Functional Verification Of Potato WRKY6 Gene Enhancing Cadmium Tolerance In Arabidopsi

Plant growth,development and adaptability to adversity are widely regulated by members of the WRKY transcription factor family.However,there are very few studies on WRKY transcription factor family of potato（Solanum tuberosum）.Our previous study showed that potato WRKY6 gene（StWRKY6）can alleviate cadmium toxicity to root length,growth and germination of Arabidopsis thaliana.In order to further explore the mechanism of StWRKY6 alleviating the toxic effect of Cadmium on Arabidopsis under cadmium stress,Columbia wild-type Clo-0（WT）and StWRKY6-overexpressed（StWRKY6-OE）Arabidopsis were used as materials.The phenotypes of WT and StWRKY6-OE were observed and recorded before and after cadmium stress（100μMCd Cl₂）.The activities of active oxygen scavenging enzymes,malondialdehyde（MDA）and cadmium content in plants related to Cd toxicity were measured,and the effects of StWRKY6 on these indexes were investigated.Bioinformatics（protein sequence,interacting protein and cis-acting element）and subcellular localization analysis of StWRKY6 were conducted to clarify its structural characteristics.Combined with transcriptomic sequencing analysis,the potential regulatory pathway of StWRKY6 enhancing cadmium tolerance was explored,in order to lay a molecular foundation for the genetic breeding of cadmium low accumulation potato.The main research results of this paper are as follows:（1）Analysis of qRT-PCR indicated that the highest expression level of StWRKY6 in potato roots and stems was 1.78 and 2.05 times of 0 h under Cd stress for12 h,respectively.While in leaf tissues,under Cd stress condition,StWRKY6 reached the highest expression level at 6 h,which was 8.17 times of 0 h.All these results showed that StWRKY6 was expressed in potato root,stem and leaf,but mainly in leaf tissue.（2）Analysis of phenotype difference revealed that StWRKY6 increased the SPAD value of StWRKY6-OE,enhanced its photosynthesis,and significantly reduced leaf chlorosis caused by Cd toxicity.Besides,the activities of reactive oxygen species scavenging enzymes（SOD,POD and CAT）of StWRKY6-OE were significantly higher than those of WT under Cd stress.On the contrary,the MDA and Cd contents of StWRKY6-OE were extremely significantly（p<0.01）reduced compared with WT.（3）The similarity between StWRKY6 sequencing results and CDS sequences in the NCBI database was as high as 99.43%.And subcellular localization results showed that the GFP signal of StWRKY6 was detected only in the nucleus,indicating that StWRKY6 functions as a transcription factor in the nucleus.（4）Bioinformatics analysis results showed that StWRKY6 only contained a conserved heptapeptide"WRKYGQK"domain,and it’s zinc finger structure type was C-X₇-C-X₂₃-H-X₁-C（C₂HC）,so it was classified as WRKY group III.In addition,StWRKY6 also contains W box（TTGAC[C/T]）,light-responsive element（GB/box,chs-CMA1a and Box 4）,salicylic acid-responsive element（TCA-element）,transcription initiation regulatory element（TATA-box,CAAT-box）and among others,which can be widely involved in plant growth,development and stress resistance.（5）Importantly,transcriptome analysis revealed that Cd-induced expression of StWRKY6 mediated significant up-regulation of genes,such as APR2,DFRA,ABCG1,VSP2,ERF013,SAUR64/67,BBX20,among others.APR2 and DFRA are responsible for chelating cadmium,VSP2 and PDF1.4 are involved in plant defense,ABCG1 is related to toxic effluency,BBX20 and SAur64/67 are involved in photomorphogenesis and autin signaling respectively,and they coordinately regulate Cd tolerance in StWRKY6-OE.In conclusion,under cadmium stress,StWRKY6 significantly enhanced the activities of SOD,POD and CAT in Arabidopsis thaliana compared with WT,and significantly reduced the content of MDA and cadmium.Furthermore,transcriptome analysis revealed a potential regulatory pathway by which StWRKY6 enhances cadmium tolerance in Arabidopsis.