Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network.

JPROT-01780; No of Pages 16 JOURNAL OF P ROTEOM IC S XX ( 2014) X XX– X XX

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Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network☆ Rajiv Kumar, Amit Kumar, Pratigya Subba, Saurabh Gayali, Pragya Barua, Subhra Chakraborty, Niranjan Chakraborty⁎ National Institute of Plant Genome Research, New Delhi-110067, India

AR TIC LE I N FO

ABS TR ACT

Article history:

Nucleus, the control centre of eukaryotic cell, houses most of the genetic machineries required

Received 22 October 2013

for gene expression and their regulation. Post translational modifications of proteins,

Accepted 2 April 2014

particularly phosphorylation control a wide variety of cellular processes but its functional connectivity, in plants, is still elusive. This study profiled the nuclear phosphoproteome of a grain legume, chickpea, to gain better understanding of such event. Intact nuclei were

Keywords:

isolated from 3-week-old seedlings using two independent methods, and nuclear proteins

Nuclear phosphopreoteome

were resolved by 2-DE. In a separate set of experiments, phosphoproteins were enriched

IMAC

using IMAC method and resolved by 1-DE. The separated proteins were stained with

Pro-Q diamond

phosphospecific Pro-Q Diamond stain. Proteomic analyses led to the identification of 107

Mass spectrometry

putative phosphoproteins, of which 86 were non-redundant. Multiple sites of phosphorylation

Non-model plant

were predicted on several key elements, which included both regulatory and functional

Protein-kinase interactome

proteins. The analysis revealed an array of phosphoproteins, presumably involved in a variety of cellular functions, viz., protein folding (24%), signalling and gene regulation (22%), DNA replication, repair and modification (16%), and metabolism (13%), among others. These results represent the first nucleus-specific phosphoproteome map of a non-model legume, which would provide insights into the possible function of protein phosphorylation in plants. Biological significance Chickpea is grown over 10 million hectares of land worldwide, and global production hovers around 8.5 million metric tons annually. Despite its nutritional merits, it is often referred to as ‘orphan’ legume and has remained outside the realm of large-scale functional genomics studies. While current chickpea genome initiative has primarily focused on sequence information and functional annotation, proteomics analyses are limited. It is thus important to study the proteome of the cell organelle particularly the nucleus, which harbors most of the genetic information and gene expression machinery. Phosphorylation-dependent modulation of gene expression plays a vital role but the complex networks of phosphorylation are poorly

☆ This article is part of a Special Issue entitled: Proteomics of non-model organisms. ⁎ Corresponding author at: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India. Tel.: +91 11 26735178; fax: +91 11 26741658. E-mail address: [email protected] (N. Chakraborty).

http://dx.doi.org/10.1016/j.jprot.2014.04.002 1874-3919/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Kumar R, et al, Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.002

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JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX– XX X

understood. This inventory of nuclear phosphoproteins would provide valuable insights into the dynamic regulation of cellular phenotype through phosphorylation. This article is part of a Special Issue entitled: Proteomics of non-model organisms. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Post-translational modifications (PTMs) of proteins provide the structural and functional plasticity of the eukaryotic proteome [1]. These modifications cause covalent alterations in the protein’s primary structure thus altering their properties such as charge, conformation or activity. Protein phosphorylation, due to its rapid and reversible mode of regulation is the most biologically relevant PTM. The mechanism of phosphoregulation is known to participate in a myriad number of cellular functions including signal transduction, protein subcellular localization, growth and development, and intercellular communication among others [2,3]. Approximately one-third of proteins are presumed to be phosphorylated during their life cycle in eukaryotic cells [4,5]. It is estimated that the functions of more than 5% of the proteins in Arabidopsis are directly related to phosphorylation [6]. Currently, a significant number of plant phosphoproteome reports are available on unfractionated whole cell lysates [7–9], while studies on the subcellular structures are still limited. Among the few examples reported from the subproteomes are those from plasma membrane [10], chloroplast [11], mitochondria [12] and vacuolar membrane [13]. The nucleus is the most fundamental component of the cellular microenvironment that has been extensively developed during the process of evolution. It not only hosts the genome but also administers its transcription and the regulated expression of proteins, thereby playing a critical role as a modulator of cellular phenotype. Nuclear proteins are predicted to comprise about 10–25% of the total cellular proteins, suggesting their involvement in diverse functions. Although approximately 40% of the phosphorylation events in plants have been predicted to occur on nuclear proteins [7], only a single report on the nuclear phosphoproteome data is available to date [14]. Two-dimensional gel electrophoresis (2-DE) is a powerful method for the separation of proteins and has long been recognized as a key technology in proteome research [15]. This technique in conjunction with fluorescence-based detection technology has recently proven to be a very useful tool to visualize protein phosphorylation [16,17]. The fluorescent phosphor sensor dye (Pro-Q Diamond) is one such dye that has been used for detection and semi-quantitative analysis of protein phosphorylation [18–20]. However, due to the inherent limitations of 2-DE [21,22], this technique is often combined with 1-DE approach to maximize the number of protein identifications [16]. Also, due to the complexity of biological samples that consist of both, phosphorylated as well as non-phosphorylated proteins/peptides, a preliminary enrichment step to isolate phosphorylated components is deemed essential to increase their relative concentration. One such strategy involves the use of IMAC (immobilized metal affinity chromatography) that exploits the high affinity of phosphate groups of the phosphorylated amino acids for cations such as Fe3+, Ga3+ and titanium dioxide (TiO2) [22–24]. Recent

advances in mass spectrometry accompanied with phosphoprotein/peptide enrichment methods have paved the way for high-throughput, large-scale analysis of protein phosphorylation events. Besides the use of model plants viz., Arabidopsis, Nicotiana and Medicago, the crop plant rice is often considered as the plant reference for genetic studies. However, several non-model plants that are agriculturally important with unique biological characteristics remained neglected and need to be investigated individually. Chickpea, often regarded as an ‘orphan’ legume [25], provides an interesting system for their rich source of nitrogen, enhancing the soil fertility and is a valuable source of human dietary protein. In a previous study, we had reported the nuclear proteome of this legume and identified several components involved in a variety of biological functions [26]. In this study, an attempt was made to catalogue the nuclear phosphoproteins by combining two different methods of intact nuclear isolation along with complementary proteomic approaches including 1-DE, 2-DE and. Using these techniques, we identified 107 putative phosphoproteins and categorized them functionally. This nuclear phosphoproteome is of interest as it will provide an insight into the functional role of this organelle and also for future cellular and molecular biology studies of chickpea and other legume crops.

