analysis of nuclear pro teo me in...

Chapter 2

Analysis of Nuclear Pro teo me in Chickpea

2.1 Introduction

The nucleus (pl. nuclei; from Latin nucleus or nuculeus, or kernel) is a

membrane-enclosed organelle common to all eukaryotic cells. It houses most of the

cell's genetic material, organized as multiple long linear DNA molecules in complex

with a large variety of proteins, such as histones, to form chromosomes. The genes

within these chromosomes constitute the cell's nuclear genome. The nucleus contains

the components and enzymes necessary to maintain, transcribe, and replicate genetic

material in a selective manner. Further, it is responsible for the synthesis and/or

assembly of components used to translate genetic material, although translation of the

code contained in mRNA is performed in the cytoplasm.

The main structures making up the nucleus are the nuclear envelope, a double

membrane that encloses the entire organelle and separates its contents from the

cellular cytoplasm, and the nuclear lamina, a meshwork within the nucleus that adds

mechanical support; much like the cytoskeleton supports the cell as a whole. Because

the nuclear membrane is impermeable to most molecules, nuclear pores are required

to allow movement of molecules across the envelope. These pores cross both of the

membranes, providing a channel that allows free movement of small molecules and

ions. The movement of larger molecules such as proteins is carefully controlled, and

requires active transport regulated by carrier proteins. Nuclear transport is crucial to

cell function, as movement through the pores is required for both gene expression and

chromosomal maintenance.

Although the interior of nucleus does not contain any membrane-bound

subcompartments, its contents are not uniform, and a number of subnuclear bodies

exist, made up of unique proteins, RNA molecules, and particular parts of the

chromosomes. The best known of these is the nucleolus, which is mainly involved in

the assembly of ribosomes. After being produced in the nucleolus, ribosomes are

exported to the cytoplasm where they translate mRNA.

2.1.1 History

The nucleus was the first organelle to be discovered, and was first described by

Franz Bauer in 1804 (Harris, 1999). It was later described in more detail by Scottish

botanist Robert Brown in 1831 in a talk at the Linnean Society of London. Brown was

studying orchids microscopically when he observed an opaque area, which he called

45

the areola or nucleus, in the cells of the flower's outer layer (Brown, 1866). He did not

suggest a potential function. In 1838 Matthias Schleiden proposed that the nucleus

plays a role in generating cells, thus he introduced the name "Cytoblast" (cell builder).

He believed that he had observed new cells assembling around "cytoblasts". Franz

Meyen was a strong opponent of this view having already described cells multiplying

by division and believing that many cells would have no nuclei. The idea that cells

can be generated de novo, by the "cytoblast" or otherwise, contradicted work by

Robert Remak (1852) and RudolfVirchow (1855) who decisively propagated the new

paradigm that cells are generated solely by cells ("Omnis cellula e cellula"). The

function of the nucleus remained unclear (Cremer, 1985).

Between 1876 and 1878 Oscar Hertwig published several studies on the

fertilization of sea urchin eggs, showing that the nucleus of the sperm enters the

oocyte and fuses with its nucleus. This was the first time it was suggested that an

individual develops from a (single) nucleated cell. This was in contradiction to Ernst

Haeckel's theory that the complete phylogeny of a species would be repeated during

embryonic development, including generation of the first nucleated cell from a

"Monerula", a structureless mass of primordial mucus ("Urschleim"). Therefore, the

necessity of the sperm nucleus for fertilization was discussed for quite some time.

However, Hertwig confirmed his observation in other animal groups, e.g. amphibians

and molluscs. Eduard Strasburger produced the same results for plants (1884). This

paved the way to assign the nucleus an important role in heredity. In 1873 August

Weismann postulated the equivalence of the maternal and paternal germ cells for

heredity. The function of the nucleus as carrier of genetic information became clear

only later, after mitosis was discovered and the Mendelian rules were rediscovered at

the beginning of the 20th century; the chromosome theory of heredity was developed

(Cremer, 1985).

2.1.2 Function

The main function of the cell nucleus is to control gene expressiOn and

mediate the replication of DNA during the cell cycle. The nucleus provides a site for

genetic transcription that is segregated from the location of translation in the

cytoplasm, allowing levels of gene regulation that are not available to prokaryotes.

46

2.1.2.1 Cell Compartmentalization

The nuclear envelope allows the nucleus to control its contents, and separate

them from rest of the cytoplasm where necessary. This is important for controlling

processes on either side of the nuclear membrane. In some cases where a cytoplasmic

process needs to be restricted, a key participant is removed to the nucleus, where it

interacts with transcription factors to downregulate the production of certain enzymes

in the pathway. This regulatory mechanism occurs in the case of glycolysis, a cellular

pathway for breaking down glucose to produce energy. Hexokinase is an enzyme

responsible for the first the step of glycolysis, forming glucose-6-phosphate from

glucose. At high concentrations of fructose-6-phosphate, a molecule made later from

glucose-6-phosphate, a regulator protein removes hexokinase to the nucleus

(Lehninger et a!., 2000), where it forms a transcriptional repressor complex with

nuclear proteins to reduce the expression of genes involved in glycolysis (Moreno et

al., 2005).

