Abstract
WRKY transcription factors (TFs), which make up one of the largest families of TFs in the plant kingdom, are key players in modulating gene expression relating to embryogenesis, senescence, pathogen resistance, and abiotic stress responses. However, the phylogeny and grouping of WRKY TFs and how their binding ability is affected by the flanking regions of W-box sequences remain unclear. In this study, we reconstructed the phylogeny of WRKY across the plant kingdom and characterized the DNA-binding profile of Arabidopsis thaliana WRKY (WRKY54) based on its W-box recognition sequence. We found that WRKY TFs could be separated into five clades, and that the functional zinc-finger motif at the C-terminal of WRKY appeared after several nucleotide substitutions had occurred at the 3′-end of the zinc-finger region in chlorophytes. In addition, we found that W-box flanking regions affect the binding ability of WRKY54 based on the results of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and quartz crystal microbalance (QCM) analysis. The great abundance of WRKY TFs in plants implicates their involvement in diverse molecular regulatory networks, and the flanking regions of W-box sequences may contribute to their molecular recognition mechanism. This phylogeny and our findings on the molecular recognition mechanism of WRKY TFs should be helpful for further research in this area.
Highlights
WRKY transcription factors (TFs) are named based on the conserved residue WRKY, which constitutes an integral part of their DNA-binding domain (DBD) and is approximately 60 residues in length
We identified no WRKY TFs in P. umbilicalis, and only two in Micromonas pusilla
There were no signals detected in the T6, T8, or T10 pairs. These results showed that a flanking region with at least three nucleotides at the 50 end of TTGACT is required for the binding of the WRKY54 DBD
Summary
WRKY transcription factors (TFs) are named based on the conserved residue WRKY, which constitutes an integral part of their DNA-binding domain (DBD) and is approximately 60 residues in length. Previous research has indicated that WRKY TFs should be classified into three groups based on the number of WRKY domains and the type of zinc-finger motif they contain [2]. TFs contain one WRKY domain with a C2H2 zinc-finger motif, whereas the group 3 TFs contain one WRKY domain with a C2HC zinc-finger motif. This grouping of WRKY TFs has been widely used, but molecular evidence has shown that it is inconsistent with phylogeny [2–4]. The WRKY genes from Arabidopsis thaliana can be grouped into group Ia, comprising AtWRKY1, -2, -3, -4, -10, -25, -26, -32, -33, -34, -44, and -58, and group Ib, comprising AtWRKY8, -12, -13, -23, -24, -28, -43, -45, -48, -56, -57, -68, -71, and
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