2. Materials and methods 2.1. Plant materials Chickpea (Cicer arietinum L. cv. JG-62) seeds were germinated in pots containing a mixture of soil and soilrite (2:1, w/w) under fluorescent white light (270 μmol m−2 s−1, 16 h photoperiod) at 25 ± 2 °C and 50 ± 5% relative humidity in an environmentally controlled growth chamber. The aerial parts of 3-weekold seedlings were harvested by quick freezing in liquid nitrogen and stored at − 80 °C until further use.

2.2. Isolation of nuclei, extraction of nuclear proteins and purity analysis Intact nuclei from chickpea seedlings were isolated using two independent methods. The first method involves the use of a hyperosmotic sucrose buffer (HSB) [26] whereas the second method utilizes a percoll density gradient (PDG) [27]. In the PDG method, tissues were ground to fine powder using liquid nitrogen and resuspended in homogenization buffer [2 M hexylene glycol, 20 mM PIPES-KOH (pH 7.0), 10 mM MgCl2, 5 mM β merceptoethanol, 1% Triton X-100, 1 mM PMSF and 10 mM ascorbic acid]. The contents were gently stirred for 30 min and then filtered through four layers of cheesecloth followed by two layers of Miracloth (Calbiochem) into ice-cold round bottomed centrifuge tubes. The Percoll gradient was prepared by layering 6 mL of 80% Percoll onto


JOURNAL OF P ROTEOM IC S XX ( 2014) X XX– X XX

6 mL of 30% Percoll. The lysate was gently layered on top of the gradient and centrifuged using swinging bucket rotor (2000 g, 30 min). The nuclear fraction was gently recovered from the interface and again layered on top of 6 mL 30% Percoll and centrifuged (2000 g, 10 min). All steps were performed on ice or at 4 °C. Isolated nuclei were stained with DAPI and visualized by fluorescence microscopy. The chlorophyll contents were determined using a spectrophotometric assay as described previously [26]. Nuclear proteins were extracted from the nuclei-enriched pellet using TriPure reagent (Roche) according to the manufacturer’s instructions. Immunoblotting was carried out with 50 μg protein resolved into 12.5% SDS-PAGE and electrotransferred onto nitrocellulose membranes. The blots were probed with primary antibodies against nuclear marker proteins histone and fibrillarin (Abcam Limited, U.K.) whereas cytochrome c oxidase was used as mitochondrial marker. The antibody-bound proteins were detected by incubating with secondary antibodies (Abcam Limited, U.K.) conjugated to alkaline phosphatase or horseradish peroxidase.

2.3. 2-DE and detection of phosphoproteins The nuclear proteins were resuspended in isoelectric focusing (IEF) buffer [8 M urea, 2 M thiourea, 2% CHAPS (w/v), 1% carrier ampholytes, 20 mM DTT and 0.001% bromophenol blue] and centrifuged (10000 g, 5 min) to remove undissolved contents. Aliquots of 250 μL protein (300 μg) were applied to IPG strips (13 cm, pH 4 − 7) for passive rehydration. IEF was carried out using IPGphor system (GE Healthcare) at 20 °C for 35 KVh. The focused strips were subjected to reduction with 1% DTT (w/v) 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% iodoacetamide (w/v) in the same buffer. Proteins were separated into 12.5% SDS-PAGE using SE 600 (Hoefer). To detect the putative phosphoproteins, gels were stained with Pro-Q Diamond (Molecular Probes) as described earlier [22]. Briefly, the gels were fixed in fixative solution (50% methanol and 10% acetic acid) overnight and then washed thrice with distilled water with gentle agitation (200 mL, 20 min each). The gels were then stained with Pro-Q Diamond, destained [20% acetonitrile and 50 mM sodium acetate (pH 4.0)] and scanned (Typhoon 9210) with 532 nm laser excitation and 555 nm band pass emission filter. The same gels, following image acquisition, were stained with Sypro Ruby (Molecular Probes). Proteins spots were detected using laser scanner with 488 nm excitation and 555 nm band pass emission filter. The spots that stained consistently with Pro-Q Diamond in the experimental replicates were considered as putative phosphoproteins. Three biological and three experimental replicates were performed. Spots were excised manually for mass spectrometric analysis. The PDQuest version 7.2.0 (Bio-Rad, CA) was used to generate standard image from three replicate 2-DE gels.

2.4. Enrichment of phosphoproteins The IMAC procedure was performed as described earlier [22] with few modifications. Nuclear proteins were dissolved in IMAC buffer (8 M urea, 2 M thiourea, 2% CHAPS) and 6 mg

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protein was diluted with wash buffer [8 M urea, 50 mM sodium acetate (pH4.0), 0.25% CHAPS] to 1 mg/mL. Proteins were then incubated with Gallium charged chelating sepharose fast flow beads (GE Healthcare) packed into homemade micro columns for 3 h at 4 °C (10 mg protein/mL bed volume). Beads were washed with 20 column volumes of wash buffer and putative phosphoproteins were eluted using 3 column volumes of elution buffer [8 M urea, 50 mM Tris acetate (pH 7.4), 0.1 M EDTA, 0.1 M EGTA, 0.25% CHAPS]. The eluted proteins were precipitated with 5 volumes of methanol and resolved into SDS-PAGE, and the profile was compared with other protein fractions. The bands stained with Pro-Q Diamond were excised and processed for mass spectrometric analysis.

2.5. In-gel digestion and mass spectrometry In-gel digestion of protein bands or spots was performed using trypsin (Sigma, USA) according to standard techniques [28]. Mass spectrometric analysis was carried out using LC-ESIMS/MS or MALDI-TOF/TOF. LC-ESI-MS/MS analysis was carried out using Q-STAR Elite mass spectrometer (Applied Biosystems) coupled with an ultimate 3000 HPLC system (Dionex). The tryptic peptides were extracted and loaded onto C18 PepMap 100, 3 μm (LC Packings), and separated with a linear gradient of water/ACN/0.1% (v/v) formic acid. The acquired spectra were analysed using Analyst Software version 1.4.1 (Applied Biosystems), and peptide identification was performed using Mascot search engine version 2.1. The following parameters were used: type of search, MS/MS ion search; ion mass range, 400 - 1800 m/z; charge state, +1/+2/+3; database, MSDB version 250509 (3,239,079 sequences; 1,079,594,700 residues); enzyme, trypsin; missed cleavage, 1; taxonomy, Viridiplantae (Green Plants, 247346 sequences); variable modifications, oxidation (M); peptide tolerance, ±1.2 Da; and fragment mass tolerance, ±0.8 Da. We considered only those proteins which had the probability scores greater than the Mascot’s default threshold, which estimates significance at p < 0.05 for individual peptides. For MALDI-TOF/TOF analysis, the eluted peptides were spotted on MALDI plate and spectra were generated on 4800 Proteomics Analyzer (Applied Biosystems). Identification was performed using GPS Explorer, version 3.5 (Applied Biosystems). The parameters were as follows: enzyme: trypsin with one missed cleavage; MS peak filtering: 800−4000 m/z interval, monoisotopic, minimum S/N 10, mass tolerance ±100 ppm; MS/MS peak filtering: monoisotopic, M + H+, minimum S/N = 3, MS/MS fragment tolerance ±0.4 Da; database used: MSDB 20050227 (1942918 sequences; 629040812 residues); and taxonomy: Viridiplantae (Green Plants, 247346 sequences). The fixed amino acid modification was carbamidomethyl, and variable amino acid modification was oxidation (M). We combined the PMF and MS/MS peak lists of each band/spot for identification. Peptides with confidence interval (C.I.) >95% were considered as positive identification. To identify the site/s of phosphorylation, nuclear phosphoproteins were enriched using the IMAC strategy mentioned above and 100 μg of the samples were resolved onto SDS-PAGE. The bands were trypsin-digested and the peptides were finally reconstituted in 2% acetonitrile containing 0.1%


