Studying On The Function Of WRKLY12 In Response To Iron Deficiency In Arabidopsis Thaliana

Iron(Fe)is an essential micronutrient in plants.It plays an important role in many activities of life,such as photosynthesis and respiration.Even though the content of iron in soil is high,iron usually exists in the form of stable insoluble iron complex in neutral and alkaline soil,which leads to the decrease of plant iron absorption capacity and inhibits its growth and development,thus reducing crop yield and nutritional quality.Therefore,it is of great theoretical significance and potential application value to discover and identify new genes that regulate the response of plants to iron deficiency stress and to study their mechanism of action.In this study,we identified WRKY12,a transcription factor involved in the regulation of iron deficiency response in Arabidopsis thaliana,and studied its possible mechanism.The main results are as follows,(1)The results of iron deficiency induced WRKY12 expression and Pro WRKY12 staining showed that WRKY12 expression was up-regulated after iron deficiency induction,and GUS staining showed that after iron deficiency induction WRKY12 expression was up-regulated in Arabidopsis roots,indicating that WRKY12 gene may be involved in the regulation of plant iron deficiency response.(2)The picture of wrky12 mutant plants concerned that there was no distinction between wrky12 mutant and WT plants in normal environment,but wrky12 mutant plants were more tolerant to iron stress than WT plants,indicating that WRKY12 gene negatively regulated the response to iron deficiency stress.(3)Further phenotypic analysis of 35S:WRKY12 overexpression plants displayed that there was no inequality between the phenotypes of 35S:WRKY12 overexpression and wild type under normal conditions,but 35S:WRKY12 overexpression was more sensitive than wild type under iron deficiency.(4)Analysis of iron content in roots and stems of wild type,wrky12 mutant and 35S:WRKY12 overexpression plants showed that under normal conditions,there was no significant difference in iron content in roots or stems of wrky12 mutant,35S:WRKY12 overexpression plants and wild type plants,but under iron deficiency stress,iron content in roots and stems of wrky12 mutant plants was significantly higher than that of wild type plants,while that of 35S:WRKY12 overexpression plants was significantly higher than that of wild type plants The iron content in the neutral stem was significantly lower than that in the root and stem of the wild type.(5)The expression of FIT,IRT1,FRO2,bHLH38 and bHLH39 genes in wrky12 mutant was analyzed by fluorescence quantitative PCR.The results showed that compared with wild type,the expression of FIT,IRT1,FRO2,bHLH38 and bHLH39 genes in wrky12 mutant was significantly increased,while the expression of BTS and BTSL1 genes was decreased,It is suggested that wrky12 may be involved in the regulation of FIT,IRT1,FRO2,b HLH38,bHLH39,BTS and BTSL1 gene expression,thus regulating the response of Arabidopsis to iron deficiency stress.(6)Furthermore,the transient expression experiment in tobacco showed that WRKY12,which encodes E3 ubiquitin ligase,could activate the expression of BTSL1.(7)In order to further determine whether WRKY12 directly binds to BTSL1 promoter,yeast one hybrid experiment showed that WRKY12 protein can bind to W-box in BTSL1 promoter.Combined with the results of tobacco transient expression experiment,WRKY12 activated the transcription of BTSL1 gene by directly binding to the promoter of BTSL1.In conclusion,WRKY12 gene is involved in plant response to iron deficiency stress,and its regulatory mechanism may be to regulate the expression of downstream related genes by activating the expression of btsl1 gene,so maintain iron homeostasis in plants.This study not only helps to reveal the new mechanism of plant response to iron deficiency stress,but also provides new gene resources and technical approaches for crop mineral nutrition molecular breeding.