ABA激活的拟南芥气孔保卫细胞质膜Ca2+通道的鉴定和活性调控机理研究

Drought stress and ABA closes stomata by triggering cytosolic Ca2+ increases and oscillation, which are mainly resulted from external Ca2+ influx mediated by ABA-activated Ca2+ channels in the plasma membrane(PM) of guard cells in higher plants. However, the genetic identities of the ABA-activated PM Ca2+ channels as well as the regulationg mechanism of the Ca2+ channel activity are still largely unknown. Several CNGCs(cyclic nucleotide gated channels) have been characterized as Ca2+ permeable channels. Therefore, CNGCs have been proposed as important candidates of the ABA-activated PM Ca2+ channels in Arabidopsis guard cells. We generated a loss-of-function CNGC6 by modifying a few essential pore forming resdues, and named it as CNGC6D. We generated dominant negative transgenic Arabidopsis lines named as CNGC6DN by overexpressing CNGC6D in Arabidopsis guard cells. We found that the CNGC6DN lines showed a strong hypersensitive phenotype to drought stress, and the stomatal closure is strongly insensitive to ABA. These data indicate that the ABA-activated PM Ca2+ channels are encoded by CNGCs. This project will clarify the genetic identities of ABA-activated PM Ca2+ channels in Arabidopsis guard cells be generating multiple cngc mutants combining the application of plant molecular genetic and electrophysiological techniques, and other techniques, and further explore the regulating mechanims of the ABA-activated PM Ca2+ channel activity in Arabidopsis guard cells.

干旱胁迫和ABA通过激活气孔保卫细胞质膜内向Ca2+通道引起胞外Ca2+内流和胞内Ca2+升高和震荡，进一步引起气孔关闭。但该质膜Ca2+通道的遗传身份和活性调控机理至今依然很不清楚。多个拟南芥CNGC已经被鉴定为Ca2+通道。因此，CNGCs被认为是植物细胞质膜Ca2+通道的重要候选蛋白。申请人课题组通过人为修饰的方法获得了离子通道功能受到严重削弱的CNGC6，将其在拟南芥气孔保卫细胞中过表达，获得了CNGC6的DN（dominant negative）转基因植株。该DN植株具有对干旱胁迫超敏感的明显表型，且气孔关闭运动对ABA不敏感。这些初步结果说明，ABA激活的气孔保卫细胞质膜Ca2+通道确实由CNGC家族成员组成。本课题拟循着该研究线索，通过构建多突变体的方法，结合多种研究手段，真正搞清楚ABA激活的Ca2+通道的遗传身份，并深入解析该Ca2+通道活性调控的分子机理。

气孔是植物吸收CO2、散失水分和释放氧气的主要渠道，围绕气孔的保卫细胞是植物感知外界环境刺激并通过气孔开闭而做出反应的重要器官。其中干旱和ABA通过诱导气孔关闭而减少水分散失，从而提高植物的耐旱能力。近些年来，来自多个国家的多个课题组的研究，成功解析出一条不依赖于细胞质Ca2+信号的ABA信号转导通路。另一方面，2000年前后的多篇经典研究论文反复确证了细胞质Ca2+信号参与ABA信号的传递，在ABA和干旱诱导的气孔关闭运动中发挥积极作用。但Ca2+依赖性信号通路的组成至今不清楚。其中，鉴定ABA激活的质膜Ca2+通道是解决该科学问题的关键步骤之一。本项目针对这一科学问题，在过去的几年中做了大量的研究，取得了很好的研究进展。我们成功分离出了一株具有对ABA不敏感的突变体，克隆了相关基因，并对其编码蛋白的功能进行了分析。研究发现，该蛋白为典型的质膜Ca2+选择性和通透性离子通道蛋白，在拟南芥气孔保卫细胞和其他多个组织均有分布。进一步的研究，发现该突变体细胞质Ca2+信号异常。这些结果说明该蛋白为ABA下游的关键组分，负责Ca2+信号的产生和调控。