Mol Biol Rep (2012) 39:5069–5083 DOI 10.1007/s11033-011-1302-4
Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome Abu Hena Mostafa Kamal • Kun Cho • Setsuko Komatsu • Nobuyuki Uozumi • Jong-Soon Choi • Sun Hee Woo
Received: 21 April 2011 / Accepted: 30 November 2011 / Published online: 11 December 2011 Ó Springer Science+Business Media B.V. 2011
Abstract We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from
Electronic supplementary material The online version of this article (doi:10.1007/s11033-011-1302-4) contains supplementary material, which is available to authorized users. A. H. M. Kamal S. H. Woo (&) Department of Crop Science, Chungbuk National University, 410 Seongbong-ro, Heungdeok-gu, Cheongju, Chungbuk 361-763, Korea e-mail:
[email protected] K. Cho Mass Spectrometry Research Center, Korea Basic Science Institute, Chungbuk 863-883, Korea S. Komatsu National Institute of Crop Science, NARO, Tsukuba 305-8517, Japan N. Uozumi Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan J.-S. Choi Division of Life Science, Korea Basic Science Institute, Daejeon 305-333, Korea J.-S. Choi Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Korea
the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat. Keywords Wheat chloroplast Thylakoid LTQ-FTICR-MS Proteomics Abbreviations BSA Bovine serum albumin cTP Chloroplast transit peptide FT Fourier transform ICR Ion cyclotron resonance LTQ Linear quadruple trap PIR Protein information resources
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SP TMD
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Signal peptide Transmembrane domain
Introduction In plants, algal and cyanobacterial photosynthesis uses carbon dioxide and water, releasing oxygen as a waste product. Photosynthesis is vital for life on earth. Nearly all life depends on it either directly as a source of energy or indirectly as the ultimate source of the energy in its food [1]. In plants, different sorts of proteins are held inside organelles called chloroplasts, while in bacteria they are embedded in the plasma membrane. Some of the light energy gathered by chlorophylls is stored as adenosine triphosphate (ATP). The rest of the energy is used to remove electrons from a substance such as water. These electrons are then used in the reactions that turn carbon dioxide into organic compounds. In plant, photosynthesis takes place in organelles; chloroplast is the best-known plastid type and contains a thylakoid membrane system that carries the photosynthetic electron transport chain converting light into chemical energy. Four multi-subunit protein complexes such as photosystem I (PSI), photosystem II (PSII), the ATP-synthase complex, and the cytochrome b6/f complex, which together compose 75–100 proteins in Arabidopsis, perform the photosynthetic process [2]. Additionally supporting photosynthesis, chloroplasts also synthesize hormones, fatty acid, lipids, amino acids, vitamins (B1, K1, and E), nucleotides, and secondary metabolites such as alkaloids and isoprenoids, and are required for nitrogen and sulfur assimilation [3]. To understand chloroplast function, biogenesis, and its many biosynthetic pathways, it is critical to characterize the chloroplast proteome as a complementary transcriptome profile. Proteomics includes the determination of the protein expression levels of the chloroplast proteome composition, and the relative expression levels of chloroplast proteins are not static but vary depending on the development state as well as environmental conditions [4]. Subcellular proteomic studies are essential to gain access to the protein location with their function [5]. Plant proteomics exemplifies this functional dimension perfectly with the recent explosion of proteomic initiatives, which are more and more focused on the analyses of subcellular compartments [6]. Plant mitochondria [7, 8], chloroplast [9, 10], chloroplast thylakoid membrane [11], plasma membrane [12–14], peroxisome [15], endoplasmic reticulum [16], the cell wall [17], ribosome [18], and envelope membranes [19] have been studied using proteomic approaches. The sub-proteome sample complexity can also be reduced for a more accurate protein location. For instance, the chloroplast can be subdivided into the
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envelope membranes, the stroma, and the thylakoids. Some papers describe both a systematic proteomic and an in silico approach aimed at the identification of the thylakoid luminal and peripheral proteins [20, 21]. Proteomic studies using 1D-PAGE and two-dimensional (2D) gel electrophoresis have provided useful insights on soluble proteins of the lumen and stroma, as well as peripheral [22, 23] and integral thylakoid membrane proteins [24, 25] by using different kinds of high-throughput mass spectrometry such as matrix-assisted laser desorption/ionization-times of flight (MALDI-TOF), liquid chromatography- mass spectrometry (LC–MS/MS), and Edman Sequencing. Interestingly, mass spectrometry (MS) is a powerful tool for analysis of isoforms and secondary modifications of proteins such as glycosylation, phosphorylation, isoprenylation, methylation, and so on [26]. In this study, we present detailed reproducible 1D-PAGE (used as Tricine-SDS-PAGE) patterns of the envelope and stroma, thylakoid, lumen, and integral and peripheral membrane proteins of chloroplast from well-developed wheat leaf using LTQ-FTICR mass spectrometry. From mass spectrometric analyses at low mass accuracy using a LTQ ion trap, false positive rates can be minimized by filtering of peptides, but not focusing at their expected iso-electric point (pI). Analysis using a LTQ-FTICR mass spectrometer delivers low false positive rates by itself due to the high mass accuracy and dynamic range of mass spectra [27]. Apart from the MS platform, the best performance was observed with the LTQ-FTICR data set (96.9% sensitivity), followed by the quadrupole time of flight (QTOF) and LTQ (95.7%), and then the LCQ and Agilent ion trap, with 83.4% and 81.4% sensitivity, respectively [28]. The aim of the present work was to enhance our understanding of the biochemical machinery of the chloroplast thylakoids and sub-organelle membrane of developed wheat leaves. The identification of these proteins are extensively discussed with respect to their sub-cellular location and their implications in the chloroplast metabolism as photosynthesis and energy transportation, which is provided a new insight of the wheat chloroplast metabolism, and also low abundance proteins have been detected by LTQ-FTICR mass spectrometry using proteomic approach.
Materials and methods Growth of plants Experiments were carried out using the facilities of College of Agriculture, Life and Environments green house, Chungbuk National University, South Korea at 10th August 2009. Wheat (Triticum aestivum L.) cv. Keumgang
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plants were grown on sandy-loam soil for 20 days in a greenhouse at 26°C/21°C day/night temperatures with 10–11-h natural light and 13–14-h darkness per day. The fully developed leaves were collected and used for chloroplast and sub-organelle compartment isolation.