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formic acid. HPLC separation was performed by a splitless Ultra 2D Plus (Eksigent) coupled with the TripleTOF 5600 using a Nanospray III source (AB SCIEX). The composition of buffer A was 2% acetonitrile and 0.1% formic acid whereas buffer B was composed of 98% acetonitrile and 0.1% formic acid. The gradient program was set for 68 min (5− 30% buffer B over 20 min, 30− 50% buffer B over 20 min, 60− 90% buffer B over 6 min, hold buffer B at 90% for 10 min, and 90 − 5% B over 12 min). Peptide separation was performed using a Zorbax 300 C18 (75 μm ID × 150 mm) column (Agilent), and analyzed in positive ion mode by electrospray ionization (spray voltage = 2.2 kv). For IDA (information dependent acquisition), survey scans were acquired in 250 ms and 12 product ion scans were collected in 100 ms/per scan. MS data were interrogated using Mascot to identify the proteins, phosphopeptides and their corresponding site/s of phosphorylation. The search parameters were as follows: database, NCBI; taxonomy, viridiplantae (664452 sequences); enzyme, trypsin; allowed trypsin missed cleavage, 1; fixed modification, carbamidomethyl (C); variable modifications, oxidation (M) and phosphorylation (STY). The mass tolerances were specified as 50 ppm for peptides and 0.2 Da for the MS/MS fragments.

2.6. Functional classification, nuclear localization and proteinkinase interactome The identified putative phosphoproteins were classified into functional categories using Blast2GO version 2.6.0 [31], which assigned two GO terms, molecular functions and biological process to each protein. Also, the identified proteins were functionally classified into different classes using protein function database Pfam (http://www.sanger.ac.uk/software/ Pfam/) or Interpro. The prediction of nuclear localization was performed using Nucpred (http://www.sbc.su.se/~maccallr/ nucpred/) and YLoc programs (http://abi.inf.uni-tuebingen. de/Services/YLoc/webloc.cgi) [29,30]. The phosphorylation site prediction was performed by NetPhos program (http://www.cbs. dtu.dk/services/NetPhos/). Protein-kinase interaction map was generated using human phosphoproteome data in NetworKIN [32]. Visualization and analysis was carried out using Cytoscape 2.8.1 (http://www.cytoscape.org/).

2.7. Alkaline phosphatase treatment Dephosphorylation assay was carried out with alkaline phosphatase (New England Biolabs, Inc.) 1 h prior to rehydration according to the manufacturer's instructions. Following IEF and second-dimension SDS-PAGE, gels were stained with Pro-Q Diamond and imaged.

3. Results and discussion 3.1. Experimental strategy and assessment of nuclear integrity The experimental strategy for systematic analysis of chickpea nuclear phosphoproteome has been depicted in Fig. 1. Intact nuclei were isolated using two approaches viz., HSB and PDG methods. The integrity of the isolated nuclei was

Fig. 1 – Experimental workflow for the analysis of nuclear phosphoproteins. No-IMAC and IMAC strategies were employed independently to the nuclear proteins prepared by HSB (hyperosmotic sucrose buffer) and PDG (Percoll density gradient) methods.

assessed using DAPI staining. The fluorescence micrograph revealed uniform spheres with an average diameter of 20 μm with no apparent organellar impurities from other compartments (Fig. 2A and B). To check for possible chloroplast contamination in the nuclear fraction, spectrophotometric analysis was carried out. The supernatant retained most of the chlorophyll, whereas no or negligible chlorophyll was present in the nuclei fraction (Fig. 2C). Nuclear proteins, essentially devoid of contaminating nucleic acids, were extracted from the purified nuclei as described earlier [26]. To visualize the nuclear protein profile with other fractions, proteins were separated by 1-DE and visualized using CBB R-250 staining (Fig. 2D). The analysis revealed a distinct profile for each fraction, besides an enrichment of lower molecular (LMW) weight proteins (12− 18 KDa range) in the nuclear fraction. Mass spectrometric analysis of these LMW bands revealed the identity of various histone variants viz., H2B, H3 and H4 of the nucleosome core particle (Supplementary Table



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Fig. 2 – Analysis of chickpea nuclear fraction and determination of purity. (A) Purified nuclear fraction was stained with DAPI and visualized by fluorescence microscopy at 358 nm absorption and 461 nm emission wavelengths. (B) Phase contrast micrograph of the intact nuclei. (C) Determination of total chlorophyll content at different stages of purification of nuclear fraction. The amount of chlorophyll was determined in whole cell extract (CE), supernatant (S), and nuclear fraction (N). The values were the average of three independent experiments. (D) Whole cell extract (CE), supernatant (S) and nuclear (N) proteins were separated by 1D-SDS-PAGE and stained with CBB. (E) Immunoblot analysis of cell extract, supernatant and nuclear fraction. An aliquot of 50 μg protein was resolved, electroblotted onto nitrocellulose membrane and probed with anti-histone, anti-fibrillarin and anti-cyc-c-ox antibodies.

S1) which confirms the enrichment of nuclear proteins. To monitor further the enrichment of nuclear proteins, immunoblot analysis was performed using anti histone and anti-fibrillarin antibodies. Negative immunoblotting was also performed to check the presence of non-nuclear protein cytochrome c oxidase. Whereas signals from histone and fibrillarin were detected in the nuclear fraction, the signal from cytochrome c oxidase was detected in the total protein extract indicating the enrichment of nuclear proteins (Fig. 2E). These results altogether indicate the high quality nuclear protein preparation with no detectable level of cross contamination.

