Proteomic analysis reveals the diversity and complexity of membrane proteins in chickpea (Cicer arietinum L.)
- Dinesh Kumar Jaiswal1,
- Doel Ray†1,
- Pratigya Subba†1,
- Poonam Mishra1,
- Saurabh Gayali1,
- Asis Datta1,
- Subhra Chakraborty1 and
- Niranjan Chakraborty1Email author
© Jaiswal et al.; licensee BioMed Central Ltd. 2012
Received: 26 April 2012
Accepted: 25 September 2012
Published: 2 October 2012
Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades.
To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains.
The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses.
KeywordsGrain legume Membrane-associated proteins 2-DE Mass spectrometry Transmembrane domain
Membranes are highly organized structures specially adapted to perform multiple functions in eukaryotic cells. They constitute the interface between the various cellular compartments and play a critical role in the exchange of substances and signals. Cell membranes consist of dynamic lipid-protein matrices wherein the lipid component provides a barrier to solute movement, and membrane-associated proteins perform unique biological roles in development as well as stress adaptation. The composition and dynamics of membrane proteins reflect their diverse function, and their nature and relative amount vary from one organellar membrane to another. The membranes associated with different organelles not only play important roles in maintaining the homeostasis within organelles, but also at the whole cell level. Approximately 30% of the cellular proteome is represented by membrane proteins  . These proteins perform some of the most important functions, including the regulation of cell signaling, cell-cell interactions, and intracellular compartmentalization  .
Due to the presence of highly specialized organelles such as plastids and vacuoles, the plant membrane proteome is more complex compared to that of animal cells. The pivotal role played by membrane proteins in many cellular processes makes their study imperative. However, the proteomic analysis of membrane proteins has always been hampered due to their recalcitrance to standard methodologies . Indeed, membrane proteins are under-represented in large-scale proteomics and are challenging to work with. The proteomic analysis of membrane proteins has been impeded due to their hydrophobic nature, lesser abundance and physicochemical heterogeneity . While much progress has been made in animal membrane proteomics, far fewer attempts have been made to characterize the plant membrane proteome [5–8].
Legumes are valuable agricultural crops that serve as important nutrient sources for human diet and animal feed worldwide. They serve as an important protein-rich food and have increasingly become a commercial commodity. The members of this family have unique features such as biological nitrogen fixation and symbioses with mycorrhizal fungi , which make them good experimental models for various studies. Chickpea is the most important grain legume and ranks third in terms of total global production [10, 11]. The world’s total production of chickpea hovers around 8.5 million metric tons annually and is grown over 10 million hectares of land. Despite the importance of chickpea and its role in nutrition requirement in humans, it has remained outside the realm of large-scale functional genomics studies. Although in recent years, much attention has been given to chickpea genomics [12–16], there is still little information on its protein complement. In previous proteomic studies, we had developed an extracellular matrix- and nucleus-specific proteome of chickpea [17, 18]. We report here the development of membrane proteome with an aim to use the reference map for more comprehensive characterization of the regulation and function of membrane proteins. Over 300 proteins were resolved using two-dimensional gel electrophoresis (2-DE), and MALDI-TOF/TOF and LC-ESI-MS/MS techniques were used to identify the proteins. The identified proteins were classified on the basis of their putative functions. Membrane-association of the identified proteins was validated by assessing their hydropathicity and the presence of transmembrane domains (TMDs). It is notable that ~70% of the dataset were predicted to be integral membrane proteins, which is considerably higher than other gel-based membrane proteomic analyses thus far. We have identified many candidates that are integral membrane proteins as well as membrane-associated proteins including those not reported earlier. This study may facilitate comparative proteomics as well as functional genomics studies of plant development and adaptation to various stresses.
Results and discussion
Isolation of membrane proteins
Development of membrane proteome
Proteins identified in membrane fraction using 2-DE coupled with MS/MS analysis
Spot No. a
gi No. b
GRAVY Value d
Signaling and stress response
Serine threonine kinase homolog COK-4
Putative serine/threonine kinase
Membrane protein CH1-like
Calcineurin-like phospho-esterase family protein
Putative quinone oxidoreductase
Guanine nucleotide-binding protein subunit beta-like
Disease resistance protein (Fragment)
Chaperonin 60 alphaf subunit
Luminal binding protein, BiP
Putative GloEL protein Chaperonin 60 kDa
Chaperonin 60 alphaf subunit
Chaperonin 60 alphaf chain precursor,
Protein synthesis and degradation
Putative 60S ribosomal protein L1
40S ribosomal protein S12
40S ribosomal protein S12
Putative glutamate-tRNA ligase
Putative 60S acidic ribosomal protein P0
Putative 60S acidic ribosomal protein P0
Proteasome subunit alpha type
Mitochondrial processing peptidase beta subunit
Cytochrome b6-f complex iron-sulfur subunit