Isolation of chloroplasts Intact chloroplasts were isolated and purified from fully developed leaves on Percoll gradients according to D’Amici et al. [23] with some modifications. For the preparation of intact chloroplasts, 50 g of wheat leaves were washed with deionized water and then drained of excess water; the leaves were then cut into small (2–3 cm) pieces. For optimal yield of intact chloroplasts, the plant leaves were kept in darkness at 4°C overnight before the preparation to avoid starch accumulation. Grinding buffer (150 ml) (330 mM sorbitol, 20 mM tricine, 5 mM EGTA, 5 mM EDTA, 10 mM Na2CO3, 0.1% (w/v) BSA, 1.9 mM ascorbic acid) was added to the pieces of leaves in four blender strokes (3 min) for homogenization with minimal production of froth. Filtering of the macerated plants was done through 3 layers of miracloth filter paper; the filtrate was then divided between 4-ml super centrifuge tubes (about 35 ml for each tube) and centrifuged for 3 min at 3,0009g (4°C). The white pellets were discarded, and the supernatant was transferred into four clean tubes and centrifuged for 7 min at 9,0009g (4°C). The white pellet precipitates were again discarded, and the supernatant was transferred into scrupulously clean 50-ml tubes and centrifuged for 7 min at 4,0009g (4°C). The green pellet was re-suspended in grinding buffer using a little brush to avoid foaming and to achieve uniform suspension. Both intact and broken chloroplasts were obtained in the green pellet. The chloroplast suspension was carefully overlaid on 40% Percoll solution, with centrifugation for 10 min at 8,0009g (4°C) using Mega 17R by Hanil Science Industrial, Seoul, South Korea. After centrifugation, the broken chloroplast forms a large green band on top of the Percoll layer, whereas the intact chloroplasts sediment to the bottom as a small green pellet. The green pellet is very fragile; therefore, all the upper phases and the Percoll layer should be carefully removed using a Pasteur pipette. The green band should be re-suspended in 500-ll storage solution [grinding buffer but without 0.1% (w/v) BSA] and stored at -80°C until use. For functional assays, all steps should be performed in the dark at 4°C, and intact chloroplasts should be used as early as possible to prevent loss of protein and enzyme activity. The total chlorophyll in chloroplast was estimated as described previously [29]. Protein concentration was measured by Bio-Rad protein assay as a standard of BSA according to the manufacturer’s instructions.
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Fractionation of sub-organellar compartments Sub-organelle compartments such as stroma, envelope, thylakoids, thylakoid membranes and lumen were fractionated from intact chloroplast (protein concentration at 1.06 mg/ml protein) using a combination of differential gradient centrifugation [20]. For the preparation of suborganelles, chloroplasts were ruptured by osmotic shock in lysis medium (50 mM Tris–HCl, pH 8.0, and 5 mM MgCl2) at a chlorophyll concentration of 0.08 mg/ml for 10 min at 4°C. This lysis medium and all solutions used in subsequent steps contained a protease inhibitor cocktail for plant cell and tissue extracts (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride, bestatin hydrochloride, E-64 protease inhibitor, leupeptin hemisulfate salt, pepstatin A and 1, 10-phenanthroline). Thylakoids were recovered by ultra-centrifugation (Rotor; SW-28; Beckman Coulter, Inc., Germany) at 16,0129g for 30 min at 4°C, washed three times at 16,0129g for 10 min at 4°C with 10 mM Tris–HCl (pH 8.0), and re-suspended in lysis medium. The clear supernatant from chloroplast, containing stroma and envelope proteins, was concentrated to 3.4 mg/ml protein in an Amicon cell (3-kD cut-off filter). To liberate the soluble lumenal proteins, we then sonicated the thylakoids at 10 times for 30 s each at 4°C (power 10.0; Sonics and Materials, Inc., USA). The thylakoid membranes were separated from the soluble lumenal proteins by ultra-centrifugation (Rotor; Type 70 Ti) for 1 h at 129,9569g at 4°C and concentrated to 1.39 mg/ml protein. The clear supernatant containing the lumenal proteins was concentrated in an Amicon (Beverly, MA) cell (3-kD cutoff filter) to a protein concentration of 1.11 mg/ml protein. For the isolation of the peripheral thylakoid proteins, the sonicated and pelleted thylakoid membranes were washed once with 10 mM Tris-HCl (pH 8.0), centrifuged (Rotor; Type 70 Ti) at 129,9569g for 30 min at 4°C, and re-suspended in 25 mM Mes (pH 6.5) and 0.5 M CaCl2. This thylakoid suspension was gently stirred for 30 min at 4°C and centrifuged (Rotor; Type 70 Ti) for 1 h at 194,1329g at 4°C to separate the extracted peripheral thylakoid proteins from the remaining thylakoid membranes. In these fractionation steps, we performed ultracentrifugation (Beckman Optima LE-80K, Brea, CA 92822-8000 USA) at 4°C under dark conditions. The clear supernatant containing the peripheral proteins was concentrated to 3.10 mg/ml protein in an Amicon cell (3-kD cut-off filter) while reverting to 10 mM Tris–HCl (pH 8.0). The yield of integral membrane was concentrated to 0.52 mg/ml protein from isolated thylakoid membrane. The extracted proteins from the supernatant and pellet (chloroplast and sub-organelles) were precipitated with 100% cold acetone at -20°C for 1 h and collected by centrifugation at 10,0009g for 30 min at 4°C. The pellet
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was washed with 10 mM Tris–HCL (pH 8.0), collected by centrifugation at 10,0009g for 10 min at 4°C with drying under speed vacuum, and re-suspended in solubilization buffer (6 M Urea, 2 M Thio-urea, 10 mM Tris–HCL (pH 8.0) 2% SDS, 5 mM TBP, 2% CHAPS, 50 mM DTT, and Protease Inhibitor Cocktail for plant tissue and tissue extracts). Purity of chloroplast fractions The purity of chloroplast proteins was determined by calculating the percentage of chloroplast proteins and impurities from other origins because of 4 sub-fractions investigated from SDS-PAGE gel. After LTQ-FTICR-MS based identification of this chloroplast fractions, 767 unique proteins from 1043 proteins were assigned in 4 chloroplast sub-fractions (thylakoid membrane, lumen, integral, and peripheral membrane) representing a purity of 73.5% (Supplementary Table 1) using WolF PSORT and PSORT sub-cellular location predictor based on UNIPROT databases. From chloroplast fractions, we identified 276 proteins, which is located in other sub-cellular organs. Out of 276 proteins, 45 proteins were identified in cytoskeleton (cysk), 128 in cytosol (cyto), 1 in endoplasmic reticulum (ER), 4 in extracellular (extr), 17 in mitochondria (mito), 71 in nuclear (nucl), 2 in peroxisome (pero), and 8 in vacuolar membrane (vac), which is representing an impurity of 26.5% (Supplementary Table 2). Four chloroplast sub-organelles were analyzed and each sample was carried out with two methodological replications [30]. Tricine-SDS-PAGE Proteins from the differentially purified samples were heated at 100°C for 3 min and centrifuged at 15,0009g before separation using Tricine-SDS-PAGE. Proteins were separated on 16 cm 9 16 cm Tricine-SDS-PAGE gels (gradient 14–16% acrylamide) as described previously [31], with minor modifications. The electrophoresis conditions were set and run at 30 V for 1 h, 150 V for 1 h, and 230 V until Coomassie Brilliant Blue R-250 (CBB) dye reached the lower part of the gel. The gels were fixed and the proteins visualized by either high-sensitivity silver staining (GE Healthcare) or CBB R-250. Gel images were taken with the Digital Imaging System (Lab Solutions, Seoul, South Korea).
