ZMBP Project Group Ulrike Zentgraf
Leaf senescence of annual plants

Research | Group members | Publications | Resources

Ulrike Zentgraf
Department of Genetics
University of Tübingen
Auf der Morgenstelle 32
72076 Tübingen
Germany

Tel: 07071/29-78833
Fax: 07071/29-5042
ulrike.zentgrafspam prevention@zmbp.uni-tuebingen.de

Research interest

Senescence is a key process that underpins many important agronomic traits including yield outcomes and pre- and post-harvest quality. Senescence is a highly regulated and organized process during which macromolecules of the mature green leaf are dismantled and remobilized for further use by the plant, either for growth or for deposition in developing fruits and seeds. Efficient senescence is essential for the overall success of the plant and its following generations; however, premature senescence, induced by stress, leads to reduced yield and quality of crop plants. Abiotic stress is estimated to be the primary cause of crop loss worldwide, with the potential to cause a reduction of more than 50% in the average yield of the main crops.

In general, selective activation of gene expression but also the decrease of certain RNAs and/or proteins initiate and regulate this process. The gene products are involved in the breakdown of macromolecules and the mobilization of nutrients out of the senescing tissues. The rapid loss of chlorophyll, the lowering of protein and RNA levels, activation of autophagy or the leakiness of the cell membranes are used as senescence markers. Despite the importance of the senescence processes in plants, our knowledge on the signals and regulatory mechanisms is still limited.

In order to characterize factors which are involved in the onset and regulation of senescence two main approaches are followed:

a) Involvement of reactive oxygen species, especially hydrogen peroxide, in regulating leaf senescence
b) Senescence-associated gene expression: Regulatory network controlling expression, activity, and degradation of the transcription factor WRKY53 in Arabidopsis leaves.

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 coupled to YFP (HyPER), which was directed to the cytoplasm, into the chloroplasts as well as into the peroxisomes, led to delayed senescence but to different extends. Obviously, cytoplasmic hydrogen peroxide appears to be more effective in senescence signaling than peroxisomal hydrogen peroxide (Bieker et al., 2012). Moreover, chloroplastic hydrogen peroxide has a high impact on senescence progression while cytoplasmic and peroxisomal hydrogen peroxide appear to control the onset.

Senescence phenotype of wild type plants and HyPER (OyxR-RD) overexpressing lines directed to different compartments. Leaves are sorted according to their age and an Automated colorimetric assay (ACA) was applied which was developed in our lab to analyze leaf color pixelwise (Bresson et al., 2018).

Moreover, RNA-Seq analyses of these plants uncovered severe differences in gene expression, which were also dependent on the developmental stage. Here, plants with low chloroplastic H₂O₂ show more severe and often even opposite changes in gene expression compared to plants with lower cytoplasmic H₂O₂, which appear to be more similar in gene expression to wildtype plants. Strikingly, higher expression of CAT and APX genes in plants with low chloroplastic H₂O₂was observed. Moreover, expression of defensin genes is increased under low chloroplastic H₂O₂ but decreased under low cytoplasmic H₂O₂. In wildtype plants, the expression of the pathogen-related defensin genes and genes encoding H₂O₂ scavenging proteins CAT and APX is triggered by oxidative stress but not by low H₂O₂. In the same chain of evidence, formation of stromules clearly increased under low chloroplastic H₂O₂ which, again, should be opposite and rise after SA or H₂O₂ treatment. These opposing expression patterns together with the unexpected increase in stromule formation prompted us to speculate that not the actual H₂O₂ concentration is sensed by the plants but more likely the ratio between the cytoplasmic and the chloroplastic H₂O₂. This would mean that low chloroplastic H₂O₂ resembles “cytoplasmic” oxidative stress (Zentgraf et al., 2022)

A) High number of stromules was observed in the chloroplastic HyPER overexpressing line, which is normally induced by high H2O2 treatment but not by low H2O2. Therefore, a model for H2O2 sensing was developed. B) Balanced H2O2 contents in wild type plants (WT) in normal situations without exogenous stress. C) Imbalanced H2O2 contents between chloroplast and cytoplasm in two different situations: (left) wild type versus HyPER expression in chloroplasts or (right) wild type under control conditions versus wild type under stress conditions. In both cases, H2O2 content is imbalanced in the same way and high stromule numbers as well as expression of genes induced by oxidative stress are observed, indicating that the ratio between the H2O2 contents of the cytoplasm and the chloroplasts might be the parameter which is sensed by the cells. This is still a hypothesis and has to be further analyzed.

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).

Matrix of leaf material used for chip hybridization and cluster analyses. Genes that are upregulated in leaf No. 6 according to their plant age, are also upregulated in leaf No. 12; however, the same genes show a different expression pattern within a 6-week-old rosette when the leaves are analyzed according to their age.

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).

ZMBP Project Group Ulrike Zentgraf Leaf senescence of annual plants

ZMBP Project Group Ulrike Zentgraf
Leaf senescence of annual plants