School of Life Sciences, Shaanxi Normal University, Xi'an, 710062, China; School of Chemistry and Bioengineering, Hechi University, Yizhou, 546300, China.
Plant J. 2015 Apr;82(2):280-301. doi: 10.1111/tpj.12815.
Brassinosteroids (BRs) are essential for plant growth and development; however, whether and how they promote stomatal closure is not fully clear. In this study, we report that 24-epibrassinolide (EBR), a bioactive BR, induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering a signal transduction pathway including ethylene synthesis, the activation of Gα protein, and hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) production. EBR initiated a marked rise in ethylene, H(2)O(2) and NO levels, necessary for stomatal closure in the wild type. These effects were abolished in mutant bri1-301, and EBR failed to close the stomata of gpa1 mutants. Next, we found that both ethylene and Gα mediate the inductive effects of EBR on H(2)O(2) and NO production. EBR-triggered H(2)O(2) and NO accumulation were canceled in the etr1 and gpa1 mutants, but were strengthened in the eto1-1 mutant and the cGα line (constitutively overexpressing the G protein α-subunit AtGPA1). Exogenously applied H(2)O(2) or sodium nitroprusside (SNP) rescued the defects of etr1-3 and gpa1 or etr1 and gpa1 mutants in EBR-induced stomatal closure, whereas the stomata of eto1-1/AtrbohF and cGα/AtrbohF or eto1-1/nia1-2 and cGα/nia1-2 constructs had an analogous response to H(2)O(2) or SNP as those of AtrbohF or Nia1-2 mutants. Moreover, we provided evidence that Gα plays an important role in the responses of guard cells to ethylene. Gα activator CTX largely restored the lesion of the etr1-3 mutant, but ethylene precursor ACC failed to rescue the defects of gpa1 mutants in EBR-induced stomatal closure. Lastly, we demonstrated that Gα-activated H(2)O(2) production is required for NO synthesis. EBR failed to induce NO synthesis in mutant AtrbohF, but it led to H(2)O(2) production in mutant Nia1-2. Exogenously applied SNP rescued the defect of AtrbohF in EBR-induced stomatal closure, but H(2)O(2) did not reverse the lesion of EBR-induced stomatal closure in Nia1-2. Together, our results strongly suggest a signaling pathway in which EBR induces ethylene synthesis, thereby activating Gα, and then promotes AtrbohF-dependent H(2)O(2) production and subsequent Nia1-catalyzed NO accumulation, and finally closes stomata.
油菜素内酯（BRs）对植物的生长和发育至关重要；然而，它们是否以及如何促进气孔关闭尚不完全清楚。在这项研究中，我们报告了 24-表油菜素内酯（EBR），一种生物活性 BR，通过触发包括乙烯合成、Gα 蛋白的激活以及过氧化氢（H₂O₂）和一氧化氮（NO）产生的信号转导途径诱导拟南芥（Arabidopsis thaliana）气孔关闭。EBR 在野生型中引发了乙烯、H₂O₂和 NO 水平的显著升高，这是气孔关闭所必需的。这些效应在 bri1-301 突变体中被消除，并且 EBR 未能关闭 gpa1 突变体的气孔。接下来，我们发现乙烯和 Gα 都介导了 EBR 对 H₂O₂和 NO 产生的诱导作用。在 etr1 和 gpa1 突变体中，EBR 触发的 H₂O₂和 NO 积累被取消，但在 eto1-1 突变体和 cGα 系（组成型过表达 G 蛋白 α 亚基 AtGPA1）中增强。外源 H₂O₂或硝普酸钠（SNP）挽救了 etr1-3 和 gpa1 或 etr1 和 gpa1 突变体中 EBR 诱导的气孔关闭缺陷，而 eto1-1/AtrbohF 和 cGα/AtrbohF 或 eto1-1/nia1-2 和 cGα/nia1-2 构建体对 H₂O₂或 SNP 的反应类似于 AtrbohF 或 Nia1-2 突变体。此外，我们提供了证据表明 Gα 在保卫细胞对乙烯的反应中起重要作用。CTX（Gα 激活剂）在很大程度上恢复了 etr1-3 突变体的损伤，但乙烯前体 ACC 未能挽救 gpa1 突变体中 EBR 诱导的气孔关闭缺陷。最后，我们证明了 Gα 激活的 H₂O₂ 产生对于 NO 合成是必需的。EBR 未能诱导突变体 AtrbohF 中的 NO 合成，但导致突变体 Nia1-2 中 H₂O₂ 的产生。外源 SNP 挽救了 AtrbohF 在 EBR 诱导的气孔关闭中的缺陷，但 H₂O₂ 并没有逆转 Nia1-2 中 EBR 诱导的气孔关闭的损伤。总之，我们的结果强烈表明，油菜素内酯通过诱导乙烯合成，从而激活 Gα，然后促进 AtrbohF 依赖性 H₂O₂ 产生和随后的 Nia1 催化的 NO 积累，最终关闭气孔，这是一条信号通路。