In order to control which genes are being transcribed, the cell separates some

transcription factor proteins responsible for regulating gene expression from physical

access to the DNA until they are activated by other signaling pathways. This prevents

even low levels of inappropriate gene expression. For example in the case of NF-KB

controlled genes, which are involved in most inflammatory responses, transcription is

induced in response to a signal pathway such as that initiated by the signaling

molecule TNF-a, binds to a cell membrane receptor, resulting in the recruitment of

signalling proteins, and eventually activating the transcription factor NF-KB. A

nuclear localisation signal on the NF-KB protein allows it to be transported through

the nuclear pore and into the nucleus, where it stimulates the transcription of the

target genes (Alberts et al., 2002).

The compartmentalization allows the cell to prevent translation of unspliced

mRNA (Gorlich and Ulrike, 1999). Eukaryotic mRNA contains introns that must be

removed before being translated to produce functional proteins. The splicing is done

inside the nucleus before the mRNA can be accessed by ribosomes for translation.

Without the nucleus ribosomes would translate newly transcribed (unprocessed)

mRNA resulting in misformed and nonfunctional proteins.

47

2.1.2.2 Gene Expression

Gene expression first involves transcription, m which DNA is used as a

template to produce RNA. In the case of genes encoding proteins, the RNA produced

from this process is messenger RNA (mRNA), which then needs to be translated by

ribosomes to form a protein. As ribosomes are located outside the nucleus, mRNA

produced needs to be exported (Nierhaus et al., 2004).

Since the nucleus is the site of transcription, it also contains a variety of

proteins which either directly mediate transcription or are involved in regulating the

process. These proteins include helicases that unwind the double-stranded DNA

molecule to facilitate access to it, RNA polymerases that synthesize the growing RNA

molecule, topoisomerases that change the amount of supercoiling in DNA, helping it

wind and unwind, as well as a large variety of transcription factors that regulate

expression (Nicolini, 1997).

2.1.2.3 Processing of Pre-mRNA

Newly synthesized mRNA molecules are known as primary transcripts or pre

mRNA. They must undergo post-transcriptional modification in the nucleus before

being exported to the cytoplasm; mRNA that appears in the nucleus without these

modifications is degraded rather than used for protein translation. The three main

modifications are 5' capping, 3' polyadenylation, and RNA splicing. While in the

nucleus, pre-mRNA is associated with a variety of proteins in complexes known as

heterogeneous ribonucleoprotein particles (hnRNPs). Addition of the 5' cap occurs co

transcriptionally and is the first step in post-transcriptional modification. The 3' poly

adenine tail is only added after transcription is complete.

RNA splicing, carried out by a complex called the spliceosome, is the process

by which introns, or regions of DNA that do not code for protein, are removed from

the pre-mRNA and the remaining exons connected to re-form a single continuous

molecule. This process normally occurs after 5' capping and 3' polyadenylation but

can begin before synthesis is complete in transcripts with many exons (Lodish et al.,

2004). Many pre-mRNAs, including those encoding antibodies, can be spliced in

multiple ways to produce different mature mRNAs that encode different protein

sequences. This process is known as alternative splicing, and allows production of a

large variety of proteins from a limited amount of DNA.

48

2.1.3 Nuclear Proteomics

The nuclear proteome is dynamic, changing its composition in response to

intracellular and environmental stimuli. Proteins involved in different cellular

functions, e.g., signaling, gene regulation, structure, translation, proteolysis, and

among others, a variety of RNA-associated functions have been identified in the

n~cleus. Increasing evidence suggest that nearly 27% of total cellular proteins are

localized in the eukaryotic nucleus implying a variety of protein functions in this

compartment (Kumar et al., 2002). 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 (Gindullis and Meier,

1999; Gindullis et al., 1999). Therefore, characterization of the nuclear proteome in

plants will go a long way in increasing our understanding about the gene expression

and function in a plant system.

The study on the nuclear proteome has been undertaken by several groups but

most efforts have been largely restricted to mammals, which include subnuclear

fraction from embryos of Drosophila (Fisher et al., 1982), nuclear matrix proteins in

various human cell types (Gerner and Sauermann, 1999; Gerner et al., 1999; Mattern

et al., 1997), nuclear envelope proteins from mouse neuroblastoma N2a cells (Dreger

et al., 2001), human nucleolar proteins (Andersen et al., 2002), and total nuclear

proteins from human liver (Jung et al., 2000). Proteomic analyses of nucleus for two

model plants viz., A. thaliana (Bae et al., 2003) and rice (Khan and Komatsu, 2004)

have been reported. The proteomic analysis of the Arabidopsis nuclear matrix

(Calikowski et al., 2003) as well as nucleolus (Pendle et al., 2005) has also been

published. Very recently, the nuclear proteome of Medicago truncatula was also

reported (Repetto et al., 2008). 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, pathogen-infected and/or environmentally challenged plants.

2.2 Materials and Methods

2.2.1 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

49

and maintained at 25 ± 2° C, 50± 5 %relative humidity under 16 h photoperiod (270

11mol m-2 s-1 light intensity). The 3-weeks-old seedlings were sampled as experimental

materials, harvested and stored at -80° C after quick-freezing in liquid nitrogen.

2.2.2 Isolation of Pure Nuclei

The nuclei were prepared from chickpea seedlings as described (Zhang et al.,

1995) with few modifications. About 20 g of the tissue was ground into powder in

liquid nitrogen with 0.3 % (w/w) PVPP and immediately transferred into an ice cold

500 ml beaker containing 200 ml ice-cold 1 x HB (10 mM Trizma base, 80 mM KCI,

10 mM EDTA, 1 mM spermidine, 1 mM spermine, 0.5 M sucrose, pH 9.5) plus

0.15% J3-mercaptoethanol and 0.5% Triton X-100. 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 fixed-angle rotor at

1,800 X gat 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 x HB minus Triton X-100). To

remove the particulate matter remaining in the suspension, the resuspended nuclei

were filtered into a 50 ml centrifuge tube through two layers of miracloth by gravity.