Chlorophyll a/b-bindingg protein AB96
Chlorophyll a/b bindingg protein
Oxygen-evolving enhancer protein 1h
Putative PSII-P protein
Chlorophyll a/b-binding protein 3 precursorg
Chlorophyll A-B binding proteing
Chlorophyll a/b-binding protein type III (Fragment)g
PSI type III chlorophyll a/b-binding protein (imported)
Oxygen-evolving enhancer protein 1h
Oxygen-evolving enhancer protein 2
Ferredoxin NADP reductase
LHCA3, chlorophyll binding
Light-harvesting chlorophyll-a/b binding protein Lhcb1
Putative ferredoxin-NADP(H) oxidoreductase
Ferredoxin: NADP + reductase
ATPase beta subunit (Fragment)i
ATP synthase subunit beta, chloroplastici
ATP synthase subunit alphaj
ATP synthase beta subunit (Fragment)i
ATP synthase beta subunit (Fragment)i
ATP synthase beta subunit (Fragment)i
ATP synthase subunit alphaj
ATP synthase subunit betai
ATP synthase subunit alphaj
ATP synthase subunit gamma, chloroplast precursor
ATP synthase CF1 alpha subunitj
ATP synthase subunit alphaj
ATP synthase CF1 alpha subunitj
H + −transporting two-sector ATPase alpha chain
Fructose-bisphosphate aldolase 1k
Fructose-bisphosphate aldolase (EC 4.1. 2.13) precursork
Phosphoglycerate kinase precursor like
Glyceraldehyde 3-phosphate dehydrogenasel
Glutamate-ammonia ligase (EC 18.104.22.168) delta precursor
Trypsin inhibitor, chain B (fragments)
Glutamate-ammonia ligase (EC 22.214.171.124) delta precursor
Glyceraldehyde-3-phosphate dehydrogenase Al
Dynein-1-alpha heavy chain
Activator of spomin
Ribulose-1,5-biphosphate carboxylase/oxygenase large subunit
Histone acetyltransferase GCN5
Ribulose bisphosphate carboxylase/Oxygenase large chain
Chromosome chr17 scaffold_16
Ribulose bisphosphate carboxylase large chain
Intronic ORF, similar to LAGLIDADG endonuclease
Putative uncharacterized protein
Putative uncharacterized protein
Hypothetical protein P0415D04.53
Hypothetical protein F26P21.180
Hypothetical protein OSJNBa0075N02.148
Physicochemical characteristics of membrane proteins
Integral membrane proteins identified in membrane proteomes
Total number of identified proteins
Integral proteins (having putative TMD a)
% of integral proteins identified
Medicago truncatula b
Lupinus albus c
Another valid indicator of the membrane association of proteins is their GRAVY value in the positive range, depicting their hydrophobic nature [23, 24]. GRAVY value analysis was performed at the protein (Table 1) as well as peptide level ( Additional file 4. Table S3). The positive GRAVY value of the identified proteins ranged from 0.001 to 0.128 (Figure 5B), and that of the peptides from 0.014 to 1.287 (Figure 5C). While some proteins such as membrane protein CH1-like (CaM-247) with three putative TMDs showed negative GRAVY value (−0.510), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, CaM-460) with no predicted TMD scored positive GRAVY value (0.069). It has been reported that most of the integral cytoplasmic membrane proteins are hydrophobic, while the majority of integral outer membrane proteins are hydrophilic [4, 24], causing the observed ambiguity in GRAVY values.
Analysis of functional groups of membrane proteins
We identified many candidates that are known membrane residents, and also membrane-associated proteins. The most abundant class of proteins belonged to ‘Bioenergy’. Members in this class are mainly associated with energy-production processes such as photosynthesis and oxidative phosphorylation. In green plants, chloroplast and mitochondria are the most prominent and abundant organelles. Most of the identified proteins in ‘Bioenergy’ belong to these organelles, such as the proteins involved in the formation of major complexes of photosynthetic apparatus such as light harvesting antenna complexes associated with PSI and PSII (CaM-21, 22, 69, 74 126, 127, and 184), cytochrome b6-f complex (CaM-242) and oxygen-evolving enhancer protein 1 and 2 (CaM-125 and 183). Protein spots CaM-319, 376 and 452 were identified as ferredoxin NADP reductase (FNR), which is tightly bound to the thylakoid membrane  and cause the reduction of NADP+ during photosynthetic electron transport. Multiple subunits of ATP synthase such as alpha, beta and gamma were also identified.
The class ‘Metabolism’ contained proteins like glutamine synthetase (CaM-218) and glutamate ammonia ligase delta precursor (CaM-214 and 216), which are primarily involved in nitrogen metabolism. The protein spot CaM-322 was identified as phosphoglycerate kinase (PGK), which has been found to be associated with the thylakoid membrane in higher plants . Carbonic anhydrase, known to provide inorganic carbon for improved photosynthetic efficiency, was identified from multiple spots (CaM-366, 370, 435, and 438). Although carbonic anhydrase is primarily located in the chloroplast, it is also reported to be present in the microsomal and plasma membrane fractions . It is increasingly apparent that many enzymes are not free in solution, but interact with membrane structures  or with other proteins . The presence of metabolic pathway enzymes like aldolase as identified from multiple spots (CaM-210, 257, 308, 310, and 453), phosphofructokinase (CaM-206), and GAPDH (CaM-460 and 462), among others, highlights this phenomenon. It has been reported that aldolase interacts with V-ATPase , which may provide a basis for coupling glycolysis directly to the ATP-hydrolyzing proton pump . In Arabidopsis, 5-10% of the cytosolic isoforms of each glycolytic enzyme is associated with the outer surface of mitochondria . These glycolytic enzymes associated with mitochondria are catalytically competent and constitute a functional glycolytic pathway , though the significance of this micro-compartmentation of glycolysis has not been fully understood.
