Comparison of phosphorus mobilization during monocarpic senescence in rice cultivars with sequential and non-sequential leaf senescence

The senescence pattern of the three uppermost leaves of four rice (Oryza sativa L.) cultivars viz. Ratna, Jaya, Masuri and Kalojira was analysed in terms of decline of chlorophyll and by measuring [³²P]-phosphate retention and export from leaf to grains during the reproductive development. With the advancement of reproductive development, the cultivars Masuri and Kalojira showed a sequential mode of senescence, but the cultivars Ratna and Jaya showed a non-sequential mode of leaf senescence where the flag leaf senesced earlier than the older second leaf. Foliar spraying with benzyladenine (0.5 mM) significantly delayed, and abscisic acid (0.1 mM) accelerated, leaf senescence. In untreated control plants, the second leaf had the highest export of labelled phosphate among the leaves at the grain formation stage (0–7 days) in Masuri and Kalojira. This was compensated by the flag leaf at the grain development stage (7–14 days), whereas export of [³²P]-phosphate was highest from the flag leaf of Ratna and Jaya at the grain development stage. Compared with the control, benzyladenine treatment caused higher retention of [³²P]-phosphate in the leaves and also export to the grains, but abscisic acid treatment gave lower retention and export of [³²P]-phosphate to the grains. The amount of [³²P]-phosphate export from a mother to a daughter shoot developed in the axil of the second leaf of plants with the panicle removed, was less than that to panicles remaining on control plants of all cultivars. When the panicle had been excised, the greatest export of [³²P]-phosphate took place from the second leaf to the daughter shoot in all cultivars. Excision of the panicle delayed leaf senescence as compared with intact controls and maintained an age-related leaf senescence pattern in all the four cultivars. The results presented here demonstrate that mobilization of phosphorus from leaf to grains, regardless of cultivar or age and position of the leaf, correlates well with the senescence of that leaf.     

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.     

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.     

Mechanism of monocarpic senescence in rice

During grain formation stage (90 to 110 days), the youngest flag leaf of rice (Oryza sativa L. cv. Jaya) remained metabolically most active (as indicated by cellular constituents and enzyme activities) and the third leaf the least active. At the grain development stage (110 to 120 days) the above pattern of age-related senescence of the flag leaf completely changed and it senesced at a faster rate than the second leaf which remained metabolically active even up to grain maturation time (120 to 130 days), when both the flag and the third leaf partially senesced. Removal of any leaf temporarily arrested senescence of the remaining attached leaves, that of flag leaf did not hasten senescence of the second leaf, while that of either the second or the third accelerated senescence of the flag. Removal of the inflorescence after emergence or foliar treatment of intact plant with kinetin equally delayed senescence and produced an age-related, sequential mode of senescence or leaves. Both translocation and retention of ³²P by the flag leaf were maximum at the time of grain formation and that by the second leaf was maintained even up to grain maturation time. The induction of senescence of the flag leaf was preceded by a plentiful transport of ³²P to the grains. Kinetin treatment decreased the transport of ³²P, prolonged its duration, and almost equally involved all of the leaves in this process. The pattern of senescence of isolated leaf tips was similar to that of attached leaves. The level of endogenous abscisic acid-like substance(s) maintained a close linearity with the senescence behavior of the leaves of intact and defruited plants during aging, and the rise in abscisic acid in the flag leaf was also preceded by higher ³²P transport to the grains.     

The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

Evidence for contribution of autophagy to Rubisco degradation during leaf senescence in Arabidopsis thaliana

During leaf senescence, Rubisco is gradually degraded and its components are recycled within the plant. Although Rubisco can be mobilized to the vacuole by autophagy via specific autophagic bodies, the importance of this process in Rubisco degradation has not been shown directly. Here, we monitored Rubisco autophagy during leaf senescence by fusing synthetic green fluorescent protein (sGFP) or monomeric red fluorescent protein (mRFP) with Rubisco in Arabidopsis (Arabidopsis thaliana). When attached leaves were individually exposed to darkness to promote their senescence, the fluorescence of Rubisco‐sGFP was observed in the vacuolar lumen as well as chloroplasts. In addition, release of free‐sGFP due to the processing of Rubisco‐sGFP was observed in the vacuole of individually darkened leaves. This vacuolar transfer and processing of Rubisco‐sGFP was not observed in autophagy‐deficient atg5 mutants. Unlike sGFP, mRFP was resistant to proteolysis in the leaf vacuole of light‐grown plants. The vacuolar transfer and processing of Rubisco‐mRFP was observed at an early stage of natural leaf senescence and was also obvious in leaves naturally covered by other leaves. These results indicate that autophagy contributes substantially to Rubisco degradation during natural leaf senescence as well as dark‐promoted senescence.     

