Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L

The activities of chlorophyllase, contents of pigments including chlorophyll a and b, chlorophyllide a and b, and phaeophorbide a during leaf senescence under low oxygen (0.5% O2) and control (air) were investigated in a non-yellowing mutant and wild-type leaves of snap beans (Phaseolus vulgaris L.). Chlorophyllase from leaf tissues had maximum activity when incubated at 40C in a mixture containing 50% acetone. In both mutant and wild type, chlorophyllase activity was the highest in freshly harvested non-senescent leaves and decreased sharply in the course of senescence, indicating that the loss of chlorophylls in senescing leaves is not directly related to the activity of chlorophyllase and that chlorophyllase activity is not altered in the mutant. The wild type had higher ratios of chlorophyll a to chlorophyll b than the mutant and chlorophyll a : b ratios increased during senescence in both types. In the senescent mutant leaves, accumulations of chlorophyllide a and chlorophyllide b were detected, but no phaeophorbide a was found. Chlorophyllide b had a greater accumulation than chlorophyllide a in the early stage of senescence. Low oxygen treatment not only delayed chlorophyll degradation but also enhanced the accumulations of chlorophyllide a and b and lowered the ratios of chlorophyll a to chlorophyll b.     

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.     

Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

Defect in non-yellow coloring 3, an α/β hydrolase-fold family protein, causes a stay-green phenotype during leaf senescence in rice

Chlorophyll degradation is an important phenomenon in the senescence process. It is necessary for the degradation of certain chlorophyll–protein complexes and thylakoid membranes during leaf senescence. Mutants retaining greenness during leaf senescence are known as 'stay-green' mutants. Non-functional type stay-green mutants, which possess defects in chlorophyll degradation, retain greenness but not leaf functionality during senescence. Here, we report a new stay-green mutant in rice, nyc3 . nyc3 retained a higher chlorophyll a and chlorophyll b content than the wild-type but showed a decrease in other senescence parameters during dark incubation, suggesting that it is a non-functional stay-green mutant. In addition, a small amount of pheophytin a , a chlorophyll a -derivative without Mg²⁺ ions in its tetrapyrrole ring, accumulated in the senescent leaves of nyc3 . nyc3 shows a similar but weaker phenotype to stay green ( sgr ), another non-functional stay-green mutant in rice. The chlorophyll content of nyc3 sgr double mutants at the late stage of leaf senescence was also similar to that of sgr . Linkage analysis revealed that NYC3 is located near the centromere region of chromosome 6. Map-based cloning of genes near the centromere is very difficult because of the low recombination rate; however, we overcame this problem by using ionizing radiation-induced mutant alleles harboring deletions of hundreds of kilobases. Thus, it was revealed that NYC3 encodes a plastid-localizing α/β hydrolase-fold family protein with an esterase/lipase motif. The possible function of NYC3 in the regulation of chlorophyll degradation is discussed.     

Ethylene regulates the timing of leaf senescence in Arabidopsis

The plant hormone ethylene influences many aspects of plant growth and development, including some specialized forms of programmed senescence such as fruit ripening and flower petal senescence. To study the relationship between ethylene and leaf senescence, etr1-1, an ethylene-insensitive mutant in Arabidopsis, was used. Comparative analysis of rosette leaf senescence between etr1-1 and wild-type plants revealed that etr1-1 leaves live approximately 30% longer than the wild-type leaves. Delayed leaf senescence in etr1-1 coincided with delayed induction of senescence-associated genes (SAGs) and higher expression levels of photosynthesis-associated genes (PAGs). In wild-type plants, exogenous ethylene was able to further accelerate induction of SAGs and decrease expression of PAGs. The extended period of leaf longevity in etr1-1 was associated with low levels of photosynthetic activity. Therefore, the leaves in etr1-1 functionally senesced even though the apparent life span of the leaf was prolonged.     

Isolation,characterization, and mapping of the stay green mutant in rice

Leaf color turns yellow during senescence due to the degradation of chlorophylls and photosynthetic proteins. A stay green mutant was isolated from the glutinous japonica rice Hwacheong-wx through N-methyl-N-nitrosourea mutagenesis. Leaves of the mutant remained green, while turning yellow in those of the wild-type rice during senescence. The stay green phenotype was controlled by a single recessive nuclear gene, tentatively symbolized as sgr(t). All the phenotypic characteristics of the mutant were the same as those of the wild-type lines except for the stay green trait. The leaf chlorophyll concentration of the mutant was similar to that of the wild-type before heading, but decreased steeply in the wild-type during grain filling, while very slowly in the mutant. However, no difference in photosynthetic activity was observed between the stay green mutant and the yellowing wild-type leaves, indicating that senescence is proceeding normally in the mutant leaves and that the mutation affects the rate of chlorophyll degradation during the leaf senescence. Using phenotypic and molecular markers, we mapped the sgr(t) locus to the long arm of chromosome 9 between RFLP markers RG662 and C985 at 1.8- and 2.1-cM intervals, respectively. Received: 29 April 2001 / Accepted: 17 July 2001     

Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants

We studied the effects of cytokinin benzyladenine (BA) and ethylene on the senescence in the dark of detached leaves of Arabidopsis thaliana(L.) Heynh wild-type plants and theeti-5mutant, which was described in the literature as the ethylene-insensitive one. Leaf senescence was assessed from a decrease in the chlorophyll content. The content of endogenous cytokinins (zeatin and zeatin riboside) was estimated by the ELISA technique. We demonstrated that the content of endogenous cytokinins in the leaves of the three-week-old eti-5mutants exceeded that of the wild-type leaves by an order of magnitude; in the five-week-old mutants, by several times; and in the seven-week-old plants, the difference became insignificant. Due to the excess of endogenous cytokinins in the three–five-week-old mutant leaves, their senescence in the dark was retarded and exogenous cytokinin affected these leaves to a lesser extent. The seven-week-old mutant and the wild-type leaves, which contained practically similar amounts of endogenous cytokinins, did not differ in these indices. Thus, the level of endogenous cytokinins determined the rate of senescence and the leaf response to cytokinin treatment. Ethylene accelerated the senescence of detached wild-type leaves. Ethylene action increased with increasing its concentration from 0.1 to 100 l/l. BA (10^–6M) suppressed ethylene action. Similar data were obtained for the eti-5mutant leaves. We therefore suggest that the mutant leaves comprised the pathways of the ethylene signal reception and transduction, which provided for the acceleration of their senescence.     

Silencing of the <Emphasis Type="Italic">LeSGR1</Emphasis> gene in tomato inhibits chlorophyll degradation and exhibits a stay-green phenotype

The full-length cDNA of LeSGR1 was cloned from tomato by RT-PCR and RACE. The cDNA encoded a protein of 272 amino acid residues and was deposited in GenBank (accession No. DQ100158). Northern analysis suggests that LeSGR1 gene specifically expresses in senescent leaves and mature fruits of tomatoes. Desiccation and flooding induce the expression of LeSGR1 in tomato leaves and stems. Both in ethylene-insensitive mutants (Nr) and ripening inhibitor mutants (rin), the expression of LeSGR1 is markedly decreased compared with that in the wild type. Alignment of the nucleotide sequence of SGR1 cloned from the tomato green flesh (gf) mutant with that from the wild type tomato shows a single nucleotide change leading to an amino acid substitution in gf mutant. Furthermore, LeSGR1 gene silencing by RNA interference results in inhibited chlorophyll degradation similar to the phenotype in gf mutant. Thus, we conclude that LeSGR1 is crucial to chlorophyll degradation and the mutation of SGR1 protein might be responsible for gf tomato properties.     

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     

Mutations in tomato 1-aminocyclopropane carboxylic acid synthase2 uncover its role in development beside fruit ripening

The role of ethylene in plant development is mostly inferred from its exogenous application. The usage of mutants affecting ethylene biosynthesis proffers a better alternative to decipher its role. In tomato (Solanum lycopersicum), 1-aminocyclopropane carboxylic acid synthase2 (ACS2) is a key enzyme regulating ripening-specific ethylene biosynthesis. We characterised two contrasting acs2 mutants; acs2-1 overproduces ethylene, has higher ACS activity, and has increased protein levels, while acs2-2 is an ethylene underproducer, displays lower ACS activity, and has lower protein levels than wild type. Consistent with high/low ethylene emission, the mutants show opposite phenotypes, physiological responses, and metabolomic profiles compared with the wild type. The acs2-1 mutant shows early seed germination, faster leaf senescence, and accelerated fruit ripening. Conversely, acs2-2 has delayed seed germination, slower leaf senescence, and prolonged fruit ripening. The phytohormone profiles of mutants were mostly opposite in the leaves and fruits. The faster/slower senescence of acs2-1/acs2-2 leaves correlated with the endogenous ethylene/zeatin ratio. The genetic analysis showed that the metabolite profiles of respective mutants co-segregated with the homozygous mutant progeny. Our results uncover that besides ripening, ACS2 participates in the vegetative and reproductive development of tomato. The distinct influence of ethylene on phytohormone profiles indicates the intertwining of ethylene action with other phytohormones in regulating plant development.     

