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 (15)N labeling followed by MS. Reciprocal (14)N/(15)N labeling of entire Arabidopsis thaliana plants showed that full incorporation of (15)N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and (15)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.
Highlights
Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome
To analyze changes in protein abundance occurring during early leaf senescence, we developed a comprehensive quantitative proteomics strategy comprising differential labeling of proteins from A. thaliana wt and old1–1 mutant plants using cyanine dyes (CyDyes) and 14N/15N-isotopes followed by mass spectrometry (MS) (Fig. 1)
This time point was chosen because our focus was to identify alterations in protein abundance during early leaf senescence, i.e. the stage before visible symptoms of leaf senescence occur
Summary
Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. 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 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]. Suspension cultures of Arabidopsis thaliana cells or A. thaliana plants grown in liquid culture were successfully labeled with either stable isotope-coded amino acids [13] or with 15N (14 –18)
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