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. 2012 May 31;13:215.
doi: 10.1186/1471-2164-13-215.

Proteome Dynamics and Early Salt Stress Response of the Photosynthetic Organism Chlamydomonas Reinhardtii

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Free PMC article

Proteome Dynamics and Early Salt Stress Response of the Photosynthetic Organism Chlamydomonas Reinhardtii

Guido Mastrobuoni et al. BMC Genomics. .
Free PMC article

Abstract

Background: The cellular proteome and metabolome are underlying dynamic regulation allowing rapid adaptation to changes in the environment. System-wide analysis of these dynamics will provide novel insights into mechanisms of stress adaptation for higher photosynthetic organisms. We applied pulsed-SILAC labeling to a photosynthetic organism for the first time and we established a method to study proteome dynamics in the green alga Chlamydomonas reinhardtii, an emerging model system for plant biology. In addition, we combined the analysis of protein synthesis with metabolic profiling to study the dynamic changes of metabolism and proteome turnover under salt stress conditions.

Results: To study de novo protein synthesis an arginine auxotroph Chlamydomonas strain was cultivated in presence of stable isotope-labeled arginine for 24 hours. From the time course experiment in 3 salt concentrations we could identify more than 2500 proteins and their H/L ratio in at least one experimental condition; for 998 protiens at least 3 ratio counts were detected in the 24 h time point (0 mM NaCl). After fractionation we could identify 3115 proteins and for 1765 of them we determined their de novo synthesis rate. Consistently with previous findings we showed that RuBisCO is among the most prominent proteins in the cell; and similar abundance and turnover for the small and large RuBisCO subunit could be calculated. The D1 protein was identified among proteins with a high synthesis rates. A global median half-life of 45 h was calculated for Chlamydomonas proteins under the chosen conditions.

Conclusion: To investigate the temporal co-regulation of the proteome and metabolome, we applied salt stress to Chlamydomonas and studied the time dependent regulation of protein expression and changes in the metabolome. The main metabolic response to salt stress was observed within the amino acid metabolism. In particular, proline was up-regulated manifold and according to that an increased carbon flow within the proline biosynthetic pathway could be measured. In parallel the analysis of abundance and de novo synthesis of the corresponding enzymes revealed that metabolic rearrangements precede adjustments of protein abundance.

Figures

Figure 1
Figure 1
Cell growth and incorporation of stable isotope labeled arginine. Relation between observed protein ratio and cell number at 0 h, 1 h, 3 h, 8 h and 24 h after addition of stable isotope labeled arginine upon 0 mM, 100 mM and 150 mM NaCl; [A] cell numbers, [B] mean of all protein ratios and [C] mean ratios plotted against cell numbers at all conditions.
Figure 2
Figure 2
Genome scale overview of protein abundance and relative protein synthesis rates. Plot of protein abundance index versus protein H/L ratios from a proteomic analysis of Chlamydomonas reinhardtii (strain CC-1618) grown under standard conditions and metabolic labeling of proteins with 13 C-labeled arginine for 24 hours. All proteins with more than 4 ratio counts are shown in the diagram; Histones, RuBisCO and Photosystem II proteins (PS II) were selected and highlighted.
Figure 3
Figure 3
Time dependent incorporation of stable isotope labeled arginine and protein concentration. Reported are the H/L ratios and relative protein abundance of RuBisCO large (rbcL) and small subunit (rbcS) as well as of photosystem II proteins. [A] Protein ratios of rbcL and rbcS at 0, 1, 3, 8 and 24 h from control conditions (0 mM NaCl) are shown with the standard deviations of all observed H/L ratios of all corresponding peptides. [B] Bars show the average abundance of rbcL and rbcS from the 0, 1, 3, 8 and 24 h time points (0 mM NaCl). [C] Protein ratios of photosystem II proteins at 0, 1, 3, 8 and 24 h from the control condition (0 mM NaCl). [D] Bars show the average relative abundance of photosystem II proteins from the 0, 1, 3, 8 and 24 h time points (0 mM NaCl).
Figure 4
Figure 4
Abundance and turnover of PSII and Calvin-Benson-cycle proteins. MapMan visualization of the proteins of the functional categories ‘light reactions’ and ‘Calvin-Benson-Cycle’ with their [A] respective H/L ratios and [B] relative abundance.
Figure 5
Figure 5
Relative protein synthesis rates upon salt stress. Normalized protein ratios obtained from Chlamydomonas after 24 h incubation with stable isotope labeled arginine upon salt stress (100 mM and 150 mM NaCl). Normalized protein ratios of 24 h/100 mM and 24 h 150 mM are plotted against protein ratios obtained after 24 h/0 mM NaCl. The upper and lower 5% of proteins with altered protein ratios at both salt conditions are marked with colors (red - up regulated and blue – down regulated).
Figure 6
Figure 6
Pathway from arginine to proline and stable isotope incorporation in metabolites. Scheme of the metabolic pathway from arginine to putrescine, glutamate and proline; shown are the metabolites and reactions indicated by the gene names including their corresponding JGI identifier. Putrescine, glutamate and proline are highlighted with yellow background color; for those metabolites the 13 C stable isotope incorporation was measured by GC-MS the results are shown below the pathway scheme for each experimental time point and sodium chloride concentration. All proteins shown in black including the ARG7 protein could be identified by LC-MS/MS based proteomics at the 24 h/0 mM condition.
Figure 7
Figure 7
Dynamics of metabolite adjustments to salt stress. Overview about the metabolic changes of Chlamydomonas upon salt treatment; [A] principal component analysis (PCA) visualizing the metabolic changes measured by GC/MS based metabolic profiling, [B] relative changes of metabolite levels in Chlamydomonas after 24 hours treatment with 100 mM NaCl compared to 24 h time point of the control. The colors indicate different classes of metabolites: red and pink, intermediates of glycolysis and TCA cycle; light blue; amino acids, gray; others. [C] The temporal behavior of the amino acids proline, putrescine, glutamate and ornithine is shown at all time points compared to control (0 h/0 mM).

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