Chlorophyll breakdown in higher plants

Chlorophyll breakdown is an important catabolic process of leaf senescence and fruit ripening. Structure elucidation of colorless linear tetrapyrroles as (final) breakdown products of chlorophyll was crucial for the recent delineation of a chlorophyll breakdown pathway which is highly conserved in land plants. Pheophorbide a oxygenase is the key enzyme responsible for opening of the chlorin macrocycle of pheophorbide a characteristic to all further breakdown products. Degradation of chlorophyll was rationalized by the need of a senescing cell to detoxify the potentially phototoxic pigment, yet recent investigations in leaves and fruits indicate that chlorophyll catabolites could have physiological roles. This review updates structural information of chlorophyll catabolites and the biochemical reactions involved in their formation, and discusses the significance of chlorophyll breakdown. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Mechanism and Significance of Chlorophyll Breakdown

Chlorophyll breakdown is the most obvious sign of leaf senescence and fruit ripening. A multistep pathway has been elucidated in recent years that can be divided into two major parts. In the first phase, which commonly is active in higher plants, chlorophyll is converted via several photoreactive intermediates to a primary colorless breakdown product within the chloroplast. The second part of chlorophyll breakdown takes place in the cytosol and the vacuole. During this phase, the primary colorless intermediate is modified in largely species-specific reactions to a number of similar, yet structurally different, linear tetrapyrrolic products that finally are stored within the vacuole of senescing cells. To date, most of the biochemical reactions of the first phase of chlorophyll breakdown have been elucidated and genes have been identified. By contrast, mechanisms of catabolite transport and modification during the second phase are largely unknown. This review summarizes the current knowledge on the biochemical reactions involved in chlorophyll breakdown, with a special focus on the second-phase reactions and the fate of by-products that are released from chlorophyll during its breakdown.     

Unravelling chlorophyll catabolism in higher plants

Chlorophyll metabolism is probably the most visible manifestation of life. In spite of this, chlorophyll catabolism has remained something of a mystery until about 10 years ago. At that time, the first non-green tetrapyrrolic chlorophyll breakdown products from higher plants were discovered, and the structure of the first one of them was elucidated by modern spectroscopic methods. In the meantime, the essential structural features of chlorophyll catabolites and some of the biochemistry of chlorophyll breakdown in higher plants have been uncovered, as outlined in this article.     

On the Nature of Isomeric Nonfluorescent Chlorophyll Catabolites in Leaves and Fruit – A Study with a Ubiquitous Phylloleucobilin and its Main Isomerization Product

The typical main products of chlorophyll (Chl) breakdown in higher plants are non‐fluorescent, colorless phyllobilins, named phylloleucobilins. These long elusive Chl‐catabolites are linear tetrapyrroles, whose structure elucidation has required thorough spectroscopic analyses. Interestingly, in recent LC/MS studies of leaf extracts, isomeric forms of phylloleucobilins were detected. The existence of isomeric phyllobilins may suggest incomplete stereo‐selectivity of catabolic processes, or isomerization processes in plant cells or in the analytes. Here we report a study with the phylloleucobilin NCC‐1, a basic Chl‐catabolite in extracts of leaves and fruit. NCC‐1 and its main isomerization product in aqueous solution were identified as 8²‐epimers. Formation of 8²‐epi‐NCC‐1 from NCC‐1 implies an unstable enol(ate)‐intermediate, which reverts to NCC‐1 or converts to 8²‐epi‐NCC‐1. Such reversible epimerization reactions are a non‐biological in vitro feature of typical phylloleucobilins, and probably also take place in vivo.     

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.     

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.     

Citrus chlorophyllase dynamics at ethylene-induced fruit color-break: a study of chlorophyllase expression, posttranslational processing kinetics, and in situ intracellular localization

Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break. We show, using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently N-terminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.     

Light‐induced pigment degradation in leaves and ripening fruits studied in situ with reflectance spectroscopy

Pigment breakdown mediated by activated oxygen species is a consequence and a general symptom of oxidative stress and injury to plants. We have attempted to estimate the patterns of pigment bleaching and follow pigment susceptibility to irradiation as related to the process of senescence/ripening. Light‐induced pigment breakdown was studied in situ in the leaves of a shade‐requiring plant, wax flower ( Hoya carnosa R. Br.), as well as in apple ( Malus domestica Borlh. cv. Zhigulevskoe) and lemon ( Citrus limon Burm. cv. Pavlovsky) fruits, using reflectance spectroscopy. It was found that the sensitivity of plant pigments to photobleaching increases as ripening progresses in lemon fruit. Kinetic analysis showed that in all systems a rapid breakdown of the pigment occurs after a lag‐phase. The signature analysis revealed a common pattern of chlorophyll and carotenoid changes, but degradation of the individual pigments was found to be inhomogeneous. Both in lemon and apple fruits a decrease in reflectance in the band of carotenoid absorption preceded pigment photodestruction. In the fruits, the bulk of chlorophyll b and the long‐wavelength chlorophyll a forms were degraded at early stages of the process whereas the breakdown of both chlorophylls in H. carnosa leaves was more synchronous. Prolonged irradiation induced bleaching of the main chlorophyll a band with maximum at 678 nm in the difference spectra, as well as carotenoids. Some features of reflectance spectra in the bands of chlorophyll and carotenoid absorption were found to be suitable for the differentiation of photo‐induced pigment breakdown from the transformation of the pigments taking place during senescence.     

