The Investigation On The Regulatory Relationship Among H₂O₂, Ca²⁺ And K⁺ In Maize Leaf Subsidiary Cells And Exploration In A Novel Technology In K⁺ Subcellular Localization

The stoma is the "window" for the gas exchange in plant, and its movement is not only related to its photosynthesis or transpiration status, but also closely related to the biotic and abiotic stresses. So far, researches about the mechanism of stomatal movement have been concentrated on guard cells rather than the subsidiary cells, and there is no available research being done on the H2O2, Ca2+, K+ regulation relationship among stomatal movement in subsidiary cells. Our previous study has found that the stomata movement is not only related to the H2O2 in guard cells, but also the H2O2 in subsidiary cells.Our research has been designed into two parts. In the first part, we use maize(Zhengdan 958)as materials, combined with pharmacological treatment and cytochemical localization of research methods to investigate the regulatory relationship among H2O2, Ca2+, K+ in the process of stomatal movement in maize leaves. In order to observe H2O2、Ca2+ content and distribution variation in guard cells and subsidiary cells during stomatal movement, exogenous H2O2 and NADPH oxidase inhibitor(DPI) were applied to control the content of H2O2. In order to observe Ca2+, K+ content and distribution variation in guard cells and subsidiary cells during stomatal movement, exogenous K+ and K+ channel inhibitor(BaCl2) were applied to control the content of K+. In order to observe H2O2、K+ content and distribution variation in guard cells and subsidiary cells during stomatal movement, exogenous Ca2+ and extracellular Ca2+ channel inhibitors(LaCl3) were applied to control the content of Ca2+. In the second part, in order to observe K+ distribution, we used a well developed analytically chemical reagent NaPbCo(NO2)6, which can be used to detect the trace potassium qualitative detection reagent by specifically binding to K+ and forming KPbCo(NO2)6 precipitates. This method has improved the existing K+ optical microscope detection method at a cellular level and created a novel K+ detecting method in electron microscope at a subcellular level based on the imaging principle of optical microscope and electron microscope.The results of the first part of this study are as follows:1. Exogenous H2O2 can elevate the content of Ca2+ and H2O2 and decrease the content of K+ in guard cells, while increase the content of the three elements in the subsidiary cells. Exogenous DPI treatment can decrease the content of Ca2+ and H2O2 and increase the content of K+ in guard cells, while decrease the content of the three elements in the subsidiary cells. This result indicates that H2O2 in guard cells and accessory cells is an upstream signal of Ca2+ and K+.2. Exogenous K+ can elevate the content of K+ and decrease the content of H2O2 in both guard cells and subsidiary cells. No obvious variation was observed in Ca2+ during such process. Exogenous BaCl2 treatment can decrease the content of K+ in guard cells or subsidiary cells. This result indicates that K+ in guard cells and accessory cells is a downstream signal of H2O2 and Ca2+.3. Exogenous Ca2+ can elevate the content of Ca2+ and H2O2 and decrease the content of K+ in guard cells, while increase the content of the three elements in the subsidiary cells. Exogenous LaCl3 treatment can decrease the content of Ca2+ and H2O2 and increase the content of K+ in guard cells, while decrease the content of the three elements in the subsidiary cells. This result indicates that Ca2+ in guard cells and subsidiary cells is an upstream signal of H2O2 and K+.Based on the above research results we reached a conclusion: the relationship among H2O2, Ca2+, K+ in guard cells is consistent with the current opinion. And according to the existing regulation relationship theory in guard cells, which believes Ca2+ and H2O2 is the upstream signal of K+, and their regulation is positively related and negatively related to K+, we speculated that it is the same in subsidiary cells as in guard cells, which is, Ca2+ and H2O2 is the upstream signal of K+, and the two are positive feedback regulation, but K+ is positively regulated by the two elements.The results of the second part of this study are as follows:We created a situ detection of potassium ions in submicroscopic structure by comparison a variety of experimental program. After this treatment, cell structure of plant tissue will remain relatively intact and color will show specificity. The method is highly sensitivite and according to the test result, K+ is mainly distributed in the vascular bundles, cell membrane, chloroplast, the nucleus and nucleolus, which is considerably consistent with current research results. It confirms the feasibility of this method to be used as new technology in detecting the potassium ions in plant submicroscopic structures in situ. This method can help us understand the distribution and relative differences in content of positions in the submicroscopic structure.