硫化氢参与保卫细胞离子通道及脱落酸信号调控的研究

Stomata are formed by a pair of guard cells and through which plant loss the majority of water by transpiration. Plants close their stomata in response to drought stress. Hydrogen sulfide (H2S) has been recognized as the third gasotransmitter after nitric oxide and carbon monoxide, it has adverse functions proved by many studies conducting in mammalian cells. Recently it was found that H2S has function in regulation of stomatal movement, however, the mechanisms remain largely unclear. The project presented here mainly focus on how exogenous as well endogenous H2S regulation on slow anion channel SLAC1, potassium channel and calcium channel in the plasma membranne of guard cells. Moreover, whether endogenous H2S is involved in abscisic acid regulation of guard cell ion channels as well as how H2S regulation of the cytosolic calcium and hydrogen peroxide levels will be studied. We also investigate how H2S regulation of SLAC1 activities with coexpression with protein kinase OST1 in Xenopus oocyte using two-electrod voltage clamp technique. Combined with eletrophysiology, biochemical, molecular biology and genetic methods, we will reveal the mechanisms of how H2S perticipate in guard cell signaling to regulate stomatal movement. Identifying new molecules in the regulation of stomatal movements and new componants to increase water use efficiency would have potential application for improving plant drought tolerance.

气孔由一对保卫细胞包围形成，是植物散失水分的主要通道。植物遭受干旱胁迫会诱导气孔关闭。硫化氢(H2S)被认为是继一氧化氮和一氧化碳之后的第三种气体信号分子，有关动物和医学研究发现H2S具有多种重要生理功能。最近发现植物中H2S对气孔开闭具有调节作用，然而其调节机制并不清楚。本项目主要通过遗传、生理和生化等研究方法探讨外源和内源H2S对保卫细胞质膜阴离子通道SLAC1、钾通道和钙通道的活性调节机制，以及H2S对保卫细胞脱落酸信号、胞质钙和过氧化氢信号的影响。将阴离子通道SLAC1和蛋白激酶OST1在爪蟾卵母细胞表达，利用双电极电压钳技术研究H2S对SLAC1的激活调控机理。通过上述研究阐明H2S诱导气孔关闭的分子机制。挖掘参与调控气孔运动的关键信号分子，探讨植物节水机制，对基础研究和利用分子技术提高植物抗旱性的应用研究都具有重要意义。

硫化氢（H2S）在动物中已被认为是一种重要的信号分子，植物中H2S的功能近年来也逐渐被揭示。气孔保卫细胞响应外界环境调节气孔开度，影响植物吸收二氧化碳进行光合作用，同时通过蒸腾作用散失水分。激素脱落酸（ABA）对气孔保卫细胞的信号调控已经比较清楚，而H2S对保卫细胞的信号调控，尤其是否以及如何参与ABA信号过程并不清楚。本项目在H2S调控保卫细胞离子通道方面，在前期发现H2S可以激活保卫细胞慢型阴离子通道电流基础上，我们还发现硫化氢能够抑制保卫细胞内向钾通道电流。在非洲爪蟾卵母细胞中发现，硫化氢可以增强蛋白激酶OST1对阴离子通道SLAC1的激活作用。在内源H2S与ABA信号调控方面，发现硫化氢合成酶突变体des1对ABA诱导的气孔关闭不敏感。另外，des1的叶片温度低于野生型，进一步研究发现ABA可显著提高DES1转录水平，另一方面还促进DES1的酶活。除了课题组前期发现的OST1参与硫化氢对气孔运动的调控之外，还发现CPK6也参与其中，实验发现H2S不能引起突变体cpk6保卫细胞内过氧化氢（H2O2）、一氧化氮（NO）和[Ca2+]cyt的升高，说明CPK6参与了H2S对保卫细胞的信号调控。通过本项目的实施，我们发现了经典的保卫细胞ABA信号分子如Ca2+、H2O2、NO，以及蛋白激酶OST1、CPK6等分子均参与了H2S调控的保卫细胞信号中，同时DES1也参与了ABA的信号。我们的结果阐述了保卫细胞中H2S和ABA信号的“纠缠”和“对话”机制，促进了保卫细胞信号转导的研究进展。