Involvement of Pheophytinase in Ethylene-Mediated Chlorophyll Degradation in the Peel of Harvested ‘Yali’ Pear

It was recently proposed that pheophytinase (PPH) is a key protein that mediates chlorophyll (Chl) breakdown in leaves. To study the role and regulation of PPH on Chl breakdown of peel in harvested ‘Yali’ pear (Pyrus bretschneideri Rehd. cv. ‘Yali’) fruit, the partial sequence of PbPPH was obtained from the NCBI database, and the alignment results revealed that the amino acid sequence of PbPPH shared great similarity to PPHs of Chinese flowering cabbage (Brassica rapa var. parachinensis) and Arabidopsis (Arabidopsis thaliana), indicating that these proteins have similar functions. Ethephon treatment significantly increased ethylene production of pear fruit and accelerated the proceeding of Chl breakdown. Conversely, 1-methylcyclopropene (1-MCP) treatment decreased ethylene production and delayed Chl breakdown. PbPPH expression was closely related to the process of Chl breakdown and was correlated with the expression of Chl degradation-associated genes such as pheide a oxygenase and stay-green 1. The chlorophyllase 1 expression level was well maintained by 1-MCP treatment, whereas red Chl catabolite reductase expression was inhibited by 1-MCP. Further analysis indicated that the gene expression levels of four ethylene receptors were stimulated by ethephon and suppressed by 1-MCP treatment and that these changes were strongly correlated with Chl breakdown and similar to the expression pattern of PbPPH. These results suggest that PPH is one of the enzymes responsible for the ethylene-mediated Chl degradation pathway of peel in harvested ‘Yali’ pear.     

Water deficit induces chlorophyll degradation via the ‘PAO/phyllobilin’ pathway in leaves of homoio‐ (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants

Angiosperm resurrection plants exhibit poikilo‐ or homoiochlorophylly as a response to water deficit. Both strategies are generally considered as effective mechanisms to reduce oxidative stress associated with photosynthetic activity under water deficiency. The mechanism of water deficit‐induced chlorophyll (Chl) degradation in resurrection plants is unknown but has previously been suggested to occur as a result of non‐enzymatic photooxidation. We investigated Chl degradation during dehydration in both poikilochlorophyllous (Xerophyta viscosa) and homoiochlorophyllous (Craterostigma pumilum) species. We demonstrate an increase in the abundance of PHEOPHORBIDE a OXYGENASE (PAO), a key enzyme of Chl breakdown, together with an accumulation of phyllobilins, that is, products of PAO‐dependent Chl breakdown, in both species. Phyllobilins and PAO levels diminished again in leaves from rehydrated plants. We conclude that water deficit‐induced poikilochlorophylly occurs via the well‐characterized PAO/phyllobilin pathway of Chl breakdown and that this mechanism also appears conserved in a resurrection species displaying homoiochlorophylly. The roles of the PAO/phyllobilin pathway during different plant developmental processes that involve Chl breakdown, such as leaf senescence and desiccation, fruit ripening and seed maturation, are discussed.     

Recent advances in chlorophyll biosynthesis and breakdown in higher plants

Chlorophyll (Chl) has unique and essential roles in photosynthetic light-harvesting and energy transduction, but its biosynthesis, accumulation and degradation is also associated with chloroplast development, photomorphogenesis and chloroplast-nuclear signaling. Biochemical analyses of the enzymatic steps paved the way to the identification of their encoding genes. Thus, important progress has been made in the recent elucidation of almost all genes involved in Chl biosynthesis and breakdown. In addition, analysis of mutants mainly in Arabidopsis, genetically engineered plants and the application of photo-reactive herbicides contributed to the genetic and regulatory characterization of the formation and breakdown of Chl. This review highlights recent progress in Chl metabolism indicating highly regulated pathways from the synthesis of precursors to Chl and its degradation to intermediates, which are not longer photochemically active.     