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 Catabolites in Senescent Leaves of the Plum Tree (Prunus domestica)

In cold extracts of senescent leaves of the plum tree (Prunus domestica ssp. domestica), six colorless non‐fluorescent chlorophyll catabolites (NCCs) were characterized, named Pd‐NCCs. In addition, several minor NCC fractions were tentatively classified. The structure of the most polar one of the NCCs, named Pd‐NCC‐32, featured an unprecedented twofold glycosidation pattern. Three of the NCCs are also functionalized at their 3²‐position by a glucopyranosyl group. In addition, two of these glycosidated NCCs carry a dihydroxyethyl group at their 18‐position. In the polar Pd‐NCC‐32, the latter group is further glycosidated at the terminal 18²‐position. Four other major Pd‐NCCs and one minor Pd‐NCC were identified with five NCCs from higher plants known to belong to the ‘epi’‐series. In addition, tentative structures were derived for two minor fractions, classified as yellow chlorophyll catabolites, which represented (formal) oxidation products of two of the observed Pd‐NCCs. The chlorophyll catabolites in leaves of plum feature the same basic structural pattern as those found in leaves of apple and pear trees.     

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.     

The appearance of chlorophyll derivatives in senescing tissue

Chlorophylls a-1 and b′, which are breakdown products of chlorophylls a and b respectively, were found in senescing leaves of Phaseolus vulgaris and Hordeum vulgare following excision from the plant. Chlorophyll a-1 was not detected in healthy plants, in senescing attached leaves or in chlorophyll-proteins isolated from senescent tissue. Chlorophyll a-1 formation in excised leaves increased with time for up to 10 days as chlorophyll a levels fell.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

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.     

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.     

Nitrogen metabolism and remobilization during senescence

Senescence is a highly organized and well-regulated process. As much as 75% of total cellular nitrogen may be located in mesophyll chloroplasts of C(3)-plants. Proteolysis of chloroplast proteins begins in an early phase of senescence and the liberated amino acids can be exported to growing parts of the plant (e.g. maturing fruits). Rubisco and other stromal enzymes can be degraded in isolated chloroplasts, implying the involvement of plastidial peptide hydrolases. Whether or not ATP is required and if stromal proteins are modified (e.g. by reactive oxygen species) prior to their degradation are questions still under debate. Several proteins, in particular cysteine proteases, have been demonstrated to be specifically expressed during senescence. Their contribution to the general degradation of chloroplast proteins is unclear. The accumulation in intact cells of peptide fragments and inhibitor studies suggest that multiple degradation pathways may exist for stromal proteins and that vacuolar endopeptidases might also be involved under certain conditions. The breakdown of chlorophyll-binding proteins associated with the thylakoid membrane is less well investigated. The degradation of these proteins requires the simultaneous catabolism of chlorophylls. The breakdown of chlorophylls has been elucidated during the last decade. Interestingly, nitrogen present in chlorophyll is not exported from senescencing leaves, but remains within the cells in the form of linear tetrapyrrolic catabolites that accumulate in the vacuole. The degradation pathways for chlorophylls and chloroplast proteins are partially interconnected.     

Biochemical aspects of the developing and ripening banana

The peel and pulp of the banana fruit and the pseudostem were examined for glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT) and aldolase activities and protein, phenolics, chlorophyll and starch. The peel-pulp ratio at various stages of fruit development on the plant and in detached fruits showing incipient ripening were used as an index of the physiological age of the fruit. The enzymes exhibited maximum activity at a stage corresponding to the initiation of the climacteric. GPT level at this stage was higher than that of GOT. An initial increase in the protein content was followed by a decline in both peel and pulp, the level reaching a minimum in climacteric fruits. Astringency, measured in terms of total phenolics, decreased with development; in mature fruits, peel contained 4–5 × as much phenolics as pulp. Chlorophyll in mature fruits was 10 × higher than in young fruits and decreased in ripe fruits. The onset of ripening was attended with a pronounced decrease in the starch. The various analyses were carried out also on the pseudostem removed from the plant soon after flower formation.