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.     

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.     

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.     

Involvement of a Putative Bipartite Transit Peptide in Targeting Rice Pheophorbide a Oxygenase into Chloroplasts for Chlorophyll Degradation during Leaf Senescence

Leaf senescence is one of the major factors contributing to the productivity and the grain quality in crops. The regulatory mechanism of leaf senescence remains largely unknown. Here, we report the identification and characterization of a rice early senescence 1(eas1)mutant, which displayed an early leaf senescence phenotype, accompanying by dwarfism and reduced tiller number, eventually leading to the reduction of grain yield. Map-based cloning revealed that the nuclear gene EAS1 encodes a pheophorbide a oxygenase(PaO), a key enzyme for chlorophyll breakdown. A highly conserved Thr residue of PaO was mutated into Ile in the eas1 mutant. Phylogenetic analysis indicates that PaO is an evolutionarily conserved protein, and EAS1 is 68% identical to the Arabidopsis ACCERLERATED CELL DEATH(ACD1) protein. Unlike ACD1 that contains a single transit peptide, EAS1 contains two putative transit peptides at its N-terminus, which are essential for its functionality, suggesting that targeting of EAS1 to the chloroplast is likely mediated by a putative bipartite transit peptide. Consistently, only a short version of EAS1 lacking the first putative transit peptide, but not the full-length EAS1,was capable of rescuing the Arabidopsis acd1 mutant phenotype. These results suggest that rice EAS1 represents a functional PaO, which is involved in chlorophyll degradation and may utilize a unique mechanism for its import into the chloroplast.     

The Wound-Inducible Lls1 Gene from Maize is an Orthologue of the Arabidopsis Acd1 Gene,and the LLS1 Protein is Present in Non-Photosynthetic Tissues

Previous studies indicated that the lethal leaf spot 1 lesion mimic locus of maize ( ZmLls1 ) encodes a novel cell protective function in plants. Here we show that the accelerated cell death 1 ( acd1 ) locus of Arabidopsis thaliana corresponds to gene At3g44880 on chromosome 3. Proof that the Acd1 gene is an orthologue of ZmLls1 is provided by in vivo complementation of the acd1 mutant by the ZmLls1 gene. The Atlls1 lesion mimic phenotype was delayed in a chlorophyll a oxygenase (CAO) mutant chlorina1 background which is deficient in chlorophyll b synthesis. The interpretation that the cell protective function of LLS1 is linked with the removal of a phototoxic chlorophyll intermediate is supported by the recent report that the maize Lls1 gene encodes pheophorbide a oxygenase (PaO). Western blot analysis demonstrates that the LLS1 protein is present constitutively in all photosynthetic plant tissues. A transient increase in Lls1 gene expression by about 50-fold upon physical wounding of maize leaves indicates that the function of Lls1 is regulated in response to stress. We show that the LLS1 protein is also present at low levels in non-photosynthetic tissues including etiolated leaves suggesting that the ability to degrade chlorophyll exists in a standby mode in plant cells.     

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.     

Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana

In plants, chlorophyll is actively synthesized from glutamate in the developmental phase and is degraded into non-fluorescent chlorophyll catabolites during senescence. The chlorophyll metabolism must be strictly regulated because chlorophylls and their intermediate molecules generate reactive oxygen species. Many mechanisms have been proposed for the regulation of chlorophyll synthesis including gene expression, protein stability, and feedback inhibition. However, information on the regulation of chlorophyll degradation is limited. The conversion of chlorophyll b to chlorophyll a is the first step of chlorophyll degradation. In order to understand the regulatory mechanism of this reaction, we isolated a mutant which accumulates 7-hydroxymethyl chlorophyll a (HMChl), an intermediate molecule of chlorophyll b to chlorophyll a conversion, and designated the mutant hmc1. In addition to HMChl, hmc1 accumulated pheophorbide a, a chlorophyll degradation product, when chlorophyll degradation was induced by dark incubation. These results indicate that the activities of HMChl reductase (HAR) and pheophorbide a oxygenase (PaO) are simultaneously down-regulated in this mutant. We identified a mutation in the AtNAP1 gene, which encodes a subunit of the complex for iron–sulfur cluster formation. HAR and PaO use ferredoxin as a reducing power and PaO has an iron-sulfur center; however, there were no distinct differences in the protein levels of ferredoxin and PaO between wild type and hmc1. The concerted regulation of chlorophyll degradation is discussed in relation to the function of AtNAP1.     

