The Role Of Heterotrimeric G Protein And ROS In EATP Regulated Stomotal Movement Of Arabidopsis Thaliana

Stomatal movement serves as an important process of water metabolic regulation in plants, and its regulative mechanism is also a main topic of plant physiology.Extracellular ATP (eATP) can function as a signaling agent in plants,and regulate plant viability,growth and stress responses.Here,we have studied the effect of eATP on stomatal movement and its possible signal transduction pathway.Here,by using Arabidopsis heterotrimeric G protein alpha subunit-null mutants and its overexpression lines,null mutant of NADPH Oxidase,null mutant of apyrase as material,and epidermal strip bioassay,CLSM,and RT-PCR as methods,the role of heterotrimeric G protein and ROS in eATP regulated stomotal movement of Arabidopsis thaliana was investigated.The stomatal aperture of Arabidopsis became bigger after eATP treatment and eATP induced increase of stomatal aperture in a dose-dependent manner.Additionally,the non-hydrolyzed ATP analogs such as BzATP,ATPγS and 2meATP,ADP and GTP could also promote stomatal opening,while AMP and adenosine was ineffective.These results suggest that eATP could induce stomatal opening as a signaling agent.Stomatal aperture became smaller after apyrase and hexokinase treatment,and the stomatal aperture of null mutant of apyrase was bigger than wild type in natural lighting environment,suggesting that endogenous eATP could also play a positive role in regulate the stomatal movement.After eATP treatment,the stomatal aperture of heterotrimeric G protein alpha subunit overexpression lines became bigger.However,in Ga null mutants,eATP-promoted stomatal opening was not found.The results indicate that heterotrimeric G protein was involved in eATP-induced stomatal opening.Stomatal aperture became bigger in response to exogenous low concentrated ROS, whereas became smaller after high concentrated ROS treatment.Diphenyleneiodonium(DPI), a suicide substrate inhibitor of NADPH oxidase,and dithiothreitol(DTT),a ROS distinguisher,suppressed the eATP-induced stomatal opening.Extracellular ATP had no effect on stomatal opening of NADPH Oxidase mutant.These data suggest that ROS generated by NADPH oxidase played an important role in eATP induce stomatal opening.By using H₂DCFDA as a fluorescent indicator of ROS,ROS in guard cells was visualized by CLSM.The result indicated that,ROS accumulated in guard cells of wild type after eATP treatment,but in NADPH Oxidase mutant,eATP induced ROS accumulation was not found.Heterotrimeric G protein alpha subunit-null mutants impaired in eATP induced ROS generation.In contrast,ROS accumulation induced by eATP was accelerated in heterotrimeric G protein alpha subunit overexpression lines.The results suggested that heterotrimeric G protein was involved in eATP induced ROS generation in guard cells.These results suggested that eATP promote stomatal opening of Arabidopsis thaliana as a extracellular messenger,heterotrimeric G protein and ROS played an important role in eATP promoted stomatal opening.