Oh Airi, Kimura Riku, Inoue Shinpei, Sato Taiyo, Hayashi Yuki, Sato Ayato, Takahashi Yohei, Kinoshita Toshinori
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan.
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Plant Cell Physiol. 2025 Jul 24;66(6):854-865. doi: 10.1093/pcp/pcaf013.
Plants control their stomatal apertures to optimize carbon dioxide uptake and water loss. Stomata open in response to light through the phosphorylation of the penultimate residue, Thr, of plasma membrane (PM) H+-ATPase in guard cells. Stomata close in response to drought and the phytohormone abscisic acid (ABA), and ABA suppresses the light-induced activation of PM H+-ATPase. However, the signaling pathways that regulate the stomatal aperture remain unclear. Previously, we identified a target of rapamycin (TOR) inhibitor, temsirolimus, to induce stomatal opening through chemical screening. In the present study, we further investigated other TOR inhibitors and identified PP242 as a novel stomatal opening chemical. PP242 induced stomatal opening even in the dark, as well as phosphorylation of the penultimate Thr of PM H+-ATPase in guard cells. Interestingly, PP242 completely suppressed ABA-induced stomatal closure, and inhibited ABA-induced activation of SNF1-related protein kinase 2s (SnRK2s), which are essential kinases for ABA signal transduction in guard cells. In vitro biochemical analysis revealed that PP242 did not directly inhibit SnRK2 but rather inhibited upstream ABA-signaling components, specifically B3 clade Raf-like kinases. A quadruple mutant of B3 clade Raf-like kinases exhibited an open stoma phenotype that resembled the effect of PP242. However, PP242 still induced stomatal opening in this mutant, suggesting that PP242 also targets other guard cell components. Together, these results reveal that PP242 induces stomatal opening partly by inhibiting steady-state ABA signal transduction.
植物通过控制气孔孔径来优化二氧化碳吸收和水分流失。气孔响应光照而开放，这一过程通过保卫细胞中质膜（PM）H⁺-ATPase倒数第二个残基苏氨酸（Thr）的磷酸化来实现。气孔响应干旱和植物激素脱落酸（ABA）而关闭，并且ABA会抑制光照诱导的PM H⁺-ATPase的激活。然而，调节气孔孔径的信号通路仍不清楚。此前，我们通过化学筛选鉴定出一种雷帕霉素靶蛋白（TOR）抑制剂坦西莫司可诱导气孔开放。在本研究中，我们进一步研究了其他TOR抑制剂，并确定PP242是一种新型的诱导气孔开放的化学物质。PP242即使在黑暗中也能诱导气孔开放，并且能使保卫细胞中PM H⁺-ATPase的倒数第二个Thr发生磷酸化。有趣的是，PP242完全抑制了ABA诱导的气孔关闭，并抑制了ABA诱导的SNF1相关蛋白激酶2（SnRK2s）的激活，而SnRK2s是保卫细胞中ABA信号转导所必需的激酶。体外生化分析表明，PP242并不直接抑制SnRK2，而是抑制上游ABA信号成分，特别是B3亚家族类Raf激酶。B3亚家族类Raf激酶的四突变体表现出类似于PP242作用效果的气孔开放表型。然而，PP242在该突变体中仍能诱导气孔开放，这表明PP242还靶向其他保卫细胞成分。总之，这些结果表明PP242部分通过抑制稳态ABA信号转导来诱导气孔开放。
