Genome-wide Analysis Of Wheat WRKY Transcription Factors And Differential Expression Under Water Deficit Condition

WRKY proteins,which comprise one of the largest transcription factor families in the plant kingdom,are involved in various biotic and abiotic stress responses.Their conserved WRKYGQK heptapeptide at the N-terminus can regulate gene expression by exclusively binding to the W-box.Studies of WRKYs in soybean,Arabidopsis,rice,etc.have been widely reported.The genome-wide identification and functional characterization of the WRKY in wheat will help to complete the family information,and lay the foundation for future functional investigation of WRKYs under various stresses.In this study,numerous bioinformatics methods,including phylogenetic analysis,conserved motif determination,exon-intron structure and cis-acting element analysis,were employed to comprehensively analyze the WRKY family in wheat.Twelve transcripts were selected as candidate drought responsive genes according to their orthologous WRKYs in Arabidopsis.The qRT-PCR was used to investigate the expression pattern of Ta WRKYs in flag leaves and spikes under water deficit condition.The main results are as follows:1)A total of 1113 WRKY TFs were identified from 20 selected plants.Most of terrestrial plants contained 86 to 171 WRKYs,whereas six members or less were found in aquatic algae,of which no WRKYs were found in Rhodophytes and Glaucophytes.The number of WRKY proteins increased during evolution from simpler unicellular to more complex multi-cellular forms.In wheat,171 WRKYs were obtained and named TaWRKY1-171 based on their chromosome locations.2)Based on the phylogenetic analysis and primary amino structure feature of WRKY,all of identified WRKYs were classified into three groups,and Group I,II and III contained 31,95 and 45 members,respectively.Besides the highly conserved WRKYGQK motif,three variants in TaWRKYs,namely WRKYGKK,WRKYGEK and WSKYGQK,were found.In addition,two zinc-finger form variants,C-X6-P-X23-H-X-C and C-X6-F-X23-H-X-C,were identified in TaWRKY80 and 166.3)Among the 171 Ta WRKY genes,115 were mapped onto the 21 wheat chromosomes,and the other 56 were anchored in the scaffolds(Ta WRKY115-171).More TaWRKY genes were distributed in Chromosomes 3B(18,15.7%),5B(11,9.57%),2A(9,7.8%)and 5D(9,7.8%).In contrast,chromosomes 6B,7A and 7B contained only one Ta WRKY gene(0.870%).In general,most identified Ta WRKY genes were observed in distal regions of chromosomes and only a few were observed in proximal regions.This phenomenon suggested that the Ta WRKY genes were mapped on the all chromosomes with a significantly non-random and uneven distribution.Gene duplication events were observed in 85 Ta WRKY genes.Among them,two gene clusters(Ta WRKY59/60,Ta WRKY113/114/115)were tandemly duplicated,and others have undergone segmental duplication events.4)Analysis of gene structure showed that the number of introns in TaWRKY family genes varied from 0 to 5,and 72(42.11%)Ta WRKY genes with two introns accounted for the largest proportion.The distribution pattern of introns and exons was group specific,and Ta WRKY genes belonging to the same subfamily shared a similar exon-intron structure,which further validated the result of phylogenetic tree.Two types of introns(V-type and R-type)were located in the WD.All of the TaWRKY genes in Groups IIa and IIb only contained V-type introns except TaWRKY165,which had no intron.However,R-type introns were mostly observed in all the other groups.The online tool MEME was utilized to identify the conserved motifs.Each protein contained many conserved motifs,and TaWRKYs,and TaWRKYs within the same group or subgroup shared similar motif compositions.Motifs 1 and 4 contained a WRKYGQK sequence,a basic feature of WRKYs.5)Various cis-acting elements were found in 142 TaWRKY genes based on the online tool PlantCARE.Many cis-acting elements involved in various biotic(hormones and fungus)and abiotic(light,wound,cold,heat,anaerobic induction and drought)stresses were identified in large number of TaWRKY genes.6)The expression pattern of Ta WRKY genes in flag leaves,glumes and lemmas under water deficit condition during the grain-filling period were determined using qRT-PCR.Most of selected genes were sensitive to water stress,and expression of WRKYs in distinct period showed obvious differences.A relatively large group of genes,including TaWRKY1,20,31,112,123,142 and 149 were significantly up-regulated in flag leaves at 0,3or 5 DAA;while genes in another interesting cluster,composed of Ta WRKY90,97,120 and 133,were down-regulated or slightly changed in glumes and lemmas during the early grain-filling stage,and induced during the middle or late grain-filling stage under water-deficit stress.The expression of WRKYs in wheat was tissue-specific.