Pheophorbide A Oxygenase Research Articles

Functions of the lethal leaf-spot 1 gene in wheat cell death and disease tolerance to Puccinia striiformis

Pheophorbide a oxygenase (PaO) is a key enzyme in chlorophyll catabolism that is known to suppress cell death in maize and Arabidopsis. The catalytic activity of PaO in chlorophyll degradation has been clearly demonstrated, but the function of PaO in the regulation of cell death and plant–microbe interactions is largely unknown. In this study, we characterized a PaO homologue in wheat of the lethal leaf-spot 1 gene, TaLls1, that was induced in leaves infected by Puccinia striiformis f.sp. tritici (Pst) and wounding treatment. The TaLls1 protein contains a conserved Rieske [2Fe-2S] motif and a mononuclear iron-binding site typical of PaOs. Silencing of TaLls1 by virus-induced gene silencing in wheat led to leaf cell death without pathogen attacks, possibly due to the accumulation of pheophorbide a (upstream substrate of PaO), indicating a suppressor role of TaLls1, while overexpression of TaLls1 also triggered cell death in both tobacco and wheat leaves, probably owing to the accumulation of the red chlorophyll catabolite (downstream product of PaO). Further deletion mutant analysis showed that the conserved Rieske domain, but not the iron-binding site, was essential for cell death induction. These results thus suggest a threshold for TaLls1 in maintaining cell homeostasis to adapt in various stresses, and shed new light on the role of TaLls1 in cell death regulation. Furthermore, silencing of TaLls1 in wheat did not change the disease symptoms but enhanced tolerance to Pst via an significant increase in H2O2 generation, elevated cell death occurrence, and upregulation of pathogenesis-related genes.

Nitric oxide deficiency accelerates chlorophyll breakdown and stability loss of thylakoid membranes during dark-induced leaf senescence in Arabidopsis.

Nitric oxide (NO) has been known to preserve the level of chlorophyll (Chl) during leaf senescence. However, the mechanism by which NO regulates Chl breakdown remains unknown. Here we report that NO negatively regulates the activities of Chl catabolic enzymes during dark-induced leaf senescence. The transcriptional levels of the major enzyme genes involving Chl breakdown pathway except for RED CHL CATABOLITE REDUCTASE (RCCR) were dramatically up-regulated during dark-induced Chl degradation in the leaves of Arabidopsis NO-deficient mutant nos1/noa1 that exhibited an early-senescence phenotype. The activity of pheide a oxygenase (PAO) was higher in the dark-induced senescent leaves of nos1/noa1 compared with wild type. Furthermore, the knockout of PAO in nos1/noa1 background led to pheide a accumulation in the double mutant pao1 nos1/noa1, which retained the level of Chl during dark-induced leaf senescence. The accumulated pheide a in darkened leaves of pao1 nos1/noa1 was likely to inhibit the senescence-activated transcriptional levels of Chl catabolic genes as a feed-back inhibitory effect. We also found that NO deficiency led to decrease in the stability of photosynthetic complexes in thylakoid membranes. Importantly, the accumulation of pheide a caused by PAO mutations in combination with NO deficiency had a synergistic effect on the stability loss of thylakoid membrane complexes in the double mutant pao1 nos1/noa1 during dark-induced leaf senescence. Taken together, our findings have demonstrated that NO is a novel negative regulator of Chl catabolic pathway and positively functions in maintaining the stability of thylakoid membranes during leaf senescence.

Long‐term exogenous application of melatonin delays drought‐induced leaf senescence in apple

To examine the potential roles of melatonin in drought tolerance, we tested the effects of its long-term exogenous application on 'Hanfu' apple (Malus domestica Borkh.). When 100 μm melatonin was added to soils under drought conditions, the resultant oxidative stress was eased and leaf senescence was delayed. This molecule significantly reduced chlorophyll degradation and suppressed the up-regulation of senescence-associated gene 12 (SAG12) and pheophorbide a oxygenase (PAO). Such treatment also alleviated the inhibition of photosynthesis brought on by drought stress. We also investigated quenching and the efficiency of Photosystem II (PSII) photochemistry under dark and light conditions and found that melatonin helped to maintain better function of PSII under drought. The addition of melatonin also controlled the burst of hydrogen peroxide, possibly through direct scavenging and by enhancing the activities of antioxidative enzymes and the capacity of the ascorbate-glutathione cycle. Thus, understanding this effect of melatonin on drought tolerance introduces new possibilities to use this compound for agricultural purposes.