Chlorophyll breakdown during pepper fruit ripening in the <Emphasis Type="Italic">chlorophyll retainer</Emphasis> mutation is impaired at the homolog of the senescence-inducible stay-green gene

The pepper chlorophyll retainer (cl) mutation is characterized by inhibition of chlorophyll degradation during fruit ripening. Ripe fruit of cl pepper containing chlorophyll and red carotenoids is brown, while ripe fruit containing chlorophyll and yellow carotenoids is green. In addition to the inhibitory effect during fruit ripening caused by cl, we show that chlorophyll degradation is inhibited during natural and dark-induced leaf senescence. Therefore, the cl mutation has the characteristics of the stay-green (sgr) mutants described in many other species. Upon the recent discovery of the SGR gene in various plant species, we isolated pepper SGR (CaSGR) and found that it genetically cosegregates with cl in a BC1 mapping population. Furthermore, sequencing the wild-type and mutant alleles revealed an amino-acid substitution of tryptophan (aromatic amino acid) to arginine (basic amino acid) at position 114 in the protein sequence. The single-nucleotide polymorphism (SNP) that differentiates the wild-type and mutant alleles was exploited to develop a PCR marker useful for marker-assisted selection. Expression of CaSGR as measured by semiquantitative RT-PCR was mostly induced upon fruit ripening and to a lesser extent upon leaf senescence. Taking together, our genetic, sequence and expression data all indicate that CaSGR is a candidate for controlling the cl mutation in pepper.     

Influence of cytokinins and novel cytokinin antagonists on the senescence of detached leaves of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Cytokinins N⁶-benzyladenine (BA) and 1-(2-chloropyridin-4-yl)-3-phenylurea (4PU-30) delayed the senescence of detached leaves (3^rd to 7^th leaf node) of wild and ethylene insensitive eti5 mutant of Arabidopsis thaliana. The novel anticytokinins, structural analogues of purine and phenylurea cytokinins also affected the senescence of detached rosette leaves of A. thaliana. They diminished to a significant extent the cytokinin-induced delay of chlorophyll destruction, but without a considerable difference in their action against both types of cytokinins. These results correlated with changes observed in ribonuclease (RNase) activity.     

Changes of Photosynthetic Characteristics in Relation to Leaf Senescence in Two Maize Hybrids with Different Senescent Appearance

A field experiment was conducted to investigate the changes in chlorophyll (Chl) and nitrogen (N) contents, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and phosphoenolpyruvate carboxylase (PEPC) contents and PEPC activity, and the photon-saturated net photosynthetic rate (P _Nsat), and their relationships with leaf senescence in two maize hybrids with different senescent appearance. One stay-green (cv. P3845) and one earlier senescent (cv. Hokkou 55) hybrid were used in this study, and we found that Chl and N contents and the P _Nsat in individual leaves of P3845 were greater than those in corresponding leaves of Hokkou 55 at the successive growth stages. In addition, larger contents of RuBPCO and PEPC, and a greater activity of PEPC were observed in P3845. Due to the lower rates of decrease of Chl, RuBPCO, and PEPC amounts per unit of N, and the lower net C translocation rate per unit of N in the stay-green hybrid, leaf senescence was delayed in comparison to the earlier senescent hybrid.     

NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll – protein complexes during leaf senescence

Yellowing/chlorophyll breakdown is a prominent phenomenon in leaf senescence, and is associated with the degradation of chlorophyll – protein complexes. From a rice mutant population generated by ionizing radiation, we isolated nyc4‐1, a stay‐green mutant with a defect in chlorophyll breakdown during leaf senescence. Using gene mapping, nyc4‐1 was found to be linked to two chromosomal regions. We extracted Os07g0558500 as a candidate for NYC4 via gene expression microarray analysis, and concluded from further evidence that disruption of the gene by a translocation‐related event causes the nyc4 phenotype. Os07g0558500 is thought to be the ortholog of THF1 in Arabidopsis thaliana. The thf1 mutant leaves show variegation in a light intensity‐dependent manner. Surprisingly, the F_v/F_m value remained high in nyc4‐1 during the dark incubation, suggesting that photosystem II retained its function. Western blot analysis revealed that, in nyc4‐1, the PSII core subunits D1 and D2 were significantly retained during leaf senescence in comparison with wild‐type and other non‐functional stay‐green mutants, including sgr‐2, a mutant of the key regulator of chlorophyll degradation SGR. The role of NYC4 in degradation of chlorophyll and chlorophyll – protein complexes during leaf senescence is discussed.     

