StCLE Polypeptides Regulate The Stomatal Movement Of Arabidopsis Thaliana

Accumulating data have revealed that the CLE(CLAVATA3/Embryo Surrounding Region-related)peptides are key players in modulating various plant growth and developmental processes.In addition,CLE genes have been found to perform various biological roles in response to environmental stimuli.However,the function of StCLE in stomatal development/movement remains unknown.Previously,we found that StCLE is expressed highly in different stomata cell types during various developmental stages of stomata.As a first step to investigate the role of StCLE,we studied the potential function of StCLE in the regulation of stomatal movement using a combination of epidermal strips bioassay,laser scanning confocal microscopy,GUS staining,RT-PCR and etc.Furthermore,the interactions of StCLE-mediating signaling pathway with known ABA-,NO-,and H2O2-signaling pathways were investigated.Additionally,the potential receptor(s)which perceiving the StCLE signaling were studied by testing various genetic mutants.Collectively,our results presented in this study are briefly summarized as follows:1.The expression pattern of StCLE is assessed by GUS staining of Arabidopsis transgenic plants containing the StCLE promoter-GUS fusion construct.The analysis revealed that strong GUS activity was found in meristemoid,guard mother cells and guard cells of stomata.2.The optimal concentration and time of StCLE treatment were found to be 10?M and 3h by the dose and time-course assay,we therefore use 10?M and 3h for following experiments.3.To assess the stomatal movement in response to CLE peptides,except for StCLE,the effect of CLV3(A-type CLE peptide)and CLE41/CLE46(B-type CLE peptide)on stomatal closure were examined.The results indicated that CLV3p,CLE41p and CLE46p exerted a weaker effect on the stomatal closure comparing with that of StCLE.4.To investigate the role of StCLE,we identified two T-DNA insertion mutants,stcle-10 and stcle-11,for further analysis.The expression of StCLE in the two mutants was determined by RT-PCR,which showed that the StCLE expression is not absence or substantial reduction.The T-DNAs of both mutants were found to be located in the 5'-UTR region.In addition,subsequent studies revealed that stcle-10 and stcle-11 did not exhibit any obvious phenotype inregard to stomatal closure.Collectively,these results suggested that stcle-10 and stcle-11 are not null mutants and are not useful for further functionalstudy.5.To examine whether ABA is required for the StCLE-induced stomatal closure,we firstly studied the effect of fluridone(FLU)on the StCLE-induced stomatal closure in the wild type plants.Futhermore,we investigated the StCLE-induced stomatal closure in the aba2-1 and aba2-4 mutants.The results of both experiements indicated that endogenous ABA is involved in the process.6.To identify the potential receptors for StCLE in the process of stomatal closure,the effects of StCLE peptide on stomatal closure in tmm-1,er-105,er erll,erll er12,er erll er12 were studied,respectively.The results revealed that all mutants examined exhibited no response to StCLE peptide treatment.However,ABA induced stomatal closure intmm-1,er-105,er erll,erll er12 as that of in the wild type plants.In conclusion,our results suggested that TMM and ERs,possibly forming a receptor complex,are required in the StCLE-mediated stomatal closure.7.In an early bioinformatic analysis,we found that StCLE and HT1 are highly co-expressed gene pair,which leads to a hypothesis that HT1 may participate in the StCLE-mediated stomatal movement.To test this hypothesis,we examined StCLE-induced stomatal closure in the htl-1 and htl-2 mutants.However,there were no significant differences in StCLE-induced stomatal closure between htl mutants and wild type plants,suggesting that HT1 is not involved in the StCLE-mediated stomatal closure.8.CAT and NADPH uricase inhibitor(DPI)could obviously inhibit the stomatal closure and H2O2 production in wild type Col-0 mediated by StCLE peptide.However,ASA could partly prevent wild-type stomatal closure induced by StCLE peptide.Consistently,StCLE peptide could not induce stomatal closure and H2O2 production in AtrbohD,AtrbohF and AtrbohD/F which were NADPH oxidase mutants.These results implied that H2O2 produced by NADPH oxidase is involved in the StCLE-induced stomatal closure.9.NO scavenger(c-PTIO),nitrate reductase(NR)and Na2WO4 inhibited stomatal closure and NO production induced by StCLE.However,NOS inhibitor(L-NAME)did not inhibit stomatal closure and NO production induced by StCLE peptide.In agreement with what observed above,StCLE peptide could induce stomatal closure and NO production in Nia2-1 and Atnos which were NR and NOS mutants,respectively,whereas the effect was partly inhibited in the mutants of Nia1-2 and Nia2-5/Nia1-2.In conclusion,our results therefore suggested that NO is involved in the stomatal closure induced by StCLE peptide.In summary,it was found,in the current study,that StCLE peptide induced stomatal closure,which was possibly mediated by TMM and ERs.In addition,it is likely that H2O2 and NO,as downstream components,were required for the StCLE-induced stomatal closure.We also determind that StCLE-induced stomatal closure is depend on ABA,suggesting a crosstalk between two groups of hormones.