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.     

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.     

A novel blue fluorescent chlorophyll catabolite accumulates in senescent leaves of the peace lily and indicates a split path of chlorophyll breakdown

Colorless, non-fluorescent Chl-catabolites (NCCs) are the typical, ubiquitous products of chlorophyll (Chl)-breakdown in senescent leaves. However, a fluorescent Chl-catabolite (FCC) accumulated in de-greened leaves of Spathiphyllum wallisii (Peace Lily), which showed a weak blue luminescence. The FCC, named Sw-FCC-62, was ‘hypermodified’ with an unprecedented 6-(2-[3,4-dihydroxy-phenyl]-ethyl)-β-glucopyranosidyl ester at the propionyl group. Such esters stabilize FCCs against their typical and rapid, spontaneous isomerization to NCCs. Chl-breakdown in Sp. wallisii thus branches off from the ‘common’ path in leaves, and furnishes unique and ‘persistent’ FCCs. Our findings on ‘hypermodified’ FCCs also call for attention as to possible physiological roles of Chl-catabolites in plants.     

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.     

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.     

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.     

The chlorophyllases AtCLH1 and AtCLH2 are not essential for senescence-related chlorophyll breakdown in Arabidopsis thaliana

One important reaction of chlorophyll (chl) breakdown during plant senescence is the removal of the lipophilic phytol moiety by chlorophyllase. AtCLH1 and AtCLH2 were considered to be required for this reaction in Arabidopsis thaliana. Here we present evidence against this assumption. Using green fluorescent protein fusions, neither AtCLH isoform localizes to chloroplasts, the predicted site of chlorophyll breakdown. Furthermore, clh1 and clh2 single and double knockout lines are still able to degrade chlorophyll during senescence. From our data we conclude that AtCLHs are not required for senescence-related chlorophyll breakdown in vivo and propose that genuine chlorophyllase has not yet been molecularly identified.     

Chlorophyll breakdown in tobacco: on the structure of two nonfluorescent chlorophyll catabolites

In extracts of senescent leaves of the tobacco plant Nicotiana rustica, two colorless compounds with UV/VIS characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively identified as Nr-NCCs. These two polar NCCs were found in similar amounts in the fresh extracts, and their constitutions could be determined by spectroscopic analysis. The data showed both of the two Nr-NCCs to have the same tetrapyrrolic core structure, as reported previously for all other NCCs from senescent higher plants. In the less polar catabolite, named Nr-NCC-2, this core structure was conjugated with a glucopyranose unit, as similarly discovered earlier in Bn-NCC-2, an NCC from oilseed rape (Brassica napus). The more polar NCC from tobacco leaves, Nr-NCC-1, carried an additional malonyl substituent at the 6'-OH group of the glucopyranosyl moiety. Partial (enzyme-catalyzed) hydrolysis of Nr-NCC-1 gave Nr-NCC-2, while enzyme-catalyzed malonylation of Nr-NCC-2 gave Nr-NCC-1, establishing the identity of their basic tetrapyrrole structure. In earlier work (on the polar NCCs from oilseed rape), only separate glucopyranosyl and malonyl functionalities were detected. Nr-NCC-1, thus, represents a further variant of the structures of NCCs from senescent higher plants and exhibits an unprecedented peripheral refunctionalization in chlorophyll catabolites.     

Stay-green protein,defective in Mendel’s green cotyledon mutant,acts independent and upstream of pheophorbide <Emphasis Type="Italic">a</Emphasis> oxygenase in the chlorophyll catabolic pathway

Type C stay-green mutants are defined as being defective in the pathway of chlorophyll breakdown, which involves pheophorbide a oxygenase (PAO), required for loss of green color. By analyzing senescence parameters, such as protein degradation, expression of senescence-associated genes and loss of photosynthetic capacity, we demonstrate that JI2775, the green cotyledon (i) pea line used by Gregor Mendel to establish the law of genetics, is a true type C stay-green mutant. STAY-GREEN (SGR) had earlier been shown to map to the I locus. The defect in JI2775 is due to both reduced expression of SGR and loss of SGR protein function. Regulation of PAO through SGR had been proposed. By determining PAO protein abundance and activity, we show that PAO is unaffected in JI2775. Furthermore we show that pheophorbide a accumulation in the mutant is independent of PAO. When silencing SGR expression in Arabidopsis pao1 mutant, both pheophorbide a accumulation and cell death phenotype, typical features of pao1, are lost. These results confirm that SGR function within the chlorophyll catabolic pathway is independent and upstream of PAO. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Chlorophyll breakdown and chlorophyll catabolites in leaves and fruit

Chlorophyll metabolism probably is the most visible manifestation of life. Total annual turnover of chlorophyll has been estimated to involve more than 1000 million tons. Surprisingly, chlorophyll catabolism has remained an enigma until less than twenty years ago, when a colorless chlorophyll catabolite from senescent plant leaves was identified and its structure was elucidated. In the meantime, chlorophyll breakdown products have been identified in a variety of plant leaves and their structural features have been elucidated. Most recently, chlorophyll breakdown products have also been identified in some ripening fruit. Chlorophyll breakdown in vascular plants only fleetingly involves enzyme-bound colored intermediates. The stage of fluorescent catabolites is also passed rapidly, as these isomerize further to colorless nonfluorescent tetrapyrrolic catabolites. The latter accumulate in the vacuoles of de-greened leaves and are considered the final products of controlled chlorophyll breakdown. The same tetrapyrroles are also found in ripening fruit and are effective antioxidants. Chlorophyll breakdown leads to tetrapyrroles that appear to have physiologically beneficial chemical properties, and it may thus not merely be a detoxification process.     