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methanol, and destained with 10 mM NH4CO3 in 50% ACN. The protein was reduced with 10 mM DTT in 100 mM NH4CO3 at 56°C for 1 h and alkylated with 55 mM IAA (iodoacetamide) in 100 mM NH4CO3 in the dark for 40 min. The gel pieces were minced, lypolized, and then rehydrated in 50 mM NH4CO3 with 11.9 ng/ll sequencing-grade modified trypsin (Promega Corporation, Madison, WI 53711 USA) at 37°C overnight. After tryptic digestion, the peptides were extracted four times with a solution containing 0.1% formic acid in 50% ACN. The solution containing eluted peptides was concentrated under a vacuum concentrator until dry. Gel bands were excised and subjected to in-gel digestion couple to tandem mass spectrometry (MS/MS) as described [32]. All MS/MS experiments for peptide identification was performed using a Nano-LC/MS system consisting of a Surveyor HPLC system and a 7-tesla LTQ-FTICR mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nano-ESI source. Ten microliters (10 ll) of each sample was loaded by an autosampler (Surveyor) onto a C18 trap column (I.D., 300 lm; length, 5 mm; particle size, 5 lm; LC Packings) for desalting and concentration at a flow rate of 20 ll/min. Then, the trapped peptides were back-flushed and separated on a homemade micro-capillary column (length, 100 mm) packed with C18 (particle size, 5 lm) in 75-lm silica tubing (8-lm i.d. orfice). The mobile phases A and B were composed of 0% and 80% acetonitrile, respectively, containing 0.02% (v/v) formic acid and 0.5% (v/v) acetic acid. The gradient began at 5% B for 15 min, and was ramped to 20% B for 3 min, to 50% for 47 min, to 95% for 2 min, and finally, to 95% B for 5 min. The column was equilibrated at 5% B for 6 min before the next run. Briefly, the mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. The Xcalibur software (Thermo Scientific, Germany) was enabled for each MS/MS spectrum. The target ions selected for MS/ MS were dynamically excluded for 60 s. The general mass spectrometric conditions were as follow: spray voltage, 2.2 kV; no sheath and auxiliary gas flow; ion transfer tube temperature, 220°C; collision gas pressure, 1.3 mT; normalized collision energy using wide band activation mode; and 35% for MS/MS. The ion selection threshold was 500 counts for MS/MS. An activation q = 0.25 and an activation time of 30 ms were applied in MS/MS acquisitions. Bioinformatics
In-gel digestion and mass spectrometry analysis Protein bands were manually excised from SDS-polyacryl amide gel, and in-gel digestion by trypsin was performed as described previously [32]. In brief, CBB-stained gel slices were washed several times with water and then with 30%
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To identify the peptides, MASCOT (version 2.0, Matrix Science, London, UK), operated on a local server, was used to search the viridiplantae (green plants) within the Uniprot_Sprot database (v 57.12). MASCOT was used with the monoisotopic mass selected, a peptide mass
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tolerance of 5 ppm, and a fragment ion mass tolerance of 0.5 Da. Trypsin was selected as enzyme, with one potential missed cleavage. ESI-FTICR was selected as instrument type, and carbamidomethyl cysteine and oxidized methionine were chosen as variable modifications [33]. All proteins identified by high-scoring peptides were considered true matches, and at least two peptide matches. The highscoring peptides corresponded with the peptides that were above the threshold in our MASCOT search (expected P \ 0.05, peptide score [29). Prediction of subcellular localization and functional domain analysis Functional domain analysis was performed for the identified proteins, which was the translated open reading frame using different freeware programs on the internet. WolF PSORT [http://wolfpsort.org/] [34] and PSORT [http://psort. ims.u-tokyo.ac.jp] [35] are knowledge-based programs for predicting subcellular location as chloroplast, chloroplast stroma, chloroplast thylakoid membrane, integral membrane, and chloroplast thylakoid spaces (lumen), respectively. PSORT was set to plant source of input sequence. ChloroP ver. 1.1 [http://www.cbs.dtu.dk/services/ChloroP/] [36] is a neural network method for identifying probable chloroplast transit peptide (cTP) sequences; its prediction of chloroplast targeting as Y (Yes) or N (No) output is based on the predicted presence of a cTP. SignalP ver. 3.0 [http://www. cbs.dtu.dk/services/SignalP/] [37] is based on neural network and Hidden-Markov model methods, which allow intolerance among eukaryotic, Gram-positive, and Gram-negative bacterial signal peptides (SP). TMHMM ver. 2.0 [http://www.cbs. dtu.dk/services/TMHMM/] [38] is a transmembrane domain (TMD) prediction method based on a Hidden-Markov model; it predicts transmembrane helices, and discriminates between soluble and membrane proteins with a high degree of accuracy. Protein Information Resources [http://pir.georgetown. edu] (PIR) is an integrated public bioinformatics resource that supports genomic, proteomic, and systems biology research. PIR was used for identifying gene ontology based biological functions, which is automatically classified the data set based on biological process using Batch Retrieval with iProClass database.
