A study on regulation of stomatal movements by the ROP2-RIC7 pathway

Abstract

Stomata regulate gas exchange between plants and atmosphere, optimize photosynthetic CO2 fixation, and minimize transpirational water loss. Stomata move rapidly to adjust plants to the ever-changing environment. Stomatal movement is affected by environmental and plants internal conditions, such as light and circadian clock signals, atmospheric CO2 levels, humidity, temperature, plant hormone abscisic acid (ABA), and pathogens. Stomatal opening and closing movements are manifested by guard cell volume changes, which involve membrane trafficking via endocytosis and exocytosis, ion accumulation, net water flux, and reorganization of actin cytoskeleton. Important signaling molecules that regulate these cellular responses include cytosolic Ca2+, protein kinases and phosphatases, reactive oxygen species, heterotrimeric G proteins, and ROP small G proteins. ROP GTPases (Rho-related GTPases from plants) belong to a distinct subfamily of the RHO GTPases and function as molecular switches during growth and development. Arabidopsis has 11 ROP GTPases important for pollen tube growth, seedling development, leaf epidermal pavement cell morphogenesis and lateral root initiation. There is emerging evidence that ROP GTPases negatively regulate the stomatal movements. ROP2 has been reported as negative regulator of stomatal movements because it inhibited the light-induced stomatal opening and ABA-induced stomatal closure. ROP GTPases are expected to have a number of interacting partners to mediate diverse plant responses. ROP-interactive CRIB motif-containing proteins (RICs) act as direct targets of ROP GTPases. RIC7 is a downstream target of ROP2 and functions in ROP2-mediated light-induced stomatal opening because RIC7 interacts with active-ROP2 in vitro and RIC7 overexpression via bombardment inhibits light-induced stomatal opening in V. faba, similarly as active-ROP2 overexpression does. The aim of this study is to identify RIC7 function in Arabidopsis and how ROP2-RIC7 negatively regulates stomatal movements. To understand the mechanism by which RIC7 regulates light-induced stomatal opening, I analyzed the stomatal responses of ric7 mutant Arabidopsis plants. Light-induced stomatal opening was promoted by ric7 knockout, whereas it was inhibited by RIC7 overexpression, indicating that RIC7 negatively regulates stomatal opening in Arabidopsis. Next, to decipher the mechanism of RIC7 function in stomatal movement, I searched for binding partners of RIC7 using yeast two-hybrid screen. I identified Exo70B1, a component of the exocyst complex that mediates vesicle tethering during exocytosis, as an interaction partner of RIC7, and confirmed this finding using a pull-down assay and bimolecular fluorescence complementation analysis. In V. faba guard cells, Exo70B1 was present mainly in cytoplasm under darkness, but not to the Golgi, TGN, or early endosome, while RIC7 was mostly present in the nucleus. By contrast, upon irradiation with white light, Exo70B1 and RIC7 were translocated and co-localized to the plasma membrane region. Moreover, localization of Exo70B1 in guard cells was largely overlapped with that of Exo84B, another subunit of the exocyst complex. Exo70B1 is highly expressed in Arabidopsis guard cells and Exo70B1 knockout Arabidopsis was delayed in light-induced stomatal opening and the Exo70B1 complementation lines recovered the retarded stomatal opening phenotype. These results indicate Exo70B1 is a positive regulator of light-induced stomatal opening. Furthermore, Light-induced stomatal opening was slower in RIC7/Exo70B1 double-knockout Arabidopsis than in the wild type, indicating Exo70B1 is a downstream target of RIC7. Together, I conclude that active ROP2 and RIC7 negatively regulate light-induced stomatal opening by inhibiting Exo70B1. This study revealed ROP2 and RIC7 suppress excess stomatal opening by inhibiting Exo70B1, which positively regulates stomatal opening. When recruited by active ROP2 to the plasma membrane region, RIC7 inhibits Exo70B1 function. Exo70B1 is the first member of the Exo70 family shown to be involved in stomatal movement. Moreover, Exo70B1 is a novel target of the small G protein ROP2-RIC7 signaling pathway, which modulates the kinetics of stomatal movement.