The content was centrifuged at 57 X g, 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 1,800 X g, 4 oc for 15 min in a swinging

bucket centrifuge. The pellet was washed 2 additional times by resuspension in wash

buffer followed by centrifugation at 1,800 X g, 4°C for 15 min.

2.2.3 Confocal Microscopy

The nuclear fraction was stained for 15 min with 0.1 flg/ml 4', 6' -diamidino-2-

phenylindole hydrochloride (DAPI) in 0.1 M potassium phosphate buffer (pH 7.4)

and then washed twice with PBS (phosphate buffer saline). For microscopy, a small

volume of suspension was placed on a slide and covered with coverglass. The images

were taken with and without UV-filter.

50

2.2.4 Chlorophyll Assay

The chlorophyll content in the starting homogenate, the supernatant and the

nuclei enriched fraction 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 acetone and 1 ml MQ to it and centrifuging at 1,000 X g for 5 min. The

absorbance of this sample was measured at 652 nm. The assay was done in triplicates

and the amount of chlorophyll m ml of the suspension was observed as mg

chlorophyll per ml Absorbance/34.5 (http://www.bio.com/

protocolstools/protocol.htm1). The chlorophyll amount was then calculated as mg per

g 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.

2.2.5 Nuclear Protein Extraction and Quantification

Nuclear proteins were prepared from the nuclei-enriched pellet using TriPure

Reagent (Roche) according to the manufacturer's instructions with few modifications.

The nuclear pellet was suspended in TriPure reagent by repetitive pipetting. The

sample was incubated for 5 min at room temperature to permit the complete

dissociation of nucleoprotein complexes. 0.2 ml chloroform/1 ml TriPure was added

to the sample and the tube vortexed for 15 sec so as to form a homogenous mixture.

The sample was allowed to stand for 10 min before centrifugation at 12,000 X for 15

min at 4°C. Following centrifugation, the mixture separated into a lower red (phenol

chloroform) phase, an interphase, and a colorless upper aqueous phase. RNA

remained exclusively in the aqueous phase. All of the aqueous (upper) phase was

removed completely using a pipette. 0.3 ml 100% ethanol/1 ml Tripure was added to

the tube so as to precipitate the DNA from the interphase and the organic phases.

Sample was mixed thoroughly by vigorously inverting the tubes several times. A

further incubation of 3 min at room temperature was followed by centrifugation at

2,000 X g for 5 min at 4°C. The phenol-ethanol supernatant was transferred to a fresh

tube, and the protein was precipitated by adding 1.5 ml acetone per 1 ml of TriPure

used. After incubation for 10 min at room temperature, the protein was sedimented by

centrifugation at 12,000 X g for 10 min at 4°C. The supernatant was removed and

around 2 ml of 0.3 M guanidine hydrochloride in 95% ethanol (guanidine HCl in 95%

ethanol) was added to each tube so as to cover the pellet. After 20 min incubation, the

protein pellet was collected by centrifugation at 7,500 X g for 5 min at 4°C. The pellet

51

was subjected to further 2-3 washes of guanidine hydrochloride before a final 20 min

wash in I 00% ethanol. After drying, the final protein pellet was re-suspended in IEF

sample buffer [8 M urea, 2 M thiourea, 2% (w/v) CHAPS].

The protein concentration was determined using the 2-D Quant kit (Amersham

Biosciences). A standard curve was prepared using 2 mg/ml BSA standard solution

provided with kit. Tubes were prepared containing 1-50f.ll of the sample to be

assayed. 500f.ll of precipitant was added to each tube including standard curve tubes

and vortexed for proper mixing. After incubation for 2-3 min at room temperature,

500f.ll of co-precipitant was added to each tube. The tubes were again vortexed for

proper mixing. The content was centrifuged at I 0,000 X g for 5 min and supernatant

was decanted completely. 100f.ll of copper solution and 400f.ll of distilled water was

added to each tube followed by vortexing for dissolving precipitated protein. Then I

ml of working colour reagent (1 00 parts colour reagent A: 1 part colour reagent B)

was added to each tube and incubated for 15-20 min at room temperature. The

absorbance of standard and sample was recorded at 480 nm using water as the

reference. A standard curve was generated by plotting the absorbances of the

standards against the quantity of protein. The protein concentration of the samples

was determined using this standard curve.

2.2.6 Immunoblot Analysis

For immunoblotting, proteins were subjected to SDS-PAGE on 12.5 % w/v

acrylamide Laemmli gels (7 em). The electrophoresis was performed at room

temperature and the proteins were electroblotted to Hybond-C membrane (Amersham

Biosciences, Bucks, UK) at 150 rnA for 2 h. The membrane was blocked with 5% w/v

nonfat milk in TBST buffer (0.1 M Tris pH 7.9, 0.15 M NaCl and 0.1% Tween 20).

The resolved proteins were probed with the primary polyclonal antibodies, viz.,

mouse anti-fibrillarin and sheep anti-histone antibodies (Abeam Limited, UK).

Antibody bound proteins were detected by incubation with anti-mouse/sheep

secondary antibodies (Abeam Limited, UK) conjugated to alkaline phosphatase.