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.

Effects of 1-MCP on chlorophyll degradation pathway-associated genes expression and chloroplast ultrastructure during the peel yellowing of Chinese pear fruits in storage

The peel yellowing is an important pigment physiological process of green fruit ripening, which mainly results from chlorophyll degradation in the fruit peel. In this work, two typical cultivars with different ripening speed, a slow ripening pear ‘Emerald’ (Pyrus bretschneideri Rehd. cv. Emerald) and a fast ripening ‘Jingbai’ (Pyrus ussuriensis Maxim. cv. Jingbai) were used to investigate the molecular mechanism of chlorophyll degradation in pear yellowing/ripening during postharvest storage. The fruits after harvest were treated with 1-methylcyclopropene (1-MCP), an ethylene action inhibitor at 1.0μLl−1 to determine its effect on chloroplast ultrastructure and the expression of chlorophyll degradation associated genes in peel tissues. Our results show that the pears treated with 1-MCP had a lower ethylene production rate and higher chlorophyll content compared to those of untreated fruit. The more intact chloroplasts with well-organised grana thylakoids and small plastoglobuli were maintained in the peel of 1-MCP treated fruit for up to 30 and 15d in ‘Emerald’ and ‘Jingbai’, respectively. The expression of chlorophyll degradation associated genes: pheophorbide a oxygenase (PAO), non-yellow colouring (NYC), NYC1-like (NOL), stay-green 1(SGR1), was suppressed, while no significant change was found in chlorophyllase 1 (CHL1) and red chlorophyll catabolite reductase (RCCR) in both cultivar fruits treated with 1-MCP. These results suggest that 1-MCP can delay chlorophyll degradation by inhibiting ethylene production and suppressing the gene expression of PAO, NYC, NOL and SGR1, which are closely associated with chlorophyll catabolic pathway.

Cloning and Expression Analysis of Wheat Pheophorbide a Oxygenase Gene TaPaO

Pheophorbide a oxygenase (PaO) plays an important role in plant chlorophyll degradation. In this study, a novel gene, designated as TaPaO, was isolated from hexaploid wheat (Triticum aestivum). TaPaO was predicted to encode a protein with 523 amino acids. The encoded protein contained the typical Rieske (2Fe-2S) and mononuclear iron-binding domains as well as the C-terminus CxxC motif. Amino acid sequence alignment revealed that TaPaO shared a high degree of identity with other higher plant PaO proteins. The result of expression analysis indicated that TaPaO was expressed in roots, stems and leaves, and the expression level of TaPaO was obviously higher in leaves than in stems and roots. Quantitative real-time PCR showed that the expression of TaPaO was upregulated by abscisic acid (ABA) and methyl jasmonate (MeJA), but not by salicylic acid (SA) and gibberellic acid (GA3). Also, TaPaO could be induced by wounding, low temperature, and high salinity. Furthermore, the transcripts of TaPaO increased after infection with Puccinia recondita f. sp. tritici. However, the expression of TaPaO was not affected obviously after infection with Puccinia striiformis f. sp. tritici. These results suggest that TaPaO may be involved in plant defense responses to various stresses.

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.

