Leaf Senescence in Arabidopsis thaliana and Brassica napus: From Molecular Regulation to Nutrient Remobilization Processes

Abstract

Leaf senescence is the final stage of leaf development and guarantees successful survival of a plant and its progeny. It follows a coordinated, genetically encoded program, which involves the differential expression of thousands of genes as basis of an ordered execution of events. The aim of senescence is the degradation of macromolecules so that released nutrients can be reallocated to storage organs or developing parts of the plant. Transcription factors (TFs) are generally accepted as crucial elements in senescence regulation and coordination. Ethylene response factors (ERFs) as well as WKRY TFs are superfamilies of TFs that are overrepresented in the senescence transcriptome. Furthermore, the role of hydrogen peroxide (H2O2) as signaling molecule in senescence has often been demonstrated and it induces the expression of many senescence associated genes (SAGs), amongst them WKRY53, which is a central player in senescence regulation. We identified a small sub-network between WRKY18, WRKY25 and WRKY53, in which WRKY25 is a positive and WRKY18 is a negative regulator of WRKY53 expression. All three WRKY factors are influencing senescence and moreover, H2O2 treatment enhanced expression of all three factors. In DPI-ELISAs we showed that binding of WRKY25 to the WRKY53 promoter is redox-sensitive. However, even though WRKY25 is a positive regulator of WRKY53, it is a negative regulator of leaf senescence, pointing to a more complex regulatory network with more and still unknown factors. Furthermore, the mitogen-activated kinase kinase kinase 1 (MEKK1) was found to bind to the promoter of WKRY53 and influence its expression most likely via phosphorylation of WRKY proteins. By reporter gene assays I confirmed that MEKK1 increased the effect of WRKY53 protein on the promotor activity of its own gene and I could show that MEKK1 also enhanced the positive effect of WRKY25 protein on WKRY53 driven gene expression. Moreover, MEKK1 turned the negative effect of WKRY18 on WRKY53 expression into a positive one. Taken together, this indicates that MEKK1 does not specifically interact with WRKY53 but also with other WRKY proteins. It is noteworthy, that it exhibited a positive effect on the expression of all three WRKYs. By characterizing an inducible knockdown line for MEKK1, a clear role of MEKK1 in senescence regulation was observed. ERF4 exists in two different isoforms due to alternative polyadenylation: ERF4-R, which contains an ERF-associated amphiphilic repression (EAR) motif and ERF4-A, which lacks this motif. By a reverse genetics approach, in which I analyzed the phenotype, I could show that ERF4 isoforms are both involved in leaf senescence regulation. By subsequent molecular and biochemical assays, I gained an understanding of the mode of action of ERF4 isoforms and how they possibly affect leaf senescence. Both isoforms bind to the promoter of the CATALASE3 (CAT3) gene and have antagonistic effects on CAT3 gene expression. The ratio of ERF4-A to ERF4-R changed over time. Together with visualization of CAT3 activity on native gels, this hints at a function of the ERF4 isoforms as an activator of CAT3 in earlier and as a repressor of CAT3 expression in later stages. This could foster an increase of H2O2 in later stages, which induces expression of many SAGs and contributes to degradation processes. Lastly, by analysis of transcriptome data, seed storage proteins (SSPs) were found to be expressed in vegetative tissues of Brassica napus L. (cv. Mozart). I confirmed NAPIN and CRUCIFERIN expression in leaves via quantitative real-time PCR and found a correlation between intracellular H2O2 and SSP accumulation. Moreover, their expression depended on the nitrogen (N)-supply. In addition, N-starvation induced senescence differed from developmental senescence, since H2O2 levels were not increased but reduced in these plants. This work reveals new insights in senescence regulation. It contributes to the general endeavor, to gain an understanding of the complex molecular network underlying leaf senescence regulation.