Results Gradient centrifugation and identification of proteins in thylakoid of chloroplast To determine the composition of wheat (Triticum aestivum L.) chloroplast thylakoid and address the dynamic range of proteins, intact chloroplast from well-developed leaves of
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wheat were separated, and sub-organelles were fractionated. With this protocol, an average of 1.06 mg/ml protein was isolated from 200 g of wheat leaves. An important prerequisite to obtaining a concise pattern of the suborganelles [thylakoid membrane, thylakoid space (lumen), integral membrane, and peripheral membrane] of chloroplast thylakoid was a reproducible second-dimension electrophoresis system that was capable of resolving the sub-organelles of thylakoid proteins using tricine-SDSPAGE (Fig. 1a). Following tryptic digestion, four fractions were analyzed using LTQ-FTICR-MS; 283 unique proteins out of 9,917 proteins in thylakoid membrane, 213 unique proteins out of 6,645 proteins in thylakoid space (lumen), 212 unique proteins out of 11,670 proteins in integral membrane, and 59 unique proteins out of 2,234 proteins from peripheral membrane fraction (data not shown) were detected using LTQ-FTICR-MS, which is highly sensitive and accurate, and hybrid mass spectrometry. WolF PSORT and PSORT are extensive web-based tools for predicting the subcellular localization of proteins. In WolF PSORT, sequence alignments of the query to similar proteins (14 nearest neighbors), and links to UniProt and Gene Ontology is provided. To identify chloroplast thylakoid space (lumen), we used the PSORT program because WolF PSORT could not identify this sub-organelle location. Peripheral membrane proteins are associated with membranes but do not penetrate the hydrophobic core of the membrane. They are often found in association with integral membrane proteins. According to the previous report [20], the peripheral protein ATP synthase (CF a, b, c, d, e), plastocyanin, and oxygen-evolving complex proteins were found in both peripheral and luminal fractions (Table 1; Supplementary Table 1) under flexible conditions, that the following identity scores must match those in the actual sub-organelle in chloroplast proteins. Out of 1,043 proteins using above these methods, 767 unique proteins identified among those fractions: 607 from chloroplast, 145 from chloroplast stroma, and 514 from chloroplast thylakoid. Mainly, 514 unique proteins identified from chloroplast thylakoid: 342 from chloroplast thylakoid membrane, 163 from chloroplast thylakoid space (lumen), and 166 from integral membrane. Most of the identified proteins overlapped with the sub-organelles (Supplementary Table 1; Fig. 1b). Others 276 proteins located in others locations like as cytoskeleton, cytosol, endoplasmic reticulum, extracellular, mitochondria, nuclear, peroxisome, and vacuolar membrane (Supplementary Table 2). Likewise, the proteins (peptides) were matched to the isozymes of other proteins based on their protein resemblance and peptide match against green plants. Molecular weight (MW), iso-electric point (pI), protein length, and exponential modified protein abundance index (emPAI) are the physical properties of proteins that are
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Fig. 1 Characterization of Percoll-purified chloroplast and suborganelle fractions of wheat (Triticum aestivum L.). a Coomassie Blue-stained gel showing (from left to right) standard marker (M), chloroplast (A), thylakoid (B), stroma and envelope (C), washed thylakoid (D), thylakoid membrane (E), chloroplast thylakoid space (lumen) (F), integral membrane (G), and peripheral membrane (H). E, F, G, and H were analyzed by linear quadruple trap-fourier transform ion cyclotron resonance mass spectrometry (LTQ-FTICR-MS) after tryptic digestion. b Cross-correlation of total identified proteins
among chloroplast (607 proteins), chloroplast thylakoid (514 proteins), and chloroplast stroma (145 proteins) proteomes. In chloroplast thylakoid, 514 proteins were shared among chloroplast thylakoid membrane, chloroplast thylakoid space (lumen), and integral membrane proteins. All proteins were analyzed using ChloroP for predicting chloroplast transit peptide (cTP), SignalP for signal peptide (SP), TMHMM for transmembrane domains (TMD), and indicated by the number of each fraction
fundamentally important in function. Here, the plots exhibited a bimodal distribution with a lower peak appearing at about pI 3.79 and a higher peak at about pI 12.25. We identified 699 proteins (91%) in pI 3–10 and 68 proteins (9%) in pI [10, which shows a direct correlation with molecular weight. A total of 722 proteins (94%) have 0.8–100 kDa molecular weight, and only 46 proteins (6%) have [100 kDa MW. However, MW and
emPAI are important guidelines for measuring absolute protein content (weight %) [39]. Of the total identified proteins, 727 proteins (94.8%) revealed an emPAI; unfortunately, the emPAI of 40 proteins (5.2%) could not be detected using FTICR mass spectrometry. emPAI values of 0.01–0.9 were detected in 610 proteins (83.9%), while 117 proteins (16.1%) had a value [1 (Supplementary Table 1).
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ATP synthase subunit beta, chloroplastic ATP synthase gamma chain, chloroplastic
ATP synthase gamma chain 1, chloroplastic
ATP synthase delta chain, chloroplastic
ATP synthase epsilon chain, chloroplastic
ATP synthase subunit b, chloroplastic
ATP synthase subunit c, chloroplastic
ATP synthase subunit a, chloroplastic
Q01908
Q07300
P69443
A1E9I7
P0C2Z9
A1E9I5
Cytochrome b6-f complex iron-sulfur subunit, chloroplastic
Cytochrome b6-f complex iron-sulfur subunit 1, chloroplastic
Cytochrome b6-f complex iron-sulfur subunit 2, chloroplastic
Cytochrome b6-f complex subunit 4 Cytochrome b6-f complex subunit 5
Q7X9A6
P30361
Q02585
A0ZZ66 P69463
Ferredoxin, chloroplastic
Ferredoxin–NADP reductase, leaf isozyme, chloroplastic
P00228
P10933
Photosystem I reaction center subunit IV, chloroplastic
P13194
Hordeum vulgare
Photosystem I reaction center subunit II, chloroplastic
Photosystem I reaction center subunit II-1, chloroplastic Photosystem I reaction center subunit II-2, chloroplastic
P36213
Photosystem I iron-sulfur center
P0C359
Q9S7H1 Q9SA56
Oryza sativa
Photosystem I P700 chlorophyll a apoprotein A2
A1E9J0
Hordeum vulgare
Arabidopsis thaliana Arabidopsis thaliana
Hordeum vulgare
Photosystem I P700 chlorophyll a apoprotein A1
Hordeum vulgare
Pisum sativum
Triticum aestivum
Oryza sativa Japonica Group
Gossypium barbadense Triticum aestivum
Nicotiana tabacum
Nicotiana tabacum
Triticum aestivum
Drimys granadensis
Triticum aestivum
Hordeum vulgare
Oryza sativa
Hordeum vulgare
Triticum aestivum
Sorghum bicolor
Arabidopsis thaliana
Triticum aestivum Chlamydomonas reinhardtii
Triticum aestivum
Taxonomy
A1E9J1
Photosystem I
Plastocyanin, chloroplastic
Q0DFC9
Photosynthetic electron transport
Apocytochrome f