3.2. 2-DE profile of nuclear phosphoproteome Nuclear proteins were separated using 2-DE to generate phosphoprotein reference map. The gels were first stained with Pro-Q Diamond and counterstained with Sypro Ruby to visualize the total proteins (Supplementary Fig. S1). Three replicate gel images from HSB and PDG methods were computationally combined to generate a “standard image” (Fig. 3). The spots included in the analysis were of high quality (quality score of 30 or above) and the correlation coefficient value (CV) was at least 0.8 among the replicate gels.


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Fig. 3 – Nuclear phosphoproteome profile from the representative Pro-Q stained 2D gels. The proteins were isolated from the purified nuclei and resolved by classical 2-DE. The experiments were repeated thrice for each protein prepared by HSB (A) and PDG (B) methods that were then computationally combined into a representative standard gel.

Analysis of the Pro-Q Diamond gel images displayed a maximum number of putative spots in pH range 5 −6 in the HSB method whereas a uniform distribution was observed in pH range 4 − 7 in the PDG method. The molecular weights ranged from 28− 100 KDa in both the methods. A comparison of the Pro-Q Diamond images with their Sypro Ruby stained images indicated minimal phospho-staining of abundant nuclear proteins (Supplementary Fig. S1). A number of spots displayed diffused Pro-Q Diamond signal and were thus disregarded. Although the number of proteins (~400) detected on the Sypro Ruby stained gels were comparable in both the methods, the number of spots detected on the Pro-Q Diamond images differed slightly (110 in HSB and 90 in PDG methods). To maximize the number of identifications, the MSDB was used for the analysis. A total of 51 proteins, 31 from HSB and 20 from PDG methods, were identified with high confidence (Table 1). While 19 proteins were unique to the HSB method, 15 were unique to the PDG method. The corresponding spots are indicated on the image (Supplementary Fig. 1), and numbered as CaNP-X wherein the letters ‘Ca’ indicates Cicer arietinum, ‘N’ represents nucleus, and ‘P’ stands for phosphoprotein while ‘X’ indicates the spot number. In a number of cases, the same protein was identified in more than one spot indicating a probable protein modification and/or degraded protein.

3.3. Enrichment of phosphoproteins Due to the low stoichiometry of phosphoproteins in biological samples, enrichment is required to detect the low abundant phosphoproteins. IMAC is a widely used enrichment technique that utilizes the affinity of phosphate groups to trivalent metal ions. In this study, putative phosphoproteins were enriched from the nuclear lysate of both the HSB and PDG methods using Ga3+ charged columns, and the efficiency of the enrichment process was analyzed by 1-DE. The gels were sequentially stained with Pro-Q Diamond followed by CBB-R250. The protein profiles revealed differential patterns of protein representation in the nuclear extract, flow-through and elute fractions (Fig. 4). The Pro-Q Diamond signals detected in the elute fraction showed enhanced intensity suggesting the enrichment of phosphoproteins. The gel bands were analyzed using LC-ESI-MS/MS (Fig. 4). The analysis led to the identification of 56 proteins, 31 from HSB and 25 from PDG methods (Table 1).

3.4. Functional classification and subcellular localization of nuclear phosphoproteins Using a combination of 1-DE and 2-DE systems, 107 putative phosphoproteins were identified which would have



been underrepresented if only a single method was used (Table 1). Several of the gel spots/slices indicated more than a single protein. The molecular weight and pI of most of these proteins were not distinctly different, suggesting that a spot/ slice contained several proteins. The identified proteins were used to create a non-redundant dataset of 86 putative phosphoproteins. We performed a search using the NetPhos phosphorylation site prediction program (http://www.cbs.dtu. dk/services/NetPhos/) to identify residues predicted to be phosphorylated. Supplementary Table S2 summarizes the residues considered most likely to be phosphorylated based on the ranking by the NetPhos algorithm. The proteins were sorted into functional categories according to their ontology as determined from the GO annotation term and functions (Fig. 5). The most abundant category according to biological and molecular functions were ‘Response to stress’ (38%) and ‘Nucleotide binding’ (49%), respectively. According to Pfam or Interpro the most abundant class was ‘Protein folding’ (24%) followed by ‘Signaling and gene regulation’ (22%). The other categories included ‘DNA replication, repair and modification’ (16%), ‘Translation’ (9%), ‘Metabolism’ (13%), ‘Uncharacterized’ (5%), ‘Protein degradation’ (7%), ‘Structure’ (3%) and ‘RNA metabolism’ (1%). It must be noted that since the current GO classification system may classify a single protein into more than one GO category, the cumulative percentage is over 100% (Fig. 5). To investigate the subcellular localization, we employed two prediction tools viz., NucPred and YLoc. Seventy eight percent of the proteins displayed positive output by either/ both of the prediction tools accounting for more than two-thirds of the identified proteins (Table 1). Further, the proteins which did not show nuclear localization cannot be ruled out as nuclear residents because currently available prediction tools for cellular localization have limitations, more so for distinguishing members of multi-protein families having different localization.

3.5. Nuclear phosphoproteome map of developing chickpea The nucleus, along with harboring the genetic information, is known to participate in several other functions like cell division, growth and reproduction and controlling metabolic functions. Therefore an understanding of the nuclear phosphoproteome will provide an insight into its functional diversity. The class ‘Protein folding’ comprised several heat shock proteins including Hsp90, Grp94, heat shock cognate protein, Hsp Cognate 80, Hsp81-2, chaperone protein ClpC1, Hsp70, dnaK-type molecular chaperone, putative CCT chaperonin gamma subunit and dnaK-type molecular chaperone CSS1. In humans, the phosphorylation of Hsp90 occurs on tyrosine residue which helps it to modulate the action of its kinase counterpart [33]. Further, its role in the nucleus to maintain the activity of cyclin dependent kinase has also been established [34]. The role of Hsp70 in translocating unfolded proteins to the subnuclear compartment, nucleolus, has been reported as an additional step in protecting the stressed cells [35]. The phosphorylation site of Hsp70 from Arabidopsis has recently been deposited in P3DB, a plant specific phosphorylation database [36]. The phosphorylated Hsp70, a GlcNAc specific lectin in rats, was reported to act as a shuttle for the

