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Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models.

Plastoglobules (PGs) in chloroplasts are thylakoid-associated monolayer lipoprotein particles containing prenyl and neutral lipids and several dozen proteins mostly with unknown functions. An integrated view of the role of the PG is lacking. Here, we better define the PG proteome and provide a conceptual framework for further studies. The PG proteome from Arabidopsis (Arabidopsis thaliana) leaf chloroplasts was determined by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids, and stroma. Scanning electron microscopy showed the purity and size distribution of the isolated PGs. Compared with previous PG proteome analyses, we excluded several proteins and identified six new PG proteins, including an M48 metallopeptidase and two Absence of bc1 complex (ABC1) atypical kinases, confirmed by immunoblotting. This refined PG proteome consisted of 30 proteins, including six ABC1 kinases and seven fibrillins together comprising more than 70% of the PG protein mass. Other fibrillins were located predominantly in the stroma or thylakoid and not in PGs; we discovered that this partitioning can be predicted by their isoelectric point and hydrophobicity. A genome-wide coexpression network for the PG genes was then constructed from mRNA expression data. This revealed a modular network with four distinct modules that each contained at least one ABC1K and/or fibrillin gene. Each module showed clear enrichment in specific functions, including chlorophyll degradation/senescence, isoprenoid biosynthesis, plastid proteolysis, and redox regulators and phosphoregulators of electron flow. We propose a new testable model for the PGs, in which sets of genes are associated with specific PG functions.

Chloroplasts in angiosperms contain at least seven nucleus-encoded members of the DEAD box RNA helicase family. Phylogenetic analysis shows that five of these plastid members (RH22, -39, -47, -50, and -58) form a single clade and that RH3 forms a clade with two mitochondrial RH proteins (PMH1 and -2) functioning in intron splicing. The function of chloroplast RH3 in maize (Zea mays; ZmRH3) and Arabidopsis (Arabidopsis thaliana; AtRH3) was determined. ZmRH3 and AtRH3 are both under strong developmental control, and ZmRH3 abundance sharply peaked in the sink-source transition zone of developing maize leaves, coincident with the plastid biogenesis machinery. ZmRH3 coimmunoprecipitated with a specific set of plastid RNAs, including several group II introns, as well as pre23S and 23S ribosomal RNA (rRNA), but not 16S rRNA. Furthermore, ZmRH3 associated with 50S preribosome particles as well as nucleoids. AtRH3 null mutants are embryo lethal, whereas a weak allele (rh3-4) results in pale-green seedlings with defects in splicing of several group II introns and rRNA maturation as well as reduced levels of assembled ribosomes. These results provide strong evidence that RH3 functions in the splicing of group II introns and possibly also contributes to the assembly of the 50S ribosomal particle. Previously, we observed 5-to 10-fold upregulation of AtRH3 in plastid Caseinolytic protease mutants. The results shown here indicate that AtRH3 up-regulation was not a direct consequence of reduced proteolysis but constituted a compensatory response at both RH3 transcript and protein levels to impaired chloroplast biogenesis; this response demonstrates that cross talk between the chloroplast and the nucleus is used to regulate RH3 levels.

Plant plastids and mitochondria have dynamic proteomes. Protein homeostasis in these organelles is maintained by a proteostasis network containing protein chaperones, peptidases, and their substrate recognition factors. However, many peptidases, as well as their functional connections and substrates, are poorly characterized. This review provides a systematic insight into the organellar peptidase network in Arabidopsis thaliana. We present a compendium of known and putative Arabidopsis peptidases and inhibitors, and compare the distribution of plastid and mitochondrial peptidases to the total peptidase complement. This comparison shows striking biases, such as the (near) absence of cysteine and aspartic peptidases and peptidase inhibitors, whereas other peptidase families were exclusively organellar; reasons for such biases are discussed. A genome-wide mRNA-based coexpression data set was generated based on quality controlled and normalized public data, and used to infer additional plastid peptidases and to generate a coexpression network for 97 organellar peptidase baits (1742 genes, making 2544 edges). The graphical network includes 10 modules with specialized/enriched functions, such as mitochondrial protein maturation, thermotolerance, senescence, or enriched subcellular locations such as the thylakoid lumen or chloroplast envelope. The peptidase compendium, including the autophagy and proteosomal systems, and the annotation based on the MEROPS nomenclature of peptidase clans and families, is incorporated into the Plant Proteome Database.

An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one- and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were alpha-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http://cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.

