Wheat ABC1-like Kinase Gene Confers Heat And Drought Tolerance In Arabidopsis

Heat and drought stress dramatically limit growth and development of crops and cause a great production loss in wheat yield and quality every year,but the molecular mechanisms underlying wheat tolerance to extreme environments are larg]y unknown.ABC1（the activity of bcl complex）genes play essential roles in plant growth,development and responses to the environmental stresses.Therefore,it is necessary and interesting to study the potential functions of ABCI-kinase gene in response to heat and drought stresses.In this study,we identified wheat ABC1-like kinase genes and examined their expression patterns when subjected to heat and drought stresses,especially the differences between homeologs and evaluated functions of candidate genes in Arabidopsis and wheat to provide molecular mechanism for heat and drought tolerance.The main findings are as followes:We cloned and evaluated the expressions patterns of 40 AfBC1-like kinase genes from a list of candidate genes provided from the RNA-seq data and sequence information released by IWGSC and NCBI to construct a phylogenetic tree for ABC1-like proteins.On the basis of expression profiling before and after heat,drought and combined heat-drought stresses we identified TaABC1-4（Triticum aestivum L.activity of bc1 complex-4;Traes₄AL_E49650760,Traes₄BS₅CBD849D0,and Traes₄DS₇5CA84065）as a candidate triplet for further study.We find obvious structural differences among homeologs of candidate gene,particularly one mir-RNA1120 in B-homeolog.In addition,the partitioned and temporal expression patterns of A-B-and-D homeologs of candidate gene suggested that heat and drought stresses induced enhanced expression might contribute to the wide-range adaptability of hexaploid wheat.Interestingly,the homeologs appeared to have partitioned expression patterns in terns of tiusses and revealed that TaABC1-4 genes are mostly expressed in green tissues,particularly in leaves.Moreover,the transformed 35S::TaABC1-GFP construct into wheat mesophyll protoplasts confirmed the localization of TaABC1-4 in the chloroplast.Furthermore,we analyzed bisulphite sequencing of wheat to observe whether DNA methylation occurred in the TaABC1-4 gene.Consistently,bisulphite sequencing analysis of wheat DNA didn’t show any changes in the promoter region but our speculation that DNA methylation might occurred in the coding region of TaABC1-4 gene in wheat that might lead to heat and drought induced phenotypic variations by regulating gene expression.However,we cannot amplify the target fragments after bisulfite treatment,although the amplicon is easy to amplify before bisulfite treatment,which indicate DNA methylation occurred intensively in gene body region,but it needs further analysis.The phenotypic performances of Atacdol mutant in which the wheat homolog was silenced,overexpression lines and WT Arabidopsis exposed to heat and drought stresses indicated AtACDO1 silencing in mutant altered its physiology which badly affected normal phenotype compared to WT.Interestingly,Atacdol mutant showed impaired heat and drought tolerance.Expression of heat shock proteins revealed in our experiment that,heat-shock response repressed normal cellular protein synthesis and stimulated Hsp synthesis in WT but silencing AtACDO1 gene in mutant didn’t provide sufficient protein to cope with heat stress.The Western Blotting analysis also confirmed silencing of AtACDOl gene suppressed heat shock gene expression and thus indicates the necessity of AtACDO1 gene under heat stress.Expression analysis of AtACDO1 gene under heat stress in met1 mutant showed markedly higher levels of expressions compared to non stress（0 M）condition and it was 2-3 folds higher than that of WT for all the treatments imposed.In addition,heat tolerant phenotype of metl mutant over WT suggests AtACDO1 gene in metl mutant might partially contribute to its tolerance against heat stress.To examine the biological relevance of candidate genes in heat and drought stresses,we overexpressed TaABC1-4A into Arabidopsis simultaneously,constructed wheat transformants by CRISPR mediated genome editing though no mutations were observed in the target region of the gene in wheat transformants.However,after successful overexpression of candidate gene into Arabidopsis,we are generating T2 plants for phenotypic analysis.In the next step,phenotyping are required to confirm their response against heat and drought stresses.