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nucleocytoplasmic transport of O-glycoproteins between the cytosol and the nucleus [37]. Hsp80s are also known to be localized to the nucleus [38]. The second largest category comprised proteins involved in signaling and gene regulation. An interesting candidate in this category was a methyl-accepting chemotaxis protein. In bacteria, this protein is known to participate in chemosensing. Certain strains of bacteria are known to exert beneficial effects on plants through colonization in the rhizosphere using this phenomenon [39]. Also, these proteins contain coiled-coil domain/s, which are known to act as “cellular velcro” to hold together molecules, subcellular structures, and even tissues [40]. Another protein in this category was a putative RH2 protein. This protein is known to possess ATP-dependent helicase activity (http://www.uniprot. org) and predicted to reside in the nucleus (http://string-db.org). A DEAD-box ATP-dependent RNA helicase was reported from a phosphoproteomic study of maize under water-deficit condition [9]. A putative U2 snRNP protein, a small nuclear ribonucleoprotein which is a component of the spliceosome, was also identified. Its Arabidopsis counterpart contains a C-terminal domain U2A/phosphoprotein 32 family A (http:// www.Arabidopsis.org). Also, a U2 snRNP auxiliary factor, which is known to bind to U2 snRNP, has been reported to undergo phosphorylation [11]. Some kinases like phosphoglycerate kinase (PGK), phosphoribulokinase and nucleoside diphosphate kinase were also identified. PGK, apart from functioning as a glycolytic enzyme, is also known to regulate various other functions [27]. Its mode of action has been reported to be regulated through phosphorylation in response to elicitor signaling in Arabidopsis [41]. Other proteins in this class were fructose-biphosphate aldolase, transketolase and H+-transporting two-sector ATPase. The former two proteins had earlier been reported in the nuclear proteome of chickpea [42]. In the category of proteins related to DNA replication, repair and modification, several histone variants were identified. The dynamic regulation of histone variants through various kinds of protein PTMs is well documented [43–45]. Among the histone variants, histone H3 has remained in the chromatin spotlight as it is currently considered to carry the greatest number of PTMs [46]. In plants, the cell cycle dependent phosphorylation of histone H3 has been described at serine 10/28 and at threonine 3/11 [47]. Histone H3 has been reported to participate in a number of functions such as drought induced signaling [48]. H2B variants of Arabidopsis were also reported to undergo various kinds of PTMs [49]. An extensive collection of the phosphorylation sites of the histone variants are available in P3DB. DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. This protein has been reported to undergo phosphorylation in plants [8]. Cell division cycle protein 48 (CDC48) has been reported to be phosphorylated in Arabidopsis [8]. AtCDC48 the Arabidopsis homologue, is a soluble nuclear protein that has been reported to be highly expressed in dividing and expanding cells [50]. Recent reports have also suggested its role in regulation of viral replication and cell to cell movement in plants [51,52]. Several variants of ubiquitin have also been reported to undergo phosphorylation, the details of which are available in P3DB. Among these variants, the SWAP (Suppressor-of-


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Table 1 – Functional classification of identified putative phosphoproteins of chickpea nucleus. Identification

Score Spot no. Accession No of % (CaNP) no. peptides coverage

DNA replication, repair and modification Cell division cycle protein 48# d DNA-directed RNA polymerase subunit B# c RNA polymerase II second Largest subunit# c DNA topoisomerase II# c PolyA-binding protein# c Glyceraldehyde-3-phosphate dehydrogenase (NADP) (phosphorylating)# c At5g59910# c Histone H3.2# c Histone H4⁎ b Histone H4.3*b Histone 2B# b Histone H2B.4# b Histone H3# b Histone H4* a Histone H4.3*a Histone H4.3*a Histone H4.3*a Histone H4*a Dehyde-3-phosphate dehydrogenase, type I*a Signaling and gene regulation Methyl-accepting chemotaxis protein# d Transketolase# d Splicing factor, putative# c Transketolase, C-terminal-like# c Transketolase, C-terminal-like*a Transketolase, C-terminal-like*a Fructose-bisphosphate aldolase, cytoplasmic isozyme# c Aminomethyltransferase#c Cytosolic 3-phosphoglycerate kinase# b 2'-hydroxyisoflavone reductase# b Phosphoglycerate kinase# b Putative U2 snRNP protein A# b Probable H + -transporting two-sector ATPase# b Transketolase# b Nucleoside diphosphate kinase⁎ a Fructose-bisphosphate aldolase*a Fructose-bisphosphate aldolase*a Fructose-bisphosphate aldolase*a Phosphoribulokinase*a Putative U2 snRNP protein A*a ATP synthase CF1 alpha subunit [Pisum sativum]*a H + -transporting two-sector ATPase*a Fructose-bisphosphate aldolase* a Putative RH2 protein*a Protein folding HSP 90-2# d predicted protein# d Grp94# d Heat shock cognate protein# d Heat shock protein 90# d Heat shock protein 90# b Predicted protein# d Hsp Cognate 80# d Hsp 81-2# d predicted protein# d ATPase with chaperone activity# d Putative HEAT SHOCK PROTEIN 81-2# Heat shock protein 90# d Chaperone protein ClpC1# c

d

Exp MW/pI

Nuc Localization

119 166 148 183 60 57

1h 1a 1b 5c 7b 7c

38455496 75294607 122179705 122244501 122231411 120658

4 5 5 5 1 1

9 10 3 6 4 3

67530/4.89 45066/8.98 135635/7.17 82558/6.94 34089/9.29 43312/8.80

Y Y Y Y Y N

39 137 211 203 152 126 77 146 225 298 153 79 260

10d 11a 1a 1b 2a 2b 2c 1 2 4 5 6 22

122237549 73919914 75301100 51315744 563329 75102839 119690018 31433309 51315744 51315744 51315744 75301100 122192499

1 3 4 4 5 4 3 10 4 9 9 2 21

6 20 61 39 29 33 18 49 51 51 51 30 23

16439/10.00 15237/11.15 7425/10.34 11413/11.66 16606/ 10.02 14807/ 10.08 15237/ 11.15 11602/11.3 11413/11.66 11413/11.66 11413/11.66 7425/10.34 48422/6.76

Y Y Y Y Y Y Y Y Y Y Y Y N

52 91 165 496 738 187 114

3 7 2c 5a 23 24 7a

116328160 4586600 19698893 122244282 122244282 122244282 3021338

2 17 3 10 31 15 3

1 2 3 13 24 15 9

106467/5.79 17124 /5.84 87481/ 5.08 79688/6.00 80087/6.00 80087/6.00 38428/6.38

Y N Y Y Y Y N

55 99 73 193 84 75 227 162 174 136 135 196 156 220 387 787 156

7d 3 5 8 7 11 13 3 8 9 10 11b 11a 17 18 16 20b

3915699 75240376 1708425 122189781 75323449 75317803 122244282 75150707 399024 399024 1168408 75319128 75323449 295137014 231586 399024 122201844

