The Nuclear Proteome of Chickpea (Cicer arietinum L.) Reveals

The Nuclear Proteome of Chickpea (Cicer arietinum L.) Reveals Predicted and Unexpected Proteins Aarti Pandey, Mani Kant Choudhary, Deepti Bhushan, Arnab Chattopadhyay, Subhra Chakraborty, Asis Datta, and Niranjan Chakraborty* National Centre for Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India Received April 6, 2006

Nuclear proteins constitute a highly organized, complex network that plays diverse roles during cellular development and other physiological processes. The yeast nuclear proteome corresponds to about one-fourth of the total cellular proteins, suggesting the involvement of the nucleus in a number of diverse functions. In an attempt to understand the complexity of plant nuclear proteins, we have developed a proteome reference map of a legume, chickpea, using two-dimensional gel electrophoresis (2-DE). Approximately, 600 protein spots were detected, and LC-ESI-MS/MS analyses led to the identification of 150 proteins that have been implicated in a variety of cellular functions. The largest percentage of the identified proteins was involved in signaling and gene regulation (36%), while 17% were involved in DNA replication and transcription. The chickpea nuclear proteome indicates the presence of few new nuclear proteins of unknown functions vis-a` -vis many known resident proteins. To the best of our knowledge, this is the first report of a nuclear proteome of an unsequenced genome. Keywords: chickpea • nuclear proteins • proteome • 2-DE • mass spectrometry

Introduction The identification of predicted gene products at the protein level bridges the gap between genome sequencing data and protein function and is referred to as “functional genomics”. The basic problem of complexity poses a significant challenge in studies to unravel the protein complement of the genome, the proteome. Many of the estimated genes in a genome are expected to provide multiple protein products that might arise as a result of alternate splicing and post-translational modification. Proteome analysis has become an indispensable source of information about protein expression, splice variants, and erroneous or incomplete prediction of gene structures in databases. However, the application of the proteomic approach at the whole cell level is limited by several factors such as protein abundance, size, hydrophobicity, and other electrophoretic properties. One approach to the daunting prospect of cataloging entire proteomes has been to focus on protein subsets. Organelles, subcellular compartments in a dynamic intracellular membrane system, provide such subsets because they can be subfractionated.1 Furthermore, the proteomes of organelles comprise a focused set of proteins that fulfills discrete but varied cellular functions. The analyses of cell organelle proteomes provide additional important information about protein localization and pathway compartmentalization.2 The nucleus is the subcellular organelle that contains nearly all the genetic information required for the regulated expression * To whom correspondence should be addressed. Dr. Niranjan Chakraborty, National Centre for Plant Genome Research, Aruna Asaf Ali Marg, New Delhi110067, India. E-mail, [email protected]; tel., 00-91-11-26735178; fax, 00-91-11-26716658. 10.1021/pr060147a CCC: $33.50

 2006 American Chemical Society

of cellular proteins. Nuclear proteins play key roles in the fundamental regulation of genome instability, the phases of organ development, and physiological responsiveness through gene expression. Proteins involved in different cellular functions, for example, signaling, gene regulation, structure, translation, proteolysis, and, among others, a variety of RNAassociated functions have been identified in the nucleus. Increasing evidence suggest that nearly one-fourth of total cellular proteins are localized in the eukaryotic nucleus, implying a variety of proteins function in this compartment.3 Some plant components of intranuclear compartments were reported to differ greatly from those of other organisms. Only a few plant nuclear matrix proteins have been characterized, and they have no obvious homology with known nuclear proteins in yeast and mammals.4,5 Whereas the subcellular proteome research is quite advanced in animals, yeast, and Escherichia coli,6 there is relatively less information on plant subcellular proteomes. Most efforts on nuclear proteomes are restricted to yeast and mammals, which include nuclear matrix proteins in various human cell types,7-9 nuclear envelope proteins from mouse neuroblastoma N2a cells,10 human nucleolar proteins,11 and total nuclear proteins from human liver.12 Proteomic analyses of nucleus for two model plants, namely, Arabidopsis thaliana13 and rice14 have recently been published. However, there has been no report for other crop plants. Legumes are valuable agricultural and commercial crops that serve as important nutrient sources for human diet and animal feed. About one-third of human nutrition comes from legumes, and in many developing countries, legumes serve as the only source of protein. They Journal of Proteome Research 2006, 5, 3301-3311

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research articles are essentially characterized by symbiotic relationships with both nitrogen-fixing bacteria and arbuscular micorrhizal fungi.15 Many secondary metabolites in legumes have been implicated with defense and are of particular interest as novel pharmaceuticals.16 In this study, we have developed the nuclear proteome map for a legume crop, chickpea, as a basis for future proteome comparisons of genetic mutants and pathogeninfected and/or environmentally challenged plants. Approximately, 600 proteins were resolved using two-dimensional gel electrophoresis (2-DE), of which 150 proteins were identified and classified into different functional categories. We examined the global state of protein expression in the chickpea nucleus to identify the potential candidates involved in the complex regulatory networks that may function in this organelle. Established nuclear proteome and putative proteins identified from the present study will provide a foundation for future investigation of the expression and function of the nuclear proteins of chickpea and other legumes.

Experimental Section Plant Material. Chickpea (Cicer aritienum L.) seedlings were grown in pots containing a mixture of soil and soil rite (2:1, w/w) in an environmentally controlled growth room and maintained at 25 ( 2 °C and 50 ( 5% relative humidity under a 16 h photoperiod (270 µmol m-2 s-1 light intensity). The 3-week-old seedlings were sampled as experimental materials, harvested, and stored at -80 °C after quick-freezing in liquid nitrogen. Isolation of Pure Nuclei. The nuclei were prepared from chickpea seedlings as described17 with few modifications. About 20 g of the tissue was ground into powder in liquid nitrogen with 0.3% (w/w) polyvinylpolypyrrolidone (PVPP) and immediately transferred into an ice-cold 500 mL beaker containing 200 mL of ice-cold 1× HB (10 mM Trizma base, 80 mM KCl, 10 mM EDTA, 1 mM spermidine, 1 mM spermine, and 0.5 M sucrose, pH 9.5), 0.15% β-mercaptoethanol, and 0.5% Triton X-l00. The contents were gently stirred for 30 min for complete lyzing of organellar membranes. This suspension was filtered through four layers of cheesecloth and two layers of miracloth into an ice-cold 250 mL centrifuge bottle. The homogenate was pelleted by centrifugation with a fixedangle rotor at 1800g and 4 °C for 20 min. The supernatant fluid was discarded, and the pellet was gently resuspended in 30 mL of ice-cold wash buffer (1× HB without Triton X-100). To remove the particulate matter remaining in the suspension, the resuspended nuclei were filtered into a 50 mL centifuge tube through two layers of miracloth by gravity. The content was centrifuged at 57g and 4 °C for 2 min to remove intact cells and tissue residues. The supernatant fluid was transferred into a fresh centrifuge tube, and the nuclei were pelleted by centrifugation at 1800g and 4 °C for 15 min in a swinging bucket centrifuge. The pellet was washed two additional times by resuspension in wash buffer followed by centrifugation at 1800g and 4 °C for 15 min. Confocal Microscopy. The nuclear fraction was stained for 15 min with 0.1 µg/mL 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) in 0.1 M potassium phosphate buffer (pH 7.4) and then washed twice with phosphate buffer saline (PBS). For microscopy, a small volume of suspension was placed on a slide and covered with a coverglass. The images were taken with and without a UV filter. Chlorophyll Assay. The chlorophyll content in the starting homogenate, the supernatant, and the nuclei-enriched fraction 3302