Delayed senescence of apple leaves by exogenous melatonin treatment: toward regulating the ascorbate–glutathione cycle

The objectives of this study were to test the effects of exogenous melatonin on apple (Malus domestica Borkh. cv. Golden Delicious) leaves and investigate its possible physiological role in delaying leaf senescence. Detached leaves treated with 10 mm melatonin solutions clearly showed a slowing in their process of dark-induced senescence, as evidenced by both biochemical and molecular parameters. Melatonin delayed the normal reduction in chlorophyll content and maximum potential photosystem II efficiency (F(v) /F(m) ). It also suppressed the transcript levels of a key chlorophyll degradation gene, pheide a oxygenase (PAO), and the senescence-associated gene 12 (SAG12). This outcome was thought to be because of the enhanced antioxidant capabilities of melatonin. Indeed, H(2) O(2) accumulation was inhibited by exogenous melatonin, which might have resulted from direct reactive oxygen species scavenging by melatonin and a great enhancement of ascorbate peroxidase (APX; EC 1.11.1.11), which acted on both mRNA and protein activity levels. Melatonin treatment led to the maintenance of higher contents of ascorbic acid (AsA) and glutathione (GSH) but less dehydroascorbate (DHA) and oxidized glutathione (GSSG) compared with the control, possibly through its regulation of the AsA-GSH cycle.

Knockdown of Os PAO and Os RCCR1 cause different plant death phenotypes in rice

Pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR) catalyze key steps in chlorophyll degradation by opening the porphyrin macrocycle of pheophorbide a and forming the primary non-photoreactive fluorescent chlorophyll catabolite. These genes strongly participate in senescence and reportedly involved in plants’ responses to physical wounding and pathogens. In this report, a single PAO gene ( OsPAO) and two RCCR genes ( OsRCCR1 and OsRCCR2) have been isolated from rice. Expression analysis by semi-quantitative PCR or quantitative real-time PCR showed that OsRCCR1 transcripts were much more abundant than OsRCCR2, and all of these genes were upregulated during senescence and following wound treatment. RNA interference knockdown of OsPAO led to pheophorbide a accumulation in leaves (especially dark-induced senescent leaves) and leaf death from regeneration stage onwards, even transgenic plants inviability after transplantation. While, knockdown of OsRCCR1 resulted in lesion-mimic spots generation in older leaves which died off early in the transgenic plants. These results suggest that OsPAO and OsRCCR1 play key roles in senescence and are involved in wound responses.

Effects of ethylene, cytokinin and physical treatments on BoPaO gene expression of harvested broccoli

Broccoli is a highly perishable vegetable that shows enhanced postharvest senescence and intense de-greening caused by chlorophyll degradation. One of the key steps of chlorophyll catabolism is the opening of chlorophyll tretrapyrrole catalysed by pheophorbide a oxygenase (PaO). In this study the expression of a gene encoding a putative PaO was characterised under several chemical and physical treatments. A fragment of a gene encoding a PaO from broccoli (BoPaO) was cloned. The expression of BoPaO showed an important increment during postharvest senescence, in correlation with chlorophyll degradation. Furthermore, broccoli heads were treated with the hormones cytokinin and ethylene. Cytokinin delayed the increment in BoPaO expression, while ethylene accelerated the process. Also, several postharvest treatments were applied in order to evaluate their effect on BoPaO expression. Samples treated with modified atmosphere, hot air, UV-C or white light showed a delay in chlorophyll degradation and de-greening. In most cases the treatments also delayed the increment in BoPaO expression during senescence. A close correlation between chlorophyll degradation and BoPaO expression was found during broccoli senescence. This relationship was corroborated in samples treated with different hormonal and physical applications.

Accumulation of chlorophyll catabolites photosensitizes the hypersensitive response elicited by Pseudomonas syringae in Arabidopsis

