The Molecular Mechanism Investigation Of WRKY1 In Plant Drought Response

The complicated plant signaling network is essential for plant to survive in the changing environment and thus finish its life cycle,and abiotic stresses,such as drought,are key restrains for both plant growth and development.Plants have been evolved delicate mechanisms in responses to these abiotic stresses,but what are these mechanisms and how can they work are mostly still under undiscovered.The research is about the mechanism dealing with the stress which may give a way to solve the problem.The plant has a complex system with many factors to cope with abiotic stress.ABA(Abscisic acid)is the most influential phytohormone involved in the response to drought stress.One role of ABA is regulation of stomatal movement.Stomata as a transpiration channel play important roles in abiotic stress with water limited.ABA is known to inhibit the inward rectifying K+(K+in)channels in the guard cell plasma membrane,thereby inhibiting influx;the result is a brake on stomatal opening.ABA also activates various anion channels,which act to encourage solute efflux and thus to hasten stomatal closure.The mean values of stomatal aperture under external ABA are measured to evaluate the performance of plant under abiotic stress with water limited.In the study,based on the data of guard cell proteome from A.thaliana,a large number of mutants were screened by ABA regulation stomatal movement experiment.At last,a mutant with stomatal response more sensitive to ABA than wild type was found.A new function of gene deletion in the mutant have been revealed by exploring the mechanism of drought and ABA response.The main research contents and results are as follows:1.Screening of mutants and the traits of wrkylThe mutant with a T-DNA insertion on the 5'-UTR of WRKY1 is coded SALK₀16954.The insertion leads that function of WRKY1 is loss in the mutant.The mutant wrkyl is less sensitive to drought and more sensitive to ABA in the stomatal movement than wild type.Smaller stomatal aperture may lead lower water loss of wrkyl in the detached leaf experiment.Complementation of the wrky1 mutant resulted in the recovery of the wild type of phenotype.All this support that the loss of WRKY1 can lead to highly sensitive of guard cells to ABA.The promoter of WRKY1 has been cloned for constructing pWRKY1::GUS.GUS staining shows that WRKY1 can express in lots of tissues such as seedlings,the cauline leaf,the root,the inflorescence and the guard cells.It consists with the data of gene chip online(http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi Electronic Fluorescent Protein Browser(eFP)).RT-PCR also shows that WRKY1 transcription in various parts of Arabidopsis thaliana.WRKY1 is gradual reduction in transcript abundance in response to ABA and drought treatments in one hour,however the transcript abundance returns to baseline after 3 hours.The results indicates WRKY1 may mainly play roles in early stage of drought and ABA treatment.2.Electrophysiology and function analysis of WRKY1 transcription factor.Patch clamp was used to record the currents of a whole guard cell from A.thaliana.In the absence of ABA,there was no significant variation for currents between wrkyl and WT(wild type),but ABA have more strong effect to currents in wrkyl than WT.The result consists with the performance of stomatal movement in wrky1 under ABA treatment.WRKY1 functions as a negative regulatory element in ABA-mediated regulation of both K+in and anion channel activity in the guard cells.WRKY1 is a transcription factor belong to WRKY family in A.thaliana.The WRKY1 GFP fusion(p35S::WRKY1-GFP)was transformed into mesophyll protoplasts,the fusion protein was deposited exclusively in the nucleus in which transcription factor regulates transcription of genes.The sequence of WRKY1 with two conserved W-domain is a marker of group I members in WRKY family.The yeastbased assay demonstrated that two W-domain is essential for WRKY1's transcriptional activity.WRKY1 have specially binding element called W-box in promotor of gene regulated by it.Many promotors of genes own the element.Four genes(MYB2,ABI5,DREB1A and ABCG40)responded differentially in wrky1 compared to WT.ABCG40,DREB1A and MYB2 are more strongly transcribed in wrky1.However,transcription of ABI5 is decreased in wrky1.Their promotors own W-box element which may be bound by WRKY1.The application of EMSA showed that WRKY1 was able to bind to promotors of the four genes in vitro.ChIP-based qRT-PCR confirmed that WRKY1 bound directly to the genes' promoter in vivo as well.In summary,the guard cells of wrky1 mutant have highly sensitive to ABA for the loss of WRKY1.All of experiments support that WRKY1 negatively regulates stomatal movement in response to ABA treatment.The small stomatal aperture under drought stress can explain the more tolerance in mutant than WT.Four genes about ABA and response to drought are regulated by WRKY1,which play roles in later stage under drought stress.The overall conclusion is that WRKY1 has a negative role in guard cell trans-membrane ion trafficking and stomatal movement in response to ABA,and thus provokes the plant to surrender the drought.Gene ZmNRGA1 of maize was also heterologously expressed in A.thaliana,which belongs to pyruvate transporter.The phenotypic analysis of heterologous expression further proved that pyruvate transporter is related to drought resistance in A.thaliana.