Molecular Physiology of Signal Transduction in Guard Cells

用ATP水解活性对钒酸盐的敏感性检测了燕麦根质膜的纯度。钒酸盐对ATP的水解有较强的抑制作用，而NO_3^-、寡霉素和钼酸铵对ATP的水解没有明显的抑制作用，说明从燕麦中分离的质膜具有较高的纯度。在1 muM时，Ca^<2+>强烈抑制了quinacrine猝灭所测得的质子泵送活性。同样，在菠菜叶片分离的质膜中，1mum时Ca^<2+>能抑制质子泵送活性。这些结果表明，在叶片和根组织中，生理浓度的Ca^<2+>抑制了质子在质膜上的泵送活性。用激光共聚焦扫描显微镜测定了毛蚶保护细胞内Ca^<2+>的浓度。在本实验中，我们证实了钙橙和rhod2的Ca^<2+>指示剂都可用于测量保护细胞细胞质中的Ca^<2+>。当EGTA添加到表皮层时，细胞内Ca^<2+>浓度下降，从培养液中去除EGTA后，细胞内Ca^<2+>浓度恢复。结果表明，外用EGTA可能对质膜质子泵活性有调控作用。在红光照射下，保护细胞内Ca^<2+>浓度未见明显变化。在保护细胞分离的液泡中，ABA对液泡体积没有影响。

Purity of the isolated plasma membrane from oat roots was examined by the sensitivity of ATP hydrolytic activity to vanadate. Vanadate strongly inhibited the ATP hydrolysis, but NO_3^-, oligomycin and ammonium molybdate had no significant inhibition of the hydrolysis, suggesting that isolated plasma membrane from oat had a high purity. Proton pumping activity measured by quinacrine quenching was inhibited strongly by Ca^<2+> at 1 muM.Similarly, proton pumping activity was inhibited by Ca^<2+> at 1 muM in isolated plasma membrane from spinach leaves. These results suggest that the proton pumping activity across the plasma memranes was inhibited by Ca^<2+> at physiological concentrations in both leaf an root tissue.We measured the cytosolic Ca^<2+> concentrations in guard cells of Commelina communis using confocal laser scanning microscope. In this experiments, we confirmed that the Ca^<2+> indicator of calcium orange and rhod 2 were both useful to measure Ca^<2+> in the guard cell cytosol. When EGTA was added to epidermal layrs, cytosolic Ca^<2+> concentration was declined and the removal of EGTA from the incubation media restored the Ca^<2+> concentration. The results suggest that the possible regulation for the plasma membrane proton pump activity by external application of EGTA.When the guard cell was illuminated with red light, we could not find any change in cytosolic Ca^<2+> concentration in guard cells.In isolated vacuole from guard cells, ABA had no effect on the vacuolar volume.