The plastid ClpPRT protease consists of two heptameric rings of ClpP1/ClpR1/ClpR2/ClpR3/ClpR4 (the R-ring) and ClpP3/ClpP4/ClpP5/ClpP6 (the P-ring) and peripherally associated ClpT1/ClpT2 subunits. Here, we address the contributions of ClpP3 and ClpP4 to ClpPRT core organization and function in Arabidopsis (Arabidopsis thaliana). ClpP4 is strictly required for embryogenesis, similar to ClpP5. In contrast, loss of ClpP3 (clpp3-1) leads to arrest at the hypocotyl stage; this developmental arrest can be removed by supplementation with sucrose or glucose. Heterotrophically grown clpp3-1 can be transferred to soil and generate viable seed, which is surprising, since we previously showed that CLPR2 and CLPR4 null alleles are always sterile and die on soil. Based on native gels and mass spectrometry-based quantification, we show that despite the loss of ClpP3, modified ClpPR core(s) could be formed, albeit at strongly reduced levels. A large portion of ClpPR subunits accumulated in heptameric rings, with overaccumulation of ClpP1/ClpP5/ClpP6 and ClpR3. Remarkably, the association of ClpT1 to the modified Clp core was unchanged. Large-scale quantitative proteomics assays of clpp3-1 showed a 50% loss of photosynthetic capacity and the up-regulation of plastoglobules and all chloroplast stromal chaperone systems. Specific chloroplast proteases were significantly up-regulated, whereas the major thylakoid protease (FtsH1/FtsH2/FtsH5/FtsH8) was clearly unchanged, indicating a controlled protease network response. clpp3-1 showed a systematic decrease of chloroplast-encoded proteins that are part of the photosynthetic apparatus but not of chloroplast-encoded proteins with other functions. Candidate substrates and an explanation for the differential phenotypes between the CLPP3, CLPP4, and CLPP5 null mutants are discussed.

Protein (de)phosphorylation plays an important role in plants. To provide a robust foundation for subcellular phosphorylation signaling network analysis and kinase-substrate relationships, we performed a meta-analysis of 27 published and unpublished in-house mass spectrometry-based phospho-proteome data sets for Arabidopsis thaliana covering a range of processes, (non) photosynthetic tissue types, and cell cultures. This resulted in an assembly of 60,366 phospho-peptides matching to 8141 nonredundant proteins. Filtering the data for quality and consistency generated a set of medium and a set of high confidence phospho-proteins and their assigned phospho-sites. The relation between single and multiphosphorylated peptides is discussed. The distribution of p-proteins across cellular functions and subcellular compartments was determined and showed overrepresentation of protein kinases. Extensive differences in frequency of pY were found between individual studies due to proteomics and mass spectrometry workflows. Interestingly, pY was underrepresented in peroxisomes but overrepresented in mitochondria. Using motif-finding algorithms motif-x and MMFPh at high stringency, we identified compartmentalization of phosphorylation motifs likely reflecting localized kinase activity. The filtering of the data assembly improved signal/noise ratio for such motifs. Identified motifs were linked to kinases through (bioinformatic) enrichment analysis. This study also provides insight into the challenges/pitfalls of using large-scale phospho-proteomic data sets to nonexperts.

5-Aminolevulinic acid (ALA) is the first committed substrate of tetrapyrrole biosynthesis and is formed from glutamyl-tRNA by two enzymatic steps. Glutamyl-tRNA reductase (GluTR) as the first enzyme of ALA synthesis is encoded by HEMA genes and tightly regulated at the transcriptional and posttranslational levels. Here, we show that the caseinolytic protease (Clp) substrate adaptor ClpS1 and the ClpC1 chaperone as well as the GluTR-binding protein (GBP) interact with the N terminus of GluTR. Loss-of function mutants of ClpR2 and ClpC1 proteins show increased GluTR stability, whereas absence of GBP results in decreased GluTR stability. Thus, the Clp protease system and GBP contribute to GluTR accumulation levels, and thereby the rate-limiting ALA synthesis. These findings are supported with Arabidopsis (Arabidopsis thaliana) hema1 mutants expressing a truncated GluTR lacking the 29 N-terminal amino acid residues of the mature protein. Accumulation of this truncated GluTR is higher in dark periods, resulting in increased protochlorophyllide content. It is proposed that the proteolytic activity of Clp protease counteracts GBP binding to assure the appropriate content of GluTR and the adequate ALA synthesis for chlorophyll and heme in higher plants.

Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C₄ photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization. The experimental analysis was based on high-accuracy mass spectrometry, protein quantification by spectral counting, and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families, and gene duplications related to the polyploidy of maize; this avoided overidentification of proteins and resulted in more accurate protein quantification. A total of 1,105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Nearly complete coverage of primary carbon, starch, and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen, and amino acid metabolism, was obtained. This showed, for example, quantitative and qualitative cell type-specific specialization in starch biosynthesis, arginine synthesis, nitrogen assimilation, and initial steps in sulfur assimilation. An extensive overview of BS and M chloroplast protein expression and homeostasis machineries (more than 200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundance, and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database.