpressing SGRL exhibit early leaf yellowing and sgrl-1 mutants exhibit persistent green color of leaves. Under salt stress, SGR1 and SGRL act synergistically for rapid Chl degradation prior to senescence. Furthermore, SGRL forms homo- and heterodimers with SGR1 and SGR2 in vivo, and interacts with LHCII and chlorophyll catabolic enzymes. The role of SGRL under abiotic stress is discussed.     

The Stay-Green Rice like (SGRL) gene regulates chlorophyll degradation in rice

The Stay-Green Rice (SGR) protein is encoded by the SGR gene and has been shown to affect chlorophyll (Chl) degradation during natural and dark-induced leaf senescence. An SGR homologue, SGR-like (SGRL), has been detected in many plant species. We show that SGRL is primarily expressed in green tissues, and is significantly downregulated in rice leaves undergoing natural and dark-induced senescence. As the light intensity increases during the natural photoperiod, the intensity of SGRL expression declines while that of SGR expression increases. Overexpression of SGRL reduces the levels of Chl and Chl-binding proteins in leaves, and accelerates their degradation in dark-induced senescence leaves in rice. Our results suggest that the SGRL protein is also involved in Chl degradation. The relationship between SGRL and SGR and their effects on the degradation of the light-harvesting Chl a/b-binding protein are also discussed.     

NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll – protein complexes during leaf senescence

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll – protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4‐1, a stay‐green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4‐1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation‐related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity‐dependent manner. Surprisingly, the F_v/F_m value remained high in nyc4‐1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4‐1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild‐type and other non‐functional stay‐green mutants, including sgr‐2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll – protein complexes during leaf senescence is discussed.     

In vivo participation of red chlorophyll catabolite reductase in chlorophyll breakdown

A central reaction of chlorophyll breakdown, porphyrin ring opening of pheophorbide a to the primary fluorescent chlorophyll catabolite (pFCC), requires pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR), with red chlorophyll catabolite (RCC) as a presumably PAO-bound intermediate. In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). Here, we show that RCCR-deficient Arabidopsis thaliana accumulates RCC and three RCC-like pigments during senescence, as well as FCCs and NCCs. We also show that the stereospecificity of Arabidopsis RCCR is defined by a small protein domain and can be reversed by a single Phe-to-Val exchange. Exploiting this feature, we prove the in vivo participation of RCCR in chlorophyll breakdown. After complementation of RCCR mutants with RCCRs exhibiting alternative specificities, patterns of chlorophyll catabolites followed the specificity of complementing RCCRs. Light-dependent leaf cell death observed in different RCCR-deficient lines strictly correlated with the accumulation of RCCs and the release of singlet oxygen, and PAO induction preceded lesion formation. These findings suggest that RCCR absence causes leaf cell death as a result of the accumulation of photodynamic RCC. We conclude that RCCR (together with PAO) is required for the detoxification of chlorophyll catabolites and discuss the biochemical role(s) for this enzyme.     

The Divergent Roles of STAYGREEN (SGR) Homologs in Chlorophyll Degradation

Degradation of chlorophyll (Chl) by Chl catabolic enzymes (CCEs) causes the loss of green color that typically occurs during senescence of leaves. In addition to CCEs, STAYGREEN1 (SGR1) functions as a key regulator of Chl degradation. Although sgr1 mutants in many plant species exhibit a stay-green phenotype, the biochemical function of the SGR1 protein remains elusive. Many recent studies have examined the physiological and molecular roles of SGR1 and its homologs (SGR2 and SGR-LIKE) in Chl metabolism, finding that these proteins have different roles in different species. In this review, we summarize the recent studies on SGR and discuss the most likely functions of SGR homologs.     

Accumulation of the NON‐YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in Arabidopsis thaliana

Chlorophyll a and chlorophyll b are interconverted in the chlorophyll cycle. The initial step in the conversion of chlorophyll b to chlorophyll a is catalyzed by the chlorophyll b reductases NON‐YELLOW COLORING 1 (NYC1) and NYC1‐like (NOL), which convert chlorophyll b to 7‐hydroxymethyl chlorophyll a. This step is also the first stage in the degradation of the light‐harvesting chlorophyll a/b protein complex (LHC). In this study, we examined the effect of chlorophyll b on the level of NYC1. NYC1 mRNA and NYC1 protein were in low abundance in green leaves, but their levels increased in response to dark‐induced senescence. When the level of chlorophyll b was enhanced by the introduction of a truncated chlorophyllide a oxygenase gene and the leaves were incubated in the dark, the amount of NYC1 was greatly increased compared with that of the wild type; however, the amount of NYC1 mRNA was the same as in the wild type. In contrast, NYC1 did not accumulate in the mutant without chlorophyll b, even though the NYC1 mRNA level was high after incubation in the dark. Quantification of the LHC protein showed no strong correlation between the levels of NYC1 and LHC proteins. However, the level of chlorophyll fluorescence of the dark adapted plant (F_o) was closely related to the accumulation of NYC1, suggesting that the NYC1 level is related to the energetically uncoupled LHC. These results and previous reports on the degradation of chlorophyllide a oxygenase suggest that the a feedforward and feedback network is included in chlorophyll cycle.     