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was determined using a spectrophotometric assay. The sample was prepared by pipetting 1 mL of suspension into a 15 mL centrifuge tube and adding 8 mL of acetone and 1 mL of MQ to it and centrifuging at 1000g for 5 min. The absorbance of this sample was measured at 652 nm. The assay was done in triplicate, and the amount of chlorophyll in 1 mL of the suspension was observed as milligram of chlorophyll per milliliter (mg chlorophyll/mL) ) absorbance/34.5 (http:// www.bio.com/protocolstools/protocol.html). The chlorophyll amount was then calculated as milligram per gram (mg/g) of fresh tissue weight. The purity of the nuclear fraction was evaluated on the basis of the difference in chlorophyll content in supernatant and the nuclear suspension. Nuclear Protein Extraction and Quantification. Nuclear proteins were prepared from the nuclei-enriched pellet using TriPure Reagent (Roche) according to the manufacturer’s instructions. The final protein pellet was resuspended in isoelectric focusing (IEF) sample buffer [8 M urea, 2 M thiourea, and 2% (w/v) CHAPS]. The protein concentration was determined using the 2-D Quant kit (Amersham Biosciences). Immunoblot Analysis. For immunoblotting, proteins were separated on 12.5% SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes. Membranes were incubated with mouse anti-fibrillarin and sheep anti-histone antibodies (Abcam Limited, U.K.). Antibody-bound proteins were detected by incubation with secondary antibodies (Abcam Limited, U.K.) conjugated to alkaline phosphatase. Enzyme Assay. The catalase enzyme assay was performed using 10 µg of organeller protein for each reaction. The reaction mixture was prepared by adding 50 µL of protein extract to 940 µL of 70 mM potassium phosphate buffer (pH 7.5). The reaction was started by addition of 10 µL of H2O2 (3% v/v), and the decrease in absorbance at 240 nm was followed for 5 min. Baseline correction was done by subtracting the absorbance taken without addition of H2O2. The assay was done in triplicate, and the absorbance values obtained were plotted against time (http://www.hos.ufl.edu/meteng/HansonWebpagecontents/Cellfractionation.html). 2-DE of Nuclear Proteins. Isoelectric focusing (IEF) was carried out with 150 µg of protein. Aliquots of proteins were diluted with 2-D rehydration buffer [8 M urea, 2 M thiourea, 2% (w/v) CHAPS, 20 mM DTT, 0.5% (v/v) Pharmalyte (either pH 3-10, 4-7, or 6-11), and 0.05% (w/v) bromophenol blue], and 250 µL of solution was used to rehydrate immobilized pH gradient strips (13 cm; pH 3-10 and 4-7). Protein was loaded by an in-gel rehydration method onto IEF strips, and electrofocusing was performed using the IPGphor system (Amersham Biosciences, Bucks, U.K.) at 20 °C for 30 000 Vh. However, for pH 6-11 strips, anodic cup-loading was performed with a load of 100 µg of protein in 100 µL of rehydration volume, and electrofocusing was performed for 70 000 Vh. The focused 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 onto 12.5% polyacrylamide gels for SDS-PAGE. The electrophoresed proteins were stained with silver stain plus kit (Bio-Rad, CA). Gel images were digitized with a Bio-Rad FluorS equipped with a 12-bit camera. The PD Quest version 7.2.0 (Bio-Rad, CA) was used to assemble first-level matchset (master image) from three replicate 2-DE gels. Protein Identification Using MS/MS. Protein spots were excised mechanically using pipet tips and in-gel-digested with

Nuclear Proteome of Chickpea

Figure 1. Analysis of isolated chickpea nuclear fraction and determination of its purity. (A) The purified nuclear fraction was stained with DAPI and visualized by confocal microscopy. Phase contrast micrograph of the nuclei is shown in left panel, while the DAPI-stained nuclei are shown in right panel. (B) Determination of chlorophyll content at different stages of purification of nuclear fraction. The amounts of chlorophyll present in tissue homogenate, supernatant, and nuclear fraction was estimated and compared.

trypsin (Sigma, St. Louis, MO). These were analyzed by electrospray ion trap time-of-flight mass spectrometry (LC/MS/ MS) (Q-Star Pulsar i, Applied Biosystems). The spectra were analyzed by the Mascot sequence matching software (www.matrixscience.com) and the Viridiplantae (green plants) database. The proteins which gave a no-significant match with Mascot were researched using NCBI (nr) against the Viridiplantae database.

Results and Discussion Isolation of Purified Nuclei. An important criterion for compartment-specific proteome is the purity of the compartment to be analyzed. Indeed, the integrity of a subcellular proteome is largely dependent on the purification of the isolated compartment away from other cellular contaminants. The separation of high-purity nuclei from plant is a difficult task as it might compromise the yield. In this study, the nuclei were isolated from chickpea seedling using hyperosmotic sucrose buffer, and the nuclei-enriched pellet so obtained was washed repeatedly separate contaminants from other organelles. The integrity of the isolated nuclei was analyzed by DAPI staining (Figure 1A). The chickpea nuclei were uniform spheres with an average diameter of approximately 20 µm. These results indicate that the isolated nuclei were highly purified. Possible chloroplast contamination in the nuclear fraction was examined by spectrophotometric analyses of chlorophyll. As shown in Figure 1B, the supernatant retained most of the chlorophyll content, and less than 3% chlorophyll was present in the nuclei pellet.

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Figure 2. (A) Western blot analysis of extracted nuclear proteins with anti-histone core and anti-fibrillarin antibodies. Aliquots of 100 µg of protein, each from nuclear (CaN), chloroplast (CaCh), and ECM (CaE) fractions of chickpea, as well as HeLa nuclear extract (HN), were separated by 12.5% SDS-PAGE. HeLa nuclear extract was used as positive control, whereas chloroplast and ECM fractions were used as negative controls. The 1-D gel was electroblotted onto Hybond-C membrane, and histones and fibrillarin were detected. The molecular weight (kDa) of the resident proteins is indicated by arrows. (B) Determination of catalase-specific activity in the nuclear and cytosolic fractions of chickpea. The cytosolic fraction for catalase activity was used as positive control.

