Chlorophyll Catabolic Enzymes Research Articles

Involvement of BcNAC083 in 1-MCP-induced delayed yellowing of pak choi (Brassica rapa subsp. Chinensis) through the regulation of reactive oxygen species metabolism and inhibition of major chlorophyll catabolic genes

Leaf yellowing is a typical senescence feature in postharvest pak choi. Plant-specific NAC transcription factors (TFs) are crucial regulators of plant senescence. In this study, a senescence-related NAC TF, BcNAC083, was investigated to identify its transcriptional regulation effects on the yellowing of pak choi by treatment with 1-methylcyclopropene (1-MCP). Applying 5.0 µL L–1 1-MCP alleviated yellowing and maintained a low respiration rate, high chlorophyll content, and relatively complete chloroplast structure in pak choi during storage. However, 10 μL L–1 ethylene accelerated yellowing, as demonstrated by the higher respiration rate, lower chloroplast structural integrity and chlorophyll content. In contrast to ethylene treatment, 1-MCP suppressed the activity of the reactive oxygen species (ROS) metabolism-related enzymes superoxide dismutase (SOD), catalase (CAT), ascorbic peroxidase (APX), and lipoxygenase (LOX), and correspondingly reduced superoxide anion (O2·–), hydrogen peroxide (H2O2), and malondialdehyde (MDA) accumulation. 1-MCP treatment also suppressed the transcript levels of ROS metabolic genes, including BcRBOHC, BcRBOHD, BcSOD, BcCAT, BcAPX, and BcLOX, and inhibited the activity of the chlorophyll catabolic enzymes chlorophyllase (CLH), Mg-de-chelatase, and pheophytin pheophorbide hydrolase (PPH) and transcript levels of associated genes, including BcCLH1/2, BcPPH1/2, BcPAO, BcNYC1, BcSGR1/2, BcHCAR, BcRCCR, and BcCAO. The expression of BcNAC083 was probably induced by ROS and was downregulated by 1-MCP and upregulated by ethylene. Molecular biology experiments determined that BcNAC083 bound directly to and activated the promoters of the chlorophyll metabolism-related genes BcCLH1, BcPPH1, BcNYC1, and BcHCAR. Importantly, transient overexpression of BcNAC083 stimulated the endogenous genes (NbCLH1, NbPPH1, NbNYC1, and NbHCAR) expression in tobacco leaves, resulting in rapid chlorophyll breakdown and leaf degreening. Treatment with 1-MCP may alleviated the yellowing of pak choi by inhibiting ROS accumulation and the transcriptional regulation of BcNAC083, which had positive regulatory effects on major chlorophyll metabolic genes.

Receptor for Activated C Kinase1B (RACK1B) Delays Salinity-Induced Senescence in Rice Leaves by Regulating Chlorophyll Degradation.

The widely conserved Receptor for Activated C Kinase1 (RACK1) protein is a WD-40 type scaffold protein that regulates diverse environmental stress signal transduction pathways. Arabidopsis RACK1A has been reported to interact with various proteins in salt stress and Light-Harvesting Complex (LHC) pathways. However, the mechanism of how RACK1 contributes to the photosystem and chlorophyll metabolism in stress conditions remains elusive. In this study, using T-DNA-mediated activation tagging transgenic rice (Oryza sativa L.) lines, we show that leaves from rice RACK1B gene (OsRACK1B) gain-of-function (RACK1B-OX) plants exhibit the stay-green phenotype under salinity stress. In contrast, leaves from down-regulated OsRACK1B (RACK1B-UX) plants display an accelerated yellowing. qRT-PCR analysis revealed that several genes which encode chlorophyll catabolic enzymes (CCEs) are differentially expressed in both RACK1B-OX and RACK1B-UX rice plants. In addition to CCEs, stay-green (SGR) is a key component that forms the SGR-CCE complex in senescing chloroplasts, and which causes LHCII complex instability. Transcript and protein profiling revealed a significant upregulation of OsSGR in RACK1B-UX plants compared to that in RACK1B-OX rice plants during salt treatment. The results imply that senescence-associated transcription factors (TFs) are altered following altered OsRACK1B expression, indicating a transcriptional reprogramming by OsRACK1B and a novel regulatory mechanism involving the OsRACK1B-OsSGR-TFs complex. Our findings suggest that the ectopic expression of OsRACK1B negatively regulates chlorophyll degradation, leads to a steady level of LHC-II isoform Lhcb1, an essential prerequisite for the state transition of photosynthesis for adaptation, and delays salinity-induced senescence. Taken together, these results provide important insights into the molecular mechanisms of salinity-induced senescence, which can be useful in circumventing the effect of salt on photosynthesis and in reducing the yield penalty of important cereal crops, such as rice, in global climate change conditions.

