拟南芥CNGC5和CNGC6在ABA诱导的气孔关闭信号途径中的功能研究

Calcium plays essential roles in ABA- and drought-induced stomatal closure. ABA triggers the increases and oscillation of cytosolic calcium in guard cells in which Ca2+ influx mediated by the plasma membrane Ca2+ channels was proposed as main Ca2+ resource. Cyclic nucleotide gated ion channels(CNGCs) have been considered as the important candidates of cation channels in plant cells.Among 20 CNGC members in Arabidopsis,we find that the expression of AtCNGC5 and AtCNGC6 in guard cells are significant higher than other members，and induceable by ABA. This data suggests that AtCNGC5 and AtCNGC6 may function in ABA-induced stomatal closure as inward Ca2+ channels in Arabidopsis guard cells. In this project, we will investigate the functions of AtCNGC5 and AtCNGC6 in ABA-induced stomatal closure. Our preliminary results show that AtCNGC5 and AtCNGC6 localize in the plasma membrane of mesophyll protoplast; patch clamping experimental data shows that cGMP triggers siginificant inward Ca2+ currents in wild type Arabidopsis guard cells, and the mutations of AtCNGC5 and AtCNGC6 impair the cGMP-induced Ca2+ currents dramatically, indicating that AtCNGC5 and AtCNGC6 do have important functions in stomatal closure.

Ca2+是ABA和干旱诱导的气孔关闭信号途径中重要的信号分子。ABA能够引起拟南芥气孔保卫细胞胞质Ca2+浓度升高，其中经保卫细胞质膜Ca2+通道流入的Ca2+被认为是Ca2+浓度上升的主要来源。CNGCs被认为是质膜Ca2+通道的重要候选蛋白。我们分析发现，在拟南芥20个CNGC成员中，CNGC5和CNGC6在气孔保卫细胞中的表达量较高，而且其表达量受ABA诱导而升高,推测其可能作为Ca2+通道在ABA诱导的气孔关闭信号途径中发挥功能。本项目拟采用电生理学、分子生物学、细胞生物学等多种方法，深入研究拟南芥CNGC5和CNGC6在ABA诱导的气孔关闭信号途径中的功能。已有初步结果表明，这两个蛋白均定位在细胞质膜上；膜片钳实验结果发现，cGMP可以诱导野生型保卫细胞产生内向Ca2+电流，而在cngc5/cngc6双突变体中该Ca2+电流被显著抑制。这些结果表明该课题思路是可行的。

Ca2+是ABA和干旱诱导的气孔关闭信号途径中重要的信号分子。在干旱和ABA等诱导的气孔关闭过程中，存在气孔保卫细胞胞质膜Ca2+浓度的上升和震荡。其中，经保卫细胞质膜Ca2+通道介导的Ca2+内流和经保卫细胞Ca2+库释放的Ca2+被认为是胞质Ca2+浓度上升的主要来源。但是，编码保卫细胞质膜Ca2+通道的基因仍不清楚。通过该项目研究，我们发现拟南芥CNGC5和CNGC6在气孔保卫细胞中有较强表达，而且它们均定位于细胞质膜上。膜片钳实验结果显示在双突变体cngc5 cngc6中，cGMP激活的Ca2+电流显著减小，ABA激活的Ca2+电流略小于野生型中记录到的电流。这些结果说明CNGC5和CNGC6是介导拟南芥保卫细胞质膜cGMP激活的主要的Ca2+通道，并且作为Ca2+通道在ABA诱导的气孔关闭过程中发挥了一定的功能。