The G Protein beta-Subunit, AGB1, Interacts with FERONIA in RALF1-Regulated Stomatal Movement

Heterotrimeric guanine nucleotide-binding (G) proteins are composed of G alpha, G beta, and G gamma subunits, and function as molecular switches in signal transduction. In Arabidopsis thaliana there are one canonical Ga (GPA1), three extra-large G alpha (XLG1, XLG2 and XLG3), one G beta (AGB1) and three G gamma (AGG1, AGG2 and AGG3) subunits. To elucidate AGB1 molecular signaling, we performed immunoprecipitation using plasma membrane enriched proteins followed by mass spectrometry to identify the protein interactors of AGB1. After eliminating proteins present in the control immunoprecipitation, commonly identified contaminants, and organellar proteins, a total of 103 candidate AGB1-associated proteins were confidently identified. We identified all of the G protein subunits except XLG1, receptor-like kinases (RLKs), Ca2+ signaling related proteins and 14-3-3-like proteins, all of which may couple with or modulate G protein signaling. We confirmed physical interaction between AGB1 and the RLK FERONIA (FER) using bimolecular fluorescence complementation (BiFC). The RALF family of polypeptides have been shown to be ligands of FER. In this study, we demonstrate that RALF1 regulates stomatal apertures, and does so in a G protein-dependent manner, inhibiting stomatal opening and promoting stomatal closure in Col but not agb1 mutants. We further show that AGGs and XLGs, but not GPA1, participate in RALF1-mediated stomatal signaling. Our results suggest that FER acts as a G-protein coupled receptor for plant heterotrimeric G proteins.