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.     

Cleavage of Chlorophyll-Porphyrin (Requirement for Reduced Ferredoxin and Oxygen)

The chemical structures of some colorless catabolites that accumulate in senescent leaves have been established recently (B. Krautler, B. Jaun, W. Amrein, K. Bortlik, M. Schellenberg, P. Matile [1992] Plant Physiol Biochem 30: 333-346; W. Muhlecker, B. Krautler, S. Ginsburg, P. Matile [1993] Helv Chim Acta 76: 2976-2980). Such studies suggest that oxygenolytic cleavage of chlorophyll-porphyrin may occur by the action of a dioxygenase. We have attempted to demonstrate such an enzyme activity and to explore the requirements of the cleavage reaction in a reconstituted system of chloroplast (Chlpl) components prepared from senescent rape (Brassica napus L.) cotyledons. Intact senescent Chpls (also referred to as gerontoplasts) contain small amounts of two fluorescent chlorophyll catabolites, Bn-FCC-1 and Bn-FCC-2, probably representing primary cleavage products. Upon the incubation of Gpls in the presence of glucose-6-phosphate (Glc6P) or ATP, these catabolites (predominantly FCC-1) were produced in organello. In a reconstituted system of thylakoids and stroma fraction the FCCs (predominantly FCC-2) were produced in the presence of ferredoxin (Fd) and cofactors (NADPH, Glc6P) helping to keep Fd in the reduced state. Reduced Fd could not be replaced by other electron donors, suggesting that the putative dioxygenase requires Fd for the operation of its redox cycle. Production of FCC-2 did not occur in the absence of oxygen and it was inhibited by chelators of Fe2+. The contributions to the production of FCCs from both parts of the reconstituted system, thylakoids and stroma, are heat labile. The enzymic process in the thylakoids yields pheophorbide a, the presumptive precursor of FCCs. However, native senescent thylakoids could not be replaced as a "substrate" by free pheophorbide a. The stromal enzyme appears to have an affinity for senescent thylakoids; thus, "loaded" thylakoids capable of FCC production in the presence of Fd and cofactors were obtained upon homogenization of senescent cotyledons in a medium containing sorbitol and ascorbate. Such thylakoids were inactive if prepared from mature green cotyledons. As senescence was induced, the capacity to generate FCCs appeared and peaked when about half of the chlorophyll had disappeared from the cotyledons. The effectiveness of a relevant inhibitor showed that cytoplasmic protein synthesis was required for inducing the catabolic machinery in the loaded thylakoids. Thylakoids from mature Chlpls were ineffective as substrate of the stromal enzyme prepared from Gpls. However, senescent thylakoids yielded FCCs if challenged with stroma from either Chlpls or Gpls. Therefore, the stromal part of the system is likely to be a constitutive enzyme, and the pace-setting step of the pathway of chlorophyll breakdown seems to be located in the thylakoids.     

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.     

Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum

Pigments, proteins and enzyme activity related to chlorophyll catabolism were analysed in senescing leaves of wild-type (WT) Lolium temulentum and compared with those of an introgression line carrying a mutant gene from stay-green (SG) Festuca pratensis. During senescence of WT leaves chlorophylls a and b were continuously catabolised to colourless products and no other derivatives were observed, whereas in SG leaves there was an accumulation of dephytylated and oxidised catabolites including chlorophyllide a, phaeophorbide a and 13(2) OH-chlorophyllide a. Dephytylated products were absent from SG leaf tissue senescing under a light-dark cycle. Retention of pigments in SG was accompanied by significant stabilisation of light harvesting chlorophyll-proteins compared with WT, but soluble proteins such as Rubisco were degraded during senescence at a similar rate in the two genotypes. The activity of phaeophorbide a oxygenase measured in SG tissue at 3d was less than 12% of that in WT tissue at the same time-point during senescence and of the same order as that in young pre-senescent WT leaves, indicating that the metabolic lesion in SG concerns a deficiency at the ring-opening step of the catabolic pathway. In senescent L. temulentum tissue two terminal chlorophyll catabolites were identified with chromatographic characteristics that suggest they may represent hitherto undescribed catabolite structures. These data are discussed in relation to current understanding of the genetic and metabolic control of chlorophyll catabolism in leaf senescence.     

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.     

Cryptic chlorophyll breakdown in non-senescent green Arabidopsis thaliana leaves

Photosynthesis Research - Chlorophyll (Chl) breakdown is a diagnostic visual process of leaf senescence, which furnishes phyllobilins (PBs) by the PAO/phyllobilin pathway. As Chl breakdown disables...