Endogenous gibberellin levels and senescence in Taraxacum officinale

Summary The level of endogenous gibberellins (GAs) in leaf tissue of Taraxacum officinale was high during leaf growth and expansion but declined progressively during leaf senescence. In the chromatographic system used, most of the GA from Taraxacum leaves moves with the R_f of GA₃. However, several other GAs were also effective in retarding senescence in Taraxacum leaves. It is concluded that ageing of dandelion leaves is associated with a deficiency of endogenous GA.     

Premature leaf senescence 3, encoding a methyltransferase,is required for melatonin biosynthesis in rice

Premature leaf senescence in rice is one of the most common factors affecting the plant's development and yield. Although methyltransferases are involved in diverse biological functions, their roles in rice leaf senescence have not been previously reported. In this study, we identified the premature leaf senescence 3 (pls3) mutant in rice, which led to early leaf senescence and early heading date. Further investigations revealed that premature leaf senescence was triggered by the accumulation of reactive oxygen species. Using physiological analysis, we found that chlorophyll content was reduced in the pls3 mutant leaves, while hydrogen peroxide (H₂O₂) and malondialdehyde levels were elevated. Consistent with these findings, the pls3 mutant exhibited hypersensitivity to exogenous hydrogen peroxide. The expression of other senescence‐associated genes such as Osh36 and RCCR1 was increased in the pls3 mutant. Positional cloning indicated the pls3 phenotype was the result of a mutation in OsMTS1, which encodes an O‐methyltransferase in the melatonin biosynthetic pathway. Functional complementation of OsMTS1 in pls3 completely restored the wild‐type phenotype. We found leaf melatonin content to be dramatically reduced in pls3, and that exogenous application of melatonin recovered the pls3 mutant's leaf senescence phenotype to levels comparable to that of wild‐type rice. Moreover, overexpression of OsMTS1 in the wild‐type plant increased the grain yield by 15.9%. Our results demonstrate that disruption of OsMTS1, which codes for a methyltransferase, can trigger leaf senescence as a result of decreased melatonin production.     

Single base substitution in OsCDC48 is responsible for premature senescence and death phenotype in rice

A premature senescence and death 128 (psd128) mutant was isolated from an ethyl methane sulfonate‐induced rice IR64 mutant bank. The premature senescence phenotype appeared at the six‐leaf stage and the plant died at the early heading stage. psd128 exhibited impaired chloroplast development with significantly reduced photosynthetic ability, chlorophyll and carotenoid contents, root vigor, soluble protein content and increased malonaldehyde content. Furthermore, the expression of senescence‐related genes was significantly altered in psd128. The mutant trait was controlled by a single recessive nuclear gene. Using map‐based strategy, the mutation Oryza sativa cell division cycle 48 (OsCDC48) was isolated and predicted to encode a putative AAA‐type ATPase with 809 amino‐acid residuals. A single base substitution at position C2347T in psd128 resulted in a premature stop codon. Functional complementation could rescue the mutant phenotype. In addition, RNA interference resulted in the premature senescence and death phenotype. OsCDC48 was expressed constitutively in the root, stem, leaf and panicle. Subcellular analysis indicated that OsCDC48:YFP fusion proteins were located both in the cytoplasm and nucleus. OsCDC48 was highly conserved with more than 90% identity in the protein levels among plant species. Our results indicated that the impaired function of OsCDC48 was responsible for the premature senescence and death phenotype.     

Altered patterns of senescence and ripening in gf, a stay-green mutant of tomato (Lycopersicon esculentum Mill.)

The gf tomato mutant, which retains chlorophyll during ripening, has been found to be affected in leaf senescence. The leaves of the gfmutant show an absolute stay-green phenotype. As leaf senescence and fruit ripening proceed, there is a marked difference in chlorophyll content between wild-type and gf. In both attached and detached leaf studies, or after treatment with ethylene, the leaves withered and abscised in gf with only slight loss of chlorophyll and carotenoids. Total protein content declined and free amino acids increased during leaf senescence in wild-type and gf, but Western analysis showed that LHCII polypeptides were retained at higher levels in gf. Expression of senescence-related mRNAs increased normally in gf whereas those for cab, rbcS and rbcL declined in both mutant and wild-type. The mutant possesses enzyme activity for chlorophyllase, the formation of phaeophorbide a by the action of Mg-dechelatase and the oxygenolytic opening of the porphyrin macrocycle. Analysis of chlorophyll breakdown products in fruit indicated that gf, like other stay-green mutants, accumulates chlorophyllides a and b, but phaeophorbide a does not accumulate in vivo. This may indicate that, in the mutant, in vivo the action of phaeophorbide a-oxygenase is somehow presented, either by altered accessibility or transport of components required for thylakoid disassembly or the absence of another factor.     