1 2 4 3 2 4 6 5 16 4 10 7 5 18 24 23 12

2 9 12 8 4 7 8 17 23 12 24 24 20 23 25

44262/8.79 31606/5.01 35386/ 5.94 50015/ 6.64 31980/5.06 60113/6.63 79688/6.00 16331/6.30 38747/ 5.83 38747/5.83 38707/6.38 39230/5.14 31980/5.06 54649/ 5.75 60335/5.95 38747 /6.38 46202/5.98

Y N Y N Y N Y N N N Y N Y Y N Y Y

735 321 284 120 118 735 690 619 538 155 58 319

2b 1a 1b 1g 1i 2a 2c 2d 2e 4b 4d 5a 5b 4

208964722 224056837 23477636 66828255 192293658 159459822 224056837 547683 84468292 226454726 124514649 84468292 159459822 347602486

15 6 5 2 2 16 16 12 10 5 2 20 5 15

18 8 6 4

80107/4.98 93980/4.85 92900/4.93 79816/5.02 69395/4.96 80152/4.98 79976/4.95 80086/4.96 55394/5.16 91854/5.20 91749/6.02 55394/ 5.16 80152/4.98 98436/5.79

Y Y Y Y Y Y Y Y Y Y Y Y Y Y

523

21

24 20 19 21 9 3 8 9 18


9


Table 1 (continued) Identification

Score Spot no. Accession No of % (CaNP) no. peptides coverage

Protein folding Heat shock protein Hsp70# c Heat shock protein hsp70# c dnaK-type molecular chaperone# c Heat shock cognate protein# c Putative CCT chaperonin gamma subunit# b Heat shock protein Hsp70*a Heat shock protein Hsp70*a Heat shock protein Hsp70*a Heat shock protein Hsp70*a dnaK-type molecular chaperone CSS1 precurso*a Hsp70# b Translation Elongation factor 2-like Isoform 1# d Translation factor; Elongation factor G, III and V# Ribosomal protein subunit 2# c Ribosomal protein S11# c Putative ribosomal protein S14# c Ribosomal protein S11# c Ribosomal protein L10; Ribosomal protein 60S# b Translation elongation factor 1A-9# b Ribosomal protein L10; Ribosomal protein 60S*a Translation initiation factor*a Metabolism Protochlorophyllide reductase# c Pathogenesis-related protein 10# c ATP synthase subunit B# b ATP synthase beta subunit*a Sucrose Synthase# d Sucrose synthase# d 2-isopropylmalate synthase# d Phosphoenolpyruvate carboxylase# c Enoyl-[acyl-carrier-protein] reductase (NADH2)# beta-Amylase*a Phosphopyruvate hydratase*a Isocitrate dehydrogenase*a

c

b

Structure Transitional endoplasmic reticulum ATPase# d Histone H3# c Actin/actin-like# b Uncharacterised SEQUENCE 1 FROM PATENT EP0723017*a Unknown [Medicago truncatula]# d Unnamed protein product# c Unnamed protein product# b Arabidopsis thaliana genomic DNA, chromosome 3, P1 clone# b Os02g0519900 [Oryza sativa (japonica cultivar-group)]# Unnamed protein product [Glycine max]⁎ a Protein degradation ClpC protease# d ATP-dependent Clp protease ATP-binding subunit clpA homolog CD4B# d SWAP Ubiquitin# c Ubiquitin# c Ubiquitin monomer protein# c Tetra-ubiquitin# c

d

Exp MW/pI

Nuc Localization

263 243 175 163 55 121 99 117 88 471 319

5b 6a 6b 6c 12 25 7 26 27 28 16

122231878 75285584 229464991 75291720 75321952 122199230 75278390 75285681 75285681 399942 122231878

7 6 5 4 3 15 5 18 15 30 10

10 9 7 7 4 19 38 25 20 26 13

75710/5.36 70821/5.11 70559/ 5.22 71202/5.04 60860/ 6.23 71445/5.11 15216/5.36 71351/5.08 71351/5.08 75583/ 5.22 75710/5.19

Y Y Y Y Y Y N Y Y Y Y

251 612 99 140 54 81 56 139 76 277

1c 2a 9a 10b 10c 11b 4 10 13A 20a

225462164 122237984 75314569 122225477 75290476 122225477 122192113 122245733 122192113 75318611

10 17 4 3 1 3 1 5 2 20

14 16 15 16 9 23 3 10 6 27

93910/5.80 110075/5.01 22682/9.58 16353/10.45 16353/10.45 16308/ 10.6 34386/ 5.24 49389/9.14 34386/ 5.24 46184/7.05

Y Y Y Y Y Y N N N Y

85 37 774 155 218 61 55 431 63 86 224 103

8a 10e 14 15 2g 4c 6 3 6 14 19 21

266742 118933 122225319 122238761 267057 267057 217976869 13507108 75219026 3913034 1169534 2497259

2 1 23 12 5 2 2 16 2 9 13 15

5 7 30 25 7 2 2 20 4 16 22 23

43091/ 9.2 16737/ 4.94 51755/5.27 49131/ 5.33 92035/6.07 92035/ 6.07 56615/ 5.66 110640/ 5.76 41754/ 8.88 56544/4.89 48111/5.56 48695/ 6.15

Y N N N Y Y N Y N N N N

182 190 277

1d 10a 9

11265361 73919914 122190148

5 4 8

8 20 17

93550/5.37 15237/ 11.15 42286/ 5.39

Y Y Y

167 168 141 48 60

13b 1e 5d 15 17

2852018 217075889 2852018 75311590 75311590

4 4 4 2 2

7 20 6 2 2

80313/ 6.58 25995/6.33 80231/6.58 74335/ 8.3 74335/8.30

Y Y Y Y Y

162 203

1f 12

115446385 28802897

7 11

8 23

93961/5.85 42951/6.11

Y Y

376 233

2f 4a

4105131 399213

8 8

12 12

99417/8.78 102178/5.86

Y Y

345 71 68 65

2b 9b 9c 9d

122188445 159484538 122209936 75267958

8 2 2 2

10 28 14 7

89007/ 5.09 8536/6.56 17021/ 6.75 34202/6.17

Y Y Y Y

(continued on next page)


10


Table 1 (continued) Identification RNA metabolism Fibrillarin# c

Score Spot no. Accession No of % (CaNP) no. peptides coverage 73

8b

357444829

3

7

Exp MW/pI

Nuc Localization

32927/ 10.8

Y

# – ESI-QUAD-TOF, * – MALDI-TOF-TOF. a – 2D HSB, b-2D PDG, c – IMAC HSB, d – IMAC PDG.

White-APricot) ubiquitin is a RNA-binding nuclear resident, which is reported to undergo phosphorylation on a serine residue [7]. Other proteins in this class comprised of protein degradation related proteins including ClpC protease and clpA homolog CD4B. The Clp proteases represent a large, ancient ATP-dependent protease family [53]. In plants, Clp protease is one of the newly identified proteolytic systems that incorporate the activity of molecular chaperones to target specific polypeptide substrates and avoid inadvertent degradation of others [54].

