Article pubs.acs.org/jpr
Proteome Analysis of Plastids from Developing Seeds of Jatropha curcas L. Camila B. Pinheiro,†,∥ Mohibullah Shah,†,∥ Emanoella L. Soares,† Fábio C. S. Nogueira,‡ Paulo C. Carvalho,§ Magno Junqueira,‡ Gabriel D. T. Araújo,‡ Arlete A. Soares,† Gilberto B. Domont,*,‡ and Francisco A. P. Campos*,† †
Department of Biochemistry and Molecular Biology, Universidade Federal do Ceará, Bld. 907, Campus do Pici, 60455-900 Fortaleza, Ceará, Brazil ‡ Proteomic Unit, Institute of Chemistry, Universidade Federal do Rio de Janeiro, Avenida Athos da Silveira Ramos, 149 21941-909, Rio de Janeiro, Brazil § Laboratory for Proteomics and Protein Engineering, Carlos Chagas Institute, Fiocruz, Rua Prof. Algacyr Munhoz Mader, 3775 - CIC 81350-010 Curitiba, Paraná, Brazil S Supporting Information *
ABSTRACT: In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled inline to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 proteins were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in PPDB, SUBA, or PlProt databases while homologues for 13 proteins were not found in any of the three plastid proteins databases but were marked as plastidial by at least one of the three prediction programs used. Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in the plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, our study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level. KEYWORDS: Jatropha curcas, oilseed proteomics, plastid proteomics, plant proteomics
1. INTRODUCTION Plastids are essential organelles of photosynthetic eukaryotes1 that in nonphotosynthetic seeds of oil plants play a major role in carbon and nitrogen flow and are the site of the biosynthetic pathways for fatty acids, amino acids, growth regulators, and secondary metabolites such as several classes of diterpenes.1 Plastid proteomes of seeds from several species have been studied,2 but apart from rapeseed,3 plastids of other major oil crops such as soybean and castor bean have not been subjected to proteome analysis. In this context, analysis of the proteome of plastids from J. curcas seeds can contribute to the understanding of the biochemical pathways of fatty acid deposition and secondary metabolites in this species, which is considered to be a potential source of raw material for the production of biodiesel. The exploitation of this potential is © XXXX American Chemical Society
hampered not only by a lack of understanding regarding key aspects about the biosynthetic and signaling pathways related to the deposition of fatty acids during seed development4 but also due to the presence in the seeds of high amounts of toxic phorbol esters that render the cake produced from the extraction of oil unsuitable for animal feeding,5 therefore diminishing the commercial value of the crop. The growing interest in the commercial exploitation of this crop6 is being met by the publication of a flurry of large-scale transcriptomic studies focused in the expression of genes Special Issue: Agricultural and Environmental Proteomics Received: June 3, 2013
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dx.doi.org/10.1021/pr400515b | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
microtubes containing the acrylic LR white resin (London Resin) under UV light at 4 °C for 3 days. Ultrathin sections of 60 nm thickness were prepared using Leica Ultracut Ultramicrotome and loaded onto Formvar coated copper grids, then stained with 2% uranyl acetate solution at room temperature for 1 h before viewing through a Jeol 1011 transmission electron microscope, operated at 80 kV.
related to the biosynthesis of fatty acids in the developing seed.7,8 These studies were aided by the availability of the complete genome of the chloroplast9 and of a draft of the J. curcas genome that covers ∼95% of the gene-containing regions.10,11 Together, these studies created an important database to foster protein identification by proteomics of developing or germinating seeds.12,13 Here we took advantage of the availability of these databases to perform an in-depth proteome analysis of plastids isolated from developing seeds. The results we present here provide insights into the major biosynthetic pathways and into certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level and are discussed in terms of the involvement of the identified proteins in carbon and nitrogen flow, in metabolic pathways related to the biosynthesis of fatty acids, amino acids, and diterpenes, as well as in the energetic metabolism geared to provide the intermediates and reducing power to feed the biosynthetic pathways. The transport machinery required for the exchange of metabolites and proteins between plastids and other locations within the cell is also discussed.
2.3. Protein Extraction, 2-DE Separation, and in-Solution Digestion
Isolated plastids were subjected to protein extraction according to Vasconcelos et al.16 Plastid material was homogenized in pyridine buffer (50 mM pyridine, 10 mM thiourea, and 1%SDS, pH 5.0) with polyvinyl−polypyrrolidone in a proportion of 1:40:2 (w/v/w), respectively. This mixture was stirred for 2 h at 4 °C and centrifuged at 10 000g for 30 min. Proteins were precipitated from supernatant using cold 10% trichloroacetic acid (TCA) in acetone. Pellets were washed with cold acetone three times, dried under vacuum, and dissolved in 7 M urea/2 M thiourea. Protein concentration was determined by the Bradford assay.17 To evaluate the distribution pattern of plastids proteins, a 2D gel was run for the extracted proteins. For this purpose, 300 μg of the proteins was suspended in rehydration buffer (7M urea, 2 M thiourea, 1% CHAPS, 0.5% Pharmalyte 3−10, 65 mM DTT) and 11 cm Immobiline DryStrips, pH 4−7 (Amersham) were rehydrated with this solution overnight. IPGPhor IEF system from Amersham Pharmacia Biotech was used to perform the isoelectric focusing with electrical conditions as described by the supplier. Prior to separation in the second dimension, the IPG gel strips were equilibrated at room temperature in 3 mL of equilibration buffer (50 mM tris, 30% glycerol, 6 M urea, 2% SDS, and traces of bromophenol blue) containing 57.8 mg of DTT for 20 min. Strips were then alkylated for an additional 20 min in 3 mL of equilibration buffer containing 69.3 mg of iodoacetamide. After equilibration, IPG gel strips were transferred to SDS containing uniform 15% polyacrylamide separating gel (14 × 14 cm) to perform the second dimension of the electrophoresis. After 2DE, protein spots were visualized by 0.1% PhastGel Blue R-350 staining. Before trypsin digestion, 40 μg of proteins was subjected to reduction with 10 mM DTT for 30 min at 56 °C and alkylation with 55 mM iodoacetamide for 30 min at room temperature in dark. After dilution of urea concentration to
