Photosynthetic characterization at different senescence stages in an early senescence mutant of rice <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

An early senescence (es) mutant of rice Oryza sativa L. with progressing death of most of leaves before heading stage was identified in the field in Hainan province. After tillering stage, the brown striations were found in the base of green leaves randomly, and then expanded to whole leaves. No fungi, bacteria, and viruses were detected in the brown striations suggesting that it was a genetic mutant. The ultrastructure of leaf cells at the site of brown striations showed breakdown of chloroplast thylakoid membrane structures and other organelles, and condensation of the cytoplasm at severe senescence stage. The photosynthetic activity and chlorophyll (Chl) contents decreased irreversibly along with leaf senescence process.     

Composition and function of plastoglobuli

The lipid composition of whole leaves and isolated plastoglobul of beech (Fagus sylvatica) has been studied during four natural autumnal senescence stages. Chlorophylls, glycolipids, and phospholipids were extensively degraded in leaves. About 20% of the glycolipids found in leaves during summer, however, remained in the last stage of leaf senescence. Triacylglycerols, also detected in large amounts in summer leaves, were hydrolyzed during senescence. The content of free fatty acids derived from degradation of glycerolipids therefore increased. The total carotenoid and prenyl quinone content was largely unchanged during senescence, except during the last stage investigated, but the reduced forms of prenyl quinones decreased while the oxidized prenyl quinones increased. Plastoglobuli isolated from summer leaves mainly contained triacylglycerols, plastohydroquinone, and -tocopherol. The triacylglycerol content declined in plastoglobuli during senescence. Most of the triacylglycerols must be located outside the plastoglobuli throughout the stages investigated. Carotenoids liberated from thylakoids were esterified and increasingly deposited in plastoglobuli during senescence. In the last senescence stage, carotenoid esters were the main component of plastoglobuli. Prenyl quinones were also transferred into plastoglobuli. Reduced prenyl quinones were sucessively oxidized during senescence and plastoquinone (oxidized) was the predominant prenyl quinone in plastoglobuli isolated from the last senescence stage. The carotenoid and prenyl quinone distribution was identical in leaves and plastoglobuli during late senescence. The main constituents of thylakoids, glycolipids and proteins, were not deposited in plastoglobuli and therefore did not play an important role in plastoglobuli metabolism.Abbreviation PQ plastoquinone     

The Role of Abscisic Acid in Senescence of Detached Tobacco Leaves

The role of abscisic acid in the regulation of senescence was investigated in detached tobacco leaves (Nicotiana rustica L.). Leaves senesced in darkness showed a sharp rise in abscisic acid level in the early stage of aging, followed by a rapid decline later. The same trend was found when leaves were aged in light, but the rise in abscisic acid occurred four days later than in darkness. Senescence was slower in light than in darkness, while salt stress accelerated the processes. Leaves treated with kinetin which senesced in light and darkness, did not show an increase in abscisic acid. Application of kinetin led to a transformation from free to bound ABA. These results may indicate that ABA and cytokinin are involved in a trigger mechanism which regulates senescence; the stage at which this trigger is activated determines the rate of senescence.     

Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: Immunocytochemical and ultrastructural observations

Summary The possible involvement of vacuolar cysteine proteinases in degradation of ribulose-bisphosphate carboxylase (Rubisco) in senescing French bean leaves was studied by ultrastructural and immunocytochemical analyses with antibodies raised against the large subunit (LSU) of Rubisco and SH-EP, a cysteine proteinase fromVigna mungo that is immunologically identical to one of the major proteinases of French bean plants. Primary leaves of 10-day-old plants were detached and placed at 25 °C in darkness for 0, 4, and 8 days to allow their senescence to proceed. The leaves at each senescence stage were subjected to the conventional electron microscopic and immunocytochemical studies. The results indicated that the chloroplasts of senescing French bean leaves were separated from the cytoplasm of the cell periphery and taken into the central vacuole and that the Rubisco LSU in the chloroplasts was degraded by vacuolar enzymes such as an SH-EP-related cysteine proteinase that developed in senescing leaves. The present results together with the results of previous biochemical studies using vacuolar lysates support the view that Rubisco is degraded through the association of chloroplasts with the central vacuole during the senescence of leaves that were detached and placed in darkness.     

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.     