A subset of the identified proteins was presumably involved in ‘Signaling and stress response’. This class included proteins such as calcineurin-like phosphoesterase (CaM-85) and membrane protein CH1-like (CaM-247), the latter having putative SUN (S ad1/UN C-84) domain. Proteins having the SUN domain are involved in the formation of bridging structures, LINC (li nker of n ucleoskeleton and c ytoskeleton) complex that plays an important role in DNA duplication, especially in the anchorage of centrosomes and spindle pole body to the nuclear envelope [34, 35]. The discovery of SUN proteins in plant established the existence of LINC complex . In animal, SUN proteins are known to involve in the transfer of mechanical force generated in cytosol to inner nuclear membrane , but such role in plants is unknown. Calcineurin-likephosphoesterase belongs to the protein family that includes diverse range of phosphoesterases, phosphoserine phosphatases, and nucleotidases. The exact role of this protein in signaling is not known; however, it is likely to be involved in phosphate mobilization either by cleavage of phosphate from phosphate-containing compounds or by metabolic changes . The spots CaM-89 and 246 were identified as serine/threonine kinase. These kinases may act as receptor, which interact with other proteins to affect a wide array of processes, specifically in stress adaptation . CaM-436 was identified as quinone oxidoreductase, a class of membrane enzymes that catalyse the oxidation or reduction of membrane-bound quinols/quinones. This protein has been reported to be associated with the plasma membrane  and microsomal membranes . Protein spot CaM-326 was identified as cytochrome P450, one of the largest super families of enzymes. It uses electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates [42, 43]. Plant P450s are a class of proteins anchored on the cytoplasmic side to ER , but is also found in inner mitochondrial membranes  as well as in tonoplasts .
In this class, another interesting candidate (CaM-89) is the one encoded by COK-4 gene that confers resistance to anthracnose caused by the fungal pathogen Colletotrichum. In addition to displaying high similarity to the Pto kinase, one of the best characterized R gene products, the predicted COK-4 protein contains a highly hydrophobic membrane-spanning region . CaM-432 was also depicted to be a disease resistance protein containing LRR repeat. CaM-483 was identified as BiP, the ER-resident molecular chaperone involved in ER stress signaling. In plants, BiP plays a key role in attenuating ER stress and suppresses the activation of the unfolded protein response . We also identified HSPs such as chaperone DnaK (CaM-99), chaperonin-60 (CaM-94, 150, and 421), and GloEL protein (CaM-157), which prevent protein misfolding and random aggregation inside the cell. The major HSP families are necessary for the assembly and unfolding or transport of proteins through membranes [49, 50].
The class ‘Protein synthesis and degradation’ predominantly include ribosomal proteins (CaM-39, 208, 240, 208, 209 and 283), putative glutamate tRNA ligase (CaM-223), proteasome subunit alpha type (CaM-301), polyubiquitin (CaM-249), and mitochondrial processing peptidase beta subunit (CaM-407). It has been reported that polysomes are linked to actin filaments, which in turn are associated with the plasma membrane . Furthermore, ribosomes are also attached to the ER and nuclear membrane through the larger subunit. The most common pathway for degradation of cellular proteins is the ubiquitin proteasome pathway. We could identify proteins associated with these pathways such as polyubiquitin (CaM-249) and proteasome subunit alpha type (CaM-301). Ubiquitin is known to be the most conserved protein required for ATP-dependent protein degradation and involved in protein transport. These proteins were identified from the tonoplast , as well as from the plasma membrane .
The ‘Miscellaneous’ class accounted for 9% of the proteins identified. This class include activator of spomin (CaM-54), dynein-1-alpha heavy chain (CaM-155), chromosome chr17 scaffold_16 (CaM-138), and histone acetyltransferase (HAT, CaM-299), among others. HAT has been linked to transcriptional activation of various genes and is known to associate with mammalian inner nuclear membrane . The activator of spomin is known to involve in the activation of a subset of sugar-responsive genes and control the carbon flow in plants . Chromosome chr17 scaffold_16 encodes for ATP-dependent Clp protease ATP-binding subunit. Clp proteases are known to degrade both soluble and membrane-bound substrates  and are reported to be associated with the stroma as well as the inner envelope membrane in plants . The proteome map revealed 9% proteins as hypothetical or proteins with unknown function, which were subjected to domain analysis using InterProScan. The analysis led to the identification of different conserved domains, thereby providing valuable insight into their functional implications ( Additional file 5. Table S4). The putative conserved domain was bacterial transferase hexapeptide repeat in CaM-443, kinesin-related domain in CaM-56, and ATPase, AAA type core in CaM-323, among others. Intriguingly, membrane receptors, aquaporins, and transporters could not be identified, possibly due to their low abundance and complex physicochemical properties. Nevertheless, the distribution of various functional classes bear resemblance to previously reported membrane proteome datasets of rice .
Comparative analysis of membrane proteomes
Most plant membrane proteomes till date have been developed from roots, the notable exceptions being leaves , trichomes , seedlings , and aerial tissues (this study). Unlike root tissues that arise from root apical meristems, the aerial tissues are initiated by the shoot apical meristems, which show more diversity. In recent years, shoot meristems have received considerable attention in view of their importance in plant development and stress adaptation . Further, only a small fraction of integral membrane proteins have been confirmed experimentally, and most of them came from in silico analyses of genome datasets of the model plants. The difficulties associated with the study of membrane proteins in non-model plants has been demonstrated in a recent proteomic study . This underscores the importance of the study of membrane proteins in chickpea, whose genome is yet to be sequenced. This study provides a firm indication of a number of membrane-associated proteins to which function is yet to be assigned. The proteome revealed many key membrane-associated proteins, for example, serine threonine kinase homolog COK4, calcineurine-like phosphoesterase, activator of spomin, and chromosome chr17 scaffold_16, among others, which were not reported earlier.
