Ethylene mediates brassinosteroid‐induced stomatal closure via Gα protein‐activated hydrogen peroxide and nitric oxide production in Arabidopsis

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 (H₂O₂) and nitric oxide (NO) production. EBR initiated a marked rise in ethylene, H₂O₂ 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 H₂O₂ and NO production. EBR‐triggered H₂O₂ 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 H₂O₂ 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 H₂O₂ 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 H₂O₂ production is required for NO synthesis. EBR failed to induce NO synthesis in mutant AtrbohF, but it led to H₂O₂ production in mutant Nia1‐2. Exogenously applied SNP rescued the defect of AtrbohF in EBR‐induced stomatal closure, but H₂O₂ 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 H₂O₂ production and subsequent Nia1‐catalyzed NO accumulation, and finally closes stomata.