We identified several metabolism related proteins which include ATP synthase beta subunit, phosphoenolpyruvate carboxylase, beta-amylase and enoyl-[acyl-carrier-protein] reductase (NADH2). Another protein identified in this category was sucrose synthase whose phosphorylation sites are available in P3DB. This protein is known to possess UDP-glycosyltransferase activity and is involved in several biosynthetic processes such as callose deposition in phloem sieve plate, galactolipid biosynthetic process and sucrose biosynthetic process, among others.

Fig. 4 – Enrichment of nuclear phosphoproteins from chickpea using IMAC. Enriched nuclear proteins (50 μg), both for HSB (A) and PDG (B) methods, were resolved onto 12.5% SDS-PAGE and stained with Pro-Q Diamond staining. Protein bands, as indicated, were excised from the gel and subjected to MS/MS analysis. M, molecular mass marker; NE, nuclear extract; FT, flow-through; E, eluted fraction. Please cite this article as: Kumar R, et al, Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.002


3.6. Comparative analysis of the HSB and PDG methods The identified putative phosphoproteins, in the present study, can be discussed under three broad terminologies: method of isolation (HSB vs PDG), method of protein separation (1-DE vs 2-DE) and phosphoprotein enrichment (IMAC vs No-IMAC fraction) (Fig. 6). Although the number of nonredundant phosphoproteins identified in HSB (52) and PDG (40) methods were comparable (Fig. 6C), there were large amount of divergence in the proteins in each of these methods. Whereas 46 proteins were unique to the HSB method, 34 were unique to the PDG method and 6 proteins were common to both the methods (Supplementary Table S3). It is likely that differences in the methods of nuclei isolation bring about significant differences in the proteins identified suggesting that such combinatorial approaches will in future help in saturating the proteomes.

11

for all the identified phosphopeptides are available online (Supplementary Fig. S2).

3.9. Protein-kinase interaction network To investigate the interaction of the identified putative phosphoproteins with kinases, a protein-kinase interactome was

3.7. Comparison of Arabidopsis and chickpea nuclear phosphoproteomes A comparative analysis of the chickpea nuclear phosphoproteome data was carried out with that of Arabidopsis [14]. A considerable amount of diversity was observed in the two phosphoproteomes, both in terms of the functional categories as well as the proteins contained therein (Fig. 6D). In contrast to this study, 146 phosphoproteins were reported in Arabidopsis. The most dominant class in this study comprised proteins associated with folding but in Arabidopsis the most abundant class belonged to proteins associated with transcriptional regulation. A careful examination revealed that only five proteins were common to both the species (Fig. 6C) which included Hsp70, Hsp81-2, splicing factor-related, actin-related protein and CDC48. The high percent of uniqueness in the proteome datasets is indicative of the representative unsaturated proteomes. A plausible explanation would be that ecological niche of the plant species influences the protein expression pattern which might contribute to the differences in the phosphoproteome. However, it may also be reflective of the diverse nature of tissue (protoplast cell culture in Arabidopsis and aerial parts of chickpea) and species-specific nuclear proteins.

3.8. Validation of the nuclear phosphoproteome To validate the nuclear phosphoproteome data, nuclear proteins were treated with alkaline phosphatase and then stained with Pro-Q Diamond or Sypro Ruby dye. The staining intensities of the spots by the Pro-Q Diamond were greatly altered on the phosphatase treated gel (Fig. 7). Many protein spots were weakly stained and some were not stained at all. The alteration/s in spot intensity and number indicate the specificity of the staining for phosphorylated proteins. The validation of the nuclear phosphoproteome was performed by mapping the phosphorylation sites of few of the nuclear proteins (Table 2). The list includes DEAD box RNA helicase, heat shock protein 70 and a putative chaperonin 60 alpha subunit. These proteins have earlier been reported to undergo phosphorylation in Arabidopsis and are available in the P3DB plant phosphoproteome database. The spectra

Fig. 5 – Functional classification of the identified nuclear phosphoproteins. The non-redundant set of proteins was catalogued according to their gene ontology using Blast2go program for predicted biological (A) and molecular function (B). Functional classification of the identified phosphoproteins according to Pfam or Interpro database (C).


12


Fig. 6 – Comparative analysis of chickpea nuclear phosphoproteins. Number of identified phosphoproteins is indicated for HSB and PDG methods (A), Venn diagram depicting overlaps of nuclear phosphoproteins between IMAC and Non-IMAC methods (B). The overlaps of phosphoproteins between HSB and PDG isolation strategies (C) and distribution of phosphoproteins proteins between the chickpea and Arabidopsis (D). The areas in the diagram are not proportional to the number of phosphoproteins in each groups.

constructed. Kinase specific phosphorylations were predicted using NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) and the relative abundance was observed to be pS > pT. Proteins with or above the set threshold value were then used to query the existing human phosphoproteome database to investigate the conservation of phosphorylation site/s (Supplementary Table S4). Input proteins for which there was at least one phosphorylation site on the corresponding human protein sequence were considered for further analysis (Fig. 8). There were 604 interactors on the predicted map of which 37 were the input proteins. Several of these interactions were

previously reported thus validating the overall quality of the interactome. Hsp70 was found to associate strongly with a large number protein of the casein kinase I family (CSNK1E, CSNK1A1 and CSNK1D). The roles of these kinases have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. Hsp70 was also found to interact with a serine/threonine kinase (STK PIM-1). Overexpression of Hsp70 mediates proteasomal degradation of STK4 [55]. Other variants of this kinase such as STK24, STK-PAK-1 and STK4 were also found on the interactome. STK4 encodes a cytoplasmic kinase that shows structural similarity with yeast Ste20p

Fig. 7 – Nuclear phosphoprotein profile obtained with Pro-Q Diamond before and after lambda alkaline phosphatase treatment. The images show the protein spots, which were phosphorylated (A) or dephosphorylated (B). Weakly-stained protein spots are indicated by dotted circles, while spots which were not stained are indicated by solid circles. Please cite this article as: Kumar R, et al, Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.002

13


Table 2 – Phosphopeptides identified from nuclear enriched fraction of chickpea. Description DEAD box RNA helicase DEAD box RNA helicase Heat shock protein hsp70 Putative chaperonin 60 alpha subunit

Accession number 25809054 25809054 224970 29367489

Phosphopeptide

Site of phosphorylation

Peptide ion score

VHACVGG[pT]SVR VHACVGG[pT]SVREDQR M[pY]WGEGAGMGAAAGMDEDAPSGGSGAGPK LAAAVAV[pT]LGPRGR

T8 T8 Y2 T8

56 36 27 18

kinase, which acts upstream of the MAPK cascade. This protein is capable of phosphorylating myelin basic protein and is also capable of autophosphorylation. A caspase-cleaved fragment of the encoded protein has been shown to be capable of phosphorylating histone H2B and has been correlated with apoptosis [56]. Further, STK4 (=MST1) and STK3 (=MST2) were demonstrated to interact and phosphorylate Hsp under stress.

