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Research

Study of Early Leaf Senescence in Arabidopsis thaliana by Quantitative Proteomics Using Reciprocal ¹⁴N/¹⁵N Labeling and Difference Gel Electrophoresis^*,S

Romano Hebeler^,, Silke Oeljeklaus^,, Kai A. Reidegeld, Martin Eisenacher, Christian Stephan, Barbara Sitek, Kai Stühler, Helmut E. Meyer, Marcel J. G. Sturre^¶, Paul P. Dijkwel^¶ and Bettina Warscheid^,||

From the Medizinisches Proteom-Center, Zentrum fuer klinische Forschung, Ruhr-Universitaet Bochum, Universitaetsstraβe 150, 44780 Bochum, Germany and ^¶ Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands

Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic ¹⁵N labeling followed by MS. Reciprocal ¹⁴N/¹⁵N labeling of entire Arabidopsis thaliana plants showed that full incorporation of ¹⁵N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and ¹⁵N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before.