Involvement of Singlet Oxygen in the Breakdown of Photosynthetic Pigments in the Leaves of Rice Seedling Exposed to Osmotic Stress and Light

The combined effects of osmotic stress and light on the generation of singlet oxygen (102) and its relation to the breakdown of photosynthetic pigments in leaves of hybrid rice (Oryza sativa L. subsp, indica cv. Shanyou 63) seedlings were studied under the condition of incubating the leaves with –0.8 MPa polyethylene glycol (PEG) solution. Under osmotic stress and increasing light intensity, the production of ¹O2 monitored as p-nitrosodimethylaniline (NDA) bleaching were increased in chloroplasts, degradation of chloro- phyll (Chl) and carotenoid (Car) were accelerated and Car loss preceded Chl causing a significant increase of Chl/Car ratio. A close correlation was observed between ¹O2 production and the contents of Chl, Car and malondialdehyde (MDA). Pretreatment with scavengers for ¹O2. such as β-carotene (β-Car) and histidine (His) reduced MDA content and retarded the degradation of photosynthetic pigments in rice leaves exposed to osmotic stress of -0.8 MPa and light intensity of 250 μmol · m- 2 · s-1, in contrast to that with photosensitizer riboflavin (RF). These results indicate that ¹O2 which generated in chloroplast from photosensitized reactions involving triplet Chl may play a significant role in the breakdown of photo- synthetic pigments and the preferential destruction of Car in the leaves under combined osmotic stress with light.     

Molecular cloning and function analysis of the stay green gene in rice

Chloroplasts undergo drastic morphological and physiological changes during senescence with a visible symptom of chlorophyll (Chl) degradation. A stay green mutant was identified and then isolated from the japonica rice (Oryza sativa) cv. Huazhiwu by gamma-ray irradiation. The stay green mutant was characterized by Chl retention, stable Chl-protein complexes, and stable thylakoid membrane structures, but lost its photosynthetic competence during senescence. The gene, designated Stay Green Rice (SGR), was cloned by a positional cloning strategy encoding an ancient protein containing a putative chloroplast transit peptide. SGR protein was found in both soluble and thylakoid membranes in rice. SGR, like the gene for pheophorbide a oxygenase (PaO), was constitutively expressed, but was upregulated by dark-induced senescence in rice leaves. Senescence-induced expression of SGR and PaO was enhanced by ABA, but inhibited by cytokinin. Overexpression of SGR reduced the number of lamellae in the grana thylakoids and reduced the Chl content of normally growing leaves. This indicates that upregulation of SGR increases Chl breakdown during senescence in rice. A small quantity of chlorophyllide a accumulated in sgr leaves, but this also accumulated in wild-type rice leaves during senescence. Some pheophorbide a was detected in sgr leaves in the dark. According to these observations, we propose that SGR may be involved in regulating or taking part in the activity of PaO, and then may influence Chl breakdown and degradation of pigment-protein complex.     

Reverse Genetic Identification of CRN1 and its Distinctive Role in Chlorophyll Degradation in Arabidopsis

Recent identification of NYE1/SGR1 brought up a new era for the exploration of the regulatory mechanism of Chlorophyll (Chl) degradation.Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes sharing a similar expression pattern with NYE1.Further characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1 ((C)o-(r)egulated with (N)YE1).Chl breakdown was significantly restrained in crn1-1 under diversified senescence scenarios,which is comparable with that in acd1-20,but much more severe than that in nye1-1.Notably,various Chl binding proteins,especially trimeric LHCP Ⅱ,were markedly retained in crn1-1 four days after dark-treatment,possibly due to a lesion in disassociation of protein-pigment complex.Nevertheless,the photochemical efficiency of PSII in crn1-1 declined,even more rapidly,two days after dark-treatment,compared to those in Col-0 and nye1-1.Our results suggest that CRN1 plays a crucial role in Chl degradation,and that loss of its function produces various side-effects,including those on the breakdown of Ch-protein complex and the maintenance of the residual photosynthetic capability during leaf senescence.     

MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis

During leaf senescence, chlorophyll (Chl) is broken down to nonfluorescent chlorophyll catabolites (NCCs). These arise from intermediary fluorescent chlorophyll catabolites (FCCs) by an acid-catalyzed isomerization inside the vacuole. The chemical structures of NCCs from Arabidopsis (Arabidopsis thaliana) indicate the presence of an enzyme activity that demethylates the C13(2)-carboxymethyl group present at the isocyclic ring of Chl. Here, we identified this activity as methylesterase family member 16 (MES16; At4g16690). During senescence, mes16 leaves exhibited a strong ultraviolet-excitable fluorescence, which resulted from large amounts of different FCCs accumulating in the mutants. As confirmed by mass spectrometry, these FCCs had an intact carboxymethyl group, which slowed down their isomerization to respective NCCs. Like a homologous protein cloned from radish (Raphanus sativus) and named pheophorbidase, MES16 catalyzed the demethylation of pheophorbide, an early intermediate of Chl breakdown, in vitro, but MES16 also demethylated an FCC. To determine the in vivo substrate of MES16, we analyzed pheophorbide a oxygenase1 (pao1), which is deficient in pheophorbide catabolism and accumulates pheophorbide in the chloroplast, and a mes16pao1 double mutant. In the pao1 background, we additionally mistargeted MES16 to the chloroplast. Normally, MES16 localizes to the cytosol, as shown by analysis of a MES16-green fluorescent protein fusion. Analysis of the accumulating pigments in these lines revealed that pheophorbide is only accessible for demethylation when MES16 is targeted to the chloroplast. Together, these data demonstrate that MES16 is an integral component of Chl breakdown in Arabidopsis and specifically demethylates Chl catabolites at the level of FCCs in the cytosol.     

Alteration of Soybean Seedling Development in Darkness and Light by the Stay-green Mutation cytG and Gd1d2

The stay-green mutations cytG and Gd₁d₂ prevent the normal yellowingduring senescence of soybean (Glycine max) leaves and cotyledons.Because light plays such an important role in regulating morphogenesisand it promotes the formation of chlorophyll (Chl), we determinedthe effect of cytG and Gd₁d₂ (in a cv. Clark background) onthe development and some light responses of seedlings. AlthoughcytG and Gd₁d₂ seeds, particularly the cotyledons, are greenwhen mature, 44 and 71 % respectively of this Chl broke downwhen the seeds were germinated in darkness. Chlorophyllidesand phaeophytins were not present in the seeds in significantamounts. cytG and Gd₁d₂ as well as wild type (cv. Clark) seedlingsdeveloped a full etiolation syndrome (morphology and lack ofChl) in darkness. Light induced rapid Chl accumulation in thedark-grown seedlings with no apparent difference among the threeisolines. A short (8 h) exposure to light induced some Chl inthe cotyledons of dark-grown plants, and 22 h of light producedfour times more. Following return to darkness, the 8-h groupshowed very little breakdown over the next 12 d. After the 22-hgroup was returned to darkness, the wild-type lost Chl steadily,but Gd₁d₂ and eventually also cytG inhibited this breakdown.In the 22-h group, the Chl a/b ratio decreased in wild typeand cytG indicating preferential breakdown of Chl a relativeto Chl b; however, Gd₁d₂ prevented this change. cytG and Gd₁d₂seem to act preferentially on Chl breakdown rather than synthesis.Copyright1995, 1999 Academic Press Glycine max, soybean, chlorophyll, chlorophyll a/b ratio, cotyledons, etiolation, cytG, Gd₁d₂, mutations, senescence     

Role of chlorophyllase in chlorophyll homeostasis and post-harvest breakdown in Piper betle L. leaf