• The staygreen (SGR) gene encodes a chloroplast-targeted protein which promotes chlorophyll degradation via disruption of light-harvesting complexes (LHCs). • Over-expression of SGR in Arabidopsis (SGR-OX) in a Columbia-0 (Col-0) background caused spontaneous necrotic flecking. To relate this to the hypersensitive response (HR), Col-0, SGR-OX and RNAi SGR (SGRi) lines were challenged with Pseudomonas syringae pv tomato (Pst) encoding the avirulence gene avrRpm1. Increased and decreased SGR expression, respectively, accelerated and suppressed the kinetics of HR-cell death. In Col-0, SGR transcript increased at 6 h after inoculation (hai) when tissue electrolyte leakage indicated the initiation of cell death. • Excitation of the chlorophyll catabolite pheophorbide (Pheide) leads to the formation of toxic singlet oxygen ((1)O(2)). Pheide was first detected at 6 hai with Pst avrRpm1 and was linked to (1)O(2) generation and correlated with reduced Pheide a oxygenase (PaO) protein concentrations. The maximum quantum efficiency of photosystem II (F(v)/F(m)), quantum yield of electron transfer at photosystem II (φPSII), and photochemical quenching (qP) decreased at 6 hai in Col-0 but not in SGRi. Disruption of photosynthetic electron flow will cause light-dependent H(2)O(2) generation at 6 hai. • We conclude that disruption of LHCs, possibly influenced by SGR, and absence of PaO produce phototoxic chlorophyll catabolites and oxidative stress leading to the HR.

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.

Stay-green protein, defective in Mendel's green cotyledon mutant, acts independent and upstream of pheophorbide a 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.

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.

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.

The role of pheophorbide a oxygenase expression and activity in the canola green seed problem.

Under normal field growth conditions, canola (Brassica napus) seeds produce chloroplasts during early seed development and then catabolize the photosynthetic machinery during seed maturation, producing mature seeds at harvest that are essentially free of chlorophyll (Chl). However, frost exposure early in canola seed development disrupts the normal programming of Chl degradation, resulting in green seed at harvest and thereby significantly devaluing the crop. Pheophorbide a oxygenase (PaO), a key control point in the overall regulation of Chl degradation, was affected by freezing. Pheophorbide a, the substrate of PaO, accumulated during late stages of maturation in seeds that had been exposed to freezing during early seed development. Freezing interfered with the induction of PaO activity that normally occurs in the later phases of canola seed development when Chl should be cleared from the seed. Moreover, we found that the induction of PaO activity in canola seed was largely posttranslationally controlled and it was at this level that freezing interfered with PaO activation. The increased accumulation of PaO transcript and protein levels during seed development was not altered by the freezing episode, and the increase in PaO protein was small compared to the increase in PaO activity. We found that PaO could be phosphorylated and that phosphorylation decreased with increasing activity, implicating PaO dephosphorylation as an important posttranslational control mechanism for this enzyme. Two PaO genes, BnPaO1 and BnPaO2, were identified in senescing canola leaves and during early seed development, but only BnPaO2 was expressed in maturing, degreening seeds.

Regulation of Color Break in Citrus Fruits. Changes in Pigment Profiling and Gene Expression Induced by Gibberellins and Nitrate, Two Ripening Retardants

Citrus clementina fruits were repeatedly treated on-tree from mature green until breaker stages with either nitrate or gibberellin, two retardants of external ripening. The natural color break was characterized by a reduction in chlorophyll concentration, a decrease in beta,epsilon-carotenoids, beta-carotene, neoxanthin, and all-E-violaxanthin, and an increase in beta,beta-xanthophylls [mainly (9Z)-violaxanthin and beta-cryptoxanthin]. The two retardants delayed both chlorophyll depletion and total carotenoid accumulation and in addition altered carotenoid composition. Treated fruits maintained longer the typical carotenoid composition of green fruits and reduced beta,beta-xanthophyll accumulation. Natural degreening was accompanied by a marked decrease in transcript levels of 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and geranylgeranyl reductase (CHL P) while, conversely, pheophorbide a oxygenase (PaO) and phytoene synthase (PSY) gene expression increased. Gibberellin and nitrate delayed the reduction of DXS expression and the induction of PaO and PSY transcript accumulation, while no differences in CHL P were observed. The data indicate that both ripening retardants repressed natural PaO and PSY expression, suggesting a mechanistic basis for the elevated levels of chlorophyll and lower carotenoid concentration resulting from the gibberellin and nitrogen treatments and the consequent color break delay in citrus fruit peels.