The nuclear proteins were prepared from the purified nuclei using TriPure reagent (Roche) to remove the contaminating nucleic acids which might interfere during the IEF process. The enrichment of nuclear proteins was evaluated by immunoblot analysis using specific antibodies for two nuclear proteins, histone core and fibrillarin. The nuclear resident proteins histone and fibrillarin were detected in the nuclear fraction but not in the cytoplasmic or chloroplast fraction (Figure 2A). Contamination with non-nuclear proteins was monitored by measuring the activity of catalase enzyme as an indirect cytosolic marker in the nuclear fraction. While the cytosolic proteins showed high catalase activity, nuclear proteins did not show any significant catalase activity (Figure 2B). These results, altogether, suggest that the nuclear preparation had no appreciable level of chloroplast or other cytoplasmic contamination. Construction of 2-DE Map. Nuclear proteins were separated by 2D-PAGE to establish a reference map. The images were analyzed by the PD Quest software as described earlier.18 Computational analysis of the silver-stained gels reproducibly revealed 312 different spots in the pH range 3-10 (Figure 3). However, proteins in the basic pH range exhibited poor resolution. To make the reference map more comprehensive, we developed the proteome in the overlapping pH ranges 4-7 and 6-11 (Figure 4). Indeed, reproducibility of high-resolution 2-D patterns is an issue of concern for the basic pH range. Thus, sample cup-loading instead of in-gel rehydration was necessary. Consequently, 482 and 361 spots were detected at pH ranges of 4-7 and 6-11, respectively, that included the overlapping region. More than 600 exclusive spots were detected, out of which 572 spots survived the filtering process. The spots were numbered as CaN-1 to CaN-572, the letters identify the Journal of Proteome Research • Vol. 5, No. 12, 2006 3303

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Figure 3. Resolution of nuclear extracts proteins on 2-DE. Nuclear proteins (150 µg) were electrofocused on a pH 3-10 IPG strip (13-cm), separated onto 12.5% SDS-PAGE. The Silver-stained gel was visualized as described in Experimental Section.

Figure 4. 2-DE reference map of the chickpea nuclear proteins. Nuclear proteins were zoomed onto two overlapping pH ranges: (A) pH 4-7 with 150 µg of protein and (B) pH 6-11 with 100 µg of protein. The protein was loaded onto 13-cm IPG strips for IEF, and second dimension was performed on 12.5% (w/v) SDS-PAGE. Protein spots marked by arrows were identified by LC-ESI-MS/MS as detailed in Experimental Section.

organism (Cicer arietinum) and the subcellular organelle (Nucleus) from which the proteome map has been made, whereas the numerals indicate the spot numbers. A total of 160 spots with more than 30 quality score assigned by the software (based on the quality and quantity of the spots) were excised. Of these, 150 spots were identified with high confi3304


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dence (Table 1), and the corresponding protein spots were indicated on the gels (Figure 4). Functional Classification of Nuclear Proteins. To understand the function of the nuclear proteins, the identified proteins were sorted into various categories, as shown in Figure 5. Protein functions were assigned using a protein function database Pfam (http://www.sanger.ac.uk/software/Pfam/) or Inter-Pro (http://www.ebi.ac.uk/interpro/). However, the classification of proteins is only tentative, since the biological function of many proteins identified has not yet been established experimentally. It is known that the same protein may have different functions in different subcellular compartments and can act as “moonlighting proteins”, as is the case with the glycolytic pathway enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK).19 In a number of cases, the same protein was found in multiple spots in the same gel, suggesting possible alternate posttranslational modifications. Interestingly, the relative positions of a single protein on the 2-D gel indicated that the modifications affected the isoelectric point, the molecular weight, or both. About 11% of the identified proteins were grouped under the unknown category, since no information as to their potential function in the organelle was available. Other functional categories mentioned in Table 1 are explained below. The proteins involved in signaling and gene regulation (36%) were found to be the most abundant. Spots 1, 208, and 236 were identified to be an RNA-binding protein, FCA. It is a plantspecific nuclear protein that is involved in flowering time control as well as abscisic acid (ABA) signaling.20 Two isoforms of PGK, spots 180 and 183, were also found to be present in the chickpea nuclear proteome. PGK is known to function as a primer recognition protein involved in DNA synthesis and is known to possess a bipartite nuclear localization signal in the N-terminus.19 Aldolase (spots 119 and 184) was included in this category, as it was identified as a DNA-binding protein.21 Apparently, aldolase is also located in the perinuclear space and functions as a nuclear protein in plants.19 A few kinases were found to be part of the nuclear proteome of chickpea (spots 137, 148, and 450). A 14-3-3-like protein (spot 8) was also identified in the nuclear proteome. Although a majority of 14-3-3 molecules are present in the cytoplasm, it is reported that in the absence of bound ligands 14-3-3 homes to the nucleus.22 Spot 331 is an AP2/EREBP transcription factor, BABY BOOM, which functions mainly as a developmental regulator of cell/organ identity and fate.23 Quite a few other transcription factors were identified in the nuclear fraction. The other important protein identified is a RAN binding protein (spot 307, Ras-related protein in the nucleus), an intracellular signaling protein which acts as a major regulator of nucleocytoplasmic transport and shuttles between the nucleus and the cytoplasm.24 Interestingly, proteins involved in signaling and gene regulation dominated other categories, reflecting the role of the nucleus in gene expression and regulation. While in other organelles, such as chloroplast and mitochondria, the largest percentage of proteins was reported to be involved in energy production, either in electron transport or in ATP production.25 The second largest category comprised proteins involved in DNA replication and transcription. Glycine-rich RNA-binding proteins (GRPs) are the predominant proteins in this category. These proteins contain two distinct domains: an aminoterminal RNA-binding domain and a Gly-rich carboxy-terminal domain. These proteins have already been reported in the Arabidopsis nucleolar proteome.26 GAPDH was also identified

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Nuclear Proteome of Chickpea Table 1. List of MS/MS Identified Chickpea Nuclear Proteins and Functional Classification functional category

DNA Replication and Transcription

Metabolism

identification

Putative glycine-rich RNA-binding protein 2 Putative glycine-rich RNA-binding protein 2 Putative glycine-rich RNA binding protein 1 Putative glycine-rich RNA-binding protein 2 Putative glycine-rich RNA-binding protein 1 Putative glycine-rich RNA-binding protein 2 Putative glycine-rich RNA-binding protein 2 glyceraldehyde-3-phosphate dehydrogenase (NADP) (phosphorylating) (EC 1.2.1.13) B precursor Putative glycine-rich RNA-binding protein 2 glyceraldehyde-3-phosphate dehydrogenase (NADP) (phosphorylating) (EC 1.2.1.13) A precursor glyceraldehyde-3-phosphate dehydrogenase (NADP) (phosphorylating) (EC 1.2.1.13) A precursor Aspartate carbamoyltransferase glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (EC 1.2.1.12) Putative glycine-rich RNA-binding protein 2 glyceraldehyde-3-phosphate dehydrogenase (NADP) (phosphorylating) (EC 1.2.1.13) A precursor Putative glycine-rich RNA binding protein 1 Putative glycine-rich RNA-binding protein 2 Aspartate carbamoyltransferase Putative glycine-rich RNA binding protein 1 Putative glycine-rich RNA-binding protein 2 glyceraldehyde-3-phosphate dehydrogenase (NADP) (phosphorylating) (EC 1.2.1.13) B precursor Putative glycine-rich RNA-binding protein 2 Putative glycine-rich RNA-binding protein 2 S19 self-incompatibility ribonucleasec Aspartate carbamoyltransferase Aspartate carbamoyltransferase beta-1,3-glucanasec Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (Fragment) Ribulose-1,5-bisphosphate carboxylase large subunit (Fragment)

score

spot no.a

gi no.b

no. of peptides

% coverage

theoretical MW/pI (kDa)

experimental MW/pI (kDa)

