A survey of WRKY transcription factors binding sites in promoters of up-regulated genes in response to iron toxicity in rice

Cis-acting elements(CREs) are short sequences present in the promoter regions of genes and that serve as targets for transcription factors. We indentified significant occurrences of CREs that are binding sites of WRKY proteins (family of transcription factors - TFs) in a promoter (1kbp upstream) of up-regulated genes in response to iron excess. A microarray approach was utilized to verify differentially expressed genes in rice leaves (Oryza sativa L. ssp. japonica cv. Nipponbare) of seedlings grown in iron excess (7mM FeSO₄7H₂O) in nutritive solution compared with control solution. The PLACE database was used to obtain the CREs. To find the significant occurrence of CREs, a Z test was performed (p≤0.05 for significant CRE occurrence). We found 1572 up-regulated genes with described function, from which 19 are WRKY TFs. We searched in all promoters of up-regulated genes the significant occurrence of nine CREs that are binding sites of WRKY TFs: ELRECOREPCRP1, SUREAHVISO1, WRKY71OS, WBBOXPCWRKY1, WBOXNTCHN48, WBOXNTERF3, WBOXATNPR1, WBOXGACAD1A, WBOXHVISO1. Of these CREs, 7 have significant occurrence in 440 up-regulated genes. The number of significant binding sites in each gene promoter ranged from 2 to 23 whereas the number of different CREs ranged from 1 to 5. The average of binding sites in each promoter was six and the median was 4, indicating that half the genes have a simple regulation by WRKY because the 258 genes that have less than 4 binding sites show 1 or 2 different CREs. A unique pattern was found for WRKY71OS where significant occurrences are repeated from 7 to 15 times in 106 gene promoters whereas in other CREs this number did not exceed 4.