Piper betle L., a dioecious shade-loving perennial climber is one of the important Pan-Asiatic plants. More than hundred landraces having marked variation in leaf chlorophyll (Chl) content are in cultivation in India. In this study, role of chlorophyllase (Chlase) in Chl homeostasis and post-harvest breakdown was investigated in two contrasting P. betle landraces Kapoori Vellaikodi (KV) with light green and Khasi Shillong (KS) with dark green leaves. The two landraces showed negative correlation between Chl content and Chlase activity in fresh as well as stored leaves. Accumulation of chlorophyllide a (Chlid a) was correlated with the level of Chlase activity, which was higher in KV than KS. The overall response of abscisic acid (ABA) and benzylaminopurine (BAP) was similar in KV and KS, however, the time-course was different. ABA-induced Chl loss was accompanied by rise in Chlase activity in KV and KS and the delay in Chl loss by BAP was accompanied by reduction in Chlase activity. While there were significant differences in Chlase activity in KV and KS, only minor differences were observed in the enzyme properties like pH and temperature optima, Km and Vmax. No landrace-related differences were observed on the effect of metal ions and functional group reagents/amino acid effectors on Chlase activity. These results showed that despite significant differences in Chl content and Chlase activity between landraces KV and KS, the properties of Chlase were similar. The findings show that in P. betle Chlase is involved in Chl homeostasis and also in Chl degradation during post-harvest storage and responds to hormonal regulations. These findings might be useful in predicting the stability of Chl during post-harvest storage and also the shelf-life in other P. betle landraces.     

The presence of chlorophyll b in Synechocystis sp. PCC 6803 disturbs tetrapyrrole biosynthesis and enhances chlorophyll degradation

Both chlorophyll (Chl) a and b accumulate in the light in a Synechocystis sp. PCC 6803 strain that expresses higher plant genes coding for a light-harvesting complex II protein and Chl a oxygenase. This cyanobacterial strain also lacks photosystem (PS) I and cannot synthesize Chl in darkness because of the lack of chlL. When this PS I-less/chlL(-)/lhcb(+)/cao(+) strain was grown in darkness, small amounts of two unusual tetrapyrroles, protochlorophyllide (PChlide) b and pheophorbide (pheide) b, were identified. Accumulation of PChlide b trailed that of PChlide a by several days, suggesting that PChlide a is an inefficient substrate of Chl a oxygenase. The presence of pheide b in this organism suggests a breakdown of Chl b via a pathway that does not involve conversion to a-type pigments. When the PS I-less/chlL(-) control strain was grown in darkness, Chl degradation was much slower than in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain, suggesting that the presence of Chl b leads to more rapid turnover of Chl-binding proteins and/or a more active Chl degradation pathway. Levels and biosynthesis kinetics of Chl and of its biosynthetic intermediates are very different in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain versus in the control. Moreover, when grown in darkness for 14 days, upon the addition of delta-aminolevulinic acid, the level of magnesium-protoporphyrin IX increased 60-fold in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain (only approximately 2-fold in the PS I-less/chlL(-) control strain), whereas the PChlide and protoheme levels remained fairly constant. We propose that a b-type PChlide, Chl, or pheide in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain may bind to tetrapyrrole biosynthesis regulatory protein(s) (for example, the small Cab-like proteins) and thus affect the regulation of this pathway.     

Chlorosis induced by nutrient deprivation in Synechococcus sp. strain PCC 7942: not all bleaching is the same.