73

CaN-16

6911146

2

15%

16.25/7.82

28.58/4.80

46

CaN-25

6911146

1

9%

16.25/7.82

38.39/4.72

48

CaN-63

6911142

1

10%

14.15/8.71

31.41/5.25

52

CaN-67

6911146

2

14%

16.25/7.82

43.08/5.23

60

CaN-107

6911142

1

10%

14.15/8.71

26.18/5.47

52

CaN-111

6911146

1

9%

16.25/7.82

37.30/5.32

62

CaN-166

6911146

2

15%

16.25/7.82

28.85/5.64

548

CaN-269

309671

11

24%

48.06/7.57

98.51/5.91

79

CaN-277

6911146

3

22%

16.25/7.82

27.41/6.33

51

CaN-358

12159

1

2%

43.31/8.80

49.80/6.45

211

CaN-363

12159

4

10%

43.31/8.80

49.49/6.60

47 282

CaN-417 CaN-366

15796550 169091

1 6

2% 16%

42.57/6.06 36.59/6.55

39.48/7.36 46.39/6.69

74

CaN-379

6911146

2

15%

16.25/7.82

70.76/6.60

303

CaN-398

12159

6

14%

43.31/8.80

46.39/6.85

55

CaN-433

6911142

2

10%

14.15/8.71

14.40/7.25

51

CaN-487

6911146

1

9%

16.25/7.82

29.81/9.11

44 61

CaN-177 CaN-464

15796550 6911142

1 1

2% 10%

42.57/6.06 14.15/8.71

41.60/5.68 18.70/8.81

56

CaN-542

6911146

1

9%

16.25/7.82

15.49/9.97

167

CaN-297

309671

3

6%

48.06/7.57

55.03/6.15

56

CaN-442

6911146

1

9%

16.25/7.82

54.38/7.60

64

CaN-212

6911146

2

15%

16.25/7.82

75.11/5.72

87

CaN-170

59896629

1

17%

21.73/8.65

31.00/5.60

48 41 93 234

CaN-276 CaN-287 CaN-139 CaN-260

15796550 15796550 6714534 37361623

2 2 1 5

3% 3% 6% 8%

42.57/6.06 42.57/6.06 52.58/9.51 46.53/6.30

29.95/6.22 42.20/6.15 68.40/5.50 71.54/5.83

155

CaN-303

24634966

4

7%

51.74/6.14

45.54/6.34

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Table 1 (Continued) functional category

Metabolism

Miscellaneous

Protein Degradation

Protein Folding

Signaling and Gene Regulation

score

spot no.a

gi no.b

Ribulose 1,5-bisphosphate carboxylase small subunit precursor (EC 4.1.1.39) Ribulose-1,5-bisphosphate carboxylase large subunit (Fragment) Methionine synthase beta-1,3-glucanasec probable alanine-glyoxylate transaminase (EC 2.6.1.44) [imported] Glycolate oxidase like protein (Fragment) Glycolate oxidase like protein (Fragment) Ribulose 1,5-bisphosphate carboxylase small subunit precursor (EC 4.1.1.39) aminotransferase-like proteinc oxygen-evolving complex protein 1 precursor ATP synthase beta chain (Fragment) Chlorophyll a/b binding protein precursor Ribulose-1,5-bisphosphate carboxylase/oxygenase activase 2 (Fragment) Carbonic anhydrase (EC 4.2.1.1) Carbonic anhydrase (EC 4.2.1.1) Formate dehydrogenase Ferredoxin-binding subunit protochlorophyllide reductase (EC 1.3.1.33) precursor putative phytochelatin synthetasec L-ascorbate peroxidase AtpB (Fragment) peptidyl-prolyl cis-trans isomerasec

150

CaN-339

3928152

4

16%

20.38/9.03

15.00/6.60

125

CaN-370

46326394

4

8%

43.78/6.14

62.94/6.70

136 94 207

CaN-388 CaN-473 CaN-476

71000469 6714534 17044259

3 1 5

3% 6% 9%

87.75/6.05 52.58/4.73 44.18/7.69

98.50/6.49 45.00/8.66 55.30/8.50

417

CaN-503

16604394

7

22%

40.28/8.99

49.31/9.30

172

CaN-541

16604394

3

10%

40.28/8.99

15.10/9.87

109

CaN-403

3928152

3

12%

20.38/9.03

12.00/6.50

68 116

CaN-4 CaN-66

53791601 344004

1 3

6% 8%

33.00/6.20 34.87/6.25

37.37/4.35 37.33/5.20

233

CaN-81

69214424

5

9%

53.36/5.23

67.06/5.25

249

CaN-61

3928140

4

24%

28.30/5.47

30.95/5.12

67

CaN-36

12620883

2

3%

48.32/5.06

59.75/5.10

102 37 84 62 170

CaN-346 CaN-343 CaN-424 CaN-483 CaN-498

20502881 20502881 38636526 167085 81946

2 2 4 1 4

5% 5% 4% 3% 9%

35.28/6.96 35.28/6.96 40.56/6.54 21.92/9.81 43.09/9.20

30.63/6.75 30.70/6.52 51.23/7.16 24.00/9.00 40.18/9.20

154 123 47 91

CaN-557 CaN-231 CaN-105 CaN-21

54287588 71534930 6110504 51849616

1 2 3 1

9% 16% 88% 6%

51.18/8.70 13.16/9.44 10.72/12.00 41.37/5.18

28.00/10.02 33.63/5.80 29.33/5.38 31.20/4.87

440 90 44

CaN-84 CaN-229 CaN-305

52075838 38345008 21593470

8 1 2

11% 8% 4%

72.49/5.54 37.29/4.99 53.17/6.02

83.12/5.19 29.86/6.00 46.78/6.39

108 433

CaN-38 CaN-44

3790441 169023

3 9

5% 13%

61.40/5.23 75.47/5.22

78.24/4.75 93.45/4.77

213 276 91 247 50


806808 806808 21592895 71370259 32482057

5 6 2 4 1

9% 10% 7% 16% 5%

62.94/5.85 62.94/5.85 30.60/6.93 31.84/9.49 29.37/8.63

81.07/5.27 81.15/5.36 33.31/8.80 36.50/9.70 31.0/4.0

78

CaN-5

37993504

2

6%

37.84/5.05

30.32/4.66

173 73 45

CaN-8 CaN-31 CaN-37

4775555 48716939 51949820

4 1 2

13% 3% 2%

29.42/4.71 73.70/5.64 89.09/6.45

36.76/4.60 54.87/5.02 72.05/4.80

79 164 53

CaN-59 CaN-69 CaN-103

37533900 38347311 29028998

1 3 1

3% 7% 1%

22.00/4.71 42.22/5.64 69.15/9.64

29.15/5.17 47.07/5.19 30.76/5.30

55 366

CaN-119 CaN-135

16224244 18831

1 5

6% 9%

21.07/8.94 60.22/5.95

45.00/5.43 69.46/5.39

putative calcium-dependent protein kinase CPK1 adapter protein 2 14-3-3-like protein putative OsD305c Cysteine-rich polycomb-like protein 1 Centrinc OSJNBa0042F21.13 protein Gb|AAF23841.1 (Putative serine-rich protein) (At5g37010) Plastidic aldolase (Fragment) H+-transporting two-sector ATPase (EC 3.6.3.14) beta chain

