The Role Of MPK Cascade Pathway In Ethylene-induced Stomatal Closure In Arabidopsis

Ethylene is a simple gaseous plant hormone,which is involved in the regulation of plant growth and development,and response to various biotic and abiotic stresses in the external environment,it plays an indispensable role in many aspects of physiological activities of plants.Studies have shown that ethylene participates in guard cells signal transduction and has dual effects on stomatal movement,which can not only induce stomatal closure or mediate stomatal closure induced by some stimulants such as ultraviolet B(UV-B),salicylic acid and brassinolide,but also inhibit stomatal closure induced by some stimulants such as ABA and darkness.Mitogen-activated protein kinase(MPK)pathway is a conserved signal transduction pathway in eukaryotes,plants can regulate various cellular signal transduction processes by activating specific MPK cascades.Studies have shown that MPK cascades play important roles in stomatal movements induced by many stimulants,however,it is unclear whether MPK cascade is also involved in ethylene-induced stomatal closure,if it is involved,what are the specific components in MPK cascade?What are the relationships between MPK cascade and H2O2,NO,and ethylene signal elements?These questions are not clear at the moment.In order to explore the above questions,we used the Arabidopsis thaliana as the material in this study,combined with genetics,molecular biology,cell biology,biochemistry and other methods and techniques,tried to clarify the mechanism of MPK cascade pathway in ethylene-induced Arabidopsis stomatal closure,and the relationship between MPK cascade pathway and guard cell signal molecules H2O2,NO and ethylene signal elements.The main experimental results and conclusions obtained in this paper are as follows:1.100 ?M 1-aminocyclopropane-l-carboxylic acid(ACC,precursors of ethylene biosynthesis)can induce stomatal closure and activation of MPK3 and MPK6 in wildtype Arabidopsis,and the time courses of the ACC-induced MPK3/6 activation were consistent with that of ACC-induced stomatal closure.ACC-induced stomatal closure were partially inhibited in the leaves of mpk3 and mpk6 single mutant,and this effect was abolished entirely in 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine(NA-PP1)-sensitized conditional loss-of-function MPK3 and MPK6 double mutant MPK3SR and MPK6SR.These results demonstrate that both MPK3 and MPK6 were involved and positively regulate ethylene-induced stomatal closure.2.ACC-induced stomatal closure and activation of MPK3 and MPK6 were partially deficiency in mkkl and mkk3 single mutant,while complete failure in mkk1/3 double mutant;overexpression of constitutively activated MPK6(MPK6CA)in Arabidopsis wildtype and double mutant mkk1/3(MPK6CA/WT and MPK6CA/mkk1/3)induced constitutive stomatal closure with or without ACC treatment.These results indicate that both MKK1 and MKK3 have positive roles in regulating ethylene-induced stomatal closure by controlling the activation of MPK3 and MPK6 in Arabidopsis.3.The results of yeast two hybrid system(Y2H),bimolecular fluorescence complementation(BiFC)and pull-down experiments showed that MKK1 and MKK3 could interact with MPK3 and mpk6 in vivo and in vitro,respectively,indicating that MKK1/3 and MPK3/6 formed a MPK cascade.4.100 ?M ACC treatment could significantly induce production of H2O2 in guard cells of Arabidopsis wild-type,mpk3,mpk6,mkk1,mkk3 single mutant,mkkl/3 double mutant,NA-PP1-pretreated conditional loss-of-function MPK3/6 double mutant MPK3SR and MPK6SR,as well as transgenic lines of MPK6CA/WT and MPK6CA/mkk1/3,meanwhile,the activation of MPK3 and MPK6 induced by ACC was significantly inhibited by NADPH-oxidase single mutant AtrbohF and double mutant AtrbohD/F,but not by single mutant AtrbohD.The results indicate that MKK1/3-MPK3/6 cascade functioned downstream of ATRBOHF-dependent H2O2 production in ethylene-induced stomatal closure.5.ACC-induced NO production in guard cells was significantly inhibited in mpk3,mpk6,mkk1,mkk3 single mutant,mkk1/3 double mutant,and complete inhibition in NAPP 1-pretreated conditional loss-of-function MPK3/6 double mutants of MPK3SR and MPK6SR,while transgenic lines of MPK6CA/WT and MPK6CA/mkk1/3 showed constitutively NO production with or without ACC treatment;in addition,the nitrate reductase mutants nia1 and nia1/2,which blocked ethylene-induced NO generation,did not affect ACC-induced MPK3 and MPK6 activation.These results suggest that MKK1/3-MPK3/6 cascade acted upstream of NIA1-dependent NO during ethyleneinduced stomatal closure.6.Compared with Arabidopsis wild-type,the activation of MPK3 and MPK6 induced by ACC was significantly inhibited in etr1 mutants,but not in ein2 and ein3 mutants;and ctr1 mutant showed constitutive activation of MPK3 and MPK6;the phenotype of triple mutant of ctr1mkk1/3 was consistent with that of mkk1/3,which reversed the phenotype of constitutive NO production and stomatal closure of ctr1;and EIN3-overexpression transgenic lines of mkk1/3(35S:EIN3/mkk1/3)showed constitutive endogenous NO production and stomatal closure in guard cells with or without ACC treatment.These results indicate that MKK1/3-MPK3/6 cascade acted downstream of ETR1 and CTR1,and upstream of EIN2 and EIN3 in ethylene-induced stomatal closure.7.Y2H,BiFC and pull-down showed that MPK3 and MPK6 could interact with EIN2 C-terminal(EIN2C/EIN2CEND),respectively;overexpressing EIN2-GFP(GFP fused to the C terminus of full-length EIN2)and GFP-EIN2(GFP fused to the N terminus of full-length EIN2)in Arabidopsis wild-type and MKK1/3-MPK3/6 cascade mutants,respectively.ACC treatment could induce the aggregation of GFP signals in the nuclei of guard cells and hypocotyl epidermal cells of transgenic plants overexpressing EIN2-GFP in the wild-type,however,this phenomenon was not observed in the guard cells and hypocotyl epidermal cells of transgenic plants overexpressing GFP-EIN2 in wild-type or overexpressing EIN2-GFP in MKK1/3-MPK3/6 cascade mutants.These results suggest that the MKK1/3-MPK3/6 cascade promoted ethylene-induced cleavage and nuclear translocation of EIN2C by interacting with EIN2.8.ACC could significantly induce the accumulation of EIN3 protein in Arabidopsis wild-type,but this effect was significantly inhibited in MKK1/3-MPK3/6 cascade mutants,while the ctr1 mutant showed constitutively accumulation of EIN3 protein.These results indicate that MKK1/3-MPK3/6 cascade mediated ethylene-induced EIN3 protein accumulation.In summary,our data provided compelling evidence for the essential role of the MKK1/3-MPK3/6 cascade in ethylene-induced stomatal closure in Arabidopsis,and establish a potential model of the ethylene-signaling pathway in guard cells,which as follows:the binding of ethylene to ETR1 leads to the inactivation of CTR1,which results in H2O2 production from NADPH-oxidase AtRBOHF;H2O2 in guard cells activates the MKK1/3-MPK3/6 cascade,which promotes the cleavage and nuclear translocation of EIN2C,and further facilitate EIN3 protein accumulation;thus EIN3 finally induces NIA1-dependent NO production in guard cells and results in stomatal closure.