Expression and regulation AtWRKY54 and AtWRKY70

WRKY family, a transcription factor family in plant, is well-known for its regulation in biotic stresses. In recent studies, genes in WRKY family also play roles in abiotic stresses. WRKY54 and WRKY70, two members of WRKY family, are involved in the regulation of various processes including plant defense and senescence, and may also participate in plant thermotolerance. In this study, protein-DNA pull down assays were used to find the promoter sequences that are recognized by WRKY54 or WRKY70 protein and to speculate what genes would directly be regulated by WRKY54 or WRKY70. Chromatin immunoprecipitation was designed to confirm this speculation. Expression of AtWRKY54 or AtWRKY70 by transforming E.coli produces inclusion body, and no detectable AtWRKY54 and AtWRKY70 was found in the soluable phase for further study. This problem obstructs the using of protein-DNA pull down assay. In addition, chromatin immunoprecipitation in several ways was performed to find genes regulated by WRKY54 or WRKY70 without predicted primers, but it fails. In recent study, wrky54wrky70 double mutant would change several gene expression under osmotic treatment. Analysis promoters of these genes by PLACE to find if promoters of these genes contain W-Box-liked sequence. Six genes with W-Box were analyzed by RT-qPCR in WRKY54 overexpression line, WRKY70 overexpression line, wrky54 T-DNA insertion line and wrky70 T-DNA insertion line. As the result, the gene expression of these five genes, COR47(cold regulated gene 47)、ERD7(early-response to dehydration 7)、LEA14(late-embryogenesis abundant protein)、LTI78(low-temperature-induce 78) and RD2( dessication responsive protein 2), are much lower in wrky54 T-DNA insertion line and wrky70 T-DNA insertion line than wild type. These five genes are not induced by heat but induced by other abiotic stresses including light stress, salt stress, osmotic stress, drought stress and cold stress. It means that WRKY54 and WRKY70 may play roles in several abiotic stresses.