Ozone fumigation promotes antioxidant activities to retard chlorophyll degradation and cell death in ‘Jinda’ chili during storage

Chlorophyll degradation plays a crucial role in mediating the senescence and devaluing of 'Jinda' chili fruit after harvest. The objective of this study was to investigate the effect of ozone fumigation on the chili fruit quality in relation to ROS-mediated chlorophyll degradation and cell death-related processes. Green chili fumigated with ozone (50 mg L−1) for 30 min was examined for antioxidant systems, chlorophyll degradation and cell death-related enzymes during 12 d of storage. The dissipation of pericarp degreening and reddening of pericarp color were observed on day 3 of storage, which were associated with the significantly elevated levels of superoxide and hydrogen peroxide accumulations, as well as increased chlorophyll catabolic enzymes (chlorophyll peroxidase, chlorophyllase and pheophytinase activities). Moreover, both caspase-like enzymes and in situ DNA degradation, features of programmed cell death (PCD), became more obviously visible at the early period of deterioration. Fruit fumigated with ozone, enhanced the activity of antioxidant enzymes (ascorbate peroxidase, catalase and superoxide dismutase) and suppressed both chlorophyll degradation and the PCD process, which were compatible with lowering senescence in chili. Our findings showed that ozone fumigation increases antioxidant activity, which in turn helps to delay chlorophyll breakdown and cell death, hence slowing senescence in 'Jinda' chili.

The pepper MYB transcription factor CaMYB306 accelerates fruit coloration and negatively regulates cold resistance

The extensively studied MYB transcription factor (TF) family has significant functions in plant development and stress responses. However, little is known about the role of MYB TFs in fruit coloration and cold resistance in pepper (Capsicum annuum L.). In this study, we identified the novel MYB gene CaMYB306 in pepper. CaMYB306 expression was higher in red fruit. Knockdown of CaMYB306 inhibited carotenoid biosynthesis and decreased expression of carotenoid-related genes in pepper fruit. CaMYB306-overexpressing (CaMYB306-OE) tomato plants showed earlier coloration with increased carotenoid accumulation and decreased chlorophyll and anthocyanin contents. The expression levels of key carotenoid biosynthesis genes (SlPSY and SlPDS) and key chlorophyll catabolic enzymes genes (SlSGR and SlPAO) were increased in CaMYB306-OE fruit, whereas key anthocyanin biosynthesis genes (SlF3H and SlCHS) were down-regulated. Additionally, CaMYB306 expression was up-regulated under cold treatment. CaMYB306 played a negative role in cold response via the regulation of the reactive oxygen species system and the expression of cold-related genes. CaMYB306 suppressed the transcriptional activity of Calcineurin B-Like-Interacting Protein Kinase gene 13 (CaCIPK13), which was a positive regulator of cold resistance. These findings clarify the role of MYB TFs in fruit coloration and cold resistance in pepper.

Enrichment of chlorophyll catabolic enzymes in grana margins and their cooperation in catabolic reactions

During leaf senescence, chlorophyll a and b are degraded through several enzymatic reactions, including chlorophyll b reductase, 7-hydroxymethyl chlorophyll a reductase, and Mg-dechelatase. Considering that the intermediates of the chlorophyll breakdown pathway are highly photoreactive, cooperative and efficient reactions of chlorophyll metabolic enzymes may protect chloroplasts from potential photo-oxidative damage. Here, we investigated the sub-organellar localization and cooperative reactions of the enzymes involved in the chlorophyll breakdown pathway by the fractionation of thylakoid membranes and enzymatic assays using recombinant proteins. We found that these enzymes were enriched in the grana margin fraction. Furthermore, we found that chlorophyll b reductase and Mg-dechelatase efficiently catabolized chlorophylls bound to the chlorophyll–protein complexes when these two enzymes were mixed. These results suggest that the co-localization of chlorophyll catabolic enzymes enables efficient chlorophyll breakdown. The results from this study highlight a key step forward in the investigation of the photosystem breakdown process.

