A novel upstream regulator of WRKY53 transcription during leaf senescence in Arabidopsis thaliana

Arabidopsis WRKY proteins comprise a family of zinc finger-type transcription factors involved in the regulation of gene expression during pathogen defence, wounding, trichome development and senescence. To better understand the regulatory role of the senescence-related WRKY53 factor, we identified upstream regulatory factors using the yeast one-hybrid system. Among others, we identified a DNA-binding protein with a so far unknown function that contains a transcriptional activation domain and a kinase domain with similarities to HPT kinases. In vitro studies revealed that this activation domain protein (AD protein) can phosphorylate itself and that phosphorylation increases its DNA-binding activity to the WRKY53 promoter region. Using the yeast two-hybrid system, an interaction with proteins that were previously shown to bind to the WRKY53 promoter was tested. The AD protein interacted with MEKK1. The interaction with MEKK1 was confirmed in vivo by bimolecular fluorescence complementation (BiFC); however, the AD protein was not phosphorylated by MEKK1 in vitro and vice versa. This indicates that there may be competition between WRKY53 and AD protein for binding of MEKK1 at the WRKY53 promoter. Overexpression and knockout of the respective gene resulted in changes in transcription levels of WRKY53, indicating that AD protein is a positive regulator of WRKY53 expression. Expression of the AD protein gene can be induced by hydrogen peroxide treatment and reduced by jasmonic acid treatment, as previously shown for WRKY53.