3306


identification

Putative FtsH-like protein Pftf OSJNBa0052O21.11c F-box protein ZEITLUPE/FKF/ LKP/ADAGIO family Chaperonin 60 alpha subunit dnaK-type molecular chaperone CSS1 precursor probable chaperonin 60 beta chain probable chaperonin 60 beta chain Prohibitin, putative PHB2 FCA protein(fragment)


% coverage


no. of peptides

research articles

Nuclear Proteome of Chickpea Table 1 (Continued) functional category

Signaling and Gene Regulation

identification

At1g64300/F15H21_13 c calcium ion bindingc unknown proteinc At1g64300/F15H21_13c fructose-bisphosphate aldolase (EC 4.1.2.13) precursor(fragment) FCA protein (Fragment) Transketolase (Fragment) unknown protein(contains F-box domain) c F1K23.18 (lipase/hydrolase) malate dehydrogenase (EC 1.1.1.37) RAN binding protein 16-likec F18O14.30 (similar to isoflavone reductase) H+-transporting two-sector ATPase (EC 3.6.3.14) gamma chain precursor putative diacylglycerol kinasec serine carboxypeptidase-like protein AP2/EREBP transcription factor BABY BOOM ATRRAN2 auxin binding protein (ABP44); isovaleryl-CoA Dehydrogenasec H+-transporting two-sector ATPase (EC 3.6.3.14) gamma chain precursor protein bindingc glycine dehydrogenase (decarboxylating) (EC 1.4.4.2) component P precursor glycine hydroxymethyltransferase (EC 2.1.2.1) DNA-binding protein-likec transcription factor/ zinc ion binding phosphoglycerate kinase (EC 2.7.2.3) precursor Hypothetical protein At2g43170 Receptor kinase 6 fructose-bisphosphate aldolase (EC 4.1.2.13) precursor serine/threonine-specific protein kinase ARA.KIN homologue T15F16.3 putative ZF-HD homeobox proteinc unknown proteinc syringolide-induced protein (MYB)c Cytosolic phosphoglycerate kinase FCA protein (Fragment) 2'-hydroxyisoflavone reductase (EC 1.3.1.45) Cytosolic malate dehydrogenase (EC 1.1.1.37) Putative mitochondrial NAD-dependent malate dehydrogenase G5bf protein glycerate dehydrogenase (EC 1.1.1.29) Putative malate dehydrogenase glycine hydroxymethyltransferase (EC 2.1.2.1)


spot no.a

gi no.b

92

CaN-137

15983374

1

4%

78.87/8.66

80.20/5.44

91 90 90 242

CaN-140 CaN-145 CaN-148 CaN-184

42566321 15230873 15983374 169037

1 1 1 5

5% 7% 4% 13%

62.06/6.87 38.28/8.19 78.87/8.66 38.63/5.83

74.05/5.52 86.61/5.48 86.72/5.53 46.14/5.65

46 255 93

CaN-236 CaN-251 CaN-282

32482140 4586600 15230873

1 5 1

2% 32% 7%

79.49/8.85 65.37/5.84 38.28/8.19

41.32/5.89 64.47/5.97 33.36/6.28

102 247

CaN-283 CaN-293

10764859 10334493

1 5

8% 17%

42.90/4.99 35.47/5.92

36.01/6.35 43.05/6.37

96 65

CaN-307 CaN-369

8978348 8778426

2 1

3% 3%

120.38/6.22 35.37/5.58

62.09/6.18 56.92/6.52

85

CaN-298

19785

1

3%

41.42/8.16

55.03/6.15

77 71

CaN-308 CaN-310

45735901 4539658

1 1

4% 3%

54.78/6.32 72.27/5.31

58.65/6.22 58.45/6.37

41

CaN-331

58761187

2

2%

76.22/6.22

110.0/6.30

131 92

CaN-347 CaN-351

1668706 5869967

4 1

19% 7%

25.02/6.65 44.99/6.01

34.24/6.79 40.65/6.54

101

CaN-353

19785

2

7%

41.42/8.16

44.10/6.60

80 153

CaN-375 CaN-402

22330529 20741

1 6

3% 3%

77.33/4.76 114.61/7.17

80.40/6.64 108.0/6.85

352

CaN-447

169158

1

12%

57.25/8.71

65.59/7.68

79 75

CaN-453 CaN-481

42407866 15232482

1 1

8% 9%

35.64/8.48 24.75/8.37

29.38/8.22 19.20/9.25

239

CaN-183

1161600

5

14%

50.15/8.48

52.71/5.64

43 47 328

CaN-215 CaN-221 CaN-244

51970954 16040954 169037

1 1 5

1% 1% 15%

95.42/5.63 91.77/5.77 38.63/5.83

88.90/5.59 108.78/5.61 46.61/6.00

45

CaN-450

7267489

2

2%

84.92/5.29

89.67/7.47

84 98 121 106 50 54

CaN-452 CaN-489 CaN-518 CaN-180 CaN-208 CaN-291

41053151 15230873 19911579 9230771 32482140 17949

1 1 1 3 2 3

6% 7% 12% 9% 2% 9%

46.97/4.60 38.28/8.19 31.35/7.89 42.26/5.73 79.49/8.85 35.38/5.94

10.00/7.83 28.01/9.16 26.90/9.40 51.57/5.58 76.28/5.62 43.26/6.23

321

CaN-296

10334493

6

20%

35.47/5.92

45.21/6.15

63

CaN-359

21388550

2

8%

36.14/8.48

45.19/6.46

101 139

CaN-368 CaN-407

17064988 18264

2 3

5% 8%

42.59/8.65 41.68/5.95

45.00/6.76 50.88/6.96

108 369

CaN-439 CaN-446

37725953 169158

3 8

6% 16%

37.10/7.01 57.25/8.71

41.28/7.49 65.00/7.41

score

% coverage


no. of peptides


research articles

Pandey et al.