Melatonin delays yellowing of broccoli during storage by regulating chlorophyll catabolism and maintaining chloroplast ultrastructure

• Exogenous melatonin markedly extended the shelf life of broccoli. • Melatonin delayed the decline of chlorophyll content and chloroplast breakdown. • Melatonin regulated activities and gene expressions of chlorophyll catabolic enzymes. • Melatonin regulated the expressions of BoSGR1 and BoSGR2 . The effect of melatonin treatment on color, chlorophyll degradation and chloroplast structure in broccoli during storage was evaluated. One hundred μmmol L −1 melatonin markedly extended the shelf life of broccoli. Compared with control florets, melatonin treatment delayed the decline of chlorophyll content, and maintained more intact chloroplasts. Meanwhile, melatonin also suppressed the activities of chlorophyll catabolic enzymes such as chlorophyllase (CLH), pheophytinase (PPH), pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR), and down-regulated the expressions of BoNYC1 , BoNOL , BoCLH , BoPPH , BoPAO , BoRCCR and BoSGR1 involved in chlorophyll catabolism. Taken together, our results indicated that melatonin could be a good candidate for delaying yellowing in broccoli during storage at 20 ℃.

Rice (Oryza sativa) Receptor for Activated C Kinase1B (OsRACK1B) Regulates Chlorophyll Catabolism and NADPH oxidase‐dependent H2O2 Signaling Pathways during Salinity Stress

The Receptor for Activated C Kinase1B (RACK1B) is a WD‐40 type scaffold protein that regulates diverse environmental stress signal transduction pathways. Arabidopsis RACK1A has been reported to interact with various proteins in salt stress and Light‐Harvesting Complex (LHC) pathways. However, the mechanism of how RACK1 contributes to the photosystem and chlorophyll metabolism in stress conditions remains elusive. Using T‐DNA‐mediated activation tagging transgenic rice (Oryza sativa L.) lines, we show that leaves from rice RACK1B gene (OsRACK1B) gain‐of‐function (OsRACK1B‐OX) plants exhibit the stay‐green phenotype by stabilizing chlorophyll under salinity stress. In contrast, leaves from down‐regulated OsRACK1B (OsRACK1B‐UX) plants display an accelerated yellowing phenotype accompanied by chlorophyll degradation. qRT‐PCR analysis revealed that genes encoding chlorophyll catabolic enzymes (CCEs) are differentially expressed in both OsRACK1B‐OX and OsRACK1B‐UX rice plants. In addition to CCEs, STAY‐GREEN (SGR) is a key component that forms the SGR‐CCE complex in senescing chloroplasts, which causes LHCII complex instability. Transcript and protein profiling revealed a significant upregulation of OsSGR in RACK1B‐UX plants than that in RACK1B‐OX plants during salt stress. The BiFC assay demonstrated that OsRACK1B interacts with OsSGR in planta in both cytoplasm and the nucleus. Our findings suggest that overexpression of OsRACK1B negatively regulates chlorophyll degradation, delays salinity‐induced senescence and OsRACK1B functions in this process, at least in part, by antagonizing the function of OsSGR through a direct interaction. Interestingly, histochemical staining revealed a global increase of hydrogen peroxide (H2O2) in plants overexpressing OsRACK1B compared to that of the wild‐type and the trend can be reversed by pre‐treatment leaves with diphenylidonium (DPI), a NADPH oxidase (NOX) inhibitor. In gel‐activity of NOX and Reactive Oxygen Species (ROS) scavenging enzymes revealed that OsRACK1B regulates NOX and ROS scavenging enzyme activity in a salt stress dependent manner indicating a complex interplay and a novel regulatory module involving OsRACK1, NOX dependent H2O2 signaling, and a balanced ROS scavenging enzyme activity. BiFC analysis demonstrated that RACK1 also interacts with N‐terminal region of NOX at the plasma membrane. We hypothesize that elevated level of H2O2 acts as a signaling molecule to counteract salt stress and chlorophyll catabolism in OsRACK1B transgenic leaves under salinity stress. Taken together, these results provide important insights into the molecular mechanisms of chlorophyll catabolism, salinity‐induced senescence which can be useful in circumventing the effect of salt on photosynthesis and in reducing yield penalty of important cereal crops, like rice, in global climate change conditions.Support or Funding InformationThis work was partially supported by National Science Foundation (NSF), USA grant to Howard University (Grant number MCB 0542312); Dept. of Biology and Graduate School of the Howard University.Proposed working model of how OsRACK1B delays chlorophyll breakdown through regulation of Stay‐Green (SGR) protein and Rboh/NADPH‐Oxidase‐dependent H2O2 signaling to cope with high salinity stress.OsRACK1B localizes in the nucleus and the cytoplasm in rice. During abiotic stresses such as high salinity, OsRACK1B directly or indirectly represses the transcripts of chlorophyll catabolic genes and activates defense‐related genes in the nucleus. After synthesized in the cytosol, OsRACK1B shuttles to the chloroplast where it binds to the Stay‐Green (SGR) protein and limits the availability of SGR to form SGR‐CCE‐LHCII multi‐protein complex, a prerequisite for chlorophyll breakdown. Simultaneously, OsRACK1B interacts with the N‐terminal region of Rboh/NADPH oxidase in the cytoplasm and regulates the NADPH oxidase‐dependent H2O2 production via the action of CuZn‐SOD resulting in a ROS dependent systemic wave inside the cell. At a certain level, H2O2 can act as a signaling molecule to generate a positive feedback loop for chloroplast homeostasis and may trigger retrograde signaling to regulate nuclear gene expression to protect the chloroplast and enhance stress tolerance of the plant. High salinity stress also induces stress phytohormone abscisic acid (ABA). OsRACK1B can also protect the photosystem complex through negative regulation of ABA signaling pathway. Solid and dotted lines indicate actual and hypothetical regulations of the signaling effect, respectively.Figure 1