Influence of reproductive organs on plant senescence in rice and wheat

The chlorophyll and protein contents of the flag, second and third leaves gradually decreased during the reproductive development of rice (Oryza sativa L. cv. Rasi) and wheat (Triticum aestivum L. cv. Sonalika) plants, whereas proline accumulation increased up to the grain maturation stage and slightly decreased thereafter. In rice plant, the rate of decrease in chlorophyll and protein and increase in proline level were higher in the flag leaf than in the second leaf. It was opposite in wheat plant. The export of [³²P]-phosphate from leaves to grains gradually increased reaching a maximal stage at the grain development stage, and then declined. The export of this radioisotope was greater in rice than in wheat. Removal of panicle at the anthesis and grainfilling stages delayed leaf senescence of rice plant, while in wheat the ponicle removal at any stage did not have a marked effect on delaying leaf senescence. The contents of chlorophyll and protein of glumes were higher in wheat than in rice. The variation of such source-sink relationship might be one of the possible reasons for the above effect on leaf senescence.     

Selenium delays leaf senescence in oilseed rape plants

Effect of selenium on leaf senescence was studied in oilseed rape plants treated with 10 μM Na₂SeO₄ at a rosette growth stage. In addition to developmental senescence, N deficiency and leaf detachment were used for induction of senescence. Nonphotochemical quenching declined in old leaves as senescence became more advancing but rose progressively in the plants supplied by Se. The total carbohydrate and protein pools decreased with leaf age, while increased by the Se treatment. However, during senescence induced by N deficiency, Se did not change remarkably the C and N metabolism, but delayed senescence mainly through protection of plants from photoinhibitory effects. After detachment, untreated leaves became chlorotic and necrotic, while the Se-treated ones remained fairly green. Our results demonstrated that Se delayed leaf senescence by a maintaining or even improving photochemical activities. During developmental senescence, the Se effect on the extending life span of the leaves was additionally linked to the metabolic regulation of senescence.     

Characterization and fine mapping of the rice premature senescence mutant ospse1

Premature senescence can limit crop productivity by limiting the growth phase. In the present study, a spontaneous premature senescence mutant was identified in rice (Oryza sativa L.). Genetic analysis revealed that the premature senescence phenotype was controlled by a recessive mutation, which we named Oryza sativa premature senescence1 (ospse1). The ospse1 mutants showed premature leaf senescence from the booting stage and exhibited more severe symptoms during reproductive and ripening stages. Key yield-related agronomic traits such as 1,000-grain weight and seed-setting rate, but not panicle grain number, were significantly reduced in ospse1 plants. Chlorophyll content, net photosynthetic rate, and transpiration rate of ospse1 flag leaves were similar to the wild-type plants in vegetative stages, but these parameters decreased steeply in the mutant after the heading stage. Consistent with this, the senescence-associated genes OsNYC1 and OsSgr were up-regulated in ospse1 mutant during premature leaf senescence. The ospse1 locus was mapped to a 38-kb region on chromosome 1 and sequence analysis of this region identified a single-nucleotide deletion in the 3′ region of an open reading frame (ORF) encoding a putative pectate lyase, leading to a frame shift and a longer ORF. Our results suggested that the premature senescence of the ospse1 may be regulated by a novel mechanism mediated by pectate lyase.     

Leaf mass loss in wetland graminoids during senescence

Mass loss of senescing leaves is an important part of plant biomass turnover and has consequences for assessment of ecosystem productivity, ecosystem nutrient use efficiency, and plant nutrient resorption efficiency. Data, however, on mass loss are scarce, and often based on leaf area as the reference base. This leads to an underestimation of the mass loss, as leaf area itself shrinks during senescence. Furthermore, the few existing studies have almost exclusively used woody species. The purpose of the present study was twofold: i) to assess leaf mass loss during senescence in herbaceous species, with the example of five wetland graminoids and, ii) to compare two different methods of mass loss assessment (two species). Assuming that leaf length does not change during senescence, we assessed leaf mass per leaf length prior to and after senescence. We also estimated pre‐senescence leaf mass nondestructively based on leaf length, width and thickness. For Typha latifolia and Carex stricta, two species with graminoid type leaves but contrasting leaf structure, both methods delivered almost identical results. After the first assessment of leaf mass on July 7th, T. latifolia leaf mass initially increased by 13%, and then decreased to be 12% below the original mass after senescence. C. stricta leaf mass remained stable until senescence, but decreased then by 33%. In a second experiment, the mass of 100 mm pieces of leaves was measured before and after senescence. Calamagrostis canadensis, Carex rostrata and C. stricta lost 23–57% of their leaf mass during senescence, whereas Glyceria canadensis did not show any mass loss. We conclude that mass loss of senescing leaves of herbaceous plants can be considerable and should not be neglected in studies of productivity, nutrient use efficiency or nutrient resorption. For species with no shrinking leaf length during senescence, mass loss can be measured with leaf length as the base whereas for others, pre‐senescent mass can be estimated on the basis of leaf dimensions.     

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.     

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.