The chloroplast proteome contains many proteins that are of unknown function and not predicted to localize to the chloroplast. Expression of nuclear-encoded chloroplast genes is regulated at multiple levels in a pathway-dependent context. The combined shotgun proteomics and RNA profiling approach is of high potential value to predict metabolic pathway prevalence and to define regulatory levels of gene expression on a pathway scale.

We have analyzed proteome dynamics during light-induced development of rice (Oryza sativa) chloroplasts from etioplasts using quantitative two-dimensional gel electrophoresis and tandem mass spectrometry protein identification. In the dark, the etioplast allocates the main proportion of total protein mass to carbohydrate and amino acid metabolism and a surprisingly high number of proteins to the regulation and expression of plastid genes. Chaperones, proteins for photosynthetic energy metabolism, and enzymes of the tetrapyrrole pathway were identified among the most abundant etioplast proteins. The detection of 13 N-terminal acetylated peptides allowed us to map the exact localization of the transit peptide cleavage site, demonstrating good agreement with the prediction for most proteins. Based on the quantitative etioplast proteome map, we examined early light-induced changes during chloroplast development. The transition from heterotrophic metabolism to photosynthesis-supported autotrophic metabolism was already detectable 2 h after illumination and affected most essential metabolic modules. Enzymes in carbohydrate metabolism, photosynthesis, and gene expression were up-regulated, whereas enzymes in amino acid and fatty acid metabolism were significantly decreased in relative abundance. Enzymes involved in nucleotide metabolism, tetrapyrrole biosynthesis, and redox regulation remained unchanged. Phosphoprotein-specific staining at different time points during chloroplast development revealed light-induced phosphorylation of a nuclear-encoded plastid RNA-binding protein, consistent with changes in plastid RNA metabolism. Quantitative information about all identified proteins and their regulation by light is available in plprot, the plastid proteome database (http://www.plprot.ethz.ch).Plastids perform essential biosynthetic and metabolic functions in plants, including photosynthetic carbon fixation and synthesis of amino acids, fatty acids, starch, and secondary metabolites (Neuhaus and Emes, 2000;Lopez-Juez and Pyke, 2005). In response to tissue-specific and environmental signals, they differentiate into specialized plastid types that can be distinguished by their structure, pigment composition (color), and function. Examples of such different plastid types are elaioplasts in seed endosperm, chromoplasts in fruits and petals, amyloplasts in roots, etioplasts in dark-grown leaves, and chloroplasts in photosynthetically active leaf tissues (Neuhaus and Emes, 2000;Lopez-Juez and Pyke, 2005). Depending on their specific biosynthetic activity and energy metabolism, plastids are broadly classified as photosynthetic and nonphotosynthetic plant organelles. Photosynthetic chloroplasts synthesize sugar phosphates that are catabolized by oxidative metabolism to produce NADPH and ATP. Nonphotosynthetic plastid types import cytosolic sugar phosphates and ATP, which are necessary to sustain their anabolic metabolism. This difference in energy metabolism is often used to distinguish the autotrophic chloroplast from heterotro...

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We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request. Molecular & Cellular Proteomics 4:1072-1084, 2005.Plastids are plant cell organelles that have essential biosynthetic and metabolic activities. These include photosynthetic carbon fixation and synthesis of amino acids, fatty acids, starch, and secondary metabolites such as pigments. Although plastids lost their autonomy and transferred most of their genes to the nucleus during evolution (1), they have retained a small genome encoding ϳ90 proteins. Different plastid types develop in a tissue-specific manner (for a detailed review on plastid biogenesis, see Ref.2). According to their structure, pigment composition (color), and functional differentiation, plastids are classified as elaioplasts that are found in seed endosperm, chromoplasts in fruits and petals, amyloplasts in roots, etioplasts in dark-grown seedlings, and chloroplasts in photosynthetically active tissues (3). These specialized plastids types are typically the result of a differentiation program that is controlled by the cell and tissue type but also by environmental factors.Perhaps the best understood example of plastid differentiation is the light-dependent conversion of etioplasts into chloroplasts. After exposure of dark-grown seedlings to light etioplasts differentiate into photosynthetically active chloroplasts within a few hours. Chloroplast differentiation is accompanied by the assembly of the thylakoid membrane-localized electron transport system, which requires proteins encoded by genes in both nuclear and chloroplast genomes (4, 5). Although chloroplast differentiation has been investigated in detail for many years, the molecular mechanisms that control the differ...