2.2. 7 Enzyme Assay

The activities of the marker enzyme catalase (EC l.ll.l.6) (Luck, 1965), alcohol

dehydrogenase (EC 1.1.1.1) (Widholm and Kishinami, 1988), fumarate hydratase (EC

52

4.2.1.2) (Hatch, 1978), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (Simcox eta/.,

1977) were determined spectrophotometrically. The assays were done in triplicate.

The catalase enzyme assay was performed using 1 0 J..lg of organellar protein

for each reaction. The reaction mixture was prepared by adding 50 J..lL of protein

extract to 940 J..lL of 70 mM potassium phosphate buffer (pH 7 .5). Reaction was

started by addition of 10 J..lL ofH20 2 (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 H20 2. The assay was done in triplicates and the

absorbance values obtained were plotted against time. The alcohol dehydrogenase

activity was measured with ethanol as substrate by measuring NADH production from

NAD by increase in absorbance at 340 nm at 25°C. The reaction mixture contained

750 11mol Tris-HCl (pH 9.0), 3 11mol NAD, and 1% (v/v) ethanol in a final volume of

5.0 ml. The reaction was initiated by adding ethanol and the absorbance changes

noted before this addition were subtracted from the ethanol induced rate. The fumarate

hydratase activity was measured as the increase in absorbance at 340 nm due to

NADPH formation. The reaction mixture consisted of 10 mM fumarate, 25 mM

Hepes-KOH buffer, pH7.5, 0.2 U malic enzyme/ml, 0.27 mM NADP, 4 mM MgCb,

and 5 mM potassium phosphate. The reaction was initiated by adding fumarate and

incubated at 37°C. The assay for glucose-6-phosphate dehydrogenase (D-glucose-6-

P:NADP+ oxidoreductase, EC 1.1.1.49) (G6PDH) contained 10 mM MgCh, 0.1%

Triton X-100, 0.17 mm NADP+, 0.33 mm glucose-6-P (G6P), 20 mm TES-NaOH (pH

7.5) in a final volume of 3 ml. The reduction ofNADP+ was measured by monitoring

the absorbance at 340 nm.

2.2.8 2-DE of Nuclear Proteins

Isoelectric focusing (IEF) was carried out with 150 J..lg protein. Aliquots of

proteins were diluted with 2-D rehydration buffer [8 M urea, 2M thiourea, 2% (w/v)

CHAPS, 20 mM DTT, 0.5% (v/v) pharmalyte (pH either 3-10, 4-7 or 6-11) and 0.05

% (w/v) bromophenol blue] and 250 111 solution was used to rehydrate immobilized

pH gradient strips (13 em; pH 3-10 and 4-7). Protein was loaded by in-gel rehydration

method onto IEF strips and electrofocusing was performed using IPGphor system

(Amersham Biosciences, Bucks, UK) at 20°C for 30,000 Vh. However, for 6-11 pH

strips, anodic cup-loading was performed with a load of 100 llg protein in 100 111

53

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 on top of 12.5% polyacrylamide gels for SDS-PAGE. The

electrophoresed proteins were stained with silver stain plus kit (Bio-Rad, CA, USA).

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, USA) was used to assemble first level

matchset (master image) from three replicate 2-DE gels.

2.2.9 Protein Identification Using MS/MS

Protein spots were excised mechanically using pipette tips and in-gel digested with

trypsin (Sigma, St. Louis, USA) and peptides extracted according to standard

techniques (Casey et al., 2005). These were analyzed by electrospray ion trap time-of

flight mass spectrometry (LC/MS/TOF) using a Q-Star Pulsar i (Applied Biosystems).

The MS/MS data was extracted using Analyst Software v.1.4.1 (Applied Biosystems).

Peptides were identified by searching the peak-list against the MSDB 20050929

(2344227 sequences; 779380795 residues) database using the MASCOT v.2.1

(http://www.matrixsciences.com) search engine. Since, the chickpea genome

sequence is not known, a homology based search was performed. The database search

criteria were: taxonomy, Viridiplantae (Green Plants, 195693 sequences); peptide

tolerance, +/-1.2 Da; fragment mass tolerance, +/-0.6 Da; maximum allowed missed

cleavage, 1; variable modifications, methionine oxidation; instrument type, ESI

QUAD-TOF. Protein scores were derived from ions scores as a non-probabilistic

basis for ranking protein hits and the protein scores as the sum of a series of peptide

scores. The score threshold to achieve p<0.05 is set by Mascot algorithm, and is based

on the size of the database used in the search.

2.3 Results

2.3.1 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

54

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 to get rid of contaminants from other organelles. The integrity

of the isolated nuclei was analyzed by staining with DAPI and examined by

fluorescence microscopy. The chickpea nuclei were uniform spheres with an average

diameter of approximately 20 ~-tM. The DAPI-stained fluorescent image of nuclei is

perfectly superimposable with their phase contrast image, indicating integrity and the

absence of other contaminating organelles (Figure lA). 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 lB, the supernatant retained most of the chlorophyll content and less than 3%

chlorophyll was present in the nuclei pellet.

The nuclear proteins were prepared from the purified nuclei using TriPure

reagent (Roche), in order 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 assaying different

organellar marker enzyme activities that could possibly contaminate the nuclear

preparation. Catalase was used for peroxisome, alcohol dehydrogenase for cytosol,

fumarate hydratase for mitochondria and glucose-6-P dehydrogenase for plastids as

marker enzymes. The whole cell extracts showed high catalase, alcohol

dehydrogenase, fumarate hydratase and glucose-6-P dehydrogenase activity, while the

nuclear proteins did not show any significant activities of these enzymes (Figure 2B).

