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.

Oxygen free radicals are thought to play an essential role in senescence, especially those derived from peroxisomes. Therefore, the activities of different isoforms of the peroxisomal hydrogen peroxide (H 2 O 2 )-scavenging enzyme catalase (CAT) were analysed during senescence of Arabidopsis . CAT2 activity decreased with bolting time parallel with cytosolic ascorbate peroxidase 1 (APX1) activity before loss of chlorophyll could be measured. At the same time point, the H 2 O 2 content increased. Subsequently, the stress-inducible CAT3 isoform was activated and APX1 activity was recovered, accompanied by a decline of the H 2 O 2 content. In very late stages, low activities of the seed-specific CAT1 became detectable in leaves, but H 2 O 2 increased again. Further analyses of CAT expression by promoter : β β β β -glucuronidase ( GUS ) fusions in transgenic plants revealed a vasculature-specific CAT3 expression, whereas CAT2 expression turned out to be specific for photosynthetic active tissues. CAT2 expression is down-regulated during leaf senescence, while CAT3 expression is induced with age and corresponds to an accumulation of H 2 O 2 in the vascular bundles. CAT2 downregulation on the transcriptional level appears as the initial step in creating the H 2 O 2 peak during bolting time, while the decrease in APX1 activity might only be a secondary and amplifying effect.

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.

Hydrogen peroxide (H 2 O 2 ) is discussed as being a signaling molecule in Arabidopsis (Arabidopsis thaliana) leaf senescence. Intracellular H 2 O 2 levels are controlled by the H 2 O 2 -scavenging enzyme catalase in concert with other scavenging and producing systems. Catalases are encoded by a small gene family, and the expression of all three Arabidopsis catalase genes is regulated in a senescence-associated manner. CATALASE2 (CAT2) expression is down-regulated during bolting time at the onset of leaf senescence and appears to be involved in the elevation of the H 2 O 2 level at this time point. To understand the role of CAT2 in senescence regulation in more detail, we used CAT2 promoter fragments in a yeast one-hybrid screen to isolate upstream regulatory factors. Among others, we could identify G-Box Binding Factor1 (GBF1) as a DNA-binding protein of the CAT2 promoter. Transient overexpression of GBF1 together with a CAT2:b-glucuronidase construct in tobacco (Nicotiana benthamiana) plants and Arabidopsis protoplasts revealed a negative effect of GBF1 on CAT2 expression. In gbf1 mutant plants, the CAT2 decrease in expression and activity at bolting time and the increase in H 2 O 2 could no longer be observed. Consequently, the onset of leaf senescence and the expression of senescence-associated genes were delayed in gbf1 plants, clearly indicating a regulatory function of GBF1 in leaf senescence, most likely via regulation of the intracellular H 2 O 2 content.Tolerance against oxidative stress and life span are strikingly correlated in many organisms ranging from Caenorhabditis to mammals

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