Molecular Mechanism Of Jasmonates Promoting Stomatal Development

Stomata are important plant epidermal structures responsible for absorbing carbon dioxide and evaporating water.Proper epidermal stomatal density and distribution are essential for plants to adapt to complex environments.Initiation of the stomatal development,proliferation and differentiation of stomatal lineage precursor cells are under rigid regulation of three closely related bHLH transcription factors,SPCH,MUTE and FAMA.The expression and function of SPCH/MUTE/FAMA are also under the complex regulation of cell-cell signaling pathway.In this pathway,extracellular ligands from EPFs(Epidermal Patterning Factors)family are perceived by membrane-located receptors from ERs(Erects)family and their co-receptor TMM(Two Many Mouth).The extracellular signals are then transduced into cytoplasm and passed down through the MAPK(Mitogen Activated Protein Kinase)cascade.MAPK cascade downstream components MPKs directly phosphorylate SPCH and regulate its function.The hormone Jasnomates participate in the modulation of stomatal development.Our previous work demonstrated that low concentration of JA inhibits the initiation of stomatal development,which depends on the function of transcription factors MYC2/3/4 in JA signaling pathway.To further analyze the molecular mechanism of JA on stomatal development,different concentrations of exogenous MeJA were used for treatment of wild type and stomatal developmental mutants.Combined with gene expression analysis by qRT-PCR,yeast two hybrid,BiFC(Bimolecular Fluorescence Complementation),and transient gene expression in tobacco,the mechanism of JA on stomatal development was studied.The main results are as follows:1.High concentration of exogenous MeJA promotes stomatal development,while low concentration of MeJA inhibits stomatal development.2.High concentrations of MeJA could not increase the stomatal density and index of the mutant spch/mute/fama/yda-2,indicating that the promotion of MeJA on stomatal development is dependent on the function of YDA and its downstream bHLH transcriptional factors and JA functions through targeting components upstream of YDA in stomatal development signaling pathway.3.High concentration of MeJA could not increase the stomatal density and index of the mutant er2-1,tmml and epf1/2,excluding the possibility of TMM-ERs and EPF1/2 as the target(s)of MeJA.Components like other members from EPFs family like STOMAGEN might be the targets.4.Analysis of EPFs genes expression indicated that low concentration of MeJA inhibited while high concentration of MeJA promoted the transcription of STOMAGEN.5.Expression of STOMAGEN was not regulated by MYC2/3/4,because the mRNA level of STOMAGEN in myc234 and 35S:MYC2 is comparable with that of wild type.6.Interaction between JAZ1/9 and FAMA was found in the experiment aiming to screening the interaction proteins of JAZs function in stomatal development using yeast two hybrid system.Their interactions were further proved by Bimolecular Fluorescence Hybridization.7.Expression of STOMAGEN in fama-1 and 35S:JAZ1 was downregulated,which indicates FAMA and JAZ1 participate in the regulation of STOMA GEN expression.8.Transient gene expression analysis in tobacco cells confirmed that FAMA activate the expression of marker pSTOMAGEN:GFP,so does the ICE1,a protein interacting with FAMA.On the contrary,JAZ1 and JAZ9 can inhibit the expression of marker pSTOMAGEN:GFP,and antagonize the promotion effect of both FAMA and ICE1 on STOMAGEN expression.Based on these results,a model about JA function in stomatal development through modulating the expression of STOMAGEN was proposed.In this model,FAMA and ICE1 positively while JAZ1 and JAZ9 negatively regulate the expression of STOMAGEN.JAZ1 and JAZ9 can form protein complexes with FAMA and ICE1.JA fine-tunes the expression level of STOMAGEN through modulating the constituent of the JAZs-FAMA-ICE1 protein complexes.Under normal condition with low levels of JA,JAZ1/9 and FAMA/ICE1 maintain the relatively low level expression of STOMAGEN.When JA levels is high,JAZ1 and JAZ9 are rapidly degradated and released from the protein complex,thus their antagonistic effect on FAMA and ICE1 and de-repression of STOMGAN expression are released.In return,high level ofSTOMAGEN expression leads to the promotion of stomatal development.