Zentrum für Molekularbiologie der Pflanzen (ZMBP)

ZMBP Project Group Ulrike Zentgraf
Leaf senescence of annual plants

Research projects

a) Free radicals are thought to play an essential role in senescence, especially those derived from oxygen. Increased levels of activated oxygen measured in senescing tissues could either occur through an enhanced production of activated species or a decline of the various defense systems that normally afford protection against oxidative injury. Both mechanisms seem to be realized in senescing tissues, so that investigating the regulation of these scavenging systems probably offers an opportunity to characterize factors and parameters regulating senescence. Especially hydrogen peroxide is discussed to be a signaling molecule in Arabidopsis thaliana leaf senescence. Intracellular hydrogen peroxide levels are controlled by the hydrogen peroxide scavenging enzyme catalase in concert with other scavenging and producing systems. The coordinated regulation of these hydrogen peroxide scavenging enzymes on the transcriptional and posttranscriptional level creates a distinct increase of hydrogen peroxide at the time point when the plants start to bolt and a coordinated senescence process of all rosette leaves should be induced (Zimmermann et al., 2006). Conversely, it was already shown that the senescence regulating transcription factor WRKY53 and its regulators as well as other senescence-associated transcription factors and SAGs can be induced by hydrogen peroxide (Miao et al., 2004) so that the hydrogen peroxide peak during bolting time most likely serves as a signal to induce senescence-associated gene transcription. Since transcriptional down-regulation of CAT2 appears to be the initial step to create this senescence-promoting signal, we identified G-box binding factor 1 (GBF1) as a negative regulator for CAT2 transcription. In gbf1 plants, CAT2 decrease in expression and activity and the thereby created H2O2 peak disappeared. Consequently, gbf1 plants showed a delayed senescence phenotype and an affected expression of SAGs.

Moreover, manipulating intracellular hydrogen peroxide levels in Arabidopsis by using the hydrogen peroxide sensitive part of the E. coli transcription regulator OxyR, which was directed to the cytoplasm as well as into the peroxisomes, led to delayed senescence in both cases but to different extends. This clearly indicates that cytoplasmic hydrogen peroxide appears to be more effective in senescence signaling than peroxisomal hydrogen peroxide (Bieker etal., 2012).

b) Senescence associated gene expression. For detailed gene expression analyses during leaf senescence we have designed a hybridization matrix for gene profiling using the Affymetrix high density genome arrays representing approximately 8.200 genes. If we analyze gene expression according to the age of individual leaves of one plant in comparison to leaves harvested from plants with different ages we get very different expression profiles. These data indicate that gene expression is governed by two different parameters, leaf age and plant age (Zentgraf et al., 2004).

The differential expression of genes according to the age of the leaves within one rosette was also analyzed using suppression subtractive hybridization (SSH). A transcription factor of the WRKY family, WRKY53, was isolated in this screen (Hinderhofer et al., 2001).

To understand the regulatory role of the senescence related WRKY53 factor, we identified targets 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. WRKY53 can be induced by H2O2 and can regulate its own expression in a negative feed-back loop (Miao et al., 2004). Our results suggest that WRKY53 acts in a complex transcription factor signaling network regulating senescence specific gene expression and hydrogen peroxide might be involved in signal transduction.

WRKY53 is tightly regulated on different levels: i) activity, positively by phosphorylation (MEKK1) and negatively by blocking the DNA-binding (ESR/ESP), ii) gene expression which is regulated by at least four proteins that bind to the promoter of WRKY53 and iii) degradation of the protein by a HECT E3 ubiquitin ligase (Miao and Zentgraf, 2007, Miao et al., 2007, Miao et al., 2008, Miao and Zentgraf, 2010, for review see Zentgraf et al., 2010, Potschin et al., 2014).

In general, the two large transcription factor families of bZIPs and WRKYs execute two different functional strategies:

bZIPs and WRKYs are two large and important transcription factor families in regulating diverse developmental and stress-related processes. Since a partial overlap in these biological processes is obvious, it can be speculated that they fulfill non-redundant functions in a complex regulatory network. The regulatory mechanisms that are so far described for bZIPs and WRKYs revealed that both families execute two different functional strategies. bZIP factors need to homo- or heterodimerize for DNA-binding and regulation of transcription, and based on a bioinformatics approach, bZIPs can build up more than the double of protein interactions than WRKYs (B). In contrast, an enrichment of the WRKY DNA-binding motifs can be found in WRKY promoters (A), a phenomenon which is not observed for the bZIP family. Thus, the two transcription factor families follow two different functional strategies in which WRKYs regulate each other’s transcription in a transcriptional network whereas bZIP action relies on intensive heterodimerization (Marco Llorca et al, 2014).

Alexander v. Humboldt Fellow

Dr. Justine Bresson,

She was awarded the PhD in Ecophysiology from the University of Montpellier II in France in December 2013. From August 2014 to July 2015 she was supported by an Innovation Grant of the University of Tübingen and started her work on senescence regulating factors.

She is interested in the genetic control of plant senescence, growth and shade avoidance by the REVOLUTA and WRKY network. Particular emphasis will be placed on identifying the interacting partners involved in the regulation of WRKY53 expression by the REV transcription factor. Growth, development, plant productivity and shade avoidance syndromes are dissected during Arabidopsis life cycle.