Molecular Mechanism Of Plasma Membrane H~+-ATPase Regulates Nitrate Uptake In Broad Bean Under Drought Stress

As one of the most important necessary chemical elements for plants growth, nitrogen is an important component of protein, nucleic acids, phospholipids and some growth hormone construction. It has played an indispensable role in agricultural production and development, as well it reached a contribution about 40%-50%to plants’final yield. As for one kind of nitrogen species plants are uptake, NO3￣ has showed a very important role in plants early growth. Plasma membrane H+-ATPase is the most abundant expression protein in plant cells, and many important physiological processes are regulated via its activity shifts in plants. NO3- absorbing by plant needs PM H+-ATPase participation, and its activity is regulated by the 14-3-3 protein. Broad bean is a kind of large-scale cultivation crop in China. Due to the short growth cycle, larger stomata and experimental material easily obtained, it is often used as a model plant for stomatal movement mechanism research in botany field. According to Zhou Bing’s experiment conclusion in the lab,5%concentration of PEG6000 was applied for simulation drought stress to dispose Yunnan yellow soil Vicia faba(YV), to explore the molecular mechanism of PM H+-ATPase regulation nitrate nitrogen absorption under drought stress. In this study, we concluded the following results:YV as the material was cultivated for 2,5 and 12 hours with 5%PEG simulation drought stress under hydroponic condition, to analyze the changing of physiological and biochemical characteristics in plants root. The results indicated that water loss rate of the plant treated by PEG were keeping increasing, as well as the capacity of nitrate nitrogen absorption has been also significant dropped along with an increasing time. The content of osmotic regulation substances in root, including soluble protein, soluble sugar and proline were also significant increased. Meanwhile the content of nitric oxide(NO) and hydrogen peroxide(H2O2) were also showed a trend of rising with an extension of time under PEG treatment condition. In root, which plants treated by PEG the phosphorylation level of PM H+-ATPase, its interaction with 14-3-3 protein, the activity as well as H+-pump activity were decreased. These results indicated that drought stress caused H2O2 accumulation in YV root, which inhibites the phosphorylation level of PM H+-ATPase as well as the correlation with 14-3-3 protein, thus causes PM H+-ATPase activity and the ability of pumping hydrogen decrease, which has made the capacity of nitrate nitrogen absorption of root recede.In order to verify nitrate nitrogen absorption of YV was regulated by PM H+-ATPase in drought stress, exogenous activator and inhibitor of PM H+-ATPase were applied to investigate the effect of nitrate nitrogen absorption of broad bean by their application under drought stress. The results showed those after application of exogenous PM H+-ATPase activator under drought stress, in root of the plants, the phosphorylation level of PM H+-ATPase and its interaction with protein 14-3-3 were significant increased, as well as the ability of nitrate nitrogen absorption improved. With leaf transpiration rate and stomatal conductivity of the leaves keeping increasing, water loss rate of the plant has also been kept increasing. It has showed an opposite results by application of exogenous PM H+-ATPase inhibitor compared with those treated by PM H+-ATPase activator, the activity of PM H+-ATPase in root of YV was restrained, the capacity of nitrate nitrogen absorption was reduced, and in leaves leaf transpiration rate as well as stomatal conductivity were reduced, along with water loss rate of the plant. These results have reflected the conclusion that PM H+-ATPase has indeed played a role in regulation of nitrate nitrogen absorption of broad beans, and application the activator of PM H+-ATPase to broad beans is available for nitrate nitrogen absorption, while to the disadvantage of surviving in the arid environment. On the contrary, application the inhibitor of PM H+-ATPase is advantageous for broad beans surviving in the arid environment, whereas doesn’t benefit for nitrate nitrogen absorption.In order to determine whether nitrate nitrogen absorption of YV is regulated by PM H+-ATPase activity, which is related to H2O2 accumulation in root of YV under drought stress. Exogenous H2O2 and its scavenger were applied to investigate the effect of PM H+-ATPase activity and nitrate nitrogen absorption of YV by their application under drought stress. The results have showed that after application of H2O2, the concentration of H2O2 in root was obvious increased, meanwhile the phosphorylation level of PM H+-ATPase and its interaction with protein 14-3-3 were significant decreased, its activity and the ability of pumping hydrogen receded, and the ability of nitrate nitrogen absorption also got weakened. Leaf transpiration rate, stomatal conductivity of the leaves and water loss rate of the plant were decreased. Application of H2O2 scavenger has showed a different consequence compared with the former treated by H2O2, the ability of nitrate nitrogen absorption meanwhile water loss rate of the plant were increased, the accumulation of H2O2 in root was decreased, the activity of PM H+-ATPase was significant rised. Leaf transpiration rate and stomatal conductivity of the plant were kept rising with an increasing concentration of H2O2 scavenger. These results confirmed that under drought stress H2O2 is involved in regulation of PM H+-ATPase activity of YV root, thus affects the capacity of nitrate nitrogen absorption.