AtCLH2, a Typical but Possibly Distinctive Chlorophyllase Gene in Arabidopsis

Chlorophyllase （EC 3.1.1.14） is involved in the first step of chlorophyll degradation. Isolation of chlorophyllase genes greatly facilitates characterization of chlorophyllase properties and elucidation of molecular regulation of their in vivo activities. There are two chlorophyllase genes, AtCLH1 and AtCLH2, in Arabidopsis thaliana. The in vivo roles of AtCLH1 have been reported previously. However, few studies have been carried out on AtCLH2. Here, we show that purified recombinant Chlase2, encoded by AtCLH2, exhibits in vitro chlorophyllase activity. Interestingly, ＂activation＂ of in vitro activity of the recombinant Chlase2 required higher concentrations of a detergent or a polar solvent. To determine its activity in vivo, the expression of AtCLH2 was inhibited by RNA interference. RNAi plants showed decreased contents of chlorophyllide without a substantial change in the total amount of the extractable chlorophyll and consequently presented lower chlorophyllide to chlorophyll ratios in their leaves. In addition, the two AtCLHs exhibited differential expression patterns. Our results suggest that AtCLH2 might play a distinctive role in chlorophyll catabolism in vivo.     

AtCLH2, a Typical but Possibly Distinctive Chiorophyllase Gene in Arabidopsis

Chlorophyllase (EC 3.1.1.14) is involved in the first step of chlorophyll degradation. Isolation of chlorophyllase genes greatly facilitates characterization of chlorophyllase properties and elucidation of molecular regulation of their in vivo activities. There are two chlorophyllase genes, AtCLH1 and AtCLH2, in Arabidopsis thallana. The in vivo roles of AtCLH1 have been reported previously. However, few studies have been carried out on AtCLH2. Here,we show that purified recombinant Chlase2, encoded by AtCLH2, exhibits in vitro chlorophyllase activity. Interestingly,"activation" of in vitro activity of the recombinant Chlase2 required higher concentrations of a detergent or a polar solvent. To determine its activity in vivo, the expression of AtCLH2 was inhibited by RNA interference. RNAi plants showed decreased contents of chlorophyllide without a substantial change in the total amount of the extractable chlorophyll and consequently presented lower chlorophyllide to chlorophyll ratios in their leaves. In addition, the two AtCLHs exhibited differential expression patterns. Our results suggest that AtCLH2 might play a distinctive role in chlorophyll catabolism in vivo.     

Chlorophyllase versus pheophytinase as candidates for chlorophyll dephytilation during senescence of broccoli

Degradation of chlorophylls during senescence is a highly regulated process which requires the concerted action of several enzymes. Traditionally, it has been stated that the dismantling process of the chlorophyll molecule begins with a dephytilation step, followed by Mg²⁺ removal and other breakdown reactions. Recently, new evidence suggests the possibility of a rearrangement in the first two steps of this process, occurring Mg²⁺ removal prior to the loss of the phytol side chain. With the purpose of approximating to the real sequential order of these reactions and to assess if dephytilation occurs on intact (catalyzed by chlorophyllase) or Mg-free (catalyzed by pheophytinase) chlorophyll, expression of both genes was analyzed in broccoli tissue during senescence. Samples of broccoli florets treated with plant hormones, such as cytokinin and ethylene were utilized, as to assess the effect of such compounds on the expression of these genes. Results showed that chlorophyllase expression did not correlate to typical expression patterns for genes related to senescence, since a decrease in expression during senescence was found for one of the two chlorophyllase genes analyzed, and the hormonal-treatment effects on gene expression did not match those observed on chlorophyll content for both chlorophyllase genes. Pheophytinase expression patterns, on the other hand, displayed an increase in the first 3 days of induced senescence, followed by lower expression values towards the end of the experiment. Samples subjected to postharvest treatments mostly showed an inhibition of pheophytinase expression, especially in samples in which degradation of chlorophylls had been delayed. These results suggest that pheophytinase expression correlates to the visual manifestation of postharvest treatments, supporting the possibility that this enzyme is responsible for the dephytilation step in chlorophyll breakdown.     

Plant degreening: evolution and expression of tomato (Solanum lycopersicum) dephytylation enzymes

Chlorophyll is the most abundant pigment on earth and even though it is known that its high photo-excitability necessitates a tight regulation of its degradation pathway, to date there are still several steps in chlorophyll breakdown that remain obscure. In order to better understand the ‘degreening’ processes that accompany leaf senescence and fruit ripening, we characterized the enzyme-encoding genes involved in dephytylation from tomato (Solanum lycopersicum). A single pheophytinase (PPH) gene and four chlorophyllase (CLH) genes were identified in the tomato genome. A phenetic analysis revealed two groups of CLHs in eudicot species and further evolutionary analysis indicated that these enzymes are under diverse selection pressures. A comprehensive expression profile analysis also suggested functional specificity for these dephytylating enzymes. The integrated analysis allows us to propose three general roles for chlorophyll dephytylation: i) PPH, which is under high selective constraint, is responsible for chlorophyll degradation during developmentally programed physiological processes; ii) Group I CLHs, which are under relaxed selection constraint, respond to environmental and hormonal stimuli and play a role in plant adaptation plasticity; and iii) Group II CLHs, which are also under high selective constraint, are mostly involved in chlorophyll recycling.     