Cytochrome b6
P05151
Q06GW7
Cytochrome b6/f complex
ATP synthase subunit alpha, chloroplastic
P20858 P12113
Protein description
P12112
ATP synthesis
UniProt accession
PSAE
psaD1 psaD2
psaD
psaC
psaB
psaA
PETH
PETF
PETE
petD PetG
petC2
petC1
petC
petB
petA
atpI
atpH
atpF
atpE
ATPD
ATPC1
atpB ATPC
atpA
Gene name
655
113 87
3924
291
1541
440
1090
67
118
143 107
169
59
2196
447
1495
150
53
670
492
42
294
24927 132
22123
Protein score
15447
22584 22293
21919
8893
82563
83080
40169
15277
15567
17481 4170
24092
24136
23711
24079
35341
27289
7969
20964
15208
26720
40886
53824 38736
55261
Molecular weight (Da)
29
10 8
125
12
58
34
38
4
4
12 5
6
6
75
13
96
11
3
27
25
4
12
705 14
504
Peptide matches
2861
154 958
179
16
531
88
1
1933
3962
13 42
1555
1133
259
169
276
90
6222
153
321
197
273
94 782
10
Peptide queries
Table 1 General features of the identified proteins (58 proteins) that are directly responsive in the photosynthetic pathway of wheat (shown in Fig. 3)
53.7
17.3 29.4
30.7
81.5
33.4
21.7
29.7
34.3
29.9
33.1 16.9
11
37.7
77.5
56.7
63.4
42.1
50.6
49.7
83.9
23.5
19.6
89.4 34.1
62.9
Sequence coverage
9.82
9.78 9.78
9.81
6.51
6.63
6.6
8.56
4.56
5.61
6.56 4.56
8.15
7.59
8.47
9.14
8.83
5.29
4.95
9.62
5.2
4.84
8.13
5.06 9.08
6.11
PI value
6.59
0.37 0.37
3.27
8.62
1.51
0.69
1.23
0.57
0.56
0.22 0.23
0.16
0.16
18.66
1.08
10.38
0.92
0.51
5.34
11.33
0.14
0.09
66.94 0.1
13.51
emPAI value
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123
Photosystem I reaction center subunit V, chloroplastic
Photosystem I reaction center subunit VI, chloroplastic Photosystem I reaction center subunit IX
Photosystem I reaction center subunit psaK, chloroplastic
Photosystem I reaction center subunit XI, chloroplastic
Photosystem I reaction center subunit N, chloroplastic
P20143 A1E9L0
P36886
P23993
P31093
Photosystem II reaction center PSB28 protein, chloroplastic
Photosystem Q(B) protein Photosystem II CP47 chlorophyll apoprotein
Photosystem II CP43 chlorophyll apoprotein
Photosystem II D2 protein
Cytochrome b559 subunit alpha
Photosystem II reaction center protein H
Photosystem II reaction center protein L
Oxygen-evolving enhancer protein 1, chloroplastic
Oxygen-evolving enhancer protein 1-1, chloroplastic
Oxygen-evolving enhancer protein 1-2, chloroplastic
Oxygen-evolving enhancer protein 2, chloroplastic
Oxygen-evolving enhancer protein 2-2, chloroplastic
Oxygen-evolving enhancer protein 3
Oxygen-evolving enhancer protein 3-1, chloroplastic
Oxygen-evolving enhancer protein 3-2, chloroplastic
Photosystem II 10 kDa polypeptide, chloroplastic Photosystem II 22 kDa protein, chloroplastic
Photosystem II reaction center protein J
Photosystem II reaction center protein K
Photosystem II 5 kDa protein, chloroplastic
Photosystem II reaction center W protein
Photosystem II core complex proteins psbY, chloroplastic
Q0JG75
P10510 A1EA34
Q9XPS4
A1E9Z3
P0C368
A1E9V3
Q67HC0
P27665
P23321
Q9S841
Q00434
P18212
P19589
Q41048
Q41806
Q40070 P54773
P19444
P12163
Q39195
Q39194
P80470
Photosystem II
Photosystem I reaction center subunit III, chloroplastic
Q00327
Protein description
P13192
UniProt accession
Table 1 continued
Spinacia oleracea
Arabidopsis thaliana
Arabidopsis thaliana
Spinacia oleracea
Zea mays
Hordeum vulgare Solanum lycopersicum
Zea mays
Zea mays
Pisum sativum
Nicotiana tabacum
Triticum aestivum
Arabidopsis thaliana
Arabidopsis thaliana
Triticum aestivum
Allium textile
Sorghum bicolor
Oryza sativa
Agrostis stolonifera
Triticum aestivum
Secale cereale Agrostis stolonifera
Oryza sativa Japonica Group
Hordeum vulgare
Hordeum vulgare
Hordeum vulgare
Hordeum vulgare Hordeum vulgare
Hordeum vulgare
Hordeum vulgare
Taxonomy
PsbY
PsbW
PsbT
PsbK
PsbJ
PSBR PSBS
PSBQ2
PSBQ1
PSBQ
PSBP2
PSBP
PSBO2
PSBO1
PSBO
psbL
psbH
psbE
psbD
psbC
psbA psbB
PSB28
PSAN
PSAL
PSAK
PSAH psaJ
PSAG
PSAF
Gene name
157
34
96
361
120
92 6568
1600
96
38
86
24251
2070
586
11781
146
278
1693
1066
663
615 7928
67
567
473
124
260 41
204
2634
Protein score
20664
13678
11028
6749
4194
14129 29250
22829
23119
3262
28543
27253
34998
35121
34719
4458
7782
9439
39532
51968
38896 56054
19724
15490
22197
13719
14873 4742
15098
24822
Molecular weight (Da)
4
9
6
8
8
6 99
59
5
43
5
516
96
26
234
4
21
65
35
29
39 263
2
17
27
13
15 3
11
79
Peptide matches
46
78
77
35
22
270 204
45
66
3199
2570
101
67
71
67
2316
2002
301
319
174
182 531
1146
222
79
283
459 1242
16
148
Peptide queries
27.5
22.8
20.5
25.4
15.6
54.3 33
29.6
19.8
84.4
9.8
60.1
28.4
22
58.2
100
23.3
39.8
28.6
28.5
42.2 33.9
14.4
40.7
42.1
51.9
16.1 26.2
51
31.5
Sequence coverage
9.73
5.29
9.55
5.82
5.59
9.71 8.67
9.3
9.77
4.79
7.67
8.84
5.92
5.55
8.73
4.53
8.09
4.64
5.34
6.92
5.21 6.06
9.79
9.54
9.52
10.49
10.28 5.91
9.59
9.46
PI value
0.03
0.04
0.13
0.11
0.23
1.09 1.34
0.86
0.84
1.48
0.13
32.56
1.27
0.5
5.38
7.07
7.42
7.51
3.27
1.14
2.03 5.89
0.19
8.37
1.21
1.12
2.21 0.92
2.16
1.35
emPAI value
5076 Mol Biol Rep (2012) 39:5069–5083
0.17
0.48 10.74
5.59 37.7
20.5
15
30 3
6554
12
40
109 PsbZ
PsbF Coffea arabica
Photosystem II reaction center protein Z
Cytochrome b559 subunit beta
A1E9R1
A0A351
Sorghum bicolor
4481
Peptide queries UniProt accession
Table 1 continued
Protein description
Taxonomy
Gene name
Protein score
Molecular weight (Da)
Peptide matches
Sequence coverage
PI value
emPAI value
Mol Biol Rep (2012) 39:5069–5083
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Identification of functional domains in chloroplast thylakoid The total identified proteins were analyzed for the presence of a predicted chloroplast transit peptide (cTP) using ChloroP. Of the 767 total identified proteins, 324 (42.24%) were predicted to have a cTP. SignalP is the most popular and reliable web-based tool for verifying the presence of signal peptides (SP) of proteins. Using SignalP, 107 (13.95%) of the total identified proteins were predicted, which shows highly reliable SP. To verify the number of transmembrane domains (TMD) in proteins, we used the most traditional predictor TMHMM. After correction, 119 (15.51%) of the total proteins were predicted to have at least one TMD (Supplementary Table 1). Functional categorization of thylakoid proteins In this experiment, we analyzed 767 unique chloroplast proteins that are localized in different sub-organelles in chloroplast. A total of identified proteins were functionally classified using Protein Information Resources (PIR) coupled with the iProClass batch retrieval system based on gene ontology with biological processes. We sought to classify functionally the proteins detected and identified in the body of work into 103 categories using gene ontology; consequently, we found 2,293 proteins based on functional frequency. A detailed analysis of iProClass database showed that several classes are encountered when functional homogeneity is considered. In the simplest case, proteins are involved in the same biological complex or in a particular biological process. We were broadly sorted into functional categorized (14 categories) such as cell