拟南芥莲座叶片发育过程中P1基因的表达特性

该实验对CDF1类似蛋白基因(P1)在拟南芥叶片发育不同阶段的定量PCR结果显示,P1基因在拟南芥叶片发育的所有时期均可表达,但在茎生叶和衰老叶中的表达水平明显高于成熟叶和幼叶。GUS报告基因表达的组织化学染色结果显示,P1启动子在拟南芥叶片中有较高的驱动活性;在营养生长阶段的幼苗和植株(4~5周)的所有叶片中均能检测到GUS表达,但在植株转入生殖生长阶段后(6周及以后),GUS表达主要集中在逐渐衰老的叶中,并随着叶片衰老程度加剧GUS染色程度也越深,这一结果与GUS荧光定量检测结果一致。通过分析P1基因启动子上可能存在的顺式调控元件,发现茉莉酸甲酯、热压、干旱和水杨酸等均能够引起叶片衰老调控元件的响应,证实P1的表达受到这些因素的调控。研究表明,P1在拟南芥莲座叶片中很可能参与了对上游衰老信号的响应,该研究结果为进一步探究P1在叶片衰老过程中的分子功能验证奠定了基础。     

Overexpression of OsRab7B3, a Small GTP-Binding Protein Gene,Enhances Leaf Senescence in Transgenic Rice

Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the OsRab genes, OsRab7B3, closely associated with senescence in expression pattern, was chosen for functional analysis. Expression of sGFP under the control of the OsRab7B3 promoter increased in leaves when ABA and NaCl were applied or when kept in dark. In transgenic rice overexpressing OsRab7B3, the senescence-related genes were upregulated and leaf senescence was significantly enhanced under dark conditions. Moreover, leaf yellowing occurred earlier in the transgenic plants than in the wild type at the ripening stage. Hence it is suggested that OsRab7B3 act as a stress–inducible gene that plays an important role in the leaf senescence process.     

Changes in the turnover of Rubisco and levels of mRNAs of rbcL and rbcS in rice leaves from emergence to senescence

Changes in the amount of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4·1·1·39) synthesized and degraded and the levels of rbcL and rbcS mRNAs were examined in the eighth leaf blades of rice from emergence to senescence. Synthesis of Rubisco was very active during leaf expansion, became quite low at the time of full expansion and then declined further during senescence. The changes in the levels of rbcL and rbcS mRNAs co‐ordinated approximately with those in the amount of Rubisco synthesized. Thus, it is suggested that the amount of Rubisco synthesized is determined primarily by the levels of rbcL and rbcS mRNAs during the life span of the leaves. Degradation of Rubisco started just before the time of full expansion and became far more active than its synthesis during senescence. Since the synthesis of Rubisco during senescence scarcely contributed to its amount, it can be concluded that the degradation of Rubisco is the major determinant for the amount of Rubisco in senescent leaves. The decline in the level of rbcL mRNA occurred much earlier in the developmental stage and proceeded at a much faster rate than that of rbcL DNA, indicating that the level of rbcL DNA is not a major determinant for the level of rbcL mRNA in senescent leaves of rice.     

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.     

Differential effects of benzyladenine on the ultrastructure of chloroplasts in intact bean leaves according to their age

Summary Primary leaves of intact bean plants (Phaseolus vulgaris) were treated with benzyladenine (BA) at different stages of growth. Changes in the ultrastructure of chloroplasts and the contents of chlorophyll, carotenoid, and protein (soluble and insoluble) in leaves with different treatments were followed and compared. When BA was applied from an early stage, it increased the chloroplast size and the number of grana per chloroplast without any pronounced effect on the grana size. When BA treatment was stopped at the early stage, these effects remained for a while and then diminished. When BA treatment was begun at a late stage, such marked effects were not observed, suggesting that only young leaves could respond to BA in that manner. However, the late treatment efficiently prevented the process of the last stage of leaf senescence characterized by disintegration of thylakoids with concomitant increase in the plastoglobule size. Chlorophyll, carotenoid, and insoluble protein contents per leaf followed similar changes in chloroplast length and the number of grana per chloroplast section.     

Identification of proteins in leaf tissues of white clover using MALDI-TOF mass spectrometry

Protein extracts, made to leaves harvested from the stolons of the pasture legume white clover (Trifolium repens L.) at two developmental stages (newly initiated; onset of senescence) were purified further using reverse-phase and ionexchange chromatography. Fractions enriched with the ethylene biosynthetic enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase were selected for each stage and the final, partially purified fraction was subjected to twodimensional gel electrophoresis (2DE). Antibodies raised against a recombinant ACC oxidase (ACO) from white clover (antiTR-ACO2) recognised a series of spots of differing pI suggesting that ACO undergoes post-translational modifications. Further, the pattern differed between the ACO proteins partially purified from newly initiated leaves with leaves at the onset of senescence suggesting that the environmental and developmental cues that operate in each tissue influences the type and/or degree of post-translational modifications of the ACO protein. MALDI-TOF mass spectrometry was used to identify protein spots associated with the ACO proteins. Protein with identities to an ACO isoform from Oryza sativa, and a phosphoribulokinase from Arabidopsis thaliana were identified in the 2DE separations from newly initiated leaves, while an isoflavone reductase from Medicago sativa was identified in the 2DE separation of the senescent leaf extract.     

