The Role Of AtMEK1-AtMPK6 Signaling Cascade In Dark-induced Arabidopsis Stomatal Closure And Its Relationship With H₂O₂ And NO

Previous studies have shown that both hydrogen peroxide(H2O2)and nitric oxide(NO)acting as signaling molecules participate in dark-induced stomatal closure,and pharmacological data also suggest the involvement of mitogen-activated protein kinase(MPK)cascades in the dark-induced stomatal closure.However,it is still not clear which specific MPK signaling cascades are involved in dark-induced stomatal closure,and how the MPK signaling cascades interact with H2O2 and NO in darkness quard cell signaling.AtMEKl-AtMPK6 signaling pathway has been proved to mediate ABA-regulated stomatal closure,suggesting that this MPK cascade maybe plays an important role in guard cell signaling triggered by multiple stimuli.Therefore,this study employed leaves of Arabidopsis(Arabidopsis thaliana)wild type and mutants as experimental materials and used methods of stmatal bioassay and laser-scanning cofocal microscopy to mainly investigate in the dark-triggered guard cell signaling pathways:(1)the role of AtMEKl-AtMPK6 pathway;(2)the enzymatic sources of H2O2 and NO and their interrelationship;(3)the interaction of AtMEK1-AtMPK6 cascade and H2O2 and(4)the relationship between AtMEKl-AtMPK6 cascade and NO.The following main results were obtained:1.When leaves were exposed to darkness for 2.5 h,stoma closed significantly,indicating that 2.5 h is the optimal time of stomatal closure under darkness in Arabidopsis leaves.Thus,darkness treatment for 2.5 h was chosen for subsequent researches.2.Dark-induced stomatal closure was significantly inhibited by PD98059,a specific inhibitor of MEKs,suggesting that MEKs are involved in dark-induced stomatal closure.Further genetic analysis showed that homozygous T-DNA inserted mutants for both AtMEK1 and AtMPK6 were impaired in dark-induced stomatal closure,indicating that AtMEK1-AtMPK6 pathway mediates dark-induced stomatal closure in Arabidopsis.3.Dark-induced H2O2 production in guard cells and subsequent stomatal closure in wild-type Arabidopsis were significantly inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride(DPI)but not by the cell wall peroxidase inhibitor salicylhydroxamic acid(SHAM).Furthermore,the NADPH oxidase gene null mutants AtrbohF and AtrbohD/AtrbohF were also defective in dark-induced H2O2 generation and stomatal closure.These results not only indicate that H2O2 is involved in the signal transduction pathway of dark-induced stomatal closure in Arabidopsis leaves,but also further show that dark-induced H2O2 generation in guard cells is via NADPH oxidase AtrbohF,but not via cell wall peroxidase.Meanwhile,stomata of single mutant Atnoal and Nial and double mutant Nia1/Nia2 failed to produce NO and close upon darkness treatment,resembling the response of the wild type pretreated with the NO synthase(NOS)inhibitor NG-nitro-L-arginine methyl ester(L-NAME)or the nitrate reductase(NR)inhibitor tungstate,whereas the single mutant Nia2 responded normally to the darkness like the wild type.These data not only confirm the involvement of NO in dark-induced stomatal closure,but also indicate that NO induced by darkness in guard cells is originated from the enzymatic sources of NOS and Nial.These results are consistent with previous results.4.Exogenous H2O2-induced stomatal closure was abolished in Atnoal,nial and nial/nia2 under either light or darkness,while H2O2 generation in guard cells of these mutants was induced by darkness.The results indicate that blocking NO formation does not affect dark-induced H2O2 formation in guard cells.In other words,the generation of H2O2 does not depend on the formation of NO.However,under light or darkness,NO-releasing compound sodium nitroprusside(SNP)could induce stomatal closure in AtrbohF and AtrbohD/F,and these mutants impaired in NO production under darkness.These results show that blocking H2O2 formation affects dark-induced NO formation in guard cells.Together,the results indicate that NO functions downstream of H2O2 during the dark-induced signal transduction pathway in guard cells.5.Exogenous H2O2 could not induce stomatal closure in null mutants of AtMEKl and AtMPK6 under either light or darkness,while darkness induced H2O2 generation in guard cells of these mutants,as wild type did.These results demonstrate that H2O2 functions in the upstream of AtMEK1 and AtMPK6 in the signal transduction pathway of dark-induced stomatal closure.6.NO donor SNP could not only induce stomatal closure in mutants of AtMEKl and AtMPK6 under light,but also rescue the defect of these mutants in the dark-induced stomatal closure.Furthermore,dark-induced NO production was abolished in these mutants.These results indicate that NO acts downstream of AtMEKl and AtMPK6 in the signal transduction pathway of dark-induced stomatal closure in Arabidopsis.In a word,the results of this study not only show that dark-induced stomatal closure is mediated by the AtMEKl-AtMPK6 signaling cascade,the NADPH oxidase AtrobhF-sourced H2O2 and NO originated from both nial and nos1,but also indicates that the AtMEK1-AtMPK6 signaling cascade works downstream of H2O2 and upstream of NO in the darkness-triggered signaling pathways in guard cells of Arabidopsis leaves.