Defects in a proteolytic step of light-harvesting complex II in an<Emphasis Type="Italic">Arabidopsis</Emphasis> stay-green mutant,<Emphasis Type="Italic">ore10</Emphasis>, during dark-induced leaf senescence

During dark-induced leaf senescence (DIS), the non-functional stay-green mutantore10 showed delayed chlorophyll (Chl) degradation and increased stability in its light-harvesting complex II (LHCII). These phenomena were closely related to the formation of aggregates that mainly consisted of terminal-truncated LHCII (Oh et al., 2003). Theore10 mutant apparently lacks the protease needed to degrade the truncated LHCII. In wild-type (WT) plants, protease was found in the thylakoid fraction, but not the soluble fraction. A similar experiment using dansylated LHCII revealed that the protease degraded both WT andore10 LHCII, indicating that its stability inore10 perhaps did not result from a defect in the LHCII polypeptides themselves. Although protease activity was not present in non-senesced WT leaves, it was induced during DIS. It also was possible to diminish the high level of protease present in the thylakoids through high-salt washing, suggesting that this enzyme is extrinsically bound to the outer surface of the stroma-exposed thylakoid regions.     

Effect of phytohormones on chlorophyll degradation during aging of chloroplastsin vivo andin vitro

Summary Phytohormones like IAA and kinetin inhibit chlorophyll loss during aging of wheat chloroplasts duringin vivo andin vitro. GA, on the other hand, stimulates the pigment degradation during aging of attached leaves in contrast to its senescence inhibiting action in detached leaves and isolated chloroplasts. A shift in optimum concentration of hormone in inhibiting chlorophyll degradation suggests a differential pool size of endogenous hormone regulating aging of chloroplastsin vivo andin vitro. The retardation of chlorophyll loss by kinetin, IAA and GA during aging of chloroplastsin vitro would indicate that the effect of hormones in preventing yellowing of senescing leaves may be mediated through their direct action on chloroplasts.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: implications of the stay-green mutation for photosynthesis, growth and nitrogen nutrition

Mutation of the nuclear gene sid disables chlorophyll degradation during leaf senescence in the pasture grass Festuca pratensis. This study investigated the effect of the mutation on photosynthesis and on leaf and whole plant growth under a range of nitrogen regimes. When plants were cultivated in a static hydroponic system, the chlorophyll content of fourth leaves of the stay-green mutant Bf993 remained virtually unchanged from full expansion to complete senescence, while tissue of the wild-type (cv. Rossa) became completely yellow. The retention of chlorophyll in Bf993 was not associated with maintenance of photosynthetic activity as shown by rates of light-saturated CO₂ fixation and apparent quantum efficiency. Higher levels of total N in senescing leaves of Bf993 than in Rossa indicated reduced nitrogen remobilization in the mutant. When using a range of [NH₄NO₃], dry matter production and tillering Mere lower for Bf993 at all but the highest [NH₄NO³, which was supra-optimal for the wild type. In contrast to the static system, where fluctuations in N supply occurred, growth and [NO₃^?] uptake were similar in mutant and wild type when [NO₃^?] was continuously maintained by a flowing solution culture system. The results are discussed in relation to the role of N supply and the effect of the stay-green mutation on N recycling.     

Consequences of CO2 and light interactions for leaf phenology, growth, and senescence in Quercus rubra

We investigated how light and CO₂ levels interact to influence growth, phenology, and the physiological processes involved in leaf senescence in red oak (Quercus rubra) seedlings. We grew plants in high and low light and in elevated and ambient CO₂. At the end of three years of growth, shade plants showed greater biomass enhancement under elevated CO₂ than sun plants. We attribute this difference to an increase in leaf area ratio (LAR) in shade plants relative to sun plants, as well as to an ontogenetic effect: as plants increased in size, the LAR declined concomitant with a decline in biomass enhancement under elevated CO₂ Elevated CO₂ prolonged the carbon gain capacity of shade‐grown plants during autumnal senescence, thus increasing their functional leaf lifespan. The prolongation of carbon assimilation, however, did not account for the increased growth enhancement in shade plants under elevated CO₂. Elevated CO₂ did not significantly alter leaf phenology. Nitrogen concentrations in both green and senesced leaves were lower under elevated CO₂ and declined more rapidly in sun leaves than in shade leaves. Similar to nitrogen concentration, the initial slope of A/C_i curves indicated that Rubisco activity declined more rapidly in sun plants than in shade plants, particularly under elevated CO₂. Absolute levels of chlorophyll were affected by the interaction of CO₂ and light, and chlorophyll content declined to a minimal level in sun plants sooner than in shade plants. These declines in N concentration, in the initial slope of A/C_i curves, and in chlorophyll content were consistent with declining photosynthesis, such that elevated CO₂ accelerated senescence in sun plants and prolonged leaf function in shade plants. These results have implications for the carbon economy of seedlings and the regeneration of red oak under global change conditions.