Correlation of leaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested Chinese flowering cabbage (Brassica rapa var. parachinensis)

Chinese flowering cabbage is one of the main leafy vegetables produced in China. They have a rapid leaf yellowing due to chlorophyll degradation after harvest that limits their marketing. In the present study, leaf senescence of the cabbages was manipulated by ethylene and 6-benzyl aminopurine (6-BA) treatment to investigate the correlation of leaf senescence and chlorophyll degradation related to gene expression/activities in the darkness. The patterns of several senescence associated markers, including a typical marker, the expression of senescence-associated gene SAG₁₂, demonstrated that ethylene accelerated leaf senescence of the cabbages, while 6-BA retarded this progress. Similar to the trends of BrSAG₁₂ gene expression, strong activation in the expression of three chlorophyll degradation related genes, pheophytinase (BrPPH), pheophorbide a oxygenase (BrPAO) and red chlorophyll catabolite reductase (BrRCCR), was detected in ethylene treated and control leaves during the incubation, while no evident increase was recorded in 6-BA treated leaves. The overall dynamics of Mg-dechelatase activities in all treatments displayed increasing trends during the senescence process, and a delayed increase in the activities was observed for 6-BA treated leaves. However, chlorophyllase activity as well as the expression of BrChlase1 and BrChlase2 decreased with the incubation in all treatments. Taken together, the expression of BrPPH, BrPAO and BrRCCR, and the activity of Mg-dechelatase was closely associated with the chlorophyll degradation during the leaf senescence process in harvested Chinese flowering cabbages under dark conditions.     

The control of chlorophyll catabolism and the status of yellowing as a biomarker of leaf senescence

The pathway of chlorophyll catabolism during leaf senescence is known in a fair amount of biochemical and cell biological detail. In the last few years, genes encoding a number of the catabolic enzymes have been characterized, including the key ring-opening activities, phaeophorbide a oxygenase (PaO) and red chlorophyll catabolite reductase (RCCR). Recently, a gene that modulates disassembly of chlorophyll–protein complexes and activation of pigment ring-opening has been isolated by comparative mapping in monocot species, positional cloning exploiting rice genomics resources and functional testing in Arabidopsis. The corresponding gene in pea has been identified as Mendel's I locus (green/yellow cotyledons). Mutations in this and other chlorophyll catabolic genes have significant consequences, both for the course of leaf senescence and senescence-like stress responses, notably hypersensitivity to pathogen challenge. Loss of chlorophyll can occur via routes other than the PaO/RCCR pathway, resulting in changes that superficially resemble senescence. Such 'pseudosenescence' responses tend to be pathological rather than physiological and may differ from senescence in fundamental aspects of biochemistry and regulation.     

Defects in a proteolytic step of light-harvesting complex II in an<Emphasis Type="Italic">Arabidopsis</Emphasis> stay-green mutant,<Emphasis Type="Italic">ore10</Emphasis>, during dark-induced leaf senescence

During dark-induced leaf senescence (DIS), the non-functional stay-green mutantore10 showed delayed chlorophyll (Chl) degradation and increased stability in its light-harvesting complex II (LHCII). These phenomena were closely related to the formation of aggregates that mainly consisted of terminal-truncated LHCII (Oh et al., 2003). Theore10 mutant apparently lacks the protease needed to degrade the truncated LHCII. In wild-type (WT) plants, protease was found in the thylakoid fraction, but not the soluble fraction. A similar experiment using dansylated LHCII revealed that the protease degraded both WT andore10 LHCII, indicating that its stability inore10 perhaps did not result from a defect in the LHCII polypeptides themselves. Although protease activity was not present in non-senesced WT leaves, it was induced during DIS. It also was possible to diminish the high level of protease present in the thylakoids through high-salt washing, suggesting that this enzyme is extrinsically bound to the outer surface of the stroma-exposed thylakoid regions.