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 mutantbri1-301,and EBR failed to close the stomata ofgpa1mutants. 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 theetr1andgpa1mutants, but were strengthened in theeto1-1mutant and the cGα line (constitutively overexpressing the G protein α-subunitAtGPA1). Exogenously applied H2O2 or sodium nitroprusside (SNP) rescued the defects ofetr1-3andgpa1oretr1andgpa1mutants in EBR-induced stomatal closure, whereas the stomata ofeto1-1/AtrbohFand cGα/AtrbohForeto1-1/nia1-2and cGα/nia1-2constructs had an analogous response to H2O2 or SNP as those ofAtrbohForNia1-2mutants. 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 theetr1-3mutant, but ethylene precursor ACC failed to rescue the defects ofgpa1mutants 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 mutantAtrbohF,but it led to H2O2 production in mutantNia1-2.Exogenously applied SNP rescued the defect ofAtrbohFin EBR-induced stomatal closure, but H2O2 did not reverse the lesion of EBR-induced stomatal closure inNia1-2.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.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.