No In The Role And Mechanism Of Light Delay The Leaf Senescence Of Wheat In Vitro Study

Plant senescence is a natural death process, in which an organ or whole plant physiological function gradually declines and eventually dies. Plant leaf senescence is a final stage of plant development. Due to delaying leaf senescence has an important application value, previous researchers have extensively studied on the regulation of leaf senescence and its mechanism. Light could significantly delay leaf senescence, one reason is that light affects plant photosynthesis, another is related to the light acting as a signal which has an important role in the regulation of the plant aging, but the study of the light signal transduction pathway in delaying leaf senescence has known little. Treated plant tissue with exogenous NO can enhance the activity of antioxidant enzymes, lower levels of reactive oxygen species (ROS) and delay plant tissue's maturity and senescence. Although the previous studies have showed that light could delay plant senescence, NO also slowed down plant senescence, whether there is the involvement of NO in the process of light-delayed plant leaf senescence is still unknown. In the present study, the detached wheat leaves were used as plant materials, through the experiment that treated the detached wheat leaves in light combined with NO scavenger hemoglobine(Hb),nitrate reductase (NR) inhibitor sodium tungstate (Na₂WO₄) and nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester(L-NAME) to control the level of endogenous NO, we measured the effects of NO on the parameters of plant senescence; At the same times, in order to open out the functions of NO and enzymatic sources of NO in the process of light-delayed leaf senescence, the NO specific fluorescence probe DAF-2DA was used to label NO in the detached wheat leaves, then revealed the changes of endogenesis NO by laser scanning Confocal microscopy technology. This study not only has an important theoretic significance to illuminate the signal transduction pathway in light-delayed leaf aging, but also abundances and perfects the function of NO in plant; at the same time has a guidance value to agriculture.Followings are the main results:1. Compared to dark treatment, the contents of chlorophyll and soluble protein have been remarkably increased, and MDA content was evidently decreased in detached wheat leaves under light alone, indicating that light could delay the detached wheat leaf senescence. When the leaves were treated by light combined with the NO scavenger hemoglobin (Hb), the content of chlorophyll and soluble protein were evidently decreased. MDA content was clearly increased, which suggest that endogenous NO is participated in the process of light-delayed plant leaf senescence. In addition. the effects of NR inhibitor Na₂WO₄ or NOS inhibitor L-NAME on the cntents of chlorophyll, soluble protein and MDA was similar to that of the NO scavenger Hb, suggesting that the light-induced NO was produced through NR and NOS pathways.2. Because the above pharmacological experiments have suggested that the activity of NR and NOS were greatly increased when the wheat leaves were treated in light. To further confirm these results, the activity of NR and NOS was directly determined. The results showed that, the highest activity of NOS was appeared in wheat leaves treated after 12h.along with the senescence of wheat leaves, the activity of NR was gradually decreased when leaves were treated under dark or light. However, the NR activity under light was higher than that under dark at all time. In addition, NR inhibitor Na₂WO₄ could significantly inhibit the light-increased NR activity. The highest activity of NOS was appeared in wheat leaves treated after 36h. while the NOS activity under light was always higher than that under dark treatment. By the way, NOS inhibitor L-NAME could greatly inhibit the light-enhanced NOS activity. These results conformed that the NR and NOS activities in wheat leaves were indeed increased by light, also suggested that the light-induced NO in early stage of wheat leaf scensence was mainly produced via NR pathway, and the light-induced NO in later stage of wheat leaf scensence was mainly via NOS pathway. In addition, the data of the two inhibitors indicated that the concentration of Na₂WO₄ or L-NAME used in our experiments is appropriate.3. The results of endogenesis NO measurement showed that the level of endogenesis NO in Leaves treated by light alone was greatly higher than that treated by dark. When leaves were treated under light combined with NO scavenger Hb,NR inhibitor Na₂WO₄ or NOS inhibitor L-NAME, the level of endogenesis NO in leaves was markedly decreased when compared to treatment of light alone. The results indicated that NO generation in wheat leaves was indeed promoted by light via NR and NOS pathways.4. Compared to dark treatment, light treatment increased activities of SOD, CAT. POD and APX. and decreased MDA content in wheat leaves, indicating that light alleviates leaves senescence through enhancing activities of antioxidant enzymes, and thereby decreasing the level of membrane lipid peroxidation. Compared to light treatment alone, light treatment combined with NO scavenger Hb, NR inhibitor Na₂WO₄ or NOS inhibitor L-NAME decreased the activities of SOD. CAT. POD and APX. and enhanced MDA content, suggesting that NO mediated the light-inhibited leaves senescence by enhancing the activities of antioxidant enzymes and resulting the decrease of membrane lipid peroxidation.