These results altogether suggest that the nuclear preparation had no appreciable level

of peroxisome, chloroplast, mitochondria or other cytosolic contamination.

2.3.2 Construction of 2-DE Reference Map

Nuclear proteins were separated by 2D-PAGE in order to establish a reference

map. The images were analyzed by the PD Quest software as described earlier

(Bhushan et al., 2006). 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

55

A

B

0 1.4

c 1.2 0

()

~ 1 J: g. 0.8 .... .2 0.6 J: ()

0.4 G> >

':;:0 0.2 cu G>

c:: 0

'I' ... T 1

Crude Supernatant homogenate

r+-1 Nuclei fraction

Figure 2.1. 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.

A

B

Fibrillarin 34.0-+

120 • Homogenate

100 ell E >. 80 N c ell -0

~ 60

·::;:: ;::

40 0 <(

~ 0

20

0

Catalase ADH

Hi stones 16.47 20.63 14.94 13.40 12.42

• Nuclear fraction

FH

Enzyme

Glu-6-P DH

Figure 2.2. A. Western blot analysis of extracted nuclear proteins with antihistone: core and anti-fibrillarin antibodies. An aliquot of 100 Jlg 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 marker enzymes activity specific for microbody (catalase), cytosol (alcohol dehydrogenase, ADH), mitrochondria (fumarate hydratase), plastid (glucose-6-phosphate dehydrogenase) in the homogenate and the nuclear fraction of chickpea. The activity in the homogenate was considered as 100%.

I

pH3

66.2

45.0

31.0

21.5

14.4

Figure 2.3. Resolution of nuclear proteins on 2-DE. Nuclear proteins (150 !lg) were electro focused on a pH 3-10 IPG strip ( 13-cm), separated onto 12.5% SDS-PAGE. The Silver-stained gel was visualized as described in Materials and Methods.

comprehensive, we developed the proteome in the overlapping pH ranges 4-7 and 6-

11 (Figure 4A and 48). 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

range 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 alphabets identify the

organism (Cicer g_rietinum) and the subcellular organelle (Nucleus) from which the

proteome map has been made, whereas the numerals indicate the spot numbers. A

total of 200 spots with more than 30 quality score assigned by the software (based on

the quality and quantity ofthe spots) were excised. Of these, 170 spots were identified

with high confidence (Table 2.1) and the corresponding protein spots are indicated on

the gels (Figure 4A and 48)

2.3.3 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.uklinterpro/). 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) (Anderson

eta!., 1995). 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 17%

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 are signaling and gene regulation, DNA replication and

transcription, stmcture, translation, protein folding, protein degradation, metabolism

and transport. The rest of the identified proteins were grouped as miscellaneous class

(9% ), as mentioned in Table 2.1.

56

---

A kDa

97.4

45.0

31.0

21.5

14.4

pH6 pH11 kDa B 97.4

66.2

45.0

31.0

14.4

Figure 2.4. 2-DE reference map of the chickpea nuclear proteins. Nuclear proteins were zoomed onto two overlapping pH ranges: (A). pH 4-7 with 150 11g of protein and (B). pH 6-·11 with 100 11g 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-ESIMS/MS as detailed in Materials and Methods.

Unknown• (17%)

Transport

Signaling and gene • regulation

(32%)

• (4%)Metabolism • 0

Protein degradation (5%) Structure DNA replication (2%)

(4%) and transcription (18%)

Figure 2.5. Functional classification of chickpea nuclear proteins. The proteins identified from the nucleus were grouped into ten classes based on their putative functions.

Table 2.1. List ofMS/MS identified chickpea nuclear proteins and functional classification

Functional Spot no. Number

% Theoretical

Experimental category

Identification Score a giNo." of Coverage

Mw/pl Mw/pl(kDa)

peptides (kDa) DNA

replication Aspartate 41 CaN-4 15796550 I 2% 42.57/6.06 37.37/4.35

and carbamoyl transferase transcription

Putative glycine-rich 73 CaN-16 6911146 2 15% 16.25/7.82 28.58/4.80

RNA-binding protein 2 Putative glycine-rich

46 CaN-25 6911146 I 9% 16.25/7.82 38.39/4.72 RNA-binding protein 2

Putative glycine-rich 48 CaN-63 6911142 I 10% 14.15/8.71 31.41/5.25

RNA binding protein I Putative glycine-rich

52 CaN-67 6911146 2 14% 16.25/7.82 43.08/5.23 RNA-binding protein 2


RNA-binding protein I Putative glycine-rich

52 CaN-Ill 6911146 I 9% 16.25/7.82 37.30/5.32 RNA-binding protein 2


RNA-binding protein 2 . Putative glycine-rich

62 CaN-166 6911146 2 15% 16.25/7.82 28.85/5.64 RNA-binding protein 2 S 19 self-incompatibility

87 CaN-170 59896629 I 17% 21.73/8.65 31.00/5.60 ribonuclease

Aspartate 44 CaN-177 15796550 I 2% 42.57/6.06 41.60/5.68

carbamoyltransferase Retrotransposon protein,

putative, Tyl-copia 43 CaN-208 77555208 2 2% 91.96/9.33 76.28/5.62 subclass

Glyceraldehyde-3-phosphate dehydrogenase 226 CaN-242 77540212 5 II% 48.20/7.10 52.63/5.92

