NADPH Oxidase Is Involved In The ROS Production Of Tobacco BY-2 Cultured Cells Under Salt Stress

The output of crop has been intensively threatened by the soil salinification. Many studies investicated the involvement of reactive oxygen species (ROS) under salt stress that cause oxidative damage to plant cells. It is proved that plants grown in high salt environment may produce more reactive oxygen species than in normal. But the origination of the ROS under salt stress has not been understood clearly. Considering the important roles of ROS under anti-environment, the source of the ROS has been aroused the highly attention. It has proved that one plasma-membrane protein NADPH oxidase was involved in the ROS production during the period of the plant-pathogen interaction. Then the question is that whether the NADPH oxidase is involved in the ROS release during the salt stress.In my research by using tobacco cultured cells(BY-2) I proved that salt stress may inhibit the growth of the cells and under 200mM NaCl the tobacco cells could no longer grow. Typan blue staining proved that salt stress may result the cell death. The cell genomic DNA after 24 hour salt disposal will degradate. Salt stress may result the H₂O₂ production of the tobacco cells and mainly within 60 minutes through DAB staining. By adding DPI, -- an NADPH oxidase inhibitor, we proved that NADPH oxidase is involved in the release of ROS of tobacco cells under salt stress. We also found that the plant hormone ABA also can induce the cells producing H₂O₂ through NADPH oxidase route by using DCFH-DA staining.RT-PCR demonstrates that expression of NADPH oxidase gene may be induced by salt. Western-blot analysis demonstrates that expression of NADPH oxidase gene may be induced by salt and ABA, but under normal conditiones we could not detect the protein expression of NADPH oxidase gene. I also acquired the tobacco cells overexpressed the NADPH oxidase gene by Agrobacterium transformation measure. Through the analysis of salt stress between normal and transgenic cells we found that the transgenic cells are sensitive to the salt.