Cell coloration changes from normal blue-green to yellow or yellow-green when the cyanobacterium Synechococcus sp. strain PCC 7942 is deprived of an essential nutrient. We found that this bleaching process (chlorosis) in cells deprived of sulfur (S) was similar to that in cells deprived of nitrogen (N), but that cells deprived of phosphorus (P) bleached differently. Cells divided once after N deprivation, twice after S deprivation, and four times after P deprivation. Chlorophyll (Chl) accumulation stopped almost immediately upon N or S deprivation but continued for several hours after P deprivation. There was no net Chl degradation during N, S, or P deprivation, although cellular Chl content decreased because cell division continued after Chl accumulation ceased. Levels of the light-harvesting phycobiliproteins declined dramatically in a rapid response to N or S deprivation, reflecting an ordered breakdown of the phycobilisomes (PBS). In contrast, P-deprived cultures continued to accumulate PBS for several hours. Whole PBS were not extensively degraded in P-deprived cells, although the PBS contents of P-deprived cells declined because of continued cell division after PBS accumulation ceased. Levels of mRNAs encoding PBS polypeptides declined by 90 to 95% in N- or S-deprived cells and by 80 to 85% in P-deprived cells. These changes in both the synthesis and stability of PBS resulted in a 90% decline in the PC/Chl ratio of N- or S-deprived cells and a 40% decline in the PC/Chl ratio of P-deprived cells. Therefore, although bleaching appears to be a general response to nutrient deprivation, it is not the same under all nutrient-limited conditions and is probably composed of independently controlled subprocesses.     

Investigation of the Mechanism of Action of a Chlorosis-Inducing Toxin Produced by Pseudomonas phaseolicola

A toxin that induced chlorotic haloes (typifying haloblight disease) on primary leaves of Phaseolus vulgaris L. (var. Canadian Wonder) was partially purified from culture filtrates of the causative agent Pseudomonas phaseolicola (Burkh.) Dowson. This material was used to investigate chlorosis induction. Haloes could only be induced in those bean leaves that were expanding and synthesizing chlorophyll (Chl); the toxin, therefore, does not promote Chl breakdown. Chl, carotene, and xanthophyll synthesis were inhibited in sections of greening barley (Hordeum vulgare L.) leaves, irrespective of the irradiance level. In parallel experiments, the toxin decreased the level of 5-aminolevulinic acid by amounts sufficient to account for toxin-inhibition of Chl synthesis. Electron microscopy revealed no difference between the transformation of etioplasts into chloroplasts in toxin-treated and control tissue, despite a 60% reduction in Chl in the former. The incorporation of [¹⁴C]acetate into lipid by greening barley leaf sections and by isolated Pisum sativum chloroplasts in the light and the dark was inhibited about 60% by the toxin. The distribution of radioactivity among the spectra of acyl residues was the same in the control and toxin-treated material. It is suggested that the toxin interferes with an early process common to the synthesis of different lipids, including Chl.     

Partial Purification and Characterization of Red Chlorophyll Catabolite Reductase,a Stroma Protein Involved in Chlorophyll Breakdown

Red chlorophyll (Chl) catabolite (RCC) reductase, which catalyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was characterized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction depended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family.     

Chlorophyll Breakdown in Senescent Chloroplasts (Cleavage of Pheophorbide a in Two Enzymic Steps)

The cleavage of pheophorbide (Pheide) a into primary fluoescent chlorophyll (Chl) catabolites (pFCCs) in senescent chloroplasts was investigated. Chloroplast preparations isolated from senescent canola (Brassica napus) cotyledons exhibited light-dependent production of pFCC when assay mixtures were supplemented with ferredoxin (Fd). pFCC production in detergent-solubilized membranes was dependent on the presence of an Fd-reducing system. Pheide a cleavage required the action of two proteins, Pheide a oxygenase and a stroma protein. In the absence of stroma protein, Pheide a oxygenase converted Pheide a into a red Chl catabolite (RCC), the presumptive intermediary product of Pheide a cleavage. Incubation of the stroma protein (RCC reductase) together with chemically synthesized RCC resulted in the production of three different FCCs. Two of these catabolites were identical to the pFCCs from canola or barley (Hordeum vulgare) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. Thus, the conversion of Pheide a to pFCC could be demonstrated to proceed in two consecutive steps, and both reactions depended on reduced Fd as the source of electrons. The function of Fd in Chl breakdown in vivo is corroborated by the marked retention of this protein until the late stages of senescence, as demonstrated by immunoblotting.     