Quantitative proteomics analysis of proteins involved in leaf senescence of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Delaying leaf senescence and prolonging the available time for photosynthesis is one of the important approaches to increase grain yield of rice (Oryza sativa L.). Here, iTRAQ-based quantitative proteomics approach was used to comparative analyze the expression profiles of proteins in rice leaves in response to senescence. Totally 5067 proteins were identified. Compared with the proteins in the flag leaves at early stage of grain-filling in rice Liang-You-Pei 9 (LYP9), 240 and 188 proteins were up-regulated and down-regulated in the flag leaves at middle stage of grain-filling, and 387 and 202 proteins were up-regulated and down-regulated in the flag leaves at late stage of grain-filling, respectively. In addition, 39 and 18 identified proteins were constantly up-regulated and down-regulated in the leaves from early to middle and late stages of grain-filling, respectively. Among them, chloroplast chaperonin 10, geranylgeranyl diphosphate reductase, Mg chelatase subunit ChLD, porphobilinogen deaminase, protochlorophyllide reductase B and thioredoxin-like protein CITRX might have involved in the senescence of leaves. This study provided important information for understanding the age-sensitive mechanism of LYP9, and offered a foundation for future studying and improving it.     

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.     

年龄效应对香樟古树程序性衰老的影响

以福建省三明市6个树龄阶段(0~49、50~149、150~349、350~549、550~749和750~900 a)香樟为研究对象,通过分析其生长发育规律、形态学和生理特征的变化,结合曲线拟合与遗传算法筛选表征衰老的相关指标,评价树龄与衰老的相关性,探讨香樟古树衰老机制,为香樟年龄预测、古树复壮和保护提供理论依据。结果表明:(1)年龄极显著地影响着古香樟的植株形态、叶片解剖结构及生理机能等生长发育指标,并以生理指标的年龄效应最大,其次为植株形态指标,最小的是解剖结构指标。(2)香樟呈现出程序性衰老的特征为:叶片细胞在130 a、叶片解剖结构和生理代谢在400 a、树皮厚度和新梢粗度在450 a先后进入衰老阶段,但香樟树体的离心生长(梢长、冠幅和胸径)在0~900 a内仍一直处于旺盛生长,还未进入衰老阶段。(3)冠幅和新叶超氧化物歧化酶(SOD)活性可作为单指标来评价香樟独立木的衰老程度,树皮厚度、冠幅、新叶SOD活性、多酚氧化酶(PPO)活性和栅栏组织与海绵组织比为参数构成的模型可作为古香樟年龄预测模型。研究认为,古香樟的保护和复壮,应先从叶片细胞着手,提高其生理功能,特别是提高其抗氧化能力,保护叶细胞结构完整,以保障叶片处在持续高效的光合状态。     

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.     

Physiological and proteomic analysis of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) in flag leaf during flowering stage and milk stage under drought stress

Hybrid rice has the inferiority of early senescence during reproductive stage. Early senescence of leaves induced by drought causes a constraint on crop productivity. Hence, to better understand traits of drought responsive mechanism in hybrid rice flag leaves during sensitive reproductive stage, we performed physiological measurements and conducted 2-D electrophoresis to investigate proteomic profile of rice flag leaves at flowering and milk stage. 43 proteins showed significant changes in silver stained gels were identified by MS/MS at flowering stage and 54 proteins at milk stage. For flowering stage, inactive CO₂ assimilation and ATP synthesis as well abnormal floral development could be postulated from decreased proteins. Additionally, higher levels of defense-related proteins and antioxidases in drought-stressed leaves suggested active ROS scavenging system. For milk stage, both photosynthesis and CO₂ assimilation were impaired. Disrupted oxidative defense and proteolysis system indicated redox imbalance. Upregulation of NADP-MDH facilitated for CO₂ assimilation and antioxidant system. In conclusion, drought stress during reproductive stage impacted a lot on biological processes in rice leaves at protein level, especially energy metabolism and redox balance.