The Role Of Ethylene And Heterotrimeric G Protein In SA Induction Of Stomatal Closure In Arabidopsis Thaliana And Its Relationship With H 2 O 2 And NO

Salicylic acid(SA)is a phytohormone produced by plants in response to various environmental stimulus,and it plays an essential role in plant growth and development,and it is also involved in the regulation of stomatal movement.But it is still unclear how SA regulates stomatal movement.Previous studies have shown that SA could induce the production of ethylene in Arabidopsis thaliana leaves in response to pathogens.Also,it was reported that SA,ethylene and G proteins are involved in stomatal immune response.Our previous studies have also shown that Ga is involved in the signaling transduction pathway of ethylene-induced stomatal closure in Arabidopsis,and Ga functions at the upstream of hydrogen peroxide(H2O2)and nitric oxide(NO).The above studies indicated that ethylene,G protein and the related signaling components may be involved in the signaling transduction pathway of SA-triggered stomatal movement.However,the following questions remain to be addressed:(1)How SA stimulates ethylene biosynthesis;(2)What are the functions of ethylene-signaling components,G proteins,H2O2 and NO in SA-induced stomatal closure;(3)Which sources of specific enzyme pathways for H2O2 and NO are involved in SA-induced stomatal closure.To answer the above questions,we firstly studied the effects of SA on the expression of ethylene biosynthesis key enzyme(ACC synthase,ACS)genes and ethylene production,using genetic assay,stomatal bio-assay,gas chromatography,inverted fluorescence microscope and RT-PCR(Reverse Transcription PCR).Then,we examined the effects of ethylene synthesis inhibitor aminoethoxyvinylglycine(AVG)on SA-induced stomatal closure and ethylene production levels,which further supported the roles of ethylene in SA-induced stomatal closure.Finally,by examining the SA induced stomatal closure of the mutants of ethylene signal transduction components and G protein sub-units,and the production of H2O2 and NO in guard cells,we further clarify the roles of ethylene signal transduction components,G protein and their relationships with signaling molecules H2O2 and NO in SA-induced stomatal closure in Arabidopsis.We obtained the following results and conclusions:1.The optimal concentration and treating time for SA treatment were found by the dose and time-course assay,and the maximal effects of wild-type Arabidopsis stomatal closure were achieved by treating the leaves using 100 ?M SA for 3 h.So we chose 3 h and 100 ?M SA treatment for the following experiments.2.In wild-type Arabidopsis leaves,SA significantly enhance the gene expression levels of ACC synthase ACS6 and ACS11,and ethylene production was significantly improved by SA.Also,after the treatment of SA,it only took 2 hours for the peaking of ACS6 and ACS11 gene expression and ethylene production levels,while it took 3 hours for SA-induced stomatal closure,which showed SA firstly enhance the production of ethylene,and then ethylene induce the stomatal closure.Also,AVG could significantly suppress SA-induced stomatal closure and ethylene formation,furthering suggesting that ethylene is involved in the signaling transduction pathway of SA-stimulated stomatal closure,and SA induced the production of ethylene by enhancing the gene expression levels of ACC synthase ACS6 and ACS11.3.SA and ethylene synthesis precursor 1-aminocyclopropane-l-carboxylic acid(ACC)either alone or together could induce stomatal closure of wild-type Arabidopsis as well as ethylene receptor mutants etr2,ers2 and ein4,but could not induce the stomatal closure of mutants for copper transporter ran1 as well as ethylene perception and signalling component mutants etrl,ersl,ein2,ein3 and arr2.Also,the stomatal aperture of ethylene-signalling component ctr1,which was very small in visible light,was not effected by SA and ACC treatment.These results suggest that ETR1,ERS1,RANT,EIN2,EIN3 and ARR,as positive regulators,and CTR1,as a negative regulator of ethylene responses,are all involved in ethylene mediated signaling transduction pathway of SA-stimulated stomatal closure,while the ethylene receptors ETR2,ERS2 and EIN4 are not involved.4.SA could not induce the stomatal closure