Pheophytin Pheophorbide Hydrolase (Pheophytinase) Is Involved in Chlorophyll Breakdown during Leaf Senescence in Arabidopsis

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll → pheophytin → pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.     

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.     

Chlorophyll breakdown in oilseed rape

Chlorophyll catabolism accompanying leaf senescence is one of the most spectacular natural phenomena. Despite this fact, the metabolism of chlorophyll has been largely neglegted until recently. Oilseed rape has been used extensively as a model plant for the recent elucidating of structures of chlorophyll catabolites and for investigation of the enzymic reactions of the chlorophyll breakdown pathway. The key reaction which causes loss of green color is catalyzed in a two-step reaction by pheophorbide a oxygenase and red chlorophyll catabolite reductase. In this Minireview, we summarize the actual knowledge about catabolites and enzymes of chlorophyll catabolism in oilseed rape and discuss the significance of this pathway in respect to chlorophyll degradation during Brassica napus seed development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Crystal Structures of the Substrate-Bound Forms of Red Chlorophyll Catabolite Reductase: Implications for Site-Specific and Stereospecific Reaction

Red chlorophyll catabolite reductase (RCCR) catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite (RCC), the catabolic intermediate produced in chlorophyll degradation. The crystal structure of substrate-free Arabidopsis thaliana RCCR (AtRCCR) demonstrated that RCCR folds into a characteristic α/β/α sandwich, similar to that observed in the ferredoxin-dependent bilin reductase (FDBR) family. Here we have determined the crystal structures of RCC-bound AtRCCR, RCC-bound F218V AtRCCR, and substrate-free F218V AtRCCR, a mutant protein that produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position. RCC is bound to the pocket between the β-sheet and the C-terminal α-helices, as seen in substrate-bound FDBRs, but RCC binding to RCCR is much looser than substrate binding to FDBRs. The loose binding seems beneficial to the large conformational change in RCC upon reduction. Two conserved acidic residues, Glu154 and Asp291, sandwich the C20/C1 double bond of RCC, suggesting that these two residues are involved in site-specific reduction. The RCC in F218V AtRCCR rotates slightly compared with that in wild type to fill in the space generated by the substitution of Phe218 with valine. Concomitantly, the two carboxy groups of Glu154 and Asp291 move slightly away from the C20/C1 double bond. The geometrical arrangement of RCC and the carboxy groups of Glu154 and Asp291 in RCCR would appear to be essential for the stereospecificity of the RCCR reaction.     

Crystal Structure of Red Chlorophyll Catabolite Reductase: Enlargement of the Ferredoxin-Dependent Bilin Reductase Family

The key steps in the degradation pathway of chlorophylls are the ring-opening reaction catalyzed by pheophorbide a oxygenase and sequential reduction by red chlorophyll catabolite reductase (RCCR). During these steps, chlorophyll catabolites lose their color and phototoxicity. RCCR catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite. RCCR appears to be evolutionarily related to the ferredoxin-dependent bilin reductase (FDBR) family, which synthesizes a variety of phytobilin pigments, on the basis of sequence similarity, ferredoxin dependency, and the common tetrapyrrole skeleton of their substrates. The evidence, however, is not robust; the identity between RCCR and FDBR HY2 from Arabidopsis thaliana is only 15%, and the oligomeric states of these enzymes are different. Here, we report the crystal structure of A. thaliana RCCR at 2.4 Å resolution. RCCR forms a homodimer, in which each subunit folds in an α/β/α sandwich. The tertiary structure of RCCR is similar to those of FDBRs, strongly supporting that these enzymes evolved from a common ancestor. The two subunits are related by noncrystallographic 2-fold symmetry in which the α-helices near the edge of the β-sheet unique in RCCR participate in intersubunit interaction. The putative RCC-binding site, which was derived by superimposing RCCR onto biliverdin-bound forms of FDBRs, forms an open pocket surrounded by conserved residues among RCCRs. Glu154 and Asp291 of A. thaliana RCCR, which stand opposite each other in the pocket, likely are involved in substrate binding and/or catalysis.     

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.     