B subunit Glyceraldehyde-3-

phosphate dehydrogenase 309671

(NADP) 548 CaN-269 II 24% 48.06/7.57 98.51/5.91 (phosphorylating) (EC 1.2.1.13) B precursor

Aspartate 48 CaN-276 15796550 2 3% 42.57/6.06 29.95/6.22

carbamoyltransferase Putative glycine-rich


Aspartate 41 CaN-287 15796550 2 3% 42.57/6.06 42.20/6.15

carbamoyl transferase Glyceraldehyde-3-

phosphate dehydrogenase (NADP) 167 CaN-297 309671 3 6% 48.06/7.57 55.03/6.15

(phosphorylating) (EC 1.2.1.13) B precursor Putative glycine-rich

61 CaN-339 6911144 I 8% 16.25/7.82 15.00/6.60 RNA binding protein 2

Glyceraldehyde-3-phosphate dehydrogenase

(NADP) 51 CaN-358 12159 I 2% 43.31/8.80 49.80/6.45 (phosphorylating) (EC 1.2.1.13) A precursor Glyceraldehyde-3-

phosphate dehydrogenase (NADP) 211 CaN-363 12159 4 10% 43.31/8.80 49.49/6.60

(phosphorylating) (EC 1.2.1.13) A precursor Glyceraldehyde-3-

phosphate dehydrogenase 282 CaN-366 169091 6 16% 36.59/6.55 46.39/6.69

(phosphorylating) (EC 1.2.1.12)


RNA-binding protein 2 Putative glycine-rich


Glyceraldehyde-3-phosphate dehydrogenase

(NADP) 303 CaN-398 12159 6 14% 43.31/8.80 46.39/6.85 (phosphorylating) (EC 1.2.1.13) A precursor

Aspartate 47 CaN-417 15796550 I 2% 42.57/6.06 39.48/7.36

carbamoyltransferase Putative glycine-rich

55 CaN-433 6911142 2 10% 14.15/8.71 14.40/7.25 RNA binding protein I Putative glycine-rich

56 CaN-442 6911146 I 9% 16.25/7.82 54.38/7.60 RNA-binding protein 2 Putative glycine-rich

61 CaN-464 6911142 I 10% 14.15/8.71 18.70/8.81 RNA binding protein 1 Putative glycine-rich

51 CaN-487 6911146 I 9% 16.25/7.82 29.81/9.11 RNA-binding protein 2 Putative glycine-rich


Metabolism Beta-! ,3-glucanase 93 CaN-139 6714534 I 6% 52.58/9.51 68.40/5.50

Ribulose-! ,5-bisphosphate

234 CaN-260 37361623 5 8% 46.53/6.30 71.54/5.83 carboxylase/oxygenase

large subunit (Fragment) Ribulose-! ,5-

bisphosphate carboxylase !55 CaN-303 24634966 4 7% 51.74/6.14 45.54/6.34 large subunit (Fragment)

Methionine synthase 136 CaN-388 71000469 3 3% 87.75/6.05 98.50/6.49

Ribulose I ,5-bisphosphate carboxylase

109 CaN-403 3928152 3 12% 20.38/9.03 12.00/6.50 small subunit precursor

(EC 4.1.1.39)

Beta-! ,3-glucanase 94 CaN-473 6714534 I 6% 52.58/4.73 45.00/8.66

Probable alanine-glyoxylate transaminase 207 CaN-476 17044259 5 9% 44.18/7.69 55.30/8.50 (EC 2.6.1.44) [imported]

Glycolate oxidase like 417 CaN-503 16604394 7 22% 40.28/8.99 49.31/9.30

protein (Fragment) Glycolate oxidase like

172 CaN-541 16604394 3 10% 40.28/8.99 15.10/9.87 protein (Fragment)

Miscellaneous 2-cys peroxiredoxin-like

62 CaN-6 47027073 I 7% 21.84/4.93 27.90/4.71 protein (Fragment)

Chlorophyll alb binding 249 CaN-61 3928140 4 24% 28.30/5.47 30.95/5.12

protein precursor Oxygen-evolving

344004 complex protein I 116 CaN-66 3 8% 34.87/6.25 37.33/5.20

precursor A TP synthase beta chain

233 CaN-81 69214424 5 9% 53.36/5.23 67.06/5.25 (Fragment)

AtpB (Fragment) 47 CaN-105 6110504 3 88% 10.72/12.00 29.33/5.38

NADPH oxidase 41 CaN-158 87116554 2 2% 105.19/9.12 105.77/5.49

Glyoxalase/bleomycin resistance 62 CaN-176 92887944 2 6% 32.22/5.13 39.69/5.64

protein/dioxygenase

L-ascorbate peroxidase 123 CaN-231 71534930 2 16% 13.16/9.44 33.63/5.80

ATPase beta subunit 40 CaN-235 3676294 I 1% 59.87/5.83 44.71/5.91

Carbonic anhydrase (EC 37 CaN-343 20502881 2 5% 35.28/6.96 30.70/6.52

4.2.1.1) superoxide dismutase

180 CaN-344 6006619 4 12% 25.44/8.62 27.33/6.63 (EC 1.15.1.1) (Mn) I

Carbonic anhydrase (EC 102 CaN-346 20502881 2 5% 35.28/6.96 30.63/6.75

4.2.1.1)