CHLOROPHYLL DEGRADATION DURING SENESCENCE

The catabolic pathway of chlorophyll (Chl) during senescence and fruit ripening leads to the accumulation of colorless breakdown products (NCCs). This review updates an earlier review on Chl breakdown published here in 1999 ( 69 ). It summarizes recent advances in the biochemical reactions of the pathway and describes the characterization of new NCCs and their formation inside the vacuole. Furthermore, I focus on the recent molecular identification of three chl catabolic enzymes, chlorophyllase, pheophorbide a oxygenase (PAO), and red Chl catabolite reductase (RCCR). The analysis of Chl catabolic mutants demonstrates the importance of Chl breakdown for plant development and survival. Mutants defective in PAO or RCCR develop a lesion mimic phenotype, due to the accumulation of breakdown intermediates. Thus, Chl breakdown is a prerequisite to detoxify the potentially phototoxic pigment within the vacuoles in order to permit the remobilization of nitrogen from Chl-binding proteins to proceed during senescence.

Cloning and expressional characterization of soybean GmLls1 gene

Maize Lls1 (lethal leaf-spot 1) gene and its Arabidopsis orthologue AtAcd1 have been suggested to encode pheide a oxygenase (PaO), a key enzyme catalyzing chlorophyll breakdown. To further elucidate the molecular mechanism that regulates chlorophyll catabolism during soybean leaf senescence, a soybean Lls1 homolog was cloned and designated as GmLls1 (GenBank Accession No. DQ154009). Database searches using the deduced protein sequence revealed that it was highly homologous to Lls1 genes or Lls1 orthologues in Arabidopsis, maize, cowpea and tomato. Structural analysis of the predicted GmLLS1 protein revealed typical Rieske [2Fe-2S] and mononuclear iron-binding domains as well as the C-terminus CxxC motif that were conserved in and featured PaO homologues. RT-PCR results showed that the transcription of GmLls1 was up-regulated in all the three tested senescence systems: the natural leaf senescence process, the dark-induced primary leaf senescence and senescing cotyledons. We have previously described the involvement of an LRR receptor-like kinase (RLK), RLPK2 in regulation of soybean leaf senescence. Here we report that the expression of GmLls1 gene was dramatically down-regulated by the RNAi-mediated suppression of rlpk2 and, as expected, greatly up regulated by the CaMV 35S promoter derived overexpression of this RLK gene. These results suggested that the expression of GmLls1 was controlled by the RLPK2-mediated senescence signaling pathway. The observation that the detached rlpk2-RNAi transgenic leaves exhibited light-dependent necrotic lesions, which featured the lls1 mutation, further supported this hypothesis.

Chlorophyll Catabolism Pathway in Fruits of Capsicum annuum (L.): Stay-Green versus Red Fruits

The aim of the present study is to investigate the chlorophyll catabolism pathway of wild-type red and stay-green mutants of Capiscum annuum (L.) fruits. In the wild-type red lines chlorophyll catabolism is concomitant with the start of carotenogenesis, whereas in the stay-green mutant lines the chlorophylls coexist with that process, even in over-ripe fruit. During the first stages of ripening, the chlorophyll a/chlorophyll b ratio is similar for both genotypes, but as ripening proceeds, the ratio in the stay-green lines becomes very high as a result of a blocked degradation of chlorophyll a while chlorophyll b is degraded at a normal rate. The absence of dephytylated chlorophylls in the wild-type lines distinguishes these from the mutant lines, in which there is a sequential accumulation of chlorophyllide a and pheophorbide a. Allomerized chlorophylls (13(2)-OH-chlorophyll a and b) have also been identified in the catabolic process of the mutant lines, but are absent from the wild type. Consequently, an alteration in pheophorbide a oxygenase (PaO) activity seems to be responsible for the stay-green genotype in the lines of pepper analyzed in this study.