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 a-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.
Tetradecameric Clp protease core complexes in nonphotosynthetic plastids of roots, flower petals, and in chloroplasts of leaves of Arabidopsis thaliana were purified based on native mass and isoelectric point and identified by mass spectrometry. The stoichiometry between the subunits was determined. The protease complex consisted of one to three copies of five different serine-type protease Clp proteins (ClpP1,3-6) and four non-proteolytic ClpR proteins (ClpR1-4). Three-dimensional homology modeling showed that the ClpP/R proteins fit well together in a tetradecameric complex and also indicated unique contributions for each protein.Lateral exit gates for proteolysis products are proposed. In addition, ClpS1,2, unique to land plants, tightly interacted with this core complex, with one copy of each per complex. The three-dimensional modeling show that they do fit well on the axial sites of the ClpPR cores. In contrast to plastids, plant mitochondria contained a single ϳ320-kDa homo-tetradecameric ClpP2 complex, without association of ClpR or ClpS proteins. It is surprising that the Clp core composition appears identical in all three plastid types, despite the remarkable differences in plastid proteome composition. This suggests that regulation of plastid proteolysis by the Clp machinery is not through differential regulation of ClpP/R/S gene expression, but rather through substrate recognition mechanisms and regulated interaction of chaperone-like molecules (ClpS1,2 and others) to the ClpP/R core.Plastids are essential organelles of prokaryotic origin that are present in every plant cell and differentiate from proplastids into non-photosynthetic plastids in roots and flowers and photosynthetic plastids in leafs and stems. Plastids are responsible for synthesis of key molecules required for the architecture and functions of plant cells.To maintain a correct stoichiometry between different proteins and pathways, to remove and recycle damaged or misfolded proteins, and to control gene expression by proteolysis of transcription or translation factors, different proteolytic systems are present in the plastid. Members of at least five protease families are present in plastids, but their structures, functions, substrates, and biological importance are poorly understood (2).A very prominent group of proteases in plants is the Clp protease family. Our latest analysis of the Arabidopsis thaliana nuclear genome indicates the presence of at least 26 Clp-related genes, with 15 genes encoding for plastid-localized proteins (3) (Fig.
This study presents an analysis of the stromal proteome in its oligomeric state extracted from highly purified chloroplasts of Arabidopsis thaliana. 241 proteins (88% with predicted cTP), mostly assembled in oligomeric complexes, were identified by mass spectrometry with emphasis on distinguishing between paralogues. This is critical because different paralogues in a gene family often have different subcellular localizations and/or different expression patterns and functions. The native protein masses were determined for all identified proteins. Comparison with the few well characterized stromal complexes from A. thaliana confirmed the accuracy of the native mass determination, and by extension, the usefulness of the native mass data for future in-depth protein interaction studies. Resolved protein interactions are discussed and compared with an extensive collection of native mass data of orthologues in other plants and bacteria. Relative protein expression levels were estimated from spot intensities and also provided estimates of relative concentrations of individual proteins. No such quantification has been reported so far. Surprisingly proteins dedicated to chloroplast protein synthesis, biogenesis, and fate represented nearly 10% of the total stroma protein mass. Oxidative pentose phosphate pathway, glycolysis, and Calvin cycle represented together about 75%, nitrogen assimilation represented 5-7%, and all other pathways such as biosynthesis of e.g. fatty acids, amino acids, nucleotides, tetrapyrroles, and vitamins B 1 and B 2 each represented less than 1% of total protein mass. Several proteins with diverse functions outside primary carbon metabolism, such as the isomerase ROC4, lipoxygenase 2 involved in jasmonic acid biosynthesis, and a carbonic anhydrase (CA1), were surprisingly abundant in the range of 0.75-1
Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen-derived carotenoid b-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyllipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wildtype PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.
Gibberellins (GAs) are involved in the regulation of flowering and fruit-set in grapes (Vitis vinifera L.), but the molecular mechanisms behind this process are mostly unknown. In this work, the family of grapevine GA oxidases involved in the biosynthesis and deactivation of GAs was characterized. Six putative GA 20-oxidase (GA20ox), three GA 3-oxidase (GA3ox), and eight GA 2-oxidase (GA2ox) proteins, the latter further divided into five C19-GA 2ox and three C20-GA2ox proteins, were identified. Phylogenetic analyses suggest a common origin of the GA3ox and C19-GA2ox groups and challenge previous evolutionary models. In vitro analysis revealed that all GA3ox and GA20ox enzymes prefer substrates of the non-13-hydroxylation pathway. In addition, ectopic expression of GA2ox genes in Arabidopsis thaliana confirmed the activity of their encoded proteins in vivo. The results show that bioactive GA1 accumulates in opening grapevine flowers, whereas at later developmental stages only GA4 is detected in the setting fruit. By studying the expression pattern of the grapevine GA oxidase genes in different organs, and at different stages of flowering and fruit-set, it is proposed that the pool of bioactive GAs is controlled by a fine regulation of the abundance and localization of GA oxidase transcripts.
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