Identification of Catabolites of Chlorophyll-Porphyrin in Senescent Rape Cotyledons

Developing shoots of rape seedlings (Brassica napus L.) were excised and fed with 4-[14C]5-aminolevulinic acid to label the pyrroles in chlorophyll (Chl) synthesized during the final phase of expansion and greening of the cotyledons. About 80% of 14C taken up into the cotyledons was incorporated into Chl. The subsequent incubation of labeled shoots in permanent darkness caused the rapid loss of labeled Chl while increasing proportions of 14C appeared in the fraction of water-soluble compounds. Reversed-phase high performance liquid chromatography resolved three nonfluorescent polar catabolites of Chl-porphyrin that were progressively accumulated as senescence advanced. At intermediate stages of senescence, the cotyledons contained a fluorescent radio-active derivative of Chl that was also detectable, together with traces of other putative fluorescent catabolites, in isolated senescent chloroplasts. The nonfluorescent catabolites, identified by means of radiolabeling, were also found to accumulate in attached cotyledons senescing under photoperiod; under these conditions, one of the compounds, NCC-1, was particularly abundant. The catabolites of rape exhibited the same ultraviolet spectra, characterized by a maximum at 320 nm, as a previously reported secoporphinoid catabolite from barley (B. Krautler, B. Jaun, W. Amrein, K. Bortlik, M. Schellenberg, P. Matile [1992] Plant Physiol Biochem 30: 333-346). Different polarities suggest, however, that the structures may be different. A terminology for Chl catabolites is proposed because present knowledge suggests that a large number of different structures results from species-specific processing of breakdown products and may require a suitable nomenclature.     

Tetrapyrrole metabolism is involved in lesion formation, cell death, in the Arabidopsis lesion initiation 1 mutant

The Arabidopsis lesion initiation 1 (len1) mutant develops lesions on leaves without pathogen attack. The len1 plants display lesion formation as they grow under short-day conditions (SD), but not under long-day conditions (LD). This study was conducted to examine how lesion formation, viz., cell death, in len1 plants occurs under SD. I present genetic and physiological data to show that tetrapyrrole metobolism is necessary for lesion formation in len1 plants. Lesion formation was suppressed in the len1lin2 double mutant under SD. lesion initiation 2 (lin2) is another lesion mimic mutant with a defect in tetrapyrrole biosynthesis. Suppression of lesion formation in len1 plants was also observed when they were crossed with the mutants that had defects in other steps in tetrapyrrole metabolism. Suppression was correlated with reduced chlorophyll (Chl) levels in the double mutants. Furthermore, I found that dark-to-light transition caused a bleached phenotype in len1 plants, as in the case of antisense ACD1 (acd, accelerated cell death) plants. ACD1 encodes pheophorbide a oxygenase (PaO), which is involved in Chl catabolism in Arabidopsis. These results suggest that tetrapyrrole metabolism, especially Chl breakdown, might be involved in lesion formation in len1 plants.     

Effects of benzyladenine and abscisic acid on the disassembly process of photosystems in anArabidopsis delayed-senescence mutant,ore9

Using wild-type (WT) leaves and those from anore9 delayed-senescenceArabidopsis mutant, we investigated the delaying and accelerating effects of benzyladenine (BA) and abscisic acid (ABA), respectively, on the degradation process of the photosynthetic apparatus during dark-induced senescence (DIS). In the mutant, delays were seen for both the breakdown of chlorophyll (Chl) and the decrease in photochemical efficiency of photosystem II (Fv/Fm). Moreover, each step was prolonged in the disassembly process of the Chl-protein complexes. In the presence of BA, Chl degradation was retarded to a similar extent for both the mutant and the WT, but the decrease in Fv/Fm was not. However, in the presence of ABA, the two processes were accelerated in both genotypes. Therefore, although theore9 mutation causes this functional delayed-senescence, it may not be related to the non-functional delay that happens afterwards. In contrast, BA seems to affect both processes.