拟南芥类囊体跨膜电位调节的分子机理及生理学意义研究

According to the chemiosmosis hypothesis proposed by Peter Mitchell in 1961, the proton motive force (pmf) can be parshed into the transmembrane proton gradient (ΔpH) and the electric potential gradient (ΔΨ), which are thermodynamically equivalent. It has been shown that K+ efflux and Cl- influx accompanying the light-driven influx protons into the thylakoid lumen are required for the precise regulation of ΔΨ and also for optimizing photosynthetic activity. Recently, the potassium channel protein TPK3 was proved to be responsible for the K+ efflux in thylakoids during photosynthesis. However, the gene encoding the chloride channel in thylakoids has not been identified. By using forward genetics, we have isolated an Arabidopsis mutant, nclc, which is defective in the efficient regulation of ΔΨ. Protein alignment indicates that NCLC shows high similarity to a novel type of chloride channel. The overall aim of this proposal is to characterize the molecular function of NCLC and the physiological significance of the ΔΨ regulation in thylakoids. The research objectives and strategies used in this work are: 1) Determination electrophysiologic characteristics of NCLC by patch clamp and non-invasive micro-test technology. 2) To determine which process of photosynthesis was affected by analyzing photosynthetic characteristics in nclc. 3) To elucidate the physiological significance of the ΔΨ regulation by construction tpk3 nclc double mutant and analyzing photosynthetic properties of various mutants. The results will provide new insights into the molecular mechanisms and physiological significance of the ΔΨ regulation during photosynthesis.

根据化学渗透学说，质子驱动力（pmf）由跨膜质子梯度（ΔpH）和跨膜电位（ΔΨ）两部分组成。在光合电子传递过程中，类囊体膜囊腔内累积H+的同时必须进行K+的外排和Cl-的内流，以实现ΔΨ的精密调节，这是光合活性优化的重要方式之一。最新研究发现TPK3是介导K+外排的离子通道蛋白，但负责Cl-内流的离子通道蛋白还没有被发现。我们通过正向遗传学策略，筛选到一个ΔΨ调节异常的拟南芥突变体nclc，其突变基因编码一个新型的Cl-通道蛋白NCLC。本项目以NCLC为切入点，通过膜片钳和非损伤微测等技术，研究NCLC的电生理学特性；通过测定nclc的光合参数，确定Cl-内流缺失对光合作用的影响；通过构建tpk3 nclc等完全无法进行ΔΨ调节的双突变体，测定不同生长条件下的光合指标，确定ΔΨ精密调节的生理学意义。相关结果将阐明叶绿体ΔΨ调节的分子基础和生理学功能，加深人们对光合作用调控机理的认识。

在光合作用过程中，类囊体膜囊腔内累积H+的同时必须进行K+的外排和Cl-的内流，以实现ΔΨ的精密调节，这是光合功能调控的重要方式。详细研究光合跨膜电位调节、阐明叶绿体ΔΨ调节的分子基础和生理学功能对于充分认识光合作用调控机理具有重要的科学意义。本研究研究了一个调控光合膜跨膜电位调节的氯离子通道蛋白AtBest的生物学功能和一个钾离子/质子逆向转运蛋白对ΔΨ调节和光合调控的作用，获得了多项成果：1）发现了AtBest蛋白作为氯离子通道蛋白参与了光合膜跨膜电位调节；2）AtBest蛋白介导的跨膜电位调节对于光保护具有重要功能；3）KEA3蛋白也和AtBest蛋白相互协作参与了光合跨膜电位调节。根据这些结果，我们在国内著名期刊JIPB发表文章1篇；在研究生培养方面，培养博士研究生3名，硕士研究生2名。项目圆满完成制定的研究目标。