Plant materials and growth condition
Chickpea (Cicer arietinum L.) seeds were soaked in water, kept overnight in dark and grown in pots (10 seedlings/1.5 L capacity pots with 18 cm diameter) containing a mixture of soil and soilrite (2:1, w/w). The seedlings were maintained at 25 ± 2°C, 50 ± 5% relative humidity under 16 h photoperiod (270 μmol m-2 s-1 light intensity) as described previously . The pots were provided with 100 ml of water every day that maintained the soil moisture content to approx. 30%. The aerial parts (stem and leaves) of 3-week-old seedlings were sampled as experimental materials.
Isolation of membrane fraction
The membrane i.e., microsomal fraction was isolated as described earlier  with few modifications. Approximately, 10 g tissue was ground into powder in liquid nitrogen with 1% (w/w) polyvinylpolypyrroledone (PVPP). The tissue powder was homogenized in homogenizing buffer [0.25 mM sucrose, 3 mM EDTA, 5 mM DTT, 10 mM ascorbic acid, 70 mM Tris-MES (pH 8.0), 0.25 mM PMSF and protease inhibitor cocktail (Sigma)] using a homogenizer (PRO Scientific, USA). The cell debris was removed from the homogenate by filtration through four-layered cheese cloth and the filtrate was centrifuged at 6000 × g for 10 min at 4°C. The supernatant was recovered and centrifuged at 150000 × g for 45 min at 4°C. The resulting pellet containing the membrane fraction was suspended in suspension buffer (1.1 mM glycerol, 5 mM DTT, and 10 mM Tris-MES, pH 8.0).
Marker enzyme assays
The presence of different subcellular membranes was determined by assaying activity their respective marker enzymes. The activities of orthovanadate-, azide-, nitrate-sensitive ATPase and latent IDPase were measured for plasma membrane, mitochondrial membrane, tonoplast and Golgi membrane, respectively [63, 64]. In brief, 30 μg membrane proteins were suspended in 30 mM Tris-MES [pH 6.5 (vanadate-sensitive ATPase), pH 8.0 (nitrate and azide-sensitive ATPase), and pH 7.5 (latent IDPase)]. The reactions were performed in 1 ml solution containing 3 mM MgSO4, 0.1 mM sodium molybdate, 50 mM KCl and 3 mM ATP, with or without ATPase inhibitor (100 μM Na3VO4, 50 mM KNO3, and 1 mM NaN3) at 38°C to minimise the hydrolysis of ATP. To subtract the residual Pi in isolated fraction, the reactions were also performed without addition of ATP. The released Pi was determined as described previously . The reaction was stopped by the addition of 1 ml Ames’s colour reagent [1 part ascorbic acid (10%) and 6 parts ammonium molybdate (0.42% in 1 N H2SO4)] containing 0.1% (w/v) SDS. The colour was allowed to develop for 20 min. After termination with 10% (w/v) sodium citrate, the absorbance was measured at 820 nm. For latent IDPase, the reaction was carried out with 3 mM IDP-salt, either in presence or absence of 0.1 mM sodium molybdate. The activity was determined on freshly isolated membrane fraction then after 5 days of incubation at 4°C.
Extraction and quantification of membrane proteins
Membrane proteins were extracted using the organic solvent mixture of chloroform/methanol as described previously . The microsomal fraction was suspended in 1 ml of suspension buffer and divided in 10 sub-fractions of 0.1 ml. The aliquots were slowly added to 0.9 ml of cold chloroform/methanol mixtures (0:9 to 9:0, v/v) and kept on ice for 15 min. Intermittent vortexing of samples was carried out during the incubation. The mixtures were centrifuged at 12000 × g for 20 min at 4°C. The pellet fractions containing insoluble proteins were retrieved carefully and to the respective supernatants, 2–5 volumes of chilled acetone was added, followed by incubation at −20°C and the precipitated proteins collected by centrifugation. The soluble and insoluble fractions’ membrane proteins were resuspended in the buffer containing 7 M urea, 2 M thiourea, and 4% CHAPS (w/v). The protein concentration was determined using the 2-D Quant kit (GE Healthcare).
Measurement of phytopigments
2-DE of membrane proteins and data analysis
Since the organic solvent extraction at 6:3 ratio of chloroform/methanol was found to be optimal for membrane proteins, isoelectric focusing (IEF) was carried out with 100 μg of proteins from the pellet fraction. The immobilized gel strips (13 cm, pH 3–10 or 4–7, GE Healthcare) were rehydrated overnight in 250 μl of rehydration buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 60 mM DTT, 2% (v/v) pharmalyte (pH 3–10 or 4–7), and 0.05% (w/v) bromophenol blue]. During cup-loading, proteins were diluted with rehydration buffer to final concentration of 1 μg/μl and loaded either at anodic or cathodic ends. Initially, proteins were electrofocused at lower voltage and later with higher voltage up to 85000 VhT at 20°C using IPGphor system (GE Healthcare). After IEF, the strips were subjected to reduction with 1% (w/v) DTT in 10 ml of equilibration buffer [6 M urea, 50 mM Tris–HCl (pH 8.8), 30% (v/v) glycerol and 2% (w/v) SDS], followed by alkylation with 2.5% (w/v) iodoacetamide in the same buffer. The strips were then loaded on top of 12.5% polyacrylamide gels for SDS-PAGE. The electrophoresed proteins were stained with silver stain plus kit (Bio-Rad). The silver stained gels were scanned with Bio-Rad FluorS equipped with a 12-bit camera. The 2-DE gels were analyzed with PDQuest version 7.2.0 (Bio-Rad). Three replicate 2-DE gels, corresponding to at least two biological replicates were matched together to generate a composite image conventionally known as first level matchset. Protein spots present in at least two of the three gels were considered for analysis. Experimental molecular mass and pI for each protein were determined from 2-DE image using standard molecular mass protein marker (Bio-Rad).