organization and biogenesis (257 proteins or 11.21%), developmental process (39 proteins or 1.70%), DNA and RNA metabolism (52 proteins or 2.27%), electron transport or energy (522 proteins or 22.76%), other biological process (156 proteins or 6.80%), other metabolic process (501 proteins or 21.85%), oxidative phosphorylation (34 proteins or 1.48%), protein folding (22 proteins or 0.96%), protein metabolism (128 proteins or 5.58%), response to stress (149 proteins or 6.50%), transcription (13 proteins or 0.50%), transport (232 proteins or 10.12%), translation (87 proteins or 3.79%), unclassified (101 proteins or 4.40%) (Fig. 2). All identified proteins are associated with electron transport or energy, metabolic process, cell organization and biogenesis, other biological process, which is routinely classified by iPro-Class out of the total identified proteins. Firstly, most of proteins are involved in photosynthesis, chloroplast biogenesis as well as plastid organization (e.g., photosystem I & II, cytochrome b6/f complex, and ATP synthase) in electron transport or energy. Second correspondence protein classes annotated with 22 other
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Fig. 2 Frequency distribution of the total identified proteins to functional categories based on biological processes displayed as bar graph. The functional classification using the iProClass database and the assignment to more detailed functional categories are listed based on gene ontology in Supplementary Table 1
metabolic processes in chloroplast. These classes have multifunctional proteins that are involved in different metabolic processes (e.g., alcohol, amine, lipid, organic acid, phosphorus, and so on). The third most abundant protein class according to biological process is involved in cell organization and biogenesis, which are associated with cell organelle organization by cell division, growth, and cell wall organization. These functional classes are annotated with 27 classes in our proteomic survey. Another abundant and important class is involved in transportation facilitation (8 classes), that is, the movement of substances (such as fluids, hormones, hydrogen, lipid, proteins, and ions) into, out of, within, or among cells, or within chloroplast by some external agent such as a transporter or channel. We identified 119 transmembrane proteins, which act as transporters in chloroplast as integral membrane proteins (Fig. 1b). The iPro-Class database in PIR identified some unknown classified proteins (101 proteins) in chloroplast that are roughly involved in seed storage (e.g., 13S globulin seed storage protein), stress (e.g., calmodin, ubiquitin), and cellular organization-related (e.g., luminal binding proteins) or responsive proteins. These assignments, which are listed in Supplementary Table 1, were grouped based on biological process using the iProClass database of PIR. Proteins involved in the photosynthetic pathway of wheat (Triticum aestivum L.) We identified 58 abundant proteins that are known to be involved in electron transport reaction during photosynthesis and carbon metabolism, as identified on 1D-PAGE following LTQ-ESI-FT-ICR mass spectrometry analysis. Out of 58 identical proteins, 8 proteins were identified in
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F-type ATP synthase excluding 1 isoform ATP synthase C1 (ATPC1), followed by 5 proteins in cytochrome b6/f complex excluding 2 isoform (petC1 & C2), 3 proteins in photosynthetic electron transport, 12 proteins in photosystem I excluding 2 isoform (psaD1 & D2), and 20 proteins in photosystem II excluding 5 isoform (PSBO1 & O2, PSBP2, PSBQ1 & Q2). By this method, we cannot identify 6 proteins which are low abundance proteins. Interestingly, we identified 8 proteins involved in ATP synthase in the photosynthetic pathway and also covered the whole ATP synthase responsive proteins. The 767 recognized proteins represent the chloroplast of the thylakoid proteome of wheat, specially the 58 proteins that are directly responsive in the photosynthetic pathway of the thylakoid of wheat (C3 plant). Since FTICR mass spectrometry identification is biased towards the abundant proteins (Table 1; Supplementary Table 1), our results most likely reflect the revealed of the photosynthetic pathway in chloroplast thylakoid that is most active during photosynthesis of C3 wheat plant in our experiments, which directly supports the findings on Arabidopsis and Oryza (C3 plant) according to the KEGG pathway databases (http://www.kegg.com/kegg/ pathway.html) (Table 1; Fig. 3). In Supplementary Table 1, the light-harvesting complex (LHC) proteins, which absorb and transfer photon during photosynthesis light reactions, that were identified in abundance in our study, usually located in the lumenal parts of chloroplast. Fifty-six unique LHC proteins were identified, in direct association with photosystems I and II, and treated to integral membrane proteins, as in [40]. NADH dehydrogenase (13 proteins) such as ndhA, ndhD, ndhE, ndhF, ndhH, ndhI, ndhM and so on, the transfer of electrons through a series of electron donors and acceptors, generating energy that is ultimately used for synthesis of
Mol Biol Rep (2012) 39:5069–5083
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Fig. 3 Possible photosynthetic pathways predicted from a proteomic analysis of thylakoids from the wheat chloroplast. The identified proteins are depicted in green, missing components in red, and those not in C3 according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.kegg.com) in gray. (Color figure online)
Photosystem II
D1 PsbA
PsbL PsbQ PsbY
D2 PsbB
PsbJ PsbR PsbZ
cp43 PsbC
PsbK PsbS Psb27
cp47 PsbB
PsbM PsbT Psb28
PsbE
PsbH PsbU Psb28-2
Cytochrome b6/f complex
cytb559 PsbF
PsbI PsbV
PetB
MSP PsbO PsbW
OEC PsbP PsbX
PC PetE
beta PsaB PsaJ
PsaC PsaK
PsaD PsaL
ATP, were identified in chloroplast, which is contain transmembrane domain and integral membrane proteins [41]. We identified 35 Rubisco family proteins such as Ribulose bisphosphate carboxylase small chain (RBCS), Ribulose bisphosphate carboxylase small chain (SSU), Ribulose bisphosphate carboxylase/oxygenase activase (RCA), Ribulose-phosphate 3-epimerase (RPE), RING-H2 finger protein ATL3G, Rubisco large subunit-binding protein subunit beta (CPN60) with isoform, which is a metabolic process in which carbon (usually derived from carbon dioxide) is incorporated into organic compounds (usually carbohydrates), e.g., those proteins found on the stromal side of chloroplast and that directly help carbon metabolism [20]. Glyceraldehyde-3-phosphate dehydrogenase, glucose and ribitol dehydrogenase, glucose-1-phosphate adenylyltransferase small subunit, glucose-6-phosphate 1-dehydrogenase, fructose-1,6-bisphosphatase, fructose-bisphosphate aldolase, and sedoheptulose-1,7-bisphosphatase were detected in chloroplast that were responsive and related to carbon metabolism in the Calvin cycle [42].
Discussion The analysis of chloroplast, especially of the sub-compartments of the thylakoid proteome of wheat reported here, has produced one of the most comprehensive protein lists for a cell organelle available to date. Our tactics
PetA
PetC
PetL
PetM
PetN
PetG
Fd PetF
FNR PetH
a
b
cyt c6 PetJ
F-type ATPase
Photosystem I
PsaA PsaI
PetD
Photosynthetic electron transport
PsaE PsaM
PsaF PsaN
PsaG PsaX
alpha
gamma
delta
epsilon
c
PsaH
focused on the identification of as many Arabidopsis [11], Spinacia, and Hordeum [23] chloroplast proteins as possible without fractionating the organelles into specific subproteomes. A comparison of our tactics with recent reports of proteins identified from isolated thylakoid membrane [11], thylakoid lumen, peripheral membrane [20–22], and chloroplast envelope membrane [19, 43] shows that our protein separation coupled with MS/MS shotgun proteomics is sensitive and results in a similar coverage of the identified protein. The linear ion trap of the LTQ-FTICR allows greater ion capacity and dynamic range than the prior generation of ion traps embodied by the LCQ-MS/MS, has the two electron multipliers to enhance sensitivity, and scans about five times faster than the LCQ-MS/MS [44]. Our methods did not identify all proteins, where as responsive in chloroplast biogenesis and development, which is also regulated the biosynthesis of chloroplast of wheat compare to Arabidopsis and Oryza biogenesis process. The total identified in the chloroplast thylakoid of wheat was 342 proteins in chloroplast thylakoid membrane, followed by 163 proteins in chloroplast thylakoid space (lumen) and 166 proteins in integral membrane proteins; there were also peripheral proteins such as ATP synthase, oxygen-evolving proteins, and so on. These results are highly similar to some suggested reports [13, 20, 45, 46]. We found many proteins that were highly matched with chloroplast proteins against green plants with high protein sequence coverage. The study also had a major limitation due to the lack of genome
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sequence of hexaploid wheat, which influenced the accuracy of low-abundance protein identification. In our study, we predicted chloroplast sub-compartment proteins such as chloroplast, chloroplast stroma, chloroplast thylakoid, thylakoid membrane, lumen, and integral membrane (See Supplementary Table 1; Fig. 1b). pI values have long been a standard measure for distinguishing among proteins. In the relation between pI and subcellular localization, the trimodal character in eukaryotes is likely to be a general property of proteomes and is associated with the need for different pI values depending on the subcellular localization [47]. In large-scale proteome analysis, MW and emPAI are essential features for estimating the protein content in a digestive sample [39]. Finally, pI, MW, and emPAI is the important facial appearance for predicting the proteins, which is also aids to identify the accurate proteins. The neural network programs ChloroP and SignalP were most successful in the prediction of localization, cTP, and SP in chloroplast proteins using plant amino acid sequences [20], with 42.24% of cTP proteins and 13.95% of SP proteins identified to the chloroplast, especially the thylakoid of wheat. ChloroP was trained with a positive test set of cTP proteins containing chloroplast [36]. The chloroplast transit peptides of a set of 324 nonredundant proteins were analyzed using ChloroP according to the experimentally determined cleavage sites. However, several of these features were more pronounced in chloroplast transit peptides. These include the presence of prolines at the end of the hydrophobic domain, most of which had nearly complete conservation of alanine and glutamic acid. The secretory avoidance motif [48, 49] does not seem to be a basic residue directly after the hydrophobic domain but is more likely to be the overall hydrophobicity, as was recently discussed for E. coli [50]. A higher hydrophobicity (i.e., more leucines) in the signal peptide is likely to favor targeting via the secretory pathway. These features taken together should make it possible to predict, with high confidence, the chloroplast transit peptides of plant proteins on a large scale. The adaptation of SignalP for chloroplast proteins might make this possible. Note that for these programs to work correctly, the starting methionine needs to be correctly assigned in the database; we observed several cases in which the assignment was incorrect [36, 37]. An updated research about genome-wide protein analysis of Arabidopsis reported that the chloroplast proteome contains 520 proteins with one or more TMDs when using TMHMM as predictor. When using the consensus prediction listed in the latest release of the Aramemnon database, 728 proteins with TMDs were found [11]. In our survey, 119 TMDs were identified in chloroplast of wheat, including a single TMD containing 52 proteins and more than 2 TMDs with 67 proteins. Fifty-five proteins (46.22%)
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out of 119 TMD proteins were identified in integral membrane proteins, while 64 TMD proteins (53.78%) were identified in chloroplast, stroma, lumen, and thylakoid membrane (Supplementary Table 1). To identify additional integral membrane or hydrophobic proteins that are tightly bound to the thylakoid membrane via lipid anchor or short helical structures parallel to the membrane system, we specified using TOPPRED [51], which is verified our TMHMM results [52]. Using TOPPRED, we verified hydrophobicity and topology, which play key roles in organizing the self-assembly of protein molecules and are entirely plausible because some amino acid residues are abundantly water-soluble while others are only sparingly so [53]. The algorithms for the prediction of organelle targeting are not perfect [4]. Analogous proteins may be targeted to different organelles [54], and sometimes, single genes encode proteins targeted to more than one organelle [55]. In addition, genomic or cDNA sequences for many of the wheat proteins were not available, so many web-based tool (ChloroP, SignalP, PSORT) predictions were based on related genes, mainly from Oryza. Thus, it is possible that a wheat protein was encoded by a gene for a plastid-specific protein but the homolog was predicted to be non-plastid or vice versa. As a result, these assignments should be treated with caution [56]. With the latest information from several sources such as UniProtKB database, iProClass provides much richer protein annotation than is found in the UniProtKB database. It presents comprehensive views of protein sequences and super families in terms of different protein information, such as structure, function, gene, genetics, disease, ontology, taxonomy, and literature, with cross-references to relevant molecular databases and executive summary lines, as well as a graphic display of domain and motif sequence regions [57]. We also used gene ontology-based biological processes, which were also used as functional classifications in our survey. After obtaining 103 groups according to biological process, all groups were again manually classified into 14 categories (Supplementary Table 1; Fig. 2). According to the annotation information in iProClass using the UniProt database, 767 proteins (2293 functional frequency) were classified into 103 classes, as shown in Supplementary Table 1. These were farther manually classified into 14 categories that cover the 103 classes (Fig. 2). The chloroplast thylakoid is the center of the lightdependent reactions of photosynthesis, and the regulation and proceeds of its proteins are expected to play a significant role in the right functioning of photosynthesis [10]. In the present study, 514 thylakoid proteins (67% of the total identified proteins) were identified; this is infrequently achieved using SDS-PAGE couple to high-throughput hybrid LTQ-FTICR-MS, showing that our technique is more powerful and highly accurate for membrane protein