Characterization of the pheophorbide a oxygenase/phyllobilin pathway of chlorophyll breakdown in grasses.

Although the PAO/phyllobilin pathway of chlorophyll breakdown is active in grass leaf senescence, the abundance of phyllobilins is far below the amount of degraded chlorophyll. The yellowing of fully developed leaves is the most prominent visual symptom of plant senescence. Thereby, chlorophyll is degraded via the so-called pheophorbide a oxygenase (PAO)/phyllobilin pathway to a species-specific set of phyllobilins, linear tetrapyrrolic products of chlorophyll breakdown. Here, we investigated the diversity and abundance of phyllobilins in cereal and forage crops, i.e. barley, rice, ryegrass, sorghum and wheat, using liquid chromatography-mass spectrometry. A total of thirteen phyllobilins were identified, among them four novel, not yet described ones, pointing to a rather high diversity of phyllobilin-modifying activities present in the Gramineae. Along with these phyllobilins, barley orthologs of known Arabidopsis thaliana chlorophyll catabolic enzymes were demonstrated to localize in the chloroplast, and two of them, i.e. PAO and pheophytin pheophorbide hydrolase, complemented respective Arabidopsis mutants. These data confirm functionality of the PAO/phyllobilin pathway in grasses. Interestingly, when comparing phyllobilin abundance with amounts of degraded chlorophyll in senescent leaves, in most analyzed grass species only minor fractions of chlorophyll were recovered as phyllobilins, opposite to A. thaliana where phyllobilin quantities match degraded chlorophyll rather well. These data show that, despite the presence and activity of the PAO/phyllobilin pathway in barley (and other cereals), phyllobilins do not accumulate stoichiometrically, implying possible degradation of chlorophyll beyond the phyllobilin level.

An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1

BackgroundPerennial ryegrass (Lolium perenne L.) is an important temperate grass used for turf and forage purposes. With the increasing accumulation of genomic and transcriptomic data of perennial ryegrass, an efficient protoplast and transient gene expression protocol is highly desirable for in vivo gene functional studies in its homologous system.ResultsIn this report, a highly efficient protoplast isolation (5.6 × 107 protoplasts per gram of leaf material) and transient expression (plasmid transformation efficiency at 55.2%) was developed and the detailed protocol presented. Using this protocol, the subcellular locations of two ryegrass proteins were visualized in chloroplasts and nuclei, respectively, and protein–protein interaction between two chlorophyll catabolic enzymes (LpNOL and LpNYC1) was recorded in its homologous system for the first time.ConclusionThis efficient protoplast isolation and transformation protocol is sufficient for studies on protein subcellular localization and protein–protein interaction, and shall be suitable for many other molecular biology applications where the mesophyll protoplast system is desirable in perennial ryegrass.

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.

7-Hydroxymethyl chlorophyll a reductase functions in metabolic channeling of chlorophyll breakdown intermediates during leaf senescence

During natural or dark-induced senescence, chlorophyll degradation causes leaf yellowing. Recent evidence indicates that chlorophyll catabolic enzymes (CCEs) interact with the photosynthetic apparatus; for example, five CCEs (NYC1, NOL, PPH, PAO and RCCR) interact with LHCII. STAY-GREEN (SGR) and CCEs interact with one another in senescing chloroplasts; this interaction may allow metabolic channeling of potentially phototoxic chlorophyll breakdown intermediates. 7-Hydroxymethyl chlorophyll a reductase (HCAR) also acts as a CCE, but HCAR functions during leaf senescence remain unclear. Here we show that in Arabidopsis, HCAR-overexpressing plants exhibited accelerated leaf yellowing and, conversely, hcar mutants stayed green during dark-induced senescence. Moreover, HCAR interacted with LHCII in in vivo pull-down assays, and with SGR, NYC1, NOL and RCCR in yeast two-hybrid assays, indicating that HCAR is a component of the proposed SGR-CCE-LHCII complex, which acts in chlorophyll breakdown. Notably, HCAR and NOL are expressed throughout leaf development and are drastically down-regulated during dark-induced senescence, in contrast with SGR, NYC1, PPH and PAO, which are up-regulated during dark-induced senescence. Moreover, HCAR and NOL are highly up-regulated during greening of etiolated seedlings, strongly suggesting a major role for NOL and HCAR in the chlorophyll cycle during vegetative stages, possibly in chlorophyll turnover.

