Yunnan Academy of Tobacco Agricultural Sciences, Kunming 650021, China.
Faculty of Life Science and Technology, Kunming University of Science and Technology, 650500, Kunming, Yunnan, China.
J Exp Bot. 2023 Nov 21;74(21):6735-6748. doi: 10.1093/jxb/erad308.
Stomatal movement can be regulated by ABA signaling through synthesis of reactive oxygen species (ROS) in guard cells. By contrast, ethylene triggers the biosynthesis of antioxidant flavonols to suppress ROS accumulation and prevent ABA-induced stomatal closure; however, the underlying mechanism remains largely unknown. In this study, we isolated and characterized the tobacco (Nicotiana tabacum) R2R3-MYB transcription factor NtMYB184, which belongs to the flavonol-specific SG7 subgroup. RNAi suppression and CRISPR/Cas9 mutation (myb184) of NtMYB184 in tobacco caused down-regulation of flavonol biosynthetic genes and decreased the concentration of flavonols in the leaves. Yeast one-hybrid assays, transactivation assays, EMSAs, and ChIP-qPCR demonstrated that NtMYB184 specifically binds to the promoters of flavonol biosynthetic genes via MYBPLANT motifs. NtMYB184 regulated flavonol biosynthesis in guard cells to modulate ROS homeostasis and stomatal aperture. ABA-induced ROS production was accompanied by the suppression of NtMYB184 and flavonol biosynthesis, which may accelerate ABA-induced stomatal closure. Furthermore, ethylene stimulated NtMYB184 expression and flavonol biosynthesis to suppress ROS accumulation and curb ABA-induced stomatal closure. In myb184, however, neither the flavonol and ROS concentrations nor the stomatal aperture varied between the ABA and ABA+ethylene treatments, indicating that NtMYB184 was indispensable for the antagonism between ethylene and ABA via regulating flavonol and ROS concentrations in the guard cells.
气孔运动可以通过 ABA 信号转导在保卫细胞中合成活性氧（ROS）来调节。相比之下，乙烯触发抗氧化类黄酮的生物合成，以抑制 ROS 积累并防止 ABA 诱导的气孔关闭；然而，其潜在机制在很大程度上仍然未知。在这项研究中，我们分离并鉴定了烟草（Nicotiana tabacum）R2R3-MYB 转录因子 NtMYB184，它属于类黄酮特异性 SG7 亚群。烟草中 NtMYB184 的 RNAi 抑制和 CRISPR/Cas9 突变（myb184）导致类黄酮生物合成基因下调和叶片中类黄酮浓度降低。酵母单杂交测定、转录激活测定、EMSA 和 ChIP-qPCR 表明，NtMYB184 通过 MYBPLANT 基序特异性结合到类黄酮生物合成基因的启动子上。NtMYB184 调节保卫细胞中的类黄酮生物合成以调节 ROS 平衡和气孔孔径。ABA 诱导的 ROS 产生伴随着 NtMYB184 和类黄酮生物合成的抑制，这可能加速 ABA 诱导的气孔关闭。此外，乙烯刺激 NtMYB184 的表达和类黄酮生物合成以抑制 ROS 积累并抑制 ABA 诱导的气孔关闭。然而，在 myb184 中，ABA 和 ABA+乙烯处理之间，类黄酮和 ROS 浓度以及气孔孔径都没有变化，表明 NtMYB184 通过调节保卫细胞中的类黄酮和 ROS 浓度，对于乙烯和 ABA 之间的拮抗作用是不可或缺的。
