Heterotrimeric G proteins function as key players in hydrogen peroxide (H2O2) production in plant cells, but whether G proteins mediate ethylene-induced H2O2 production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H2O2 in guard cell ethylene signalling. In wild-type leaves, ethylene-triggered H2O2 synthesis and stomatal closure were dependent on activation of Gα.GPA1mutants showed the defect of ethylene-induced H2O2 production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H2O2 production in response to ethylene. Ethylene-triggered H2O2 generation and stomatal closure were impaired inRAN1,ETR1,ERS1andEIN4mutants but not impaired inETR2andERS2mutants. Gα activator and H2O2 rescued the defect ofRAN1andEIN4mutants oretr1-3in ethylene-induced H2O2 production and stomatal closure, but only rescued the defect ofERS1mutants oretr1-1andetr1-9in ethylene-induced H2O2 production. Stomata ofCTR1mutants showed constitutive H2O2 production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata ofEIN2,EIN3andARR2mutants did not close in responses to ethylene, Gα activator or H2O2, but do generate H2O2 following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene-induced stomatal closure via H2O2 production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H2O2 signalling in guard cells.Significance Statement
This paper provided genetic and physiological evidence to show that heterotrimeric G protein α subunit mediates ethylene-induced stomatal closure via modulating NADPH oxidase-dependent H2O2 synthesis and works downstream of RAN1, ETR1, ERS1, EIN4 and CTR1, while H2O2 signalling in guard cells is initiated by ETR1 and ERS1 through EIN2, EIN3 and ARR2-dependent pathway(s). These findings will help us to further understand the ethylene, G proteins and H2O2 signalling pathways in plants.