In this interactome, STK24 was predicted to interact with histone H3.2, whereas STK2 and STK-PAK-1 were predicted to interact with the structural protein actin. Several MAPKs were found to interact with TOP2B. This protein displayed interaction with a large number of proteins such as CCT gamma chaperonin, glucose-regulated protein 94 (GRP94) and members of PRKC family. GRP94 is a Hsp90-like protein with essential

Fig. 8 – Interaction network of putative nuclear phosphoproteins of chickpea. Larger nodes with dark colour represent the higher number of interactors, while smaller nodes with bright colour represent the lower number of interactors. Edge colour and thickness is correlated with the degree of interactions. Dark and thick edges indicate strong interactions while bright colour and thin edges represent weak interactions. Please cite this article as: Kumar R, et al, Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.002

14


functions in the development and physiology of multicellular organisms [57]. Hsp90 is an important molecular chaperone which facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery [58]. Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function [58]. Phosphopeptide databases suggest that CK2 alone may be responsible for the generation of a large proportion (10–20%) of eukaryotic phosphoproteome [59]. ATP synthase beta-subunit has been previously found to be phosphorylated at CK2 phosphorylation sites [59]. This kinase was reported to phosphorylate and repress the activity of E47, a basic helix-loop-helix transcription factor known to be involved in the regulation of tissue-specific gene expression and cell differentiation [60]. Glycolytic enzymes viz, PGK, GAPDH and aldolase comprised a large hub in the network. The secondary roles of these glycolytic enzymes may be related to the fact that each contains a nucleotide binding site and may therefore bind to DNA or RNA in the nucleus.

4. Conclusions Protein phosphorylation, one of the most frequently occurring and best characterized PTMs, highly modulate protein activity and cellular localization. There is hardly any cellular process in plants which is not regulated in one or multiple ways by reversible phosphorylation. Therefore, identifying and characterizing protein phosphorylation events are the key to understanding cell signalling networks. To identify the nuclear phosphoproteins of chickpea, we investigated non-IMAC as well as the potential of IMAC in combination with tandem mass spectrometry. The functional categorization of the identified putative phosphoproteins showed a high representation of proteins with a role in signal transduction and gene regulation. Although the nuclei were of high purity, a small number of proteins were identified as possible contaminants, which included cytoplasmic proteins, such as GAPDH, fructose-biphosphate aldolase and sucrose synthase and plastidic proteins such as protochlorophyllide reductase and ClpC protease. Until recently, large-scale plant proteomics investigations have been restricted to few model species for which the genome sequence had been fully determined. However, identifying the proteins in non-model plant species even with strong agro-industrial interests remains unexplored, and is still considered challenging [61]. While the draft sequence of chickpea genome has recently been published [25,62], functional annotation is yet to be completed. With the set of chickpea nuclear phosphoproteins identified in this work, we have made two major achievements: firstly this resource will facilitate signaling research on the proteins identified and secondly the data provides biological insight into regulation patterns of protein phosphorylation in plants. Furthermore, the data provide evidence for phosphorylation events that had previously been predicted by genome annotation and constitute valuable tools for comparative studies of protein expression. Our future efforts will focus on unearthing the

dynamics associated with the nucleus-specific phosphorylation towards cell metabolic and signalling pathways under different physiological conditions. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2014.04.002.

Competing interests The authors declare that they have no competing interests.

Acknowledgement This work was supported by a grant [BT/PR/10677/PBD16/795] from the Department of Biotechnology (DBT), Govt. of India. The authors thank DBT and Council of Scientific and Industrial Research (CSIR) for providing research fellowship to RK, PB and AK, PS, respectively. The authors gratefully acknowledge International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India for providing chickpea seeds. Mr. Jasbeer Singh is thanked for manuscript layout and format, and the artwork.

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Global transcriptome analysis of developing chickpea (Cicer arietinum L.) seeds.

Determination of Photoperiod-Sensitive Phase in Chickpea (Cicer arietinum L.).

Dissecting the Root Nodule Transcriptome of Chickpea (Cicer arietinum L.).

Genetic transformation in the grain legume Cicer arietinum L. (chickpea).

Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.).

Molecular Mapping of Flowering Time Major Genes and QTLs in Chickpea (Cicer arietinum L.).

Cold stress alters transcription in meiotic anthers of cold tolerant chickpea (Cicer arietinum L.).

Synergistic effect of Pseudomonas putida and Bacillus amyloliquefaciens ameliorates drought stress in chickpea (Cicer arietinum L.).

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.).

Approaches for enhancement of N₂ fixation efficiency of chickpea (Cicer arietinum L.) under limiting nitrogen conditions.

An advanced draft genome assembly of a desi type chickpea (Cicer arietinum L.).

Colloidal Nanomolybdenum Influence upon the Antioxidative Reaction of Chickpea Plants (Cicer arietinum L.).

Immunohistochemical localization of β-glucosidases in lignin and isoflavone metabolism in Cicer arietinum L. seedlings.

Early generation yield testing versus visual selection in chickpea (Cicer arietinum L.).

Comprehensive transcriptome assembly of Chickpea (Cicer arietinum L.) using sanger and next generation sequencing platforms: development and applications.

Quality of Low Fat Chicken Nuggets: Effect of Sodium Chloride Replacement and Added Chickpea (Cicer arietinum L.) Hull Flour.

Diurnal patterns of canopy photosynthesis, evapotranspiration and water use efficiency in chickpea (Cicer arietinum L.) under field conditions.

The CarERF genes in chickpea (Cicer arietinum L.) and the identification of CarERF116 as abiotic stress responsive transcription factor.

Trichoderma inoculation augments grain amino acids and mineral nutrients by modulating arsenic speciation and accumulation in chickpea (Cicer arietinum L.).

Response of chickpea (Cicer arietinum L.) to terminal drought: leaf stomatal conductance, pod abscisic acid concentration, and seed set.

Isoflavones extracted from chickpea Cicer arietinum L. sprouts induce mitochondria-dependent apoptosis in human breast cancer cells.

Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.).

Quantitative inheritance and barriers to crossability in Cicer arietinum L.

Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea).