The role of hydrogen peroxide as signalling molecule in leaf senescence and its impact on senescence-induced nitrogen mobilisation
Zusammenfassung der Projektergebnisse
Our main goal was to verify whether H2O2 is used as a signal in senescence induction and to analyse its impact on N-mobilization processes. In Arabidopsis, H2O2 increases in the leaves during the onset of senescence by a complex regulatory interplay of the H2O2 scavenging enzymes catalase and ascorbate peroxidase, in which transcriptional down-regulation of CAT2 by the G-Box binding factor 1 (GBF1) is an essential step. This H2O2 peak is involved in the transcriptional up-regulation of important senescence-associated transcription factors like WRKY or NAC factors. By continuous watering of Arabidopsis plants with antimycin A to inhibit cytochrome C oxidase we lowered H2O2 production specifically in mitochondria through the induction of alternative oxidase. However, this decrease in H2O2 had no effect on leaf senescence indicating an impact of the cellular compartment. Therefore, a H2O2 in vivo imaging system was established using H2O2 sensing YFPs (HyPER) which were directed to the cytosol, the chloroplast, the mitochondria and the peroxisome to analyse long-term H2O2 profiles. However, all lines expressing the HyPER protein were delayed in senescence due to the H2O2 scavenging function of these proteins, and the strength of the phenotype correlated with the expression strength of HYPER protein and the cellular compartment, with the most prominent effect for the cytoplasmic line. Therefore, these lines were not suitable for the initially planned long-term in vivo imaging but clearly confirmed that H2O2 is used as signal to induce senescence in Arabidopsis. A similar H2O2 increase due to down-regulation of the H2O2 scavenging enzymes during bolting time could be observed for oilseed rape (OSR) summer variety cv Mozart. In a concerted experiment in Hohenheim using OSR cv Mozart and different N and CO2 supplies, H2O2 profiles, hormone levels and gene expression data were analysed in parallel by different groups. Here, shifting of senescence induction by high CO2 supply coincided with a shift of the hydrogen peroxide maximum clearly indicating that H2O2 is most likely also used as a signal in developmental senescence in OSR. An increase in H2O2 contents could also be confirmed for the OSR winter varieties NPZ1 and NPZ2 during developmental senescence. Under N-starvation conditions, neither Arabidopsis nor OSR showed an increase in H2O2 levels indicating that H2O2 is not used as a signal in N starvation-induced senescence. In contrast, H2O2 contents were always lower after N starvation. However, high levels of anthocyanins are produced in all these plants which might also be involved in scavenging of H2O2, maybe even to counteract senescence induction at the beginning of N starvation. The microarray analyses of developmental senescence of OSR cv Mozart revealed that 380 genes followed the H2O2 profile and that in this cluster seed storage proteins were highly enriched under high but not under low N supply. These proteins are usually expressed exclusively in seeds but were detected in leaves. The presence of the mRNA and the storage proteins were confirmed and quantification of the protein contents revealed enrichment of the seed storage proteins in leaves starting in old leaves and timely staggered in younger leaves, stem and pods pointing to the possibility that these proteins might serve in interim N storage. Promoter analyses of the different storage protein-encoding genes revealed binding motifs for different transcription factors involved in N and ROS management. OSR transformation was tested by different techniques, however, using the CaMV 35S promoter all constructs were silenced in the T2 generation. After changing the promoter to NtUbiqutin10 we were able to successfully transform OSR cv Mozart by seed transformation with the chl-HYPER construct. These plants will now be analysed for developmental and N starvation-induced senescence.
Projektbezogene Publikationen (Auswahl)
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(2010) A HECT E3 ubiquitin ligase negatively regulates Arabidopsis leaf senescence through degradation of the transcription factor WRKY53. The Plant Journal 63, 179–188 (2010)
Miao, Y., Zentgraf, U.
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(2010) G-Box Binding Factor1 reduces CATALASE2 expression and regulates the onset of leaf senescence in Arabidopsis. Plant Physiology 153, 1321-1331 (2010)
Smykowski, A., Zimmermann, P., Zentgraf, U.
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(2010) The complex regulation of WRKY53 during leaf senescence of Arabidopsis thaliana. Eur J Cell Biol 89, 133-137
Zentgraf, U., Laun, T., Miao, Y.
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(2012) Determination of the in vivo redox potential by one-wavelength spectro-microscopy of roGFP. Anal Bioanal Chem 403 (3), 737-744
Wierer, S., Peter, S., Elgass, K., Mack, H.G., Bieker, S., Meixner, A.J., Zentgraf, U., Schleifenbaum, F.
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(2012) Role of intracellular hydrogen peroxide as signaling molecule for plant senescence, In: Senescence, Tetsuji Nagata, IntechOpen
Zentgraf, U., Zimmermann, P., Smykowski, A.
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(2012) Senescence-specific alteration of hydrogen peroxide levels in Arabidopsis thaliana and oilseed rape spring variety Brassica napus L. cv. Mozart. J Integr Plant Biol 54(8), 540-554, Special issue: Senescence
Bieker, S., Riester, L. Stahl, M. Franzaring, J., Zentgraf, U.
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(2013) Plant Senescence and Nitrogen Mobilization and Signaling, In: Senescence and Senescence-Related Disorders. Ed. by Zhiwei Wang and Hiroyuki Inuzuka. ISBN: 978-953-51-0997-6
Bieker, S., Zentgraf, U.
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(2014) bZIPs and WRKYs: two large transcription factor families executing two different functional strategies. Frontiers Plant Sci. 5, 169
Marco Llorca, C., Potschin, M. Zentgraf, U.
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(2014) REVOLUTA and WRKY53 connect early and late leaf development in Arabidopsis. Development 141, 4772-4783
Xie, Y., Huhn, K., Brandt, R., Potschin, M., Bieker, S., Straub, D., Doll, J., Drechsler, T., Zentgraf, U., Wenkel, S.
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(2014) Senescence Networking: WRKY18 is an upstream regulator, a downstream target gene, and a protein interaction partner of WRKY53. J. Plant Growth Regul. 33, 106-118, Special issue: Plant Senescence
Potschin, M., Schlienger, S. Bieker, S., Zentgraf, U.
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(2015) Phosphorylation Affects DNA-Binding of the senescence-regulating bZIP transcription factor GBF1. Plants 4(3), 691-709 (2015)
Smykowski, A., Fischer, S. M., Zentgraf, U.
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(2015) The elucidation of the interactome of 16 Arabidopsis bZIP factors reveals three independent functional networks. PLoS One 10(10):e0139884
Llorca, C.M., Berendzen, K.W., Malik, W.A., Mahn, S., Piepho, H.P., Zentgraf, U.