Update on the biochemistry of chlorophyll breakdown

In land plants, chlorophyll is broken down to colorless linear tetrapyrroles in a highly conserved multi-step pathway. The pathway is termed the 'PAO pathway', because the opening of the chlorine macrocycle present in chlorophyll catalyzed by pheophorbide a oxygenase (PAO), the key enzyme of the pathway, provides the characteristic structural basis found in all further downstream chlorophyll breakdown products. To date, most of the biochemical steps of the PAO pathway have been elucidated and genes encoding many of the chlorophyll catabolic enzymes been identified. This review summarizes the current knowledge on the biochemistry of the PAO pathway and provides insight into recent progress made in the field that indicates that the pathway is more complex than thought in the past.

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.

Effects of postharvest ethanol vapor treatment on activities and gene expression of chlorophyll catabolic enzymes in broccoli florets

Postharvest yellowing of broccoli ( Brassica oleracea L. Italica Group) is an important indicator of quality deterioration and occurs with chlorophyll (Chl) degradation. Postharvest ethanol vapor treatment could delay the yellowing of broccoli florets, through the suppression of Chl degradation. As the first step in identifying the mechanism of this delay, the effects of postharvest ethanol vapor treatment on activities and gene expression of the Chl catabolic enzymes in broccoli were determined. Broccoli branchlets were placed in a perforated polyethylene bag with or without (control) an ethanol pad and stored at 20 °C in darkness. The Chl contents of the control broccoli florets decreased remarkably after three days in storage, whereas the contents of the ethanol-treated broccoli showed no significant changes except at day one. Changes in chlorophyllase activity in the ethanol-treated broccoli were similar to those of the control until three days in storage, but then the activity tended to decease. Mg-dechelatase and Chl-degrading peroxidase activities, which increased greatly with senescence in the controls, remained unchanged in the ethanol-treated broccoli. BoCLH1 expression showed changes almost similar to those of chlorophyllase activity. BoPAO expression in the control broccoli increased greatly at day one in storage and was maintained at a high level until three days. In contrast, this expression in ethanol-treated broccoli was suppressed until two days. BoRCCR expression in the control broccoli increased until two days in storage, while the expression in the ethanol-treated broccoli showed no change during storage. These results show that postharvest ethanol vapor treatment suppressed the activities and gene expression of Chl catabolic enzymes, resulting in delayed yellowing of broccoli florets.

Chlorophyll breakdown in senescent Arabidopsis leaves. Characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction.

During senescence, chlorophyll (chl) is metabolized to colorless nonfluorescent chl catabolites (NCCs). A central reaction of the breakdown pathway is the ring cleavage of pheophorbide (pheide) a to a primary fluorescent chl catabolite. Two enzymes catalyze this reaction, pheide a oxygenase (PAO) and red chl catabolite reductase. Five NCCs and three fluorescent chl catabolites (FCCs) accumulated during dark-induced chl breakdown in Arabidopsis (Arabidopsis thaliana). Three of these NCCs and one FCC (primary fluorescent chl catabolite-1) were identical to known catabolites from canola (Brassica napus). The presence in Arabidopsis of two modified FCCs supports the hypothesis that modifications, as present in NCCs, occur at the level of FCC. Chl degradation in Arabidopsis correlated with the accumulation of FCCs and NCCs, as well as with an increase in PAO activity. This increase was due to an up-regulation of Pao gene expression. In contrast, red chl catabolite reductase is not regulated during leaf development and senescence. A pao1 knockout mutant was identified and analyzed. The mutant showed an age- and light-dependent cell death phenotype on leaves and in flowers caused by the accumulation of photoreactive pheide a. In the dark, pao1 exhibited a stay-green phenotype. The key role of PAO in chl breakdown is discussed.

Chlorophyll Breakdown in Senescent Arabidopsis Leaves. Characterization of Chlorophyll Catabolites and of Chlorophyll Catabolic Enzymes Involved in the Degreening Reaction

Chlorophyll Breakdown in Senescent Arabidopsis Leaves. Characterization of Chlorophyll Catabolites and of Chlorophyll Catabolic Enzymes Involved in the Degreening Reaction