CO2-CA-G蛋白信号通路调控拟南芥气孔运动的作用与机制研究

There have many evidences showed the effects of elevated CO2 on many aspects, such as plant growth and development, plant gas exchanges, ecological system and global climate. Among various responses of plants, stomatal movement plays a vital role in plant adaptation to elevated atmospheric CO2. There has been reported that carbonic anhydrases (CA), hydrogen peroxide (H2O2) and nitric oxide (NO) are involved in CO2-regulated stomatal movement. However, the in-depth mechanisms of these elements in elevated CO2-mediated stomatal movement remain to be clarified. In plants, G proteins are important to life progresses of growth and development, cell proliferation, some hormonal responses and adaption to environmental stress. Our preliminary experiments showed that pertussis toxin (PTX), an inhibitor of G protein α subunit, inhibited CO2-induced stomatal closure significantly. Meanwhile, cholera toxin (CTX), an activator of G protein α subunit, strengthened stomatal closure of leaves. So, we speculate that G protein mediates “CO2-CA-H2O2-NO” pathway to regulate stomatal movement in Arabidopsis. Therefore, in this project, in order to construct CA-G protein signal pathway of CO2-mediated stomatal movement, we will investigate the effect of CO2-CA-G protein pathway on the stomatal aperture, endogenous generation of H2O2 and NO in molecular and cell aspects combined with pharmacology, molecular biology analysis, bimolecular fluorescence complementation (BiFC), laser confocal scanning microscope (LCSM) and RT-PCR techniques. The plant materials used in our experiments are Arabidopsis wild type and a variety of genetic mutants. Our research will provide new scientific understanding in molecular signal mechanism of CO2-regulated stomatal movement.

大气CO2浓度升高影响植物生长、气体交换和生态气候等多个方面。植物响应CO2浓度变化的关键反应是气孔运动。前期工作显示，碳酸酐酶（CA）、H2O2和NO均参与CO2调控拟南芥气孔运动。但这些因素的信号调控机制仍有待明晰。G蛋白是植物生长发育、激素应答、适应环境胁迫的重要信号蛋白。预实验显示，G蛋白α亚基（Gα）抑制剂PTX阻断CO2诱导野生型拟南芥气孔关闭，Gα活化剂CTX加强气孔关闭效应。我们推测G蛋白参与“CO2-CA-H2O2-NO”信号通路而调控拟南芥气孔运动。因此，本项目拟在分子和细胞水平上，采用药理学、分子生物学、BiFC、LCSM和RT-PCR等方法和技术，以拟南芥野生型和突变体为材料，研究“CO2-CA-G蛋白”信号通路对气孔开度、内源H2O2和NO生成的影响，构建CO2调控气孔运动的CA-G蛋白相关的信号通路，以期为CO2调控植物气孔运动的信号分子机制提供新的科学理解。

大气CO2浓度升高影响植物生长、气体交换和生态气候等多个方面。植物响应CO2浓度变化的关键反应是气孔运动。H2O2和NO是CO2调控拟南芥气孔运动的重要信号分子。G蛋白是植物生长发育、激素应答、适应环境胁迫的重要信号蛋白。G蛋白由α、β和γ三种亚基构成，其中大多数关键受体结合位点与活化位点都在α亚基上，一般认为它是功能亚基。推测G蛋白参与CO2信号通路而调控拟南芥气孔运动。据此，研究了G蛋白对高浓度CO2诱导拟南芥气孔关闭的影响，以及在高浓度CO2诱导拟南芥气孔关闭中G蛋白与H2O2和NO之间的作用关系。结果显示：Ga亚基抑制剂PTX阻断CO2诱导野生型气孔关闭，Ga亚基突变体gpa1-1、gpa1-2、gpa1-3和gpa1-4均不被CO2诱导气孔关闭，Gα亚基过表达株系wGa和组成型活化表达株系cGa被CO2诱导气孔关闭；Ga亚基活化剂CTX不能恢复CO2诱导H2O2清除剂CAT、ASA和合成抑制剂DPI处理的野生型气孔关闭，不诱导NADPH氧化酶突变体AtrbohF和AtrbohD/F气孔关闭也不恢复CO2诱导AtrbohF和AtrbohD/F气孔关闭，而H2O2诱导PTX处理的野生型和gpa1-1、gpa1-2、gpa1-3与gpa1-4气孔关闭，也恢复CO2诱导PTX处理的野生型和gpa1-1、gpa1-2、gpa1-3与gpa1-4气孔关闭，此外，CO2不诱导cGa/F、wGa/F、cGa/DF和wGa/DF气孔关闭；CTX不能恢复CO2诱导NO清除剂c-PTIO和硝酸还原酶（NR）抑制剂Tungstate处理的野生型气孔关闭，不诱导NR突变体Nia1-2和NRII气孔关闭也不恢复CO2诱导Nia1-2和NRII气孔关闭，而NO供体SNP诱导PTX处理的野生型和gpa1-1、gpa1-2、gpa1-3与gpa1-4突变体气孔关闭，也恢复CO2诱导PTX处理的野生型和gpa1-1、gpa1-2、gpa1-3与gpa1-4突变体气孔关闭，此外，CO2不诱导cGa/Nia1、wGa/ Nia1、cGa/NRII和wGa/NRII气孔关闭。这些结果表明, 高浓度CO2诱导拟南芥气孔关闭需要G蛋白，且G蛋白作用于H2O2和NO 的上游，构建了CO2调控气孔运动的“G蛋白-H2O2-NO”相关信号通路，以期为CO2调控植物气孔运动的信号分子机制提供新的科学理解。