Protein identification using MALDI-TOF/TOF and LC-ESI-MS/MS
The protein spots were excised manually, washed twice with deionized water, and trypsin in-gel digestion was performed . The peptide extract was vacuum dried. While reconstitution of the peptides for MALDI-TOF/TOF was performed in 3 μl of 50% (v/v) ACN and 0.1% (v/v) TFA, the same was performed in 7 μl of 50% (v/v) ACN and 0.1% (v/v) HCOOH for LC-MS/MS. Instruments used for analysis were 4800 MALDI TOF/TOF Analyzer (Applied Biosystems), QStar Elite coupled to Tempo nano MDLC (Applied Biosystems) equipped with ion spray source running Analyst QS software, and ultimate 3000 nano HPLC system (Dionex) coupled to a 4000 QTRAP mass spectrometer (Applied Biosystems).
The acquired mass spectra were searched using Mascot search engine (http://www.matrixscience.com). The following parameters were used: maximum allowed missed cleavage 1, fixed amino acid modification as carbamidomethyl and variable amino acid modifications either oxidation (M) or acetyl (N-term) or both, taxonomy set to Viridiplantae/Oryza/Arabidopsis, and databases used MSDB or Ludwig NR. The peptide and fragment mass tolerance for spectra obtained from MALDI and QStar were 100 ppm, 0.3 Da, and 100 ppm, 0.4 Da, respectively, while for TRAP it was 1.2 and 0.6 Da. Only those protein samples whose MOWSE score [68, 69] was above the significant threshold level (p < 0.05) as determined by MASCOT were considered. Since chickpea genome is not sequenced, therefore a homology based search was performed. The details regarding the precursor ion mass, expected molecular weight, theoretical molecular weight, delta, score, rank, charge, number of missed cleavages, peptide sequence, database, taxonomy, and spectra for proteins identified with a single peptide are given in in Additional file 7. A list of each peptide score and threshold score of identified proteins is given in Additional file 8.
The prediction of transmembrane domain (TMD) of the identified proteins were carried out using TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), DAS (http://www.sbc.su.se/~miklos/DAS/), HMMTOP (http://www.enzim.hu/hmmtop/), Sosui (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html) and SPLIT (http://split.pmfst.hr/ split/4/). Grand Average of Hydropathicity (GRAVY) value for each protein and peptide was calculated using ProtParam tool available at the Expasy server (http://au.expasy.org/tools/protparam.html). To determine the function of unknown proteins, domain analysis was performed to predict the conserved domain using the InterPro (http://www.ebi.ac.uk/interpro/) database and queried for domains in the SMART (http://smart.embl-heidelberg.de/), Panther (http://www.Pantherdb.org/), and Pfam (http://www.sanger.ac.uk/software/Pfam/) databases.
Two-Dimensional gel Electrophoresis
Grand Average of hydropathicity
Sodium Dodecyl Sulphate
Polyacrylamide Gel Electrophoresis.
This work was supported by the Department of Biotechnology (DBT) [BT/PR/10677/PBD/16/795] and the Council of Scientific and Industrial Research (CSIR) [38(1255)11/EMR-II], Govt. of India. Support of the DBT pre-doctoral fellowships to D.K.J. and P.M. and post-doctoral fellowship to D.R. is also acknowledged. The authors thank the CSIR for providing pre-doctoral fellowships to P.S. and S.G. No conflict of interest declared. The authors thank Mr. Jasbeer Singh for illustrations and graphical representations in the manuscript.
- Schwacke R, Flugge UI, Kunze R: Plant membrane proteome database. Plant Physiol Biochem 2004, 42: 1023–1034. 10.1016/j.plaphy.2004.09.011View ArticleGoogle Scholar
- Wu CC, Yates JR: The application of mass spectrometry to membrane proteomics. Nature Biotechnol 2003, 21: 262–267. 10.1038/nbt0303-262View ArticleGoogle Scholar
- Groen AJ, Lilley KS: Proteomics of total membranes and subcellular membranes. Expert Rev Proteomics 2010, 7: 867–878. 10.1586/epr.10.85View ArticleGoogle Scholar
- Santoni V, Molloy M, Rabilloud T: Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000, 21: 1054–1070. 10.1002/(SICI)1522-2683(20000401)21:6<1054::AID-ELPS1054>3.0.CO;2-8View ArticleGoogle Scholar
- Ephritikhine G, Ferro M, Rolland N: Plant membrane proteomics. Plant Physiol Biochem 2004, 42: 943–962. 10.1016/j.plaphy.2004.11.004View ArticleGoogle Scholar
- Rolland N, Ferro M, Ephritikhine G, Marmagne A, Ramus C, Brugière S, Salvi D, Seigneurin-Berny D, Bourguignon J, Barbier-Brygoo H, Joyard J, Garin J: A versatile method for deciphering plant membrane proteomes. J Exp Bot 2006, 57: 1579–1589. 10.1093/jxb/erj162View ArticleGoogle Scholar
- Komatsu S, Konishi H, Hashimoto M: The proteomics of plant cell membranes. J Exp Bot 2007, 58: 103–112.View ArticleGoogle Scholar
