The Role Of CDPKs In Ethylene-induced Stomatal Closure In Arabidopsis And Its Relationship With H₂O₂ And NO

Calcium-dependent protein kinases(CDPKs)are sensors as well as the second messenger of Ca2+ in plant cell signal transduction,they are the direct "sensors and responders" to decode Ca2+ signal in the process of plant growth and development,and response to external stimulation.There are 34 kinds of CDPKs in Arabidopsis thaliana.Previous studies have shown that a variety of CDPKs are involved in signal transduction of stomatal movement regulated by many biological and abiotic stimuli,including ABA,salicylic acid(SA),jasmonic acid(MeJA)and pathogen elicitors.Exogenous ethylene can not only induce stomatal closure,but also mediate other hormones-induced stomatal closure,such as salicylic acid and brassinolide;however,ethylene inhibits ABA-induced stomatal closure.Previous studies have shown that in the process of ethylene induced stomatal closure in Arabidopsis,the generation of hydrogen peroxide(H2O2)from NADPH oxidase AtRBOHF pathway is activated,followed by the enrichment of nitric oxide(NO)from nitrate reductase NiAl pathway,then resulted in stomatal closure.However,the role of CDPKs in ethylene-induced stomatal closure and its relationship with H2O2 and NO in Arabidopsis remain unclear.In order to explore the above issues,we used Arabidopsis thaliana and mutants of CDPKs as material in this paper,and studied the interaction between CDPKs and AtRBOHD/F in ethylene-induced stomatal closure via yeast two hybrid and complementation of two molecule fluorescence assays,the main experimental results and corresponding conclusions are as follows:1.Exogenous ethylene treatment could induce stomatal closure of Arabidopsis wild type and mutants of cpk10,cpk23 and cpk30,but did not do that in mutants of cpk3,cpk4,cpk6,cpk11,cpk21 and cpk33.These results indicate that CDPK3,CDPK4,CDPK6,CDPK11,CDPK21 and CDPK33 are positive regulators involved in ethylene-induced stomatal closure in Arabidopsis thaliana.2.Exogenous H2O2 treatment could induce stomatal closure in wild type and in mutants of cpk3,cpk4,cpk11 and cpk33,and partially or completely reverse the ethylene-induced stomatal closure of these mutants;however,exogenous H2O2 treatment could not induce stomatal closure in mutants of cpk6 and cpk21,nor reverse the ethylene-induced stomatal closure of these two mutants.Exogenous ethylene treatment could induce endogenous H2O2 production in guard cells of Arabidopsis wild type,cpk6 and cpk21,but could not induce H2O2 production in guard cells of cpk3,cpk4,cpk11 and cpk33.These results suggest that CDPK3,CDPK4,CDPK11 and CDPK33 act upstream of H2O2,and CDPK6 and CDPK21 act downstream of H2O2 in ethylene induced stomatal closure in Arabidopsis.3.SNP treatment can induce the stomatal closure in wild type and in mutants of cpk3,cpk4,cpk11 and cpk33,and partially or completely reverse the ethylene-induced stomatal closure of these mutants;however,the treatment of exogenous SNP could not induce the stomatal closure in mutants of cpk6 and cpk21,nor reverse the ethylene-induced stomatal closure of these two mutants.Exogenous ethylene treatment could induce endogenous NO production in guard cells of Arabidopsis wild type,cpk6 and cpk21,but could not induce NO production in guard cells of cpk3,cpk4,cpk11 and cpk33.The results show that CDPK3,CDPK4,CDPK11 and CDPK33 act upstream of NO,while CDPK6 and CDPK21 act downstream of NO in the signal pathway of ethylene-induced stomatal closure in Arabidopsis.4.The results of yeast two hybrid and two molecule fluorescence complementation experiments showed that CDPK3/4/11 could interact with AtRBOHF,and CDPK4/11 could also interact with AtRBOHD,but CDPK33 could not interact with AtRBOHD/F,suggesting that CDPK3/4/11 mediated ethylene induced H2O2 formation in guard cells,and these two NADPH oxidases might be phosphorylated and activated through the physical interaction between these kinases and AtRBOHD/F.