Auto- and Cross-repression of Three Arabidopsis WRKY Transcription Factors WRKY18, WRKY40, and WRKY60 Negatively Involved in ABA Signaling

Some members of the WRKY transcription factor family are known to be involved in ABA signaling. However, it remains unclear how the WRKY transcription factors cooperate to regulate ABA signaling. In the present study, we showed that three Arabidopsis (A. thaliana) WRKY proteins previously identified as ABA signaling regulators, WRKY18, WRKY40, and WRKY60, directly target the W-box regions in various domains of the promoters of all their own encoding genes WRKY18, WRKY40, and WRKY60, which was evidenced by chromatin immunoprecipitation and gel shift assays. Furthermore, we showed that the three WRKY proteins inhibit expression of all three WRKY genes, which was evidenced in both an in vivo assay of coexpression of the WRKY proteins with the three WRKY promoters and expression analysis of the three WRKY genes in various wrky mutants. Additionally and importantly, we provide new evidence, with three different testing systems, that WRKY18, WRKY40, and WRKY60 are negative, not positive, ABA signaling regulators, and that ABA treatment represses all three WRKY genes through a mechanism partly independent of the WRKY proteins, in which the response of the WRKY60 gene to ABA partly requires WRKY18 and WRKY40. These findings describe a mechanism of auto- and cross-repression of the WRKY transcription repressors that suggests a sophisticated mechanism to balance the negative functions of the WRKY transcription repressors in ABA signaling and helps to understand the WRKY-mediated complex events in ABA signaling pathways.