National Engineering Research Center of Tree Breeding and Ecological Restoration, College of Biological Sciences and Technology, Beijing Forestry University, No. 35, Qinghua East Road, Beijing 100083, China.
Int J Mol Sci. 2022 Aug 12;23(16):9002. doi: 10.3390/ijms23169002.
Plants regulate stomatal mobility to limit water loss and improve pathogen resistance. Ethyl vinyl ketone (evk) is referred to as a reactive electrophilic substance (RES). In this paper, we found that evk can mediate stomatal closure and that evk-induced stomatal closure by increasing guard cell K efflux. To investigate the role of eATP, and HO in evk-regulated K efflux, we used Arabidopsis wild-type (WT), mutant lines of , , and . Non-invasive micro-test technology (NMT) data showed that evk-induced K efflux was diminished in , , mutant, which means eATP and HO work upstream of evk-induced K efflux. According to the eATP content assay, evk stimulated eATP production mainly by MRP4. In and mutant groups and the ABC transporter inhibitor glibenclamide (Gli)-pretreated group, evk-regulated stomatal closure and eATP buildup were diminished, especially in the group. According to qRT-PCR and eATP concentration results, evk regulates both relative gene expressions of and eATP concentration in and WT group. According to the confocal data, evk-induced HO production was lower in , mutants, which implied that eATP works upstream of HO. Moreover, NADPH-dependent HO burst is regulated by DORN1. A yeast two-hybrid assay, firefly luciferase complementation imaging assay, bimolecular fluorescence complementation assay, and pulldown assay showed that the interaction between DORN1 and RBOHF can be realized, which means DORN1 may control HO burst by regulating RBOHF through interaction. This study reveals that evk-induced stomatal closure requires MRP4-dependent eATP accumulation and subsequent HO accumulation to regulate K efflux.
植物通过调节气孔运动来限制水分流失并提高对病原体的抗性。乙烯基酮（EVK）被称为反应性亲电物质（RES）。在本文中，我们发现 EVK 可以介导气孔关闭，并且 EVK 通过增加保卫细胞 K+外排来诱导气孔关闭。为了研究 eATP 和 HO 在 EVK 调节的 K+外排中的作用，我们使用了拟南芥野生型（WT）、突变体 、 、 。非侵入性微测试技术（NMT）数据表明，EVK 诱导的 K+外排在 、 、 突变体中减少，这意味着 eATP 和 HO 在 EVK 诱导的 K+外排的上游起作用。根据 eATP 含量测定，EVK 主要通过 MRP4 刺激 eATP 的产生。在 、 突变体组和 ABC 转运蛋白抑制剂格列本脲（Gli）预处理组中，EVK 调节的气孔关闭和 eATP 积累减少，尤其是在 组中。根据 qRT-PCR 和 eATP 浓度结果，EVK 调节 相对基因表达和 WT 组中的 eATP 浓度。根据共聚焦数据，EVK 诱导的 HO 产生在 、 突变体中较低，这表明 eATP 在 HO 的上游起作用。此外，NADPH 依赖性 HO 爆发受 DORN1 调控。酵母双杂交试验、萤火虫荧光素酶互补成像试验、双分子荧光互补试验和拉下试验表明，DORN1 和 RBOHF 之间的相互作用可以实现，这意味着 DORN1 可以通过与 RBOHF 的相互作用来控制 HO 爆发。本研究揭示了 EVK 诱导的气孔关闭需要 MRP4 依赖性 eATP 积累和随后的 HO 积累来调节 K+外排。
