Proteome dynamics and early salt stress response of the photosynthetic organism Chlamydomonas reinhardtii.
Proteome dynamics and early salt stress response of the photosynthetic organism Chlamydomonas reinhardtii.
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DOI：
10.1186/1471-2164-13-215
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发表时间：
2012-05-31
期刊：
影响因子：
4.4
通讯作者：
Kempa S
中科院分区：
文献类型：
--
作者：
Mastrobuoni G;Irgang S;Pietzke M;Assmus HE;Wenzel M;Schulze WX;Kempa S
细胞蛋白质组和代谢组是潜在的动态调节，允许快速适应环境的变化。对这些动态的全系统分析将为高等光合作用生物的逆境适应机制提供新的见解。我们首次将脉冲SILAC标记应用于光合作用有机体，建立了一种研究绿藻衣藻蛋白质组动力学的方法，这是一种新兴的植物生物学模式系统。此外，我们还将蛋白质合成分析与代谢谱分析相结合，研究了盐胁迫条件下植物代谢和蛋白质组周转的动态变化。为了研究从头合成蛋白质，在稳定同位素标记的精氨酸存在下，培养了一株精氨酸营养缺陷型衣藻。从3种盐浓度的时程实验中，我们可以在至少一个实验条件下检测到2500多个蛋白质及其H/L比值，对于998个蛋白质，在24 h时间点(0 mMNaC l)至少检测到3个比值计数。分离后共鉴定出3115个蛋白质，测定了其中1765个蛋白质的从头合成速率。与以前的发现一致，我们发现Rubisco是细胞中最重要的蛋白质之一；并且可以计算出小的和大的Rubisco亚基的相似的丰度和周转率。在合成速率较高的蛋白质中鉴定出了D1蛋白。在选定的条件下，衣藻蛋白的全球中位半衰期为45 h。为了研究蛋白质组和代谢组的时间共调控，我们对衣藻施加盐胁迫，并研究了蛋白质表达和代谢组变化的时间依赖性调节。对盐胁迫的主要代谢反应是氨基酸代谢。尤其是，Pro被上调了多种形式，根据这一点，可以测量到Pro生物合成途径中增加的碳流。同时，对相应酶的丰度和从头合成的分析表明，代谢重排先于蛋白质丰度的调节。
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. 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. 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.
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