Heterotrimeric G Protein-regulated Ion Fluxes Participated Eatp-regulated Stomotal Movement Of Arabidopsis Thaliana

Stomatal movement governs gas and water exchange between plants and environment, and stomatal regulating mechanism is an important topic in research of plant physiology. Extracellular ATP (eATP) modulates cell metabolism, growth and development as an extracellular signal molecule, by triggering intracellular signal cascades. Here, the effect of eATP on stomatal movement and its possible signal transduction pathway was investigated.Our previous results showed that eATP promoted stomatal opening in Arabidopsis, and heterotrimeric G protein alpha subunit (Gα)-regulated ROS accumulation participated in this process. However, the role of Gβand Gγin eATP-promoted stomatal movement is still unknown. And in eATP signal transduction pathway, downstream target of ROS, which will lead stomatal opening finally, is also unclear.To verify the role of Gβγand downstream targets in eATP signaling pathway, in this paper, Arabidopsis wild type, null mutants of heterotrimeric G protein, and null mutant of NADPH oxidase were used as material, epidermal strip bioassay, confocal laser scanning microscopy (CLSM), and the scanning ion-selective electrode technique (SIET) were used as main methods, the role of heterotrimeric G protein, ROS, Ca2+ and H+ fluxes in guard cells in eATP-promoted stomatal opening was investigated.In Gβand Gγnull mutants, added ATP promoted stomatal opening. The stomatal aperture became bigger than control, the reaction of stomata in mutants and wild type to ATP stimulation was not significantly different. ROS accumulated markedly in guard cells of both wild type and null mutants after ATP treatment. The results suggest that Gβand Gγwere not involved in eATP promoted ROS generation and stomatal opening.In order to detect the role of calcium and proton pump in eATP regulated stomatal movement, Ca2+ and H+ fluxes in Arabidopsis guard cells were measured by using the scanning ion-selective electrode technique. In wild type and Gαover-expression strain (wGα), eATP remarkably triggered transmembrane Ca2+ influx into guard cells. Similar results were also found in Gβand Gγnull mutants. While in Gαnull mutants, eATP only lead a slight increase of calcium influx. In NADPH oxidase null mutant, eATP induced Ca2+ influx was abolished. Indicating that eATP-induced Ca2+ influx may be involved in eATP-promoted stomatal opening. Heterotrimeric G protein and NADPH oxidase play important roles in eATP-induced calcium influx. In the three subunits, Gαparticipated in signal transduction of eATP, but Gβand Gγwere unlikely involved in this signal pathway.The results of H+ flux detection showed that eATP induced transmembrane H+ efflux in guard cells of wild type and Gαover-expression strain (wGα). In NADPH oxidase null mutant, eATP induced H+ efflux was not found. In Gαnull mutants, eATP induced H+ efflux was abolished. In Gβand Gγnull mutants, eATP induced H+ efflux was also found. Indicating that eATP promoted plasma membrane proton pump activation via ROS, which promoted H+ exclusion from guard cells, finally promoted stomatal opening. In this process, Gαmay be involved in signaling pathway. While, neither Gβnor Gγparticipated in this signal cascade.