Ribonuclease and Chlorophyllase Activities in Senescing Leaves

The activities of two enzymes, ribonuclease and chlorophyllase were investigated during the senescence of leaves. Ribonuclease activities were measured in primary leaves of Phaseolus vulgaris, and related to the levels of nucleic acid, protein and chlorophyll. Similarly, changes in chlorophyllase activity during senescence of leaves of Raphanus sativus were measured and related to chlorophyll. During senescence the levels of each enzyme as well as its respective substrate declined. Retardation of senescence, by excision of young tissue from intact plants or by treatment of detached leaves with cytokinins resulted in a maintainace of both the substrate and enzyme levels. It was concluded that high levels of ribonuclease and chlorophyllase activity are not linked directly with the degradation of RNA and chlorophyll during leaf senescence.     

Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L

The activities of chlorophyllase, contents of pigments including chlorophyll a and b, chlorophyllide a and b, and phaeophorbide a during leaf senescence under low oxygen (0.5% O2) and control (air) were investigated in a non-yellowing mutant and wild-type leaves of snap beans (Phaseolus vulgaris L.). Chlorophyllase from leaf tissues had maximum activity when incubated at 40C in a mixture containing 50% acetone. In both mutant and wild type, chlorophyllase activity was the highest in freshly harvested non-senescent leaves and decreased sharply in the course of senescence, indicating that the loss of chlorophylls in senescing leaves is not directly related to the activity of chlorophyllase and that chlorophyllase activity is not altered in the mutant. The wild type had higher ratios of chlorophyll a to chlorophyll b than the mutant and chlorophyll a : b ratios increased during senescence in both types. In the senescent mutant leaves, accumulations of chlorophyllide a and chlorophyllide b were detected, but no phaeophorbide a was found. Chlorophyllide b had a greater accumulation than chlorophyllide a in the early stage of senescence. Low oxygen treatment not only delayed chlorophyll degradation but also enhanced the accumulations of chlorophyllide a and b and lowered the ratios of chlorophyll a to chlorophyll b.     

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.     

Mechanistic analysis of wheat chlorophyllase

Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripening. Here, we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide a detailed mechanistic analysis of the enzyme. Purification of recombinant chlorophyllase from an Escherichia coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (k(cat) = 566 min(-1); K(m) = 63 microM) and was active over a broad temperature range (10-75 degrees C). In addition, the enzyme displays carboxylesterase activity toward p-nitrophenyl (PNP)-butyrate, PNP-decanoate, and PNP-palmitate. The pH-dependence of the reaction showed the involvement of an active site residue with a pK(a) of approximately 6.5 for both k(cat) and k(cat)/K(m) with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5 to 1.9 and from 1.4 to 1.9 on k(cat) and k(cat)/K(m), respectively, were observed. Proton inventory experiments suggest the transfer of a single proton in the rate-limiting step. Our analysis of wheat chlorophyllase indicates that the enzyme uses a charge-relay mechanism similar to other carboxylesterases for catalysis. Understanding the activity and mechanism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.     

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.     

Endogenous cytokinin in developing kiwifruit is implicated in maintaining fruit flesh chlorophyll levels

Background and Aims

Green kiwifruit (Actinidia deliciosa) retain high concentrations of chlorophyll in the fruit flesh, whereas in gold-fleshed kiwifruit (A. chinensis) chlorophyll is degraded to colourless catabolites during fruit development, leaving yellow carotenoids visible. The plant hormone group the cytokinins has been implicated in the delay of senescence, and so the aim of this work was to investigate the link between cytokinin levels in ripening fruit and chlorophyll de-greening.

Methods

The expression of genes related to cytokinin metabolism and signal transduction and the concentration of cytokinin metabolites were measured. The regulation of gene expression was assayed using transient activation of the promoter of STAY-GREEN2 (SGR2) by cytokinin response regulators.

Key Results

While the total amount of cytokinin increased in fruit of both species during maturation and ripening, a high level of expression of two cytokinin biosynthetic gene family members, adenylate isopentenyltransferases, was only detected in green kiwifruit fruit during ripening. Additionally, high levels of O-glucosylated cytokinins were detected only in green kiwifruit, as was the expression of the gene for zeatin O-glucosyltransferase, the enzyme responsible for glucosylating cytokinin into a storage form. Season to season variation in gene expression was seen, and some de-greening of the green kiwifruit fruit occurred in the second season, suggesting environmental effects on the chlorophyll degradation pathway. Two cytokinin-related response regulators, RRA17 and RRB120, showed activity against the promoter of kiwifruit SGR2.

Conclusions

The results show that in kiwifruit, levels of cytokinin increase markedly during fruit ripening, and that cytokinin metabolism is differentially regulated in the fruit of the green and gold species. However, the causal factor(s) associated with the maintenance or loss of chlorophyll in kiwifruit during ripening remains obscure.