and H2O2 production of NADPH oxidase mutants AtrbohD,AtrbohF and AtrbohD/F,which not only showed that H2O2 played a vital role in SA induced stomatal closure,but also further supported that AtrbohD/F-dependent H2O2 production is required for SA-induced stomatal closure.5.Exogenous SA could induce the stomatal closure and NO production of WT,but not of nitrate reductase double mutant nia1-2/nia2-5.Also the stomatal closure and NO synthesis induced by SA in a single mutant of nia1-2 or nia2-1 was partially suppressed in comparison with nia1-2/nia2-5.At the same time,nia1-2 had a smaller stomatal aperture and less NO production than nia2-1 under the treatment of SA.These results showed that both Nial and Nia2 are involved in the SA induced NO generation and stomatal closure,and Nial may be more effective than Nia2.6.SA could induce the stomatal closure of wild-type,deficient mutants of G?subunit agbl and Gy subunit aggl,agg2 and aggS3 but not for Ga subunit gpal mutant.And exogenous H2O2 and NO not only could induce the stomatal closure of Ga subunit gpal,but also rescue the defects of these mutants in SA-induced stomatal closure.Also,SA could induce H2O2 and NO synthesis in guard cell of wild-type,but not in guard cells of gpal mutant.These results showed that Ga subunit GPA1 is involved in SA-induced stomatal closure,and acts at the upstream of H2O2 and NO.However,G?subunit AGB1 and Gy subunit AGG1,AGG2 and AGG3 did not participate in SA-induced stomatal closure.7.With or without SA,exogenous H2O2 could induce the stomatal closure of copper transporter mutant ran1,but not for the ethylene perception and signalling mutants etrl,ers1,ein2,ein3 and arr2.Also SA could induce H2O2 production in the mutant of ein2,ein3 and arr2 as well as wild-type Arabidopsis,but not of ran1,etrl and ersl.These results showed that in the signal transduction pathway of SA-induced stomatal closure,ETR1,ERS1 and RAN1 act at the upstream of H2O2,but ethylene receptors ETR1 and ERS1 and ethylene signalling components EIN2,EIN3 and ARR2 mediate H2O2 signaling in guard cells.8.With or without SA,Ga subunit activator cholera toxin(CTX)could induce the stomatal closure and guard cell H2O2 production of the copper transporter mutant ran1,but not for the ethylene perception and signalling mutants etr1,ers1,ein2,ein3 and arr2.But SA could induce H2O2 production in guard cell of ran1,etr1,ers1,ein2,ein3 and arr2.Based on the relationships of ethylene signal transduction components and H2O2,these results showed that in the signal transduction pathway of SA-induced stomatal closure,Ga acts at the upstream of H2O2,EIN2,EIN3 and ARR2,and at the downstream of ETR1,ERS1 and RAN1.9.With or without SA,exogenous NO releasing agent-sodium nitroprusside(SNP)could induce the stomatal closure of the copper transporter mutant rant1,as well as the ethylene perception and signalling mutants etrl,ers1,ein2,ein3 and arr2.But SA could not induce the stomatal closure and NO production of the copper transporter mutant ran1,as well as the ethylene perception and signalling mutants etr1,ers1,ein2,ein3 and arr2.These results showed that in the signal transduction pathway of SA-induced stomatal closure,RAN1,ETR1 ERS1,EIN2,EIN3 and ARR2 act at the upstream of NO.Previous studies have shown the relationship of Ga and ethylene signal transduction components which participated in ethylene induced stomatal closure in Arabidopsis.Here,based on the previous studies and our results,we proposed a model of the SA signalling pathway in guard cells:(1)SA induced the expression of ACC synthases ACS6 and ACS11,and thus improved the ethylene synthesis;(2)Ethylene signalling in guard cells is initiated by ethylene receptors ETR1 and ERS1 as well as copper transporter RAN1 via inactivating CTR1 protein kinase,resulting in activation of Ga;(3)Activated Ga induced H2O2 production via NADPH oxidase;(4)H2O2 signalling in guard cells is initiated by ETR1 and ERS1 through EIN2,EIN3 and ARR2,which increased nitrate reductase-catalysed NO production and finally induced stomatal closure.These findings not only further unraveled the signaling transduction pathway of SA-induced stomatal closure,but also help us to further understand the relationship among SA,ethylene and G proteins signalling pathways in plants.