微管结合蛋白WDL3和WDL5在气孔运动中的作用

Stomatal opening and closing are crucial for plant photosynthesis and transpiration which have significant impact on water use efficiency and crop production, so the mechanisms of stomatal movement lie at the center of biological research. As we know, microtubules are essential for guard cell function, how guard cells sense the environmental stimuli and highly control the exact temporal and spatial patterns of microtubule is the fundamental question. Microtubule-associated proteins (MAPs) bind and regulate microtubules directly, however, whether and how they modulate the specific microtubule dynamics during stomatal movement remain largely unknown. Our previous work showed that both WDL3 and WDL5, two microtubule regulatory proteins of WVD2/WDL family found widely throughout the plant kingdom , participate in ABA-induced stomatal closure and Ca2+ fluxes across guard cells. Here, we focus on WDL3 and WDL5 of guard cell to elucidate the detailed molecular mechanisms underlying the regulation of microtubule behaviors by WDL3 and WDL5. WDL3 RNAi、wdl5 and YFP-TUA5 Arabidoposis would be used. With the aids of several biological approaches, such as stomatal aperture analysis, confocal laser scanning microscopy, point mutation, and non-invasive micro-test technology, we try to reveal the role of WDL3 and WDL5 in light, dark or ethylene-triggered stomatal signaling pathways and highlight their potential contributions to the regulatory mechanism of stomatal movement.

气孔运动调节植物光合、蒸腾两大生理功能，进而影响水分的利用和产量形成，因此，气孔运动调控机制的研究一直是大家关注的热点。保卫细胞微管骨架在气孔运动信息传递中具有重要作用，严格调控其空间组织排布和动态变化是精细调节气孔开度的关键。作为微管动态行为最直接的调节者，微管结合蛋白的作用不容忽视。我们前期工作发现，植物界保守的微管结合蛋白WDL3和WDL5参与ABA诱导的气孔关闭及Ca2+流动。在此基础上，本项目综合植物生理学、药理学、细胞生物学和分子遗传学等技术，以拟南芥WDL3 RNAi、wdl5(T-DNA插入)以及YFP-TUA5等为材料，进一步解析WDL3和WDL5在光、暗、乙烯等诱导的气孔运动信息传递中，对微管动态变化的调节及作用方式。从微管结合蛋白WDL3和WDL5的角度上，进一步完善气孔运动信号转导的分子调节机制。

气孔是植物与外界进行水分和CO2交换的门户，对全球水循环和碳-氧平衡以及作物产量有着重要的意义，因此，气孔运动机理的研究一直是植物生物学关注的热点。保卫细胞微管骨架动态变化在气孔运动信息传递中具有重要作用，但其进一步的分子调控机制尚待深入研究。微管结合蛋白是微管动态组装最直接的调控者，本项目以微管结合蛋白WVD2/WDL(WAVE-DAMPENED2-LIKE, WDLs)家族成员WDL3、WDL5为切入点，从叶片的失水率、气孔开度、保卫细胞微管骨架动态排布以及Ca2+、H2O2跨膜流动等不同角度，探究在脱落酸(abscisic acid, ABA)、乙烯诱导的气孔关闭信号通路中，微管结合蛋白WDL3和WDL5、微管骨架及Ca2+等信号分子之间的功能关系。结果发现，1）微管结合蛋白WDL3参与了ABA诱导的气孔关闭运动。该信号通路中, 上游Ca2+通过WDL3调节下游微管骨架的动态特征(保卫细胞微管整体的组织重排、微管密度、微管成束能力)进行信息传递；同时WDL3反过来也可调节保卫细胞内外Ca2+流动, 降低Ca2+内流, 从而抑制ABA引起的气孔关闭, 最终调控叶片的蒸腾失水。2）WDL5基因在保卫细胞内表达，并受乙烯信号上调。WDL5可以通过调节保卫细胞微管解聚聚合的动态转换速率，影响乙烯信号下微管的动态组织重排，参与乙烯诱导的气孔关闭，控制叶片蒸腾失水。并且该过程与保卫细胞内H2O2的含量变化存在一定的正负反馈关系。本项目的研究结果深入了解和阐释了气孔运动机制， 拓展了植物微管结合蛋白WDL3、WDL5在植物中的生理功能，为农业生产提供合理而可靠的理论依据。