Transcriptome wide identification and characterization of NO-responsive WRKY transcription factors in Arabidopsis thaliana L.

WRKY transcription factors are important plant-specific regulatory genes characterized by one or two conserved WRKY domain(s) usually followed by a zinc-finger motif. In this study using Arabidopsis thaliana, the RNA-Seq based transcriptomic analysis showed differential expression of 33 genes encoding WRKY TFs in response to the nitric oxide (NO) donor S-Nitrosocysteine (CySNO). Interestingly, 93.9% of these TFs were up-regulated with at least 2-fold change, suggesting their putative involvement in NO mediated gene regulation. GO- analysis of all the 33 transcriptomic elements showed their putative involvement in biological processes such as abiotic stress tolerance and defense against fungal pathogens (89.39 fold enrichment). Analysis of the NO-responsive AtWRKY TFs promoter region revealed the presence of the cis-acting elements such as ABRE, EIRE, ERE, and MBS involved in osmotic stress response, maximal elicitor-mediated activation, and drought-stress regulation. The analysis of NO-responsive AtWRKY TF motifs and their comparison with rice, soybean, and tomato orthologs suggested that members of the WRKY family belonging to the same group shared similar motifs and phylogenetic tree suggested that these TFs were highly conserved. Validation of transcriptomic data through quantitative real time-PCR showed a high correlation coefficient (0.85) indicating the high reliability and similarity of both types of analysis. Comparison of the NO-responsive and non-responsive WRKYs showed the presence of tyrosine (T) and cysteine (C) residues at a distance of 7 residues from the WRKYGQK motif which may serve as potential targets for modification by NO via tyrosine nitration and S-nitrosylation. We also validated the response of WRKYs through in vivo analysis using atwrky62 loss of function mutant and the results indicated a negative role of AtWRKY62 in plant growth. Furthermore, atwrky62 showed significantly less SNO contents compared to wild type plants indicating putative role of AtWRKY62 in NO metabolism