Formate dehydrogenase 84 CaN-424 38636526 4 4% 40.56/6.54 51.23/7.16

Ferredoxin-binding 62 CaN-483 167085 I 3% 21.92/9.81 24.00/9.00

subunit protochlorophyllide

reductase (EC 1.3.1.33) 170 CaN-498 81946 4 9% 43.09/9.20 40.18/9.20 precursor

putative phytochelatin 154 CaN-557 54287588 I 9% 51.18/8.70 28.00/10.02

synthetase Protein Putative FtsH-like protein

440 CaN-84 52075838 8 II% 72.49/5.54 83.12/5.19 degradation Pftf

Cyclin-like F-box; 37 CaN-342 92879072 I 2% 59.22/7.84 26.23/6.45

Agenet

Hypothetical protein (kelch repeat -containing 41 CaN-350 48210029 2 4% 56.81/4.93 37.23/6.47

F-box family protein) AF041258 NID (ATP-

dependent 26S 74 CaN-370 3914449 I 2% 47.52/6.43 62.94/6.70

proteasome regulatory subunit)

Protein Chaperonin 60 alpha 108 CaN-38 3790441 3 5% 61.40/5.23 78.24/4.75

folding subunit dnaK-type molecular

chaperone CSS I 433 CaN-44 169023 9 13% 75.47/5.22 93.45/4.77 precursor

probable chaperonin 60 213 CaN-82 806808 5 9% 62.94/5.85 81.07/5.27 beta chain

probable chaperonin 60 276 CaN-133 806808 6 10% 62.94/5.85 81.15/5.36

beta chain

Prohibitin, putative 91 CaN-490 21592895 2 7% 30.60/6.93 33.31/8.80

PHB2 247 CaN-563 71370259 4 16% 31.84/9.49 36.50/9.70

Signaling and Gene FCA protein( fragment) 50 CaN-! 32482057 I 5% 29.37/8.63 31.0/4.0

regulation putative calcium-

dependent protein kinase 78 CaN-5 37993504 2 6% 37.84/5.05 30.32/4.66 CPKI adapter protein 2

14-3--3-like protein 173 CaN-8 4775555 4 13% 29.42/4.71 36.76/4.60

putative Os0305 73 CaN-31 48716939 1 3% 73.70/5.64 54.87/5.02

AAA A TPase, central region; Homeodomain- 66 CaN-36 92874675 2 3% 52.13/5.42 59.75/5.10

like

Centrin 79 CaN-59 37533900 1 3% 22.00/4.71 29.15/5.17

OSJNBa0042F21.13 164 CaN-69 38347311 3 7% 42.22/5.64 47.07/5.19

protein Plastidic aldolase

55 CaN-119 16224244 1 6% 21.07/8.94 45.00/5.43 (Fragment)

H+-transporting two-sector A TPase (EC 366 CaN-135 18831 5 9% 60.22/5.95 69.46/5.39 3.6.3.14) beta chain

Atl g64300/F15H21_13 92 CaN-137 15983374 1 4% 78.87/8.66 80.20/5.44

(protein kinase)

calcium ion binding 91 CaN-140 42566321 1 5% 62.06/6.87 74.05/5.52

Atlg64300/F15H21_13 90 CaN-148 15983374 1 4% 78.87/8.66 86.72/5.53

(protein kinase) Cytosolic

106 CaN-180 9230771 3 9% 42.26/5.73 51.57/5.58 phosphoglycerate kinase phosphoglycerate kinase

239 CaN-183 1161600 5 14% 50.15/8.48 52.71/5.64 (EC 2.7.2.3) precursor fructose-bisphosphate aldolase (EC 4.1.2.13) 242 CaN-184 169037 5 13% 38.63/5.83 46.14/5.65

precursor( fragment) Hypothetical protein

43 CaN-215 51970954 I 1% 95.42/5.63 88.90/5.59 At2g43170

Receptor kinase 6 47 CaN-221 16040954 I 1% 91.77/5.77 I 08.78/5.61

Protein kinase 2 45 CaN-229 7573598 1 1% 45.30/7.11 29.86/6.00

Homeobox-leucine zipper 37 CaN-233 89257493 1 3% 30.88/8.50 33.59/6.02

protein, putative

FCA protein (Fragment) 46 CaN-236 32482140 I 2% 79.49/8.85 41.32/5.89

NBS-LRR type disease resistance protein Rps 1- 37 CaN-238 62632823 2 1% 139.71/7.29 43.42/6.09

k-1 DNA cytosine

methyltransferase Zmet3, 39 CaN-240 77553397 I 1% 49.60/5.25 51.38/5.83 putative

fructose-bisphosphate aldolase (EC 4.1.2.13) 328 CaN-244 169037 5 15% 38.63/5.83 46.61/6.00

precursor

Transketolase (Fragment) 255 CaN-251 4586600 5 32% 65.37/5.84 64.47/5.97

unknown protein( contains F-box 93 CaN-282 15230873 1 7% 38.28/8.19 33.36/6.28

domain)

FIK23.18 102 CaN-283 10764859 I 8% 42.90/4.99 36.01/6.35

(lipase/hydrolase) 2'-hydroxyisoflavone

54 CaN-291 17949 3 9% 35.38/5.94 43.26/6.23 reductase (EC 1.3.1.45) malate dehydrogenase

247 CaN-293 10334493 5 17% 35.47/5.92 43.05/6.37 (EC 1.1.1.3 71

Cytosolic malate dehydrogenase (EC 321 CaN-296 10334493 6 20% 35.47/5.92 45.21/6.15