- Kota U, Goshe MB: Advances in qualitative and quantitative plant membrane proteomics. Phytochemistry 2011, 72: 1040–1060. 10.1016/j.phytochem.2011.01.027View ArticleGoogle Scholar
- Baker DG, Bianchi S, Blondon F, Dattee Y, Duc G, Flament P, Gallusci P, Genier G, Guy P, Muel X, Tourneur J, Denarie J, Huguet T: Medicago trancatula, a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Plant Mol Biol Rep 1990, 8: 40–49. 10.1007/BF02668879View ArticleGoogle Scholar
- Singh KB, Ocampo B, Robertson LD: Diversity for abiotic and biotic stress resistance in the wild annual Cicer species. Genet Res Crop Evo 1998, 45: 9–17. 10.1023/A:1008620002136View ArticleGoogle Scholar
- Winter P, Benko-Iseppon AM, Huttel B, Ratnaparkhe M, Tullu A, Sonnante G, Pfaff T, Tekeoglu M, Santra D, Sant VJ, Rajesh PN, Kahl G, Muehlbauer FJ: A linkage map of the chickpea (Cicer arietinum L) genome based on recombinant inbred lines from a C arietinum x C reticulatum cross: localisation of resistance genes for Fusarium wilt races 4 and 5. Theor Appl Genet 2000, 101: 1155–1163. 10.1007/s001220051592View ArticleGoogle Scholar
- Molina C, Rotter B, Horres R, Udupa SM, Bessre B, Bellarmino L, Baum M, Matsumura H, Terauchi R, Khal G, Winter P: SuperSAGE: the drought stress-responsive transcriptome of chickpea roots. BMC Genomics 2008, 9: 553. 10.1186/1471-2164-9-553PubMed CentralView ArticleGoogle Scholar
- Ashraf N, Ghai D, Barman P, Basu S, Gangisetty N, Mandal MK, Chakraborty N, Datta A, Chakraborty S: Comparative analysis of genotype dependent expressed sequence tags and stress-responsive transcriptome of chickpea wilt illustrate predicted and unexpected genes and novel regulators of plant immunity. BMC Genomics 2009, 10: 415. 10.1186/1471-2164-10-415PubMed CentralView ArticleGoogle Scholar
- Flowers TJ, Gaur PM, Gowda CL, Krishnamurthy L, Samineni S, Siddique KH, Turner NC, Vadez V: Salt sensitivity in chickpea. Plant Cell Environ 2009, 33: 490–509.View ArticleGoogle Scholar
- Varshney RK, Hiremath PJ, Lekha P, Kashiwagi J, Balaji J, Deokar AA, Vadez V, Xiao Y, Srinivasan R, Gaur PM, Siddique KH, Town CD, Hoisington DA: A comprehensive resource of drought- and salinity-responsive ESTs for gene discovery and marker development in chickpea (Cicer arietinum L). BMC Genomics 2009, 10: 523. 10.1186/1471-2164-10-523PubMed CentralView ArticleGoogle Scholar
- Garg R, Patel RK, Jhanwar S, Priya P, Bhattacharjee A, Yadav G, Bhatia S, Chattopadhyay D, Tyagi AK, Jain M: Gene discovery and tissue-specific transcriptome analysis in chickpea with massively parallel pyrosequencing and web resource development. Plant Physiol 2011, 156: 1661–1678. 10.1104/pp.111.178616PubMed CentralView ArticleGoogle Scholar
- Bhushan D, Pandey A, Chattopadhyay A, Choudhary MK, Chakraborty S, Datta A, Chakraborty N: Extracellular matrix proteome of chickpea (Cicer arietinum L) illustrates pathway abundance, novel protein functions and evolutionary perspect. J Proteome Res 2006, 5: 1711–1720. 10.1021/pr060116fView ArticleGoogle Scholar
- Pandey A, Choudhary MK, Bhushan D, Chattopadhyay A, Chakraborty S, Datta A, Chakraborty N: The nuclear proteome of chickpea (Cicer arietinum L) reveals predicted and unexpected proteins. J Proteome Res 2006, 5: 3301–3311. 10.1021/pr060147aView ArticleGoogle Scholar
- Tan S, Tan HT, Chung MC: Membrane proteins and membrane proteomics. Proteomics 2008, 8: 3924–3932. 10.1002/pmic.200800597View ArticleGoogle Scholar
- Valot B, Gianinazzi S, Eliane DG: Sub-cellular proteomic analysis of a Medicago trancatula root microsomal fraction. Phytochemistry 2004, 65: 1721–1732. 10.1016/j.phytochem.2004.04.010View ArticleGoogle Scholar
- Tian L, Peel GJ, Lei Z, Aziz N, Dai X, He J, Watson B, Zhao PX, Sumner LW, Dixon RA: Transcript and proteomic analysis of developing white lupin Lupinus albus L roots. BMC Plant Biol 2009, 9: 1. 10.1186/1471-2229-9-1PubMed CentralView ArticleGoogle Scholar
- Schwacke R, Schneider A, Van der Graaff E, Fischer K, Catoni E, Desimone M, Frommer WB, Flugge U, Kunze R: ARAMEMNON, a novel database for Arabidopsis integral membrane proteins. Plant Physiol 2003, 131: 16–26. 10.1104/pp.011577PubMed CentralView ArticleGoogle Scholar
- Molloy MP: Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal Biochem 2000, 280: 1–10. 10.1006/abio.2000.4514View ArticleGoogle Scholar
- Nouwens AS, Cordwell SJ, Larsen MR, Molloy MP, Gillings M, Willcox MDP, Walsh BJ: Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1. Electrophoresis 2000, 21: 3797–3809. 10.1002/1522-2683(200011)21:17<3797::AID-ELPS3797>3.0.CO;2-PView ArticleGoogle Scholar
