Multiple cyclic nucleotide-gated channels (CNGCs) are abscisic acid (ABA)-activated Ca2+ channels in Arabidopsis (Arabidopsis thaliana) guard cells. In particular, CNGC5, CNGC6, CNGC9, and CNGC12 are essential for ABA-specific cytosolic Ca2+ signaling and stomatal movements. However, the mechanisms underlying ABA-mediated regulation of CNGCs and Ca2+ signaling are still unknown. In this study, we identified the Ca2+-independent protein kinase OPEN STOMATA1 (OST1) as a CNGC activator in Arabidopsis. OST1-targeted phosphorylation sites were identified in CNGC5, CNGC6, CNGC9 and CNGC12. These CNGCs were strongly inhibited by Ser-to-Ala mutations and fully activated by Ser-to-Asp mutations at the OST1-targeted sites. The overexpression of individual inactive CNGCs (iCNGCs) under the UBIQUITIN10 promoter in wild-type Arabidopsis conferred a strong dominant-negative-like ABA-insensitive stomatal closure phenotype. In contrast, expressing active CNGCs (aCNGCs) under their respective native promoters in the cngc5-1 cngc6-2 cngc9-1 cngc12-1 quadruple mutant fully restored ABA-activated cytosolic Ca2+ oscillations and Ca2+ currents in guard cells, and rescued the ABA-insensitive stomatal movement mutant phenotypes. Thus, we uncovered that ABA elicits cytosolic Ca2+ signaling via an OST1–CNGC module, in which OST1 functions as a convergence point of the Ca2+-dependent and -independent pathways in Arabidopsis guard cells.

中文翻译：

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拟南芥中 OPEN STOMATA1 磷酸化环状核苷酸门控通道，触发 Ca2+ 信号，导致 ABA 诱导气孔关闭

多环核苷酸门控通道 (CNGC) 是拟南芥 (Arabidopsis thaliana) 保卫细胞中脱落酸 (ABA) 激活的 Ca2+ 通道。特别是，CNGC5、CNGC6、CNGC9 和 CNGC12 对于 ABA 特异性胞质 Ca2+ 信号传导和气孔运动至关重要。然而，ABA 介导的 CNGC 和 Ca2+ 信号传导调节的机制仍不清楚。在这项研究中，我们确定了 Ca2+ 独立蛋白激酶 OPEN STOMATA1 (OST1) 作为拟南芥中的 CNGC 激活剂。在 CNGC5、CNGC6、CNGC9 和 CNGC12 中鉴定了 OST1 靶向磷酸化位点。这些 CNGC 受到 OST1 靶位点 Ser-to-Ala 突变的强烈抑制，并被 Ser-to-Asp 突变完全激活。在野生型拟南芥中，UBIQUITIN10 启动子下个体失活 CNGC（iCNGC）的过表达赋予了强烈的显性失活样 ABA 不敏感气孔关闭表型。相比之下，在 cngc5-1 cngc6-2 cngc9-1 cngc12-1 四重突变体中各自的天然启动子下表达活性 CNGC（aCNGC）完全恢复了 ABA 激活的细胞质 Ca2+ 振荡和保卫细胞中的 Ca2+ 电流，并拯救了 ABA-气孔运动不敏感突变表型。因此，我们发现 ABA 通过 OST1-CNGC 模块引发胞质 Ca2+ 信号传导，其中 OST1 充当拟南芥保卫细胞中 Ca2+ 依赖性和非依赖性途径的汇聚点。