Signaling and Gene Regulation Structure

Translation

Transport

Unknown Function


identification

score

spot no.a

gi no.b

Putative glycine hydroxymethyltransferase maturase Kc maturasec Beta-conglutin Actin Actin (Fragment) kinesin motor protein-like Histone H3 (Fragment) Putative MAR binding filament-like protein 1 Translational elongation factor Tu Putative elongation factor 1-gamma Elongation factor 1-gamma TufA Ribosomal protein subunit 2 (Fragment) AY085926 NID 40S ribosomal protein S5 VFEF1A NID Putative sorbitol transporter ATGLR2.6c amino acid transporter-like proteinc putative oligopeptide transporter Adenine nucleotide translocator Brain protein 44-like

59

CaN-517

24429608

2

3%

59.09/8.81

18.10/9.50

83 80 106 110 177 81 102 47

CaN-572 CaN-545 CaN-17 CaN-72 CaN-118 CaN-233 CaN-569 CaN-230

54021417 21388370 46451223 58533119 1498334 49388000 1213307 55296302

1 1 2 3 4 1 3 1

6% 25% 5% 8% 11% 10% 30% 1%

35.53/9.77 10.23/9.93 62.09/6.43 41.70/5.23 37.14/5.47 26.95/7.26 16.47/11.58 84.08/5.00

33.00/10.9 17.00/9.70 26.75/4.90 55.61/5.27 55.73/5.37 33.59/6.02 18.50/10.57 27.51/6.02

72 69

CaN-130 CaN-257

17225494 46806490

1 2

2% 4%

50.38/6.19 46.90/6.31

58.71/5.50 63.49/6.16

158 48 50 42 116 217 44 80 101 86 243 46

CaN-279 CaN-304 CaN-558 CaN-543 CaN-550 CaN-535 CaN-19 CaN-154 CaN-164 CaN-198 CaN-560 CaN-27

3868758 42566420 20379255 27808502 40748265 3122060 34393630 15238975 56201561 56784228 2780194 37806192

4 1 2 1 3 6 2 1 1 1 4 1

11% 2% 9% 9% 14% 10% 4% 3% 8% 5% 10% 7%

47.45/6.10 44.65/6.29 23.02/9.55 14.04/9.57 23.48/9.48 49.21/9.15 54.38/9.27 99.66/6.66 40.59/9.76 58.52/9.33 42.13/9.75 12.07/8.71

33.75/6.12 53.62/6.36 29.00/10.01 16.50/9.60 26.80/9.65 47.43/9.43 29.84/5.09 94.75/5.40 21.50/5.55 56.99/5.72 31.70/10.0 37.15/5.09

44 72 58 44 41 41 74 41 68 48 77 82 81 42 43 42

CaN-57 CaN-74 CaN-96 CaN-116 CaN-157 CaN-188 CaN-480 CaN-511 CaN-288 CaN-551 CaN-412 CaN-552 CaN-571 CaN-172 CaN-228 CaN-491

37806192 23308421 14532624 37806192 37806192 37806192 17065518 37806192 729479 1352767 49388388 45736022 48475212 37806192 37806192 37806192

1 2 2 1 1 1 1 1 2 2 1 1 1 1 1 1

7% 3% 2% 7% 7% 7% 5% 7% 4% 4% 10% 13% 23% 7% 7% 7%

12.07/8.71 51.97/5.69 85.94/5.48 12.07/8.71 12.07/8.71 12.07/8.71 18.42/9.11 12.07/8.71 40.55/8.70 51.64/6.46 23.98/7.79 20.68/12.05 11.66/12.23 12.07/8.71 12.07/8.71 12.07/8.71

98.0/5.02 59.96/5.18 97.0/5.21 51.35/5.32 97.03/5.50 52.96/5.77 11.00/4.88 11.50/9.29 41.24/6.20 25.10/9.90 29.81/7.14 23.90/10.0 28.60/10.90 31.77/5.77 15.00/6.10 33.10/9.20

Brain protein 44-like At2g39730/T5I7.3 Hypothetical protein At1g62750 Brain protein 44-like Brain protein 44-like Brain protein 44-like ATU32176 NID Brain protein 44-like VFU14956 NID DRSCPRBCB NID hypothetical proteinc hypothetical proteinc unknown proteinc Brain protein 44-like Brain protein 44-like Brain protein 44-like

% coverage


no. of peptides

a Spot number as given on the 2-D gel images. The first letters (Ca) signify the source plant, Cicer arietinum, followed by the subcellular fraction, nuclear (N). The numerals indicate the spot numbers corresponding to Figure 3. b Gene identification number as in GenBank. c The spots were researched in short peptide blast in NCBI using NCBInr database, as the identifications made in Mascot were found to be unsatisfactory.

which is an essential component of a transcriptional activator complex regulating histone expression.26 Aspartate carbamoyltransferase (ATCase) is a protein involved in de novo pyrimidine biosynthesis pathway. The ATCase activity is virtually absent from “isotonic nuclei” but present in nuclei isolated in hyperosmotic sucrose media.27

Figure 5. Functional classification of chickpea nuclear proteins. The proteins identified from the chickpea nucleus were grouped into 10 classes based on their putative functions.

as multiple protein spots. It was earlier detected in Arabidopsis and rice nuclear proteomes. Recent evidence suggests that, along with its glycolytic activity, GAPDH is a multifunctional protein with both cytoplasmic and nuclear functions, one of 3308


Structural proteins like actin (spots 72 and 108) and kinesin (spot 233) represent the third set of nuclear proteins. These proteins are known to be major constituents of the cytoskeleton in eukaryotic cells including chromatin remodeling and related processes.28-30 Interestingly, spot 569, a histone H3 protein identified in the chickpea nuclear proteome, was previously not reported in Arabidopsis or rice proteomes. Spot 230 is a putative MAR binding filament-like protein 1 (MFP1). The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is important for higherorder chromatin organization and the regulation of gene expression. The animal nuclear matrix proteins with the greatest structural similarity to MFP1 are the nuclear lamins.31

research articles

Nuclear Proteome of Chickpea Table 2. Comparison of Nuclear Proteins Identified in Chickpea, Arabidopsis, and Rice functional category

Signaling and gene regulation

Cicer arietinum

identification

Oryza sativa

AF078903 AF058902 -

Centromere protein homologue F-box protein Serine-rich protein

-

15233570 M

CaN-282 CaN-103

Ca-binding protein

CaN-140

Lipase/hydrolase H+ ATPase


15229075 M 15231752 M 15241439 M 115480 M 15236276 M 15221593 M 11135591 M 21594055 -

Cysteine synthase Serine carboxypeptidase AP2 transcription factor Phosphoglycerate kinase

CaN-331 CaN-180 CaN-183

Receptor kinase Aldolase

CaN-221 CaN-119 CaN-184 CaN-244 CaN-450 CaN-307 CaN-347 CaN-369 CaN-291 CaN-5

Ser/Thr kinase RAN binding protein Transcriptional repressor Ca-dependent protein kinase Protein kinase

CaN-137 CaN-148 CaN-293 CaN-296 CaN-359 CaN-439 CaN-8

Malate dehydrogenase

14-3-3 protein Glycine hydroxymethyltransferase

DNA replication and transcription

LEA protein Glycine decarboxylase Glyceraldehyde-3-phosphate dehydrogenase

Metabolism

β-1,3-glucanase Glycolate oxidase

Protein degradation

Arabidopsis thaliana

Protein folding

Peptidase E3 ubiquitin ligase component Chaperonin 60

Structure

DnaK-type molecular chaperone Actin

Translation

Tubulin Kinesin EF-Tu

EIF 40S ribosomal protein

CaN-447 CaN-446 CaN-517 CaN-402 CaN-269 CaN-358 CaN-363 CaN-366 CaN-398 CaN-297 CaN-139 CaN-473 CaN-503 CaN-541 CaN-229 CaN-305 CaN-38 CaN-82 CaN-133 CaN-44 CaN-72 CaN-118 CaN-233 CaN-130 CaN-257 CaN-279 CaN-304 CaN-550