- Schneeman R, Krogmann DW: Polycation interactions with spinach ferredoxin-nicotinamide adenine dinucleotide phosphate reductase. J Biol Chem 1975, 250: 4965–4971.Google Scholar
- Suss KH, Arkona C, Manteuffel R, Adler K: Calvin cycle multienzyme complexes are bound to chloroplast thylakoid membranes of higher plants in situ. Proc Natl Acad Sci USA 1993, 90: 5514–5518. 10.1073/pnas.90.12.5514PubMed CentralView ArticleGoogle Scholar
- Utsunomiya E, Muto S: Carbonic anhydrase in the plasma membranes from leaves of C3 and C4 plants. Physiol Plantarum 1993, 88: 413–419. 10.1111/j.1399-3054.1993.tb01353.xView ArticleGoogle Scholar
- Martin SW, Glover BJ, Davies JM: Lipid microdomains-plant membranes get organized. Trends Plant Sci 2005, 10: 263–265. 10.1016/j.tplants.2005.04.004View ArticleGoogle Scholar
- Uhrig JF: Protein interaction networks in plants. Planta 2006, 224: 771–781. 10.1007/s00425-006-0260-xView ArticleGoogle Scholar
- Konishi H, Maeshima M, Komatsu S: Characterization of vacuolar membrane proteins changed in rice root treated with gibberellin. J Proteome Res 2005, 4: 1775–1780. 10.1021/pr050079cView ArticleGoogle Scholar
- Lu M, Holliday LS, Zhang L, Dunn WA Jr, Gluck SL: Interaction between aldolase and vacuolar H+-ATPase: evidence for direct coupling of glycolysis to the ATP-hydrolyzing proton pump. J Biol Chem 2001, 276: 30407–30413. 10.1074/jbc.M008768200View ArticleGoogle Scholar
- Mustroph A, Sonnewald U, Biemelt S: Characterisation of the ATP-dependent phosphofructokinase gene family from Arabidopsis thaliana. FEBS Lett 2007, 7581: 2401–2410.View ArticleGoogle Scholar
- Giege P, Heazlewood JL, Roessner-Tunali U, Millar AH, Fernie AR, Leaver CJ, Sweetlove LJ: Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells. Plant Cell 2003, 15: 2140–2151. 10.1105/tpc.012500PubMed CentralView ArticleGoogle Scholar
- Kemp CA, Song MH, Addepalli MK, Hunter G, Connell KO: Suppressors of zyg-1 define regulators of centrosome duplication and nuclear association in Caenorhabditis elegans. Genetics 2007, 176: 95–113. 10.1534/genetics.107.071803PubMed CentralView ArticleGoogle Scholar
- Starr DA: A nuclear envelope bridge positions nuclei and moves chromosomes. J Cell Sci 2009, 122: 577–586. 10.1242/jcs.037622PubMed CentralView ArticleGoogle Scholar
- Graumann K, Runions J, Evans DE: Characterization of SUN-domain proteins at the higher plant nuclear envelope. Plant J 2009, 61: 134–144.View ArticleGoogle Scholar
- Wang N, Tytell JD, Ingber DE: Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus. Nat Rev Mol Cell Biol 2009, 10: 75–82.View ArticleGoogle Scholar
- Muller R, Morant M, Jarmer H, Nilsson L, Nielsen TH: Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant Physiol 2007, 143: 156–171.PubMed CentralView ArticleGoogle Scholar
- Afzal AJ, Wood AJ, Lightfoot DA: Plant receptor-like serine threonine kinases: roles in signaling and plant defense. Mol Plant-Microbe Interact 2008, 21: 507–517. 10.1094/MPMI-21-5-0507View ArticleGoogle Scholar
- Trost P, Foscarini S, Preger V, Bonora P, Vitale L, Pupillo P: Dissecting the diphenylene idonium-sensitive NADPH: quinone oxidoreductase of zucchini plasma membrane. Plant Physiol 1997, 114: 737–746.PubMed CentralGoogle Scholar
- Pupillo P, Valenti V, De Luca L, Hertel R: Kinetic characterization of reduced pyridine nucleotide dehydrogenase (duroquinone-dependent) in Cucurbita mirosomes. Plant Physiol 1986, 80: 384–389. 10.1104/pp.80.2.384PubMed CentralView ArticleGoogle Scholar
- Wreck-reichhart D, Feyereisen R: Cytochrome P450: a success story. Genome Biol 2000, 6: 30031–30039.Google Scholar
- Isin EM, Guengerich FP: Complex reactions catalyzed by cytochrome P450 enzymes. Biochim Biophys Acta 2007, 1770: 314–329. 10.1016/j.bbagen.2006.07.003View ArticleGoogle Scholar
- Bak S, Beisson F, Bishop G, Hamberger B, Höfer R, Paquette S, Werck-Reichhart D: Cytochromes P450. In The Arabidopsis Book Number 9. American Society of Plant Biologists, Rockville, MD; 2011.Google Scholar
- Tijet N, Helvig C, Feyereisen R: The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny. Gene 2001, 262: 189–198. 10.1016/S0378-1119(00)00533-3View ArticleGoogle Scholar
- Xu Y, Ishida H, Reisen D, Hanson MR: Upregulation of a tonoplast-localized cytochrome P450 during petal senescence in Petunia inflate. BMC Plant Biol 2006, 6: 8. 10.1186/1471-2229-6-8PubMed CentralView ArticleGoogle Scholar
- Melotto M, Kelly JD: Fine mapping of the Co-4 locus of common bean reveals a resistance gene candidate, COK-4, that encodes for a protein kinase. Theor Appl Genet 2001, 103: 508–517. 10.1007/s001220100609View ArticleGoogle Scholar
