Arabidopsis WRKY proteins comprise a family of plant specific zinc-finger-type transcription factors involved in the regulation of gene expression during pathogen defense, wounding, trichome development, and senescence. To understand the regulatory role of the senescence-related WRKY53 factor, we identified target genes of this transcription factor by a pull down assay using genomic DNA and recombinant WRKY53 protein. We isolated a number of candidate target genes including other transcription factors, also of the WRKY family, stress- and defence related genes, and senescence-associated genes (SAGs). WRKY53 protein could bind to these different promoters in vitro and in vivo and it could act either as transcriptional activator or transcriptional repressor depending on the sequences surrounding the W-boxes. Overexpression, RNAi and knock-out lines showed accelerated and delayed senescence phenotypes, respectively, and exhibited altered expression levels of the target genes. WRKY53 can be induced by H2O2 and can regulate its own expression in a negative feed back loop. Our results suggest that WRKY53 acts in a complex transcription factor signalling network regulating senescence specific gene expression and that hydrogen peroxide might be involved in signal transduction.

Arabidopsis WRKY proteins comprise a family of plant specific zinc-finger-type transcription factors involved in the regulation of gene expression during pathogen defense, wounding, trichome development, and senescence. To understand the regulatory role of the senescence-related WRKY53 factor, we identified target genes of this transcription factor by a pull down assay using genomic DNA and recombinant WRKY53 protein. We isolated a number of candidate target genes including other transcription factors, also of the WRKY family, stress- and defence related genes, and senescence-associated genes (SAGs). WRKY53 protein could bind to these different promoters in vitro and in vivo and it could act either as transcriptional activator or transcriptional repressor depending on the sequences surrounding the W-boxes. Overexpression, RNAi and knock-out lines showed accelerated and delayed senescence phenotypes, respectively, and exhibited altered expression levels of the target genes. WRKY53 can be induced by H2O2 and can regulate its own expression in a negative feed back loop. Our results suggest that WRKY53 acts in a complex transcription factor signalling network regulating senescence specific gene expression and that hydrogen peroxide might be involved in signal transduction.

Despite the importance of the senescence processes in plants, our knowledge on regulatory mechanisms of senescence is still poor. WRKY transcription factors have been shown to be involved in the regulation of leaf senescence. However, almost nothing is known about the upstream regulation of the senescence specific expression of WRKY factors. Therefore, we characterized proteins that bind and activate the promoter of WRKY53, which participates in leaf senescence in Arabidopsis thaliana. Surprisingly, a mitogen activated protein kinase kinase kinase (MEKK1) was identified as a DNA-binding protein. The binding motif for MEKK1 in the WRKY53 promoter could be characterized and promoter:GUS analyses revealed that this region is important for the switch of WRKY53 expression from a leaf age dependent to a systemic plant age dependent expression during bolting time. In addition to its promotor-binding activity, MEKK1 was also able to interact with the WRKY53 protein. Using bimolecular fluorescence complementation assays the complex formation of MEKK1 and WRKY53 could be localized predominately in the nucleus of Arabidopsis cells. MEKK1 could also phosphorylate WRKY53 in vitro and phosphorylation could increase DNA-binding activity of WRKY53 in vitro and transcription of a WRKY53 promoter driven reporter gene in vivo. These results suggest that MEKK1 is a bifunctional protein: it binds to the promoter of the WRKY53 gene regulating the switch from a leaf age dependent to a plant age dependent expression and it can phosopharylate WRKY53 in vitro increasing its DNA binding activity. Thus, MEKK1 might be able to take a very direct short cut in mitogen-activated protein kinase (MAPK) signalling by directly phosphorylating a transcription factor.

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Osmotic cell swelling activates Cl− channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl−swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl−swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl−swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl−swell in lymphocytes via the tyrosine kinase p56lck.