2118307 M 22326940 M 15230595 M 15219412 2129669 M U72724 -

T05703

S17916 D10207 AF073697 D17587 D10985 D50301

15221284 M 21536533

AF333275

7267238 M

-

15221781 M

D13436 D64036 -

9958055 7488270 M 15219721 M

1361987 9759623 15235745 M

D85763

-

15232660 M 15226973 M 81622 M 16974416 15222848 M

AF046884 AF010582

-

AF337174

15231850 M 15229497 M 2511592 18411417 M -

P80502 AF489695

15241849 M 71633 M 18409908 M 15230191 M 20514259 15234638 M 23397095

T05618 -

4588003 11467967 M 15218458 M 11467967 M 15225180 M

AB046415 -

AF182523 AF327413


research articles

Pandey et al.


identification

Miscellaneous

Ribosomal protein subunit ATP synthase

Unknown

Brain protein

Cicer arietinum

Arabidopsis thaliana

Oryza sativa

CaN-558 CaN-81 CaN-105 CaN-27 CaN-57 CaN-116 CaN-157 CaN-188 CaN-511 CaN-172 CaN-228 CaN-491

21537296 -

AB035347 Q01859 Q07233 D16140

-

a The first letter (Ca) signifies the source plant, Cicer arietinum, followed by the subcellular fraction, nucleus (N). The numerals indicate the spot numbers in this study. b Accession numbers according to NCBI non-redundant database.13 c Accession numbers according to Rice proteome database.14

Several metabolism-related proteins have also been identified in the chickpea nucleus. Spots 139 and 473 represent β-1, 3-glucanase and fall under this category. These proteins are members of the O-glycosyl hydrolases family that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. It is targeted to the secretory pathway but has been reported earlier also in the rice nucleus.14 Glycolate oxidase (spots 503 and 541) is another protein of this class, which is involved in the photorespiratory pathway and also reported in the Arabidopsis nuclear proteome.13 The presence of Rubisco subunits in this category could come across as possible contamination, since it is the most abundant protein in the green plants. Figure 6. Comparative analyses of plant nuclear proteome. Venn diagram depicting overlaps within Arabidopsis thaliana, Oryza sativa, and chickpea nuclear proteomes. The numbers signify the unique and/or orthologous proteins among the organisms studied.

Proteins involved in the translational machinery are standard in the case of any nuclear proteome. In our study, 5% of the total identified proteins belong to this category. Eukaryotic elongation factor (eEF-1R) plays a pivotal role in protein biosynthesis, present mainly in the cytoplasm, but a small population of eEF-1R molecules has been previously identified in the nucleus where it forms a complex with a zinc finger protein.32 The molecular chaperones account for 5% of the total nuclear proteome of chickpea that include chaperonin 60 and dnaK-type molecular chaperone (Hsp70). Under normal circumstances, Hsp70 is present mainly in the cytosol, but it translocates to the nucleus and nucleolus during physiological stress to prevent random aggregation of proteins.33 Spots 490 and 563 represent two isoforms of prohibitin, a large multimeric complex which provides protection of native peptides against proteolysis, suggesting a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins.34 Another important category of proteins identified are presumably involved in degradation mechanism. Spot 84 is a putative FtsH-like protein, Pftf, an ATP-dependent cell-division protein involved in proteolysis and peptidolysis.35 The F-box protein ZEITLUPE/FKF/LKP/ADAGIO family (spot 305) is a component of the E3 ubiquitin ligase complex in the nucleus that plays a central role in the blue-light-dependent circadian cycle.36 3310


Proteins involved in transport account for 3% of the chickpea nuclear proteome. This category included a putative sorbitol transporter (spot 19), an acyclic polyol transporter related with normal growth and development.37 However, the presence of polyols in plants has often been related to the response to different abiotic (water/cold/salt) and biotic (pathogen attack) stresses.38 A putative ligand-gated ion channel protein (spot 154), an amino-acid transporter-like protein (spot 164), a putative oligopeptide transporter (spot 198), and an adenine nucleotide translocator (spot 560) constitute the rest of the class. The miscellaneous class of proteins, in this study, account for 9% of the nuclear proteome. These proteins cover a wide range of functions from ATP synthase (spots 81 and 105) involved in energy production to a putative phytochelatin synthetase (spot 557), a protein involved in metal-ion homeostasis.39 Comparison of Arabidopsis, Rice, and Chickpea Nuclear Proteomes. In this study, a comparison between the proteins identified in the chickpea nucleus and those previously reported in the Arabidopsis13 and rice nuclei14 was attempted (Table 2 and Supporting Information table). As expected, proteins involved in signaling and gene regulation were found to be the most abundant across the three nuclear proteomes. Intereatingly, only eight proteins were found to be identical in the nucleus of Arabidopsis, rice, and chickpea, although the number of common proteins between any two systems was generally higher (Figure 6). This difference in the pattern of the nuclear proteomes may be attributed to the fact that the protein expression profile is shaped by the cellular environment and the ecological niche of the corresponding organism.40 A total of 32 proteins, including the eight proteins present ubiquitously, was common between Arabidopsis and chickpea,

research articles

Nuclear Proteome of Chickpea

and these proteins cover all the functional categories. Chickpea and rice have exclusively 11 common proteins between them, while Arabidopsis and rice share only 6 proteins. The brain protein identified in the chickpea nucleus was catalogued in the unknown category, as no function could be assigned to it in the plant nucleus. A similar protein, brain-specific protein, was also identified in the rice nucleus. The comparative data suggest that approximately 71% of the chickpea nuclear proteins identified (Figure 6) are unique or novel, which needs to be experimentally verified. Nevertheless, this signifies the necessity to compare nuclear proteomes of different organisms, and most certainly between the major lineages of higher plants, to better understand the complex role of this organelle. It must be noted that among the exclusive 107 proteins in chickpea, many were putative, and also protein redundancy was not taken into account while cataloging the proteins. Nevertheless, the differences in the plant proteomes might reflect technical issues as well as biological absence of the proteins in the organisms studied.

Concluding Remarks The present study is directed toward the systematic analysis of the nuclear proteome in a food legume, chickpea, in particular, and possible functional classification of the proteins. This approach may be used in the future to dissect biochemical pathways encompassed by the identified proteins. This will also be important in long-term efforts to develop faithful, quantitative models for plant processes.41 For a complete understanding of cellular functions, it is important to determine and characterize the proteomes at different subcellular locations and their involvement in biosynthetic and signaling pathways. A total of 150 proteins is reported that characterize the nuclear proteome of this important legume. Most of the identified proteins are verified nuclear proteins as evident by the literature. However, few of the nonclassical proteins are identified in the chickpea nucleus, which have never been associated with this compartment. These results, in part, are in agreement with the previous reports on plant nuclear proteomes, and also there is a great level of divergence in the protein classes. Nevertheless, until a much more complete survey of the proteomes of nucleus in several plants is conducted using more similar protein arraying and identification technology, it will be difficult to determine the presence/or absence of specific proteins between plant species. This is an initial attempt in the direction that will be expanded upon during future proteomic studies of plant nucleus. Our future efforts will focus on increasing the number of analyzed proteins with an aim to draw a complete functional map of the nuclear proteome. Further, we will focus on identifying the dynamics associated with the nuclear proteome toward cells metabolic and regulatory pathways at different physiological conditions.

