Physiological And Molecular Mechanisms Of Stomatal Response To ROS-mediated Stress Signals

Guard cells can accurately detect environmental signals and respond by opening and closing stomata.Hydrogen peroxide(H₂O₂)production is induced in plants following exposure to a wide variety of abiotic and biotic stimuli,including salt,drought,etc.It is now clear that H₂O₂ acts as the second messenger involved in abscisic acid(ABA)signaling in the regulation of stomatal behavior. Therefore,finding the new intermediates of H₂O₂ signaling in guard cells will facilitate further insights into the role of H₂O₂ played in signal transduction of plant cells and co-ordinated whole plant to adapt to various stress.Here,many mutants in response to H₂O₂ were identified using the high-throughput infrared thermography imaging system.Genetic analysis indicated that two of these muatants,roi30 and roi45(ROS-insensitive),were two allelic recessive Arabidopsis mutants,designated roi1-1 and roi1-2,respectively.The mutation gene of roi1 was cloned by positional cloning,which encode a protein kinase(same as OST1).Expression of ROI1 occurred mainly in guard cells and vascular tissue,and the ROI1 localized in the nucleus.A large percent of the stomata of roi1-1 and roi1-2 remained open under 45℃heat stress;comparatively,the stomata of wild type plants were almost entirely closed.Prolong treatment under 45℃to 20 h,we discover that while 95%of the wild type remain alive,the death rates of roi1-1 and roi1-2 has amounted to as much as 60%and 50% separately.The over-expression of ROI1 gene in roi1-1 plants markedly improved their tolerance to heat stress.Meanwhile,we have characterized ROI1 and heat-stress transcription factors 3(HSF3) and provide evidence that ROI1 associate with HSF3 both in vitro and in vivo by using GST Pull-Down,two-hybrid,and Spilt-YFP assays.Interestingly,we further demonstrate that ROI1 has autophosphorylation capacity and kinase activation of ROI1 is inhibited by HSF3 protein in a concentration dependant manner.Contrast to roi1 mutants,HSF3 loss-of-function mutants show increased heat tolerance,and the double mutants are sensitive to heat stress.We also identify the long-distance signal from root cells as a component of guard cell signaling by using infrared camera.The stomata of Vicia faba leaves closed when plants were treated by 300 mM NaCl in the soil for 3 h,thereby accompanying the increase of temperature of leaf surface, stomatal closing and H₂O₂ increasing in roots,transpiration stream and guard cells of Vicia faba L. The solution of transpiration stream could induce stomatal closing and inhibited by AsA. Pretreatments of seedlings of Vicia faba with H₂O₂ could induce stomatal closing,inhibit the increasing of NaCl in leaves,increases the rate of K⁺/Na⁺ of leaves.Whole-cell recording showed that H₂O₂ could abolishe inward K⁺ current of guard cell induced by NaCl.These results suggest that H₂O₂ as a long-distance signal molecule could induce stomatal closing,decrease transpiration and Na⁺ accumulation,improve tolerance of seedlings of Vicia faba L.In summary,these data suggest that ROI1 is a new component in H₂O₂ signaling in response heat-shock response in Arabidopsis,and stomata could probably act as sensor after NaCl treatment to co-ordinate whole plant to adapt to salt stress through transduction of long-distance signal in Vicia faba.