- Reis PA, Rosado GL, Silva LA, Oliveira LC, Oliveira LB, Costa MD, Alvim FC, Fontes EP: The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway. Plant Physiol 2011, 157: 1853–1865. 10.1104/pp.111.179697PubMed CentralView ArticleGoogle Scholar
- Vierling E: The role of heat shock proteins in plants. Ann Rev Plant Physiol Plant Mol Biol 1991, 42: 579–620. 10.1146/annurev.pp.42.060191.003051View ArticleGoogle Scholar
- Brugiere S, Kowalski S, Ferro M, Seigneurin-Berny D, Miras S, Salvi D, Ravanel S, Herin P, Garin J, Bourguignon J, Joyard J, Rolland N: The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions. Phytochemistry 2004, 65: 1693–1707. 10.1016/j.phytochem.2004.03.028View ArticleGoogle Scholar
- Davies E, Fillingham BD, Oto Y, Abe S: Evidence for the existence of cytoskeleton-bound polysomes in plants. Cell Biol Int Rep 1991, 15: 973–981. 10.1016/0309-1651(91)90147-BView ArticleGoogle Scholar
- Hicke L: A new ticket for entry into budding vesicles-ubiquitin. Cell 2001, 106: 527–530. 10.1016/S0092-8674(01)00485-8View ArticleGoogle Scholar
- Schirmer EC, Florens L, Guan T, Yates JR, Gerace L: Nuclear membrane proteins with potential disease links found by subtractive proteomics. Science 2003, 301: 1380–1382. 10.1126/science.1088176View ArticleGoogle Scholar
- Masaki T, Mitsui N, Tsukagoshi H, Nishii T, Morikami A, Nakamura K: Activator of Spomin::LUC1/WRINKLED1 of Arabidopsis thaliana transactivates sugar-inducible promoters. Plant Cell Physiol 2005, 46: 547–556. 10.1093/pcp/pci072View ArticleGoogle Scholar
- Kato Y, Sun X, Zhang L, Sakamoto W: Cooperative D1 degradation in the photosystem II repair mediated by chloroplastic proteases in Arabidopsis. Plant Physiol 2012, 159: 1428–1439. 10.1104/pp.112.199042PubMed CentralView ArticleGoogle Scholar
- Olinares PD, Kim J, Van Wijk KJ: The Clp protease system; a central component of the chloroplast protease network. Biochim Biophys Acta 2011, 1807: 999–1011. 10.1016/j.bbabio.2010.12.003View ArticleGoogle Scholar
- Pang Q, Chen S, Dai S, Chen Y, Wang Y, Yan X: Comparative proteomics of salt tolerance in Arabidopsis thaliana and Thellungiella halophile. J Proteome Res 2010, 9: 2584–2599. 10.1021/pr100034fView ArticleGoogle Scholar
- Van Cutsem E, Simonart G, Degand H, Faber AM, Morsomme P, Boutry M: Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response. Proteomics 2011, 11: 440–454. 10.1002/pmic.201000356View ArticleGoogle Scholar
- Mitra SK, Gantt JA, Ruby JF, Clouse SD, Goshe MB: Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques. J Proteome Res 2007, 6: 1933–1950. 10.1021/pr060525bView ArticleGoogle Scholar
- Traas J, Monéger F: Systems biology of organ initiation at the shoot apex. Plant Physiol 2010, 152: 420–427. 10.1104/pp.109.150409PubMed CentralView ArticleGoogle Scholar
- Vertommen A, Panis B, Swennen R, Carpentier SC: Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants. Planta 2010, 231: 1113–1125. 10.1007/s00425-010-1121-1PubMed CentralView ArticleGoogle Scholar
- Martinec J, Felt T, Scanlon CH, Lumsden PJ, Machavkova I: Subcellular localization of a high affinity binding site for D-myo-inositol 1,4,5-triphosphate from Chenopodium rubrum. Plant Physiol 2000, 124: 475–483. 10.1104/pp.124.1.475PubMed CentralView ArticleGoogle Scholar
- Sze H: H+-translocating ATPases: advances using membrane vesicles. Ann Rev Plant Physiol 1985, 36: 175–208. 10.1146/annurev.pp.36.060185.001135View ArticleGoogle Scholar
- Ray PM, Shininger TL, Ray MM: Isolation of beta-glucan synthetase particles from plant cells and identification with Golgi membranes. Proc Natl Acad Sci USA 1969, 64: 605–614. 10.1073/pnas.64.2.605PubMed CentralView ArticleGoogle Scholar
- Ames BN: Assay of inorganic phosphate, total phosphate and phosphatase. Methods Enzymol 1966, 8: 115–118.View ArticleGoogle Scholar
- Ferro M, Seigneurin-Berny D, Rolland N, Chapel A, Salvi D, Garin J, Joyard J: Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins. Electrophoresis 2000, 21: 3517–3526. 10.1002/1522-2683(20001001)21:16<3517::AID-ELPS3517>3.0.CO;2-HView ArticleGoogle Scholar
- Tripathy BC, Chakraborty N: 5-aminolevulinic acid induced photodynamic damage to the photosynthetic electron transport chain of cucumber (Cucumis sativus L.) cotyledons. Plant Physiol 1991, 96: 761–767. 10.1104/pp.96.3.761PubMed CentralView ArticleGoogle Scholar
- Perkins DN, Pappin DJ, Creasy DM, Cottrell JS: Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20: 3551–3567. 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2View ArticleGoogle Scholar
- Creasy DM, Cottrell JS: Error tolerant searching of uninterpreted tandem mass spectrometry data. Proteomics 2002, 2: 1426–1434. 10.1002/1615-9861(200210)2:10<1426::AID-PROT1426>3.0.CO;2-5View ArticleGoogle Scholar
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