Li Xue, Wang Lixiao, Zhang Meixiang, He Junmin, An Yuyan
College of Life Sciences, Shaanxi Normal University, Xi'an, China.
Physiol Plant. 2025 Mar-Apr;177(2):e70196. doi: 10.1111/ppl.70196.
Calcium-dependent protein kinases (CPKs) play crucial roles in plant guard cell signal transduction. Ethylene is known to induce stomatal closure, with the hydrogen peroxide (HO)-nitric oxide (NO) signalling module being pivotal to this process. However, the specific roles of CPKs in this process and their interactions with HO and NO remain unclear. In this study, we screened Arabidopsis mutants of nine CPKs and found that in the loss-of-function mutants for CPK3, CPK4, CPK6, CPK11, CPK21, and CPK33, exogenous ethylene failed to induce stomatal closure, indicating that these CPKs act as positive regulators in ethylene-induced stomatal closure. Mutants' stomatal responses to HO and NO treatment and changes of endogenous HO and NO levels in guard cells upon ethylene treatment indicated that CPK3, CPK4, CPK11, and CPK33 function upstream of the HO-NO module, while CPK6 and CPK21 act downstream. Furthermore, NADPH oxidases play critical roles in ethylene-induced HO production. We identified the interactions of CPK3, CPK4, and CPK11 with AtRBOHF, and CPK4 and CPK11 with AtRBOHD using four different assays, and exogenous ethylene enhanced these interactions. These results suggest that CPK3, CPK4, and CPK11 may mediate ethylene-induced HO formation in guard cells through their interactions with AtRBOHD/F. Additionally, exogenous ethylene significantly upregulates the expression of CPK3, CPK4, CPK6, CPK11 and CPK21, providing a potential mechanism by which ethylene modulates CPKs. Our findings not only establish the role of CPKs in ethylene guard cell signalling but also offer insights into the mechanism by which ethylene activates NADPH oxidases to initiate HO production.
钙依赖蛋白激酶（CPKs）在植物保卫细胞信号转导中起关键作用。已知乙烯可诱导气孔关闭，而过氧化氢（HO）-一氧化氮（NO）信号模块对这一过程至关重要。然而，CPKs在这一过程中的具体作用及其与HO和NO的相互作用仍不清楚。在本研究中，我们筛选了9个CPKs的拟南芥突变体，发现CPK3、CPK4、CPK6、CPK11、CPK21和CPK33的功能缺失突变体中，外源乙烯未能诱导气孔关闭，表明这些CPKs在乙烯诱导的气孔关闭中起正向调节作用。突变体气孔对HO和NO处理的反应以及乙烯处理后保卫细胞内源性HO和NO水平的变化表明，CPK3、CPK4、CPK11和CPK33在HO-NO模块的上游发挥作用，而CPK6和CPK21在下游发挥作用。此外，NADPH氧化酶在乙烯诱导的HO产生中起关键作用。我们使用四种不同的检测方法确定了CPK3、CPK4和CPK11与AtRBOHF以及CPK4和CPK11与AtRBOHD的相互作用，外源乙烯增强了这些相互作用。这些结果表明，CPK3、CPK4和CPK11可能通过与AtRBOHD/F的相互作用介导保卫细胞中乙烯诱导的HO形成。此外，外源乙烯显著上调CPK3、CPK4、CPK6、CPK11和CPK21的表达，这为乙烯调节CPKs提供了一种潜在机制。我们的研究结果不仅确立了CPKs在乙烯保卫细胞信号传导中的作用，还为乙烯激活NADPH氧化酶启动HO产生机制提供了见解。
