WRKY26, WRKY38 and MYC2 are direct upstream transcription factors regulating ORA47 gene expression in wound-induced JA signaling

Jasmonates (JAs) are plant signaling molecules that play important roles in defense of biotic stress. Many transcription factors, such as MYC2 and some ERFs are reported to be involved in the regulation of the signaling pathways mediated by JA. ORA47 is an ERF transcription factor and its expression is highly induced by wounding and JA. ORA47 is also known from our lab to upregulate many JA biosynthetic genes. My research aim is to study the potential upstream components of ORA47. Nucleotide sequence analysis revealed that the promoter of the ORA47 gene contained conserved sequence motifs, which are analogous to the MYC2 binding consensus sequences (CACGTG, or G-box) and putative WRKY26 and WRKY38 binding sequences (TTGATC, or W-like box). We therefore asked whether they could directly regulate ORA47 expression in wounding signaling. The GUS activity of ORA47 pro:GUS inoculated plants was lower in myc2 mutant line than in WT indicating wounding induced ORA47 expression is MYC2-dependent. ORA47 and several genes involved in JA biosynthesis were induced in the 35S::MYC2-GFP mutants compared with WT plant indicating MYC2 is an upstream regulator of JA biosynthesis. Chromatin immunoprecipitation (ChIP) assay showed MYC2 could interact with two closely linked G-box elements in the ORA47 promoter. Furthermore, electrophoretic mobility shift assay (EMSA) analysis showed MYC2 could only bind to G-box1 and yeast one-hybrid assay also exihibted MYC2 could interact with the G-BOX1 fragment but not G-BOX2. With respect to WRKY transcription factors, both WRKY26 and WRKY38 could bind to W-like-box2 (WL-box2), W-like-box3, W-like-box4 and much less the W-like- box1 cis-elements in the EMSA experiment. Moreover, yeast one hybrid assay showed WRKY26 and WRKY38 could interact with the WL-box1 and WL-box4 DNA fragments. ChIP assay revealed WRKY26 and WRKY38 could interact only with the WL-box1 element in the ORA47 promoter. In conclusion, MYC2, WRKY26, and WRKY38 directly regulate ORA47 in the JA signaling pathway.