What stay-green mutants tell us about nitrogen remobilization in leaf senescence

Leaf senescence has an important role in the plant's nitrogen economy. Chlorophyll catabolism is a visible symptom of protein mobilization. Genetic and environmental factors that interfere with yellowing tend to modify protein degradation as well. The chlorophyll-protein relationship is much closer for membrane proteins than it is for soluble or total leaf proteins. In stay-greens, genotypes with a specific defect in the chlorophyll catabolism pathway, soluble protein degradation during senescence may be close to normal, but light-harvesting and reaction centre thylakoid membrane proteins are much more stable. Genes for the chlorophyll catabolism pathway and its control are important in the regulation of protein mobilization. Genes for three steps in the pathway are reported to have been isolated. The gene responsible for the stay-green phenotype in grasses and legumes has not yet been cloned but a fair amount is known about it. Pigment metabolism in senescing leaves of the Festuca-Lolium stay-green mutant is clearly disturbed and is consistent with a blockage at the ring-opening (PaO) step in chlorophyll breakdown. PaO is de novo synthesized in senescence and thought to be the key enzyme in the chlorophyll a catabolic pathway. The stay-green mutation is likely to be located in the PaO gene, or a specific regulator of it. These genes may well be in the various senescence-enhanced cDNA collections that have been generated, but functional handles on them are currently lacking. When the stay-green locus from Festuca pratensis was introgressed into Lolium temulentum, a gene encoding F. pratensis UDPG-pyrophosphorylase was shown to have been transferred on the same chromosome segment. A strategy is described for cloning the stay-green gene, based on subtractive PCR-based analyses of intergeneric introgressions and map-based cloning.     

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.     

Identification of a novel chloroplast protein AtNYE1 regulating chlorophyll degradation during leaf senescence in Arabidopsis

A dramatic increase of chlorophyll (Chl) degradation occurs during senescence of vegetative plant organs and fruit ripening. Although the biochemical pathway of Chl degradation has long been proposed, little is known about its regulatory mechanism. Identification of Chl degradation-disturbed mutants and subsequently isolation of responsible genes would greatly facilitate the elucidation of the regulation of Chl degradation. Here, we describe a nonyellowing mutant of Arabidopsis (Arabidopsis thaliana), nye1-1, in which 50% Chl was retained, compared to less than 10% in the wild type (Columbia-0), at the end of a 6-d dark incubation. Nevertheless, neither photosynthesis- nor senescence-associated process was significantly affected in nye1-1. Characteristically, a significant reduction in pheophorbide a oxygenase activity was detected in nye1-1. However, no detectable accumulation of either chlorophyllide a or pheophorbide a was observed. Reciprocal crossings revealed that the mutant phenotype was caused by a monogenic semidominant nuclear mutation. We have identified AtNYE1 by positional cloning. Dozens of its putative orthologs, predominantly appearing in higher plant species, were identified, some of which have been associated with Chl degradation in a few crop species. Quantitative polymerase chain reaction analysis showed that AtNYE1 was drastically induced by senescence signals. Constitutive overexpression of AtNYE1 could result in either pale-yellow true leaves or even albino seedlings. These results collectively indicate that NYE1 plays an important regulatory role in Chl degradation during senescence by modulating pheophorbide a oxygenase activity.     

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.     

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.     

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.     

Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis

The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation.     

Functional Analysis of Wheat <Emphasis Type="Italic">TaPaO1</Emphasis> Gene Conferring Pollen Sterility Under Low Temperature

Thermosensitive male sterility plays an important role in wheat fertility and production. As a key enzyme for chlorophyll degradation, pheophorbide a oxygenase (PaO) can suppress cell death in plants. We cloned the wheat gene TaPaO1 from the thermosensitive genetic male sterile (TGMS) line BS366; it encodes a typical PaO protein, containing a conserved Rieske [2Fe-2S] iron–sulphur motif, a mononuclear non-heme iron-binding motif, and a C-terminal CxxC motif. TaPaO1 was expressed in all tissues and was upregulated during the meiosis stage of BS366 anthers under low temperature. Subcellular localization of TaPaO1 specifically labelled the surrounding of chloroplasts. TaPaO1 regulated by RD29A promoter which responded to low temperature led to pollen sterility in transgenic tobacco. Expression analysis showed that TaPaO1 exhibited a higher level of expression in the anther than in other tissues in transgenic tobacco plants during low temperature treatment. We propose that the higher senescence-related activity of TaPaO1 may lead to the cell death of anthers, which happens at an early developmental stage under low temperature. These results provide new insights into the function of PaO during the early developmental stage of anthers. PaO is closely related to cell death regardless of whether it exhibits increased activity or inactive.     

Evolution of Chlorophyll Degradation: The Significance of RCC Reductase

Abstract: In angiosperms the key process of chlorophyll breakdown in senescing leaves is catalyzed by pheophorbide a oxygenase and RCC reductase which, in a metabolically channeled reaction, cleave the porphyrin macrocycle and produce a colourless primary catabolite, pFCC. RCC reductase is responsible for the reduction of the C20/C1 double bond of the intermediary catabolite, RCC. Depending on plant species, RCC reductase produces one of the two C1 stereoisomers, pFCC-1 or pFCC-2. Screening of a large number of taxa for the type of RCCR revealed that the isomer produced is uniform within families. It also revealed that type RCCR-2 is predominant; RCCR-1 seems to represent a recent derivation which in unrelated lineages has evolved independently from RCCR-2. A third type of pFCC was produced by RCCR from basal pteridophytes and some gymnosperms; its structure is unknown. Collectively, the data suggest that the pathway of chlorophyll breakdown is very conserved in vascular plants. RCCR appears to represent a decisive addition to the catabolic pathway: it allows terrestrial plants to metabolize the porphyrin part of the chlorophyll molecule to photodynamically inactive final products that are stored in the vacuoles of senescing mesophyll cells.     

Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis

The Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) protein protects cells from programmed cell death (PCD) caused by endogenous porphyrin‐related molecules like red chlorophyll catabolite or exogenous protoporphyrin IX. We previously found that during bacterial infection, ACD2, a chlorophyll breakdown enzyme, localizes to both chloroplasts and mitochondria in leaves. Additionally, acd2 cells show mitochondrial dysfunction. In plants with acd2 and ACD2 ⁺ sectors, ACD2 functions cell autonomously, implicating a pro‐death ACD2 substrate as being cell non‐autonomous in promoting the spread of PCD. ACD2 targeted solely to mitochondria can reduce the accumulation of an ACD2 substrate that originates in chloroplasts, indicating that ACD2 substrate molecules are likely to be mobile within cells. Two different light‐dependent reactive oxygen bursts in mitochondria play prominent and causal roles in the acd2 PCD phenotype. Finally, ACD2 can complement acd2 when targeted to mitochondria or chloroplasts, respectively, as long as it is catalytically active: the ability to bind substrate is not sufficient for ACD2 to function in vitro or in vivo. Together, the data suggest that ACD2 localizes dynamically during infection to protect cells from pro‐death mobile substrate molecules, some of which may originate in chloroplasts, but have major effects on mitochondria.