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

Abstract

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 N 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. Molecular & Cellular Proteomics 7:, 108 -120.A major focus of proteome research is the simultaneous identification and quantification of proteins in cells, tissues, or organisms in dependence on the developmental stage, different physiological conditions, environmental influences, or genotypes. This quantitative, mass spectrometry (MS)1 -based description of proteomes was facilitated by the development of various stable isotope labeling techniques that have since been applied to proteomics studies in a multitude of organisms (1, 2). In plant proteomics, however, the most frequently used method for comparative, quantitative studies so far has been two-dimensional PAGE (3). In traditional two-dimensional PAGE approaches, quantitative differences in protein abundance between biological samples are revealed by comparing spot patterns in individual gels based on densitometric analysis following silver or Coomassie Blue staining. Limitations of this method regarding reproducibility, sensitivity, and dynamic range of protein quantification were improved significantly by introducing the DIGE technology (4). The DIGE technique employs spectrally resolvable fluorescent cyanine dyes (CyDyes) to label proteins prior to separation by twodimensional PAGE (5). Using the minimal labeling approach, two distinct protein samples are separately labeled with the fluorescent dyes Cy3 and Cy5, respectively, while an internal standard consisting of equa...

Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots

“…However, heavy water is toxic to organisms including plants and altered the transcriptome pattern in plants (9), so this approach may alter cell physiology and in vivo proteome-wide turnover rates. 15 N labeling had already been successfully used in labeling Arabidopsis cell culture and plants (15,19,21). Our DIGE results demonstrate that quantitative 15 N labeling does not change the proteome profile, which is consistent with other reports in plants (19,21,32).…”

“…15 N labeling had already been successfully used in labeling Arabidopsis cell culture and plants (15,19,21). Our DIGE results demonstrate that quantitative 15 N labeling does not change the proteome profile, which is consistent with other reports in plants (19,21,32). Hence, this strategy is probably the most nonintrusive way to investigate the pro-teome dynamics without changing physiological conditions in plant cells.…”

TheArabidopsis onset of leaf death5Mutation of Quinolinate Synthase Affects Nicotinamide Adenine Dinucleotide Biosynthesis and Causes Early Ageing

“…The ascorbate-deficient mutant vtc1 shows premature senescence when grown under a long photoperiod or when leaves are placed in the dark . Moreover, a proteomics study of the early senescence mutant old1 revealed that most of the highly upregulated proteins in the mutant belong to the family of glutathione S-transferases (Hebeler et al, 2008). This suggests that glutathione S-transferases in the context of leaf senescence are needed to protect cells against ROS during the process.…”

Proteomic Analyses of Cells Isolated by Laser Microdissection

“…Encouraging pioneer studies on specific subproteomes in plants have revealed candidate proteins that are phosphorylated under specific stress conditions (Oda et al, 1999;Benshop et al, 2007;Niittylä et al, 2007) or during the light independent cycle of photosynthesis (Reiland et al, 2009). Protein abundance changes have been monitored in response to heat shock (Palmblad et al, 2008), during leaf senescence (Hebeler et al, 2008) and during the protein turnover of photosynthetic proteins, monitored using pulse-chase labeling in combination with massspectrometry (Nowaczyk et al, 2006). The combination of subcellular fractionation techniques and mass-spectrometry has led to the extensive characterization of the plant subcellular proteome which in turn has led to the discovery of new metabolic pathways (Dunkley et al, 2006).…”