The Mechanism Of Receptor-like Kinase FLS2 Regulating ABA And Flg22-induced Stomatal Closure In Arabidopsis

Stomata function as a passage for CO2 assimilation and transpiration,playing important roles not only during plant growth and development but also in response to environmental stress.Stomatal movement is finely regulated by both biotic factors(such as pathogen invasion)and abiotic factors(such as drought stress and light).For example,plant hormone ABA and the converved peptide Flg22 of bacterial flagellin could induce the stomatal closure effectively.So far,some progress has been made in the field of somatal closure induced by ABA and Flg22 signaling.OST1,GHR1 and CPKs are able to regulate the anion channel acticity of SLAC1,which is the key downstream target in ABA signaling.Some reports also showed that Flg22 promotes stomatal closure by provoking the anion current of SLAC1.However,the molecular mechanism to explain Flg22-mediated SLAC1 activation is largely unknown.The crosstalk between the ABA and Flg22 signaling in guard cell still need to be inverstigated.In this study,by screening about 300 T-DNA insertion RLK mutants,we find that fls2 mutant is sensitive to drought treatment.The excised-leaf water loss rate of fls2 is faster than that of wild type,and its leaf temperature is lower.The stomata response of fls2 mutant is impaired to ABA treatment.The transcription level of FLS2 gene was induced by ABA.These results suggest that FLS2 is likely to be involved in ABA-induced stomatal closure.LCI,Co-IP and Pull-down assay indicate that FLS2 as well as its co-receptor BAK1,is able to interact with the anion channel SLAC1.Using mass spectrometry analysis and in vitro phosphorylation assay,we find that FLS2 is able to phosphorylate Ser120,Ser113 and Ser86,and BAK1 is able to phosphorylate Ser59,Ser86 and Thr142 in the N-terminal of SLAC1(N-SLAC1).These biochemical results indicate that SLAC1 may be a substrate protein of FLS2 and BAK1.Flg22-induced FLS2-BAK1 complex can activate the anion current of SLAC1 in oocytes.The Ser59 and Ser86 of N termini are important for SLAC1 activation mediated by FLS2-Flg22-BAK1.BIK1 is able to phosphorylate both the N termini and C termini of SLAC1 in intro.However,electrophysiological experiments show that SLAC1 activation induced by FLS2-Flg22-BAK1 seems to be independent of BIK1.Genetic analysis showed that ABA-and Flg22-induced stomatal closure was blocked in fls2 bakl and fls2 slacl double mutant.The In-gel assay indicates that OST1 is activated by Flg22 treatment in Arabidopsis,and this process is partly dependent on BAK1,but not on FLS2.While FLS2 mutation shifts Flg22-activated OST1 and Flg22-induced OST1 and BAK1 interaction to an early time point.OST1-activated SLAC1 can be enhanced by BAK1 in oocytes.ABI1,the negative regulator of ABA signaling,interacts with FLS2 and inhibits FLS2-mediated N-SLAC1 phosphorylation.In conclusion,this study indicates that FLS2 is the checkpoint between ABA and Flg22 signaling interaction in guard cell.The molecular mechanism of FLS2 involved in ABA-and Flg22-induced stomatal closure has been preliminarily revealed.Flg22-induced FLS2-BAK1 complex and Flg22-activated OST1 directly regulate SLAC1,promoting stomatal closure during pathogen invasion.Under drought stress,ABA releases ABI1-mediated FLS2 inhibition,and the activated FLS2 probably regulates stomatal movement by acting on SLAC1.This work is helpful to deeply understand the sophisticated signal network formed by biotic and abiotic stress in Arabidopsis guard cells.