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 G 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 G 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 cG line (constitutively overexpressing the Gprotein -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 cG/AtrbohF or eto1-1/nia1-2 and cG/nia1-2 constructs had an analogous response to H2O2 or SNP as those of AtrbohF or Nia1-2 mutants. Moreover, we provided evidence that G plays an important role in the responses of guard cells to ethylene. G 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 G-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 G, and then promotes AtrbohF-dependent H2O2 production and subsequent Nia1-catalyzed NO accumulation, and finally closes stomata. Significance Statement The data presented herein suggest a working model for BR action in guard cell response in Arabidopsis,The findings confirm the effect of BR on stomatal movement, and elucidate the crucial role of ethylene and G proteins in the guard cell BR signalling.