Mol Biol Rep (2012) 39:5069–5083
identification, especially for chloroplast proteomics. Based on our proteomic assessment, 180 proteins (58 proteins directly involved in the photosynthetic pathway, and 122 proteins involved in photosynthesis and carbon metabolism) are involved in photosynthesis; these primarily include the components of photosystem II (PSII) and photosystem I (PSI), the subunits of ATP synthase, the subunits of cytochrome b6/f complexes, photosynthetic electron transport, and proteins involved in the Calvin cycle (Table 1; Supplementary Table 1). The other 587 non-photosynthetic proteins were identified as being involved in energy metabolism, protein folding, transport, cell division, DNA and RNA metabolism, flavonoid and enzyme metabolism, translation, and transcription. Although the protein related to apolipoprotein is predicted to localize in the thylakoid membrane, stroma, lumen, and integral membrane of chloroplast thylakoid, predict its function using iProClass databases; undoubtedly more work is needed to integrate these proteins within the context of chloroplast function. The thylakoid is bordered by the soluble chloroplast stroma, a sub-organelle that contains many glowing characterized enzymes involved in carbon metabolism and other biosynthetic pathways [9]. Here, 145 chloroplast stroma proteins (19% of identified proteins) were found (Fig. 1b). Firstly, many of these are involved in the Calvin cycle and other reactions, such as: synthesis of organelle-encoded proteins, alcohol metabolism, carbohydrate metabolism, glycolysis, lipid metabolism, nitrogen metabolism, phosphorus metabolism, amino acid metabolism, hormone metabolism, co-factor and vitamin metabolism, and tetrapyrrole synthesis. Plastoglobules are lipid-containing particles in the stroma that are thought to serve as lipid reservoirs for thylakoid membranes. The thylakoid membrane encloses the thylakoid space (lumen). In recent years, many different categories of protein have been found in the thylakoid membrane, such as chaperones, carbonic anhydrases, violaxanthin de-epoxidases, peroxidases, and proteases. In this study, only 163 thylakoid space (lumen) proteins were identified: an anion-transporting ATPase family protein, peroxiredoxin Q, plastocyanin, oxygenevolving proteins, and also some identified protein function still unknown. There are, however, 101 proteins that cannot be designated as accurately functioning in the chloroplast based on the bioinformatic tools available to us. The second largest group of identified proteins include those involved in primary and secondary metabolic processes, such as pigment metabolic process (e.g., magnesiumchelatase subunit chlI); lipid (e.g., non-specific lipidtransfer protein Cw18), amino acid (e.g., ornithine carbamoyltransferase), and nucleic acid synthesis (e.g., adenylate kinase); DNA and RNA metabolism (e.g., histone, maturase); and protein synthesis (e.g., glutamate-cysteine ligase),
5081
folding, and degradation (e.g., heat shock protein). Additionally, we also identified proteins involved in the other two groups: transport (e.g., GTP-binding protein), redox regulation (e.g., isocitrate dehydrogenase), and stress response (e.g., ascorbate peroxidase, catalase, aquaporin, and peroxidase). In chloroplast, we revealed 76 ribosomal protein isoforms (30S, 40S, 50S, and 60S ribosomal proteins), which are involved in regulation and termination of transcription [10, 11]. In the above cases, those data supported the current model of a chloroplast [11, 20, 22, 42, 58] as a semiautonomous organelle, with its own genome, some of its own metabolic processes, and the ability to synthesize proteins by itself. We tested our hypothesis by analyzing the proteins for important photosynthetic activities in the thylakoid of wheat (Fig. 3). The coverage of photosynthetic function proteins was 87% of proteins in photosystem II, followed by 80% in cytochrome b6/f complex and electron transport, 92.3% in photosystem I, and 100% in F-type ATP synthase proteins, according to the C3 rice plant (Oryza sativa), which directly support [59]. Unfortunately, we could not identify six proteins (PsbM, PsbI, and Psb27 in photosystem II; PsaI in photosystem I; and PetN and PetJ in cytochrome b6/f complex and electron transport), which were mostly low-abundance proteins. Interesting, unidentified proteins molecular weight is 3–4 kDa, which is separated moderately complicated by gradient centrifugation coupled to Tricine-SDS-PAGE. Unidentified proteins also detected by different technique i.e., gene cloning [59, 60]. Because of the large dynamic range of protein amount in biological tissues, proteomic studies mainly detect those abundance proteins for which tryptic digestion produces peptides within the optimal size range for analysis [61]. Tissue fractionation improves the range of proteins detected, and many proteins were detected in the chloroplast sub-fractions that were not previously identified in a general extract of salt-soluble proteins. In our survey, 14-3-3 proteins (25 proteins) are signaling proteins that bind to phosphorylated serine and threonine residues, and interact with a variety of other proteins that are in both plants and animals [62]. They are among the better-known plant receptor kinases, such as the brassinosteroid-insensitive kinase that is involved in brassinosteroid signaling [63]. Brassinosteroids are essential plant growth regulators. BRI1 is highly phosphorylated, with more than 15 potential autophosphorylation sites within the cytoplasmic kinase domain; interestingly, these sites seem to have different functions, such as stress response against bacteria and cadmium ion [64]. One of the downstream affects of BRI1 complex formation is an increase in the levels of metabolic enzymes, including nitrate reductase.
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Conclusion We have presented the most comprehensive list of proteins (767 proteins) using a well-established chloroplast fractionation system coupled with high-throughput hybrid mass spectrometry (LTQ-FTICR-MS) of C3 wheat plant. A full description of the chloroplast thylakoid proteome is important to understand the photosynthesis system (Fig. 3) and carbon metabolism. As previously mentioned, proteomics is not only well suited to determine large-scale proteins but also to identify multi-subunit complexes. These highly accurate data in chloroplast thylakoid can be very useful tools for the plant, especially wheat, research community. Acknowledgments We would like to thank Prof. Shien Young Kang of the Department of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Korea, for conducting the ultra-centrifugation. Mainly, financial support for this study was obtained from the AGENDA (20090101036022), RDA, Korea to S. H. Woo, and also technically and financially supported by the Korea Basic Science Institute Grant (G30121) to Kun Cho and Korea Basic Science Institute K-MeP Project (T30110) to J.-S. Choi.
13.
14.
15.
16.
17.
18.
19.
20.
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