Acknowledgment. The authors thank the Council of Scientific and Industrial Research (CSIR), Govt. of India for providing Predoctoral Fellowships to A.P., M.K.C., and D.B. MS/ MS sequence analysis was performed at the Proteomics International Pty Ltd. in Australia. This work was supported by a grant (BT/PR/4016/Agr/16/327) from the Department of Biotechnology (DBT), Govt. of India, and a grant from National Centre for Plant Genome Research, New Delhi, India, to N.C. Supporting Information Available: Table comparing the nuclear proteomes of chickpea, Arabidopsis, and rice. This

material is available free of charge via the Internet at http:// pubs.acs.org.

References (1) Storrie, B.; Madden, E. A. Methods Enzymol. 1990, 182, 203-235. (2) Dreger, M. Eur. J. Biochem. 2003, 270, 589-599. (3) Kumar, A.; Agarwal, S.; Heyman, J. A.; Matson, S.; et al. Genes Dev. 2002, 16, 707-719. (4) Gindullis, F.; Meier, I. Plant Cell 1999, 11, 1117-1128. (5) Gindullis, F.; Peffer, N.; Meier, I. Plant Cell 1999, 11, 1755-1768. (6) Park, O. K. J. Biochem. Mol. Biol. 2004, 37, 133-138. (7) Gerner, C.; Sauermann, G. J. Cell. Biochem. 1999, 71, 363-374. (8) Gerner, C.; Holzmann, K.; Meissner, M.; Gotzmann, J.; Grimm, R.; Sauermann, G. J. Cell. Biochem. 1999, 74, 145-151. (9) Mattern, K. A.; van Goethem, R. E.; de Jong, L.; van Driel, R. J. Cell. Biochem. 1997, 65, 42-52. (10) Dreger, M.; Bengtsson, L.; Schoneberg, T.; Otto, H.; Hucho, F. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 11943-11948. (11) Andersen, J. S.; Lyonm, C. E.; Fox, A. H.; Leung, A. K.; et al. Curr. Biol. 2002, 12, 1-11. (12) Jung, E.; Hoogland, C.; Chiappe, D.; Sanchez, J. C.; Hochstrasser, D. F. Electrophoresis 2000, 21, 3483-3487. (13) Bae, M. S.; Cho, E. J.; Choi, E. Y.; Park, O. K. Plant J. 2003, 36, 652-663. (14) Khan, M. K.; Komatsu, S. Phytochemistry 2004, 65, 1671-1681. (15) Baker, D. G.; Bianchi, S.; Blondon, F.; Dattee, Y.; et al. Plant Mol. Biol. Rep. 1990, 8, 40-49. (16) Haridas, V.; Higuchi, M.; Jaytilake, G. S.; Baily, D.; et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5821-5826. (17) Zhang, H. B.; Zhao, X.; Ding, X.; Paterson, A. H.; Wing, R. A. Plant J. 1995, 7, 175-184. (18) Bhushan, D.; Pandey, A.; Chattopadhyay, A.; Choudhary, M. K.; et al. J. Proteome Res. 2006, 5, 1711-1720. (19) Anderson, L. E.; Wang, X.; Gibbons, J. T. Plant Physiol. 1995, 108, 659-667. (20) Razem, F. A.; El-Kereamy, A.; Abrams, S. R.; Hill, R. D. Nature 2006, 439, 290-294. (21) Ronai, Z.; Robinson, R.; Rutberg, S.; Lazarus, P.; Sardana, M. Biochim. Biophys. Acta 1992, 1130, 20-28. (22) Brunet, A.; Kanai, F.; Stehn, J.; Xu, J.; et al. J. Cell Biol. 2002, 156, 817-828. (23) Boutilier, K.; Offringa, R.; Sharma, V. K.; Kieft, H.; et al. Plant Cell 2002, 14, 1737-1749. (24) Kunzler, M.; Gerstberger, T.; Stutz, F.; Bischoff, F. R.; Hurt, E. Mol. Cell Biol. 2000, 20, 4295-4308. (25) Millar, A. H.; Sweetlove, L. J.; Giege, P.; Leaver, C. J. Plant Physiol. 2001, 127, 1711-1727. (26) Pendle, A. F.; Clark, G. P.; Boon, R.; Lewandowska, D.; et al. Mol. Biol. Cell 2005, 16, 260-269. (27) Nagy, M.; Laporte, J.; Penverne, B.; Herve, G. J. Cell Biol. 1982, 92, 790-794. (28) Grzanka, A.; Grzanka, D.; Orlikowska, M. Oncol. Rep. 2004, 11, 765-770. (29) Hu, P.; Wu, S.; Hernandez, N. Genes Dev. 2004, 18, 3010-3015. (30) Percipalle, P.; Fomproix, N.; Kylberg, K.; Miralles, F.; et al. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6475-6480. (31) Harder, P. A.; Silverstein, R. A.; Meier, I. Plant Physiol. 2000, 122, 225-234. (32) Gangwani, L.; Mikrut, M.; Galcheva-Gargova, Z.; Davis, R. J. J. Cell Biol. 1998, 143, 1471-1484. (33) Nollen, E. A.; Salomons, F. A.; Brunsting, J. F.; Want, J. J.; et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12038-12043. (34) Nijtmans, L. G. J.; de Jong, L.; Sanz, M. A.; Coates, P. J.; et al. EMBO J. 2000, 19, 2444-2451. (35) Takechi, K.; Sodmergen, G.; Murata, M.; Motoyoshi, F.; Sakamoto, W. Plant Cell. Physiol. 2000, 41, 1334-1346. (36) Yasuhara, M.; Mitsui, S.; Hirano, H.; Takanabe, R.; et al. J. Exp. Biol. 2004, 55, 2015-2027. (37) Gao, Z.; Maurousset, L.; Lemoine, R.; Yoo, S. D.; et al. Plant Physiol. 2003, 131, 1566-1575. (38) Noiraud, N.; Maurousset, L.; Lemoine, R. Plant Physiol. Biochem. 2001, 39, 717-728. (39) Grill, E.; Loffler, S.; Winnacker, E. L.; Zenk, M. H. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6838-6842. (40) Skovgaard, M.; Jensen, L. J.; Brunak, S.; Ussery, D.; Krogh, A. Trends Genet. 2001, 17, 425-428. (41) Raikhel, N.; Coruzzi, G. Plant Physiol. 2003, 132, 404-409.

PR060147A Journal of Proteome Research • Vol. 5, No. 12, 2006 3311

The Nuclear Proteome of Chickpea (Cicer arietinum L.) Reveals

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