Identification of a novel type of WRKY transcription factor binding site in elicitor-responsive cis-sequences from Arabidopsis thaliana

Using a combination of bioinformatics and synthetic promoters, novel elicitor-responsive cis-sequences were discovered in promoters of pathogen-upregulated genes from Arabidopsis thaliana. One group of functional sequences contains the conserved core sequence GACTTTT. This core sequence and adjacent nucleotides are essential for elicitor-responsive gene expression in a parsley protoplast system. By yeast one-hybrid screening, WRKY70 was selected with a cis-sequence harbouring the core sequence GACTTTT but no known WRKY binding site (W-box). Transactivation experiments, mutation analyses, and electrophoretic mobility shift assays demonstrate that the sequence CGACTTTT is the binding site for WRKY70 in the investigated cis-sequence and is required for WRKY70-activated gene expression. Using several cis-sequences in transactivation experiments and binding studies, the CGACTTTT sequence can be extended to propose YGACTTTT as WRKY70 binding site. This binding site, designated WT-box, is enriched in promoters of genes upregulated in a WRKY70 overexpressing line. Interestingly, functional WRKY70 binding sites are present in the promoter of WRKY30, supporting recent evidence that both factors play a role in the same regulatory network.     

Activation of the Oryza sativa non-symbiotic haemoglobin-2 promoter by the cytokinin-regulated transcription factor, ARR1

Using in silico methods, several putative phytohormone-responsive cis-elements in the Oryza sativa non-symbiotic haemoglobin (NSHB) 1-4 and Arabidopsis thaliana NSHB1-2 promoters have been identified. An OsNSHB2 promoter::GUS reporter gene fusion shows tissue-specific expression in A. thaliana. GUS expression was observed in roots, the vasculature of young leaves, in flowers, and in the pedicel/stem junction. In transient assays, activity of the OsNSHB2 promoter was significantly up-regulated in the presence of the cytokinin, 6-benzylaminopurine (BA). Deletion analyses indicated that the full-length promoter was required for maximal trans-activation in the presence of cytokinin. Mutation of the single cytokinin-regulated ARR1-binding element abolished promoter activation in response to cytokinin. Constitutive expression of ARR1 under the control of the 35S cauliflower mosaic virus promoter enhanced wild-type OsNSHB2 promoter activity, but had no effect on the activity of the mutated promoter in the absence of cytokinin. However, overexpression of ARR1 in the presence of cytokinin resulted in super-activation of the wild-type promoter. The mutated promoter was only moderately activated in the presence of cytokinin and ARR1, indicating that the OsNSHB2 promoter can be regulated by the ARR1 protein, but requires other cytokinin-induced factors for optimal activation. This is the first report that identifies a trans-acting factor involved in the activation of a NSHB gene.