| In rice cultivation, early senescence of leaves has significant effects on the grain filling and yield traits during the later growth stages, which restricts the growth of rice severely and even causes a large-scale dropping in yield. Therefore, researches on leaves early senescence of rice plant, especially on physiology characteristics of leaves early senescence during grain filling stage will be in favor of increasing yield and imporving quality of grains by breeding and cultivation management. Therefore, rice mutant with early leaf senescence (esl) and corresponding wild genotype were employed to study the relationships between leaf senescence and related gene expressions of PS Ⅱ reaction center, sugar signaling and its transport characteristics during leaf senescence, and the effects of H2O2on leaf secescence and key genes related to antioxidant metabolism. The main results are as following:1. The degradation of chloroplast, declining of photosynthetic rate and the value of Chlorophyll a/b are important physiological characteristics for esl mutant rice. Compared to wild type, the maximum fluorescence (Fm), variable fluorescence (Fv) and ratio of variable to maximum fluorescence (Fv/Fm) in the leaves of esl mutant decreased gradually, whereas initial fluorescence (Fo) and thermal dissipation value (D=1-Fv/Fm) incresed significantly along with leaf senescence. Meanwhile, the expressions of Cab, PsbA, PsbB, PsbC and PsbD in the leaves of esl mutant lower significantly than its wild genotype. These results indicated that early leaf senescence in esl mutant were closely related to the impairment of the activity of PS Ⅱ reaction center and the inhibition of photosythetic primary reaction, which resulted in that light energy couldn’t be converse to chemical energy effectively, and the heat dissipation of energy increased, then caused the decreasing of light use efficiency and the declining of photosynthetic rate significantly during leaf senescence.2. The experiments on sugar content, sugar signal and transport, and the temporal expression pattern of genes involved in sugar metabolism showed that hexose content in leaves of esl mutant was significantly higher than that in wild type at the beginning of leaf senescence, and then began to decrease, whereas the contents of soluble sugar, sucrose decreased gradually during leaf senescence, which led to the sugar starving in cells and resulted in promoting leaf senescening. The additional experiment using detached leaf segments incubated in sugar solutions revealed that exogenous sugar treatments delayed the process of leaf senescence, and the sugar starving promoted the leaf senescence. The Hxkl and Hxk2participated in the regulation of leaf senescence by answering to hexose signal in cytoplasm. In addition, the genes of SuSyl, SuSy2, SuSy4, CIN1and CIN4were involved in the sucrose signal perception to regulate the leaf senescence. Comparison to wild type, the expression levels of SuSyl, SuSy2and SuSy4in the leaves of esl mutant were relatively higher at the beginning of leaf senescence, and then decreased sharply. These genes of SuSy and CIN isoforms were more senesitive to sugar level, which were in charge of the sucrose signal perception. Moreover, the sucrose transport by SUTs was closely related to leaf senescence, SUT1in flag leaves of esl mutant at the early stage of senescence was favorable to the sucrose export out of leaves before senescence. Owing to descending photosynthesis, the expression levels of cyFBP, SPS1, SPS2, SPS6and SPS8in leaves of esl mutant were down-regulated, which resulted in the decrease of sucrose synthesis, meanwhile, the starch synthesis catalyzed by AGP isoforms was also restrained, whereas starch degradation was promoted by increasing expression levels of Amy2A and Amy4A, which led to the decrease of starch content in senescing leaves.3. The AsA-GSH cycle metabolism controls H2O2level in various subcellular compartments and is closely related to the stress resistance and senescence of plant tissues. Comparison with the wild type, the H2O2level in flag leaves of esl mutant was higher at the beginning of leaf senescence, and then slightly decreased, at the mid-later stage of leaf senescence, the H2O2began to increase again. The MDA content in flag leaves of esl mutant gradually increased and was higher than that in wild type. The enzyme activities of APX and GR in esl mutant were significantly higher than those in wild type, which might be related to the high H2O2level at the early stage of leaf senescence, and then the APX activity began to decrease significantly. The temporal expression patterns of OsAPX isoforms in flag leaves showed that the esl mutant exhibited significantly inducible transcripts of two cytosolic OsAPX genes(OsAPX1and OsAPX1) and chloroplastic OsAPX7, severely repressed transcripts of peroxisomal OsAPX4and thylakoid-bound OsAPX8. Additional experiment by detached leaf segments incubated in H2O2solutions revealed that the transcripts of OsAPXl and OsAPX2were upregulated by H2O2treatment, whereas the transcripts of OsAPX4and OsAPX8were down-regulated by exogenous H2O2treatment, and the OsAPX6was not sensitive to H2O2, the transcript of OsAPX7was dependent on H2O2concentration. These results suggested that the deficiency of OsAPX4and OsAPX8transcripts were strongly responsible for H2O2accumulation in the cytoplasm and stroma of chloroplasts. Excess H2O2induced the transcripts of OsAPX1, OsAPX2, and OsAPX1in the cytoplasm and chloroplast, which resulted in the promoting APX activity.4. Besides accurate control of H2O2levels in various subcellular compartments by AsA-GSH cycle, CATs mainly respond to the excess H2O2accumulation in the cytoplasm. The experiment by detached leaf segments incubated in H2O2solution revealed that CATA and CATB were more sensitive to exogenous H2O2treatment than CATC, which suggested that CATA and CATB have a more important role than CATC in H2O2inactivation during leaf senescence. The comparative analyses on the temporal expressin patterns of three CAT isoform genes in leaves showed that the expressions of CATA and CATB in esl mutant were higher than those in wild type, and then began to decrease with the process of leaf senescence. The promoting expressions of CATA and CATB in esl mutant at the early stage of leaf senescence mainly responsed to high H2O2level, whereas the down-regulating expressions of CATA and CATB at the mid-late stage of leaf senescence led to the disturbances of H2O2level in cells, which accelerated the process of leaf senescence.5. Plasma membrane NADPH oxidase (PM-NOX) is one of the main sources of O2-in plant cells, which produces H2O2by the dismutation of SOD. As an important signal molecule, H2O2plays a key role in regulating stress response and senescence. The analyses on the expression patterns of PM-NOX isoforms in leaves showed that the expressions of NOX2, N0O5, NOX6and NOX7in esl mutant were higher than in wild type, while the expressions of NOX1, NOX3and FR07in esl mutant were lower than in wild type at the beginning of leaf senescence. The experiment using exogenous ABA treated detached leaf segments showed that the expressions of NOX2, NOX5, NOX6and NOX7were induced, whereas NOX1,NOX3and FR07were inhibited by exogenous ABA, meanwhile, the exogenous ABA accelerated the O2-production rate in detached leaf segments. These results suggested that promoting ABA in esl mutant induced the expressions of NOX2,NOX5, NOX6and NOX7, which accelerated the production of O2-and H2O2, and then regulated the leaf senescence process. |
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