Ethylene,heterotrimeric G proteins,hydrogen peroxide(H2O2)and nitric oxide(NO)all function as key players in plant responses to developmental and environmental signals,but their interrelationships are not clear.In this study,the role and interrelationship of Gα protein,H2O2,NO and ethylene signaling elements in ethyleneinduced stomatal closure were studied in Arabidopsis.In wild-type leaves,ethyleneinduced H2O2 and NO generation in guard cells and subsequent stomatal closure were dependent on activation of Gα.Mutants for Gα subunit gene GPA1 showed the defect of ethylene-induced H2O2 and NO production in guard cells and stomatal closure,whereas wGα and cGα overexpression lines showed faster stomatal closure,H2O2 and NO production in response to ethylene.Ethylene-triggered generation of H2O2 and NO in guard cells and subsequent stomatal closure were impaired in RAN1,ETR1,ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants.Gα activator and H2O2 rescued the defect of RAN1 and EIN4 mutants or etr1-3 in ethylene-induced H2O2 and NO production and stomatal closure,but only rescued the defect of ERS1 mutants or etr1-1 and etr1-9 in ethylene-induced H2O2 production,while NO rescued the defect of all of the above mutants in ethylene-induced stomatal closure.Stomata of EIN2,EIN3 and ARR2 mutants did not close in responses to ethylene,Gα activator or H2O2,but closed in response to NO,and generated H2O2 but not NO following challenge of ethylene,Gα activator or H2O2.Stomata of CTR1 mutants showed constitutive H2O2 and NO production and stomatal closure,but which could be abolished by Gα inhibitor.The data indicate that Gα of the heterotrimeric G proteins participates in ethylene induced stomatal closure in Arabidopsis and functions through inducing H2O2 and NO production in guard cells.The data also indicate that ethylene signaling in guard cells is initiated by ethylene receptors ETR1,ERS1 and EIN4 as well as copper transporter RAN1 via inactivating CTR1 protein kinase and then relieving Gα-dependent H2O2 production,while H2O2 signaling in guard cells is initiated by ETR1 and ERS1 through EIN2,EIN3 and ARR2-dependent pathway(s)to induce NO generation in guard cells and subsequent stomatal closure.These findings will help us to further understand the ethylene,G proteins,H2O2 and NO signaling pathways in plants.