1.1.1.37) H+-transporting two-sector A TPase (EC

85 CaN-298 19785 I 3% 41.42/8.16 55.03/6.15 3.6.3.14) gamma chain

precursor Fructose-bisphosphate

199 CaN-299 40457267 3 9% 38.37/6.77 47.49/6.16 aldolase (EC 4.1.2.1 :32_

Serine-threonine kinase 42 CaN-305 38194927 2 5% 45.58/5.48 46.78/6.39

putative diacylglycerol 77 CaN-308 45735901 1 4% 54.78/6.32 58.65/6.22

kinase serine carboxypeptidase-

71 CaN-310 4539658 1 3% 72.27/5.31 58.45/6.37 like protein AP2/EREBP

transcription factor 41 CaN-331 58761187 2 2% 64.50/5.99 76.22/6.22 BABY BOOM

A Y045676 NID (WRKY-37 CaN-338 27363252 2 3% 55.78/8.18 22.30/6.42

DNA binding domain) auxin binding protein

5869967 (ABP44) ; isovaleryl- 92 CaN-351 I 7% 44.99/6.01 40.65/6.54 CoA Dehydrogenase H+-transporting two-sector A TPase (EC

101 CaN-353 19785 2 7% 41.42/8.16 44.10/6.60 3.6.3.14) gamma chain

precursor Putative mitochondrial

NAD-dependent malate 63 CaN-359 21388550 2 8% 36.14/8.48 45.19/6.46 dehydrogenase

G5bf protein 101 CaN-368 17064988 2 5% 42.59/8.65 45.00/6.76

Fl8014.30 (similar to 65 CaN-369 8778426 I 3% 35.37/5.58 56.92/6.52

isoflavone reductase) glycine dehydrogenase ( decarboxylating) (EC

153 CaN-402 20741 6 3% 114.61/7.17 108.0/6.85 1.4.4.2) component P

precursor glycerate dehydrogenase

139 CaN-407 18264 3 8% 41.68/5.95 50.88/6.96 (EC 1.1.1.29)

Putative malate 108 CaN-439 37725953 3 6% 37.10/7.01 41.28/7.49

dehydrogenase glycine

hydroxymethyltransferase 369 CaN-446 169158 8 16% 57.25/8.71 65.00/7.41 (EC 2.1.2.1)

glycine hydroxymethyltransferase 352 CaN-447 169158 I 12% 57.25/8.71 65.59/7.68

(EC 2.1.2.1) seri ne/threon ine-speci fi c protein kinase ARA.KIN 45 CaN-450 7267489 2 2% 84.92/5.29 89.6717.47

homolog T15F16.3 putative ZF-HD

84 CaN-452 41053151 1 6% 46.97/4.60 I 0.00/7.83 homeobox protein

DNA-binding protein-79 CaN-453 42407866 I 8% 35.64/8.48 29.38/8.22

like transcription factor/ zinc

75 CaN-481 15232482 I 9% 24.75/8.37 19.20/9.25 ion binding

unknown protein 98 CaN-489 15230873 I 7% 38.28/8.19 28.01/9.16

Putative glycine 59 CaN-517 24429608 2 3% 59.09/8.81 18.10/9.50

hydroxymethyltransferase syringolide-induced

121 CaN-518 19911579 I 12% 31.35/7.89 26.90/9.40 protein (MYB)

Maturase 80 CaN-545 21388370 I 25% 10.23/9.93 17.00/9.70

maturase K 83 CaN-572 54021417 I 6% 35.53/9.77 33.00110.9

Structure Beta-conglutin 106 CaN-17 46451223 2 5% 62.09/6.43 26.75/4.90

Actin 110 CaN-72 58533119 3 8% 41.70/5.23 55.61/5.27

Kinesin-related protein 56 CaN-103 14041829 3 1% 118.68/8.14 30.76/5.30

Actin (Fragment) 177 CaN-118 1498334 4 II% 37.14/5.47 55.73/5.37

Histone H3 (Fragment) 102 CaN-569 1213307 3 30% 16.47/11.58 18.50110.57

Putative MAR binding 47 CaN-230 55296302 I 1% 84.08/5.00 27.5116.02

filament-like protein I

Translation Translational elongation

72 CaN-130 17225494 I 2% 50.38/6.19 58.71/5.50 factor Tu

translation elongation 56.22/5.62 factor EF-Tu precursor, 345 CaN-195 20070084 8 16% 53.02/6.62

chloroplast Putative elongation factor

69 CaN-257 46806490 2 4% 46.90/6.31 63.49/6.16 1-l(amma

Elongation factor 1-158 CaN-279 3868758 4 II% 47.45/6.10 33.75/6.12

gamma

TufA 48 CaN-304 42566420 I 2% 44.65/6.29 53.62/6.36

Elongation factor-] alpha 267 CaN-535 3122060 6 10% 49.21/9.15 47.43/9.43

2

A Y085926 NID 42 CaN-543 27808502 I 9% 14.04/9.57 16.50/9.60

40S ribosomal protein S5 116 CaN-550 40748265 3 14% 23.48/9.48 26.80/9.65

Ribosomal protein 50 CaN-558 20379255 2 9% 23.02/9.55 29.00110.0 I

subunit 2 (Fragment)

Transpor1 Putative sorbitol

transporter 44 CaN-19 34393630 2

4% 54.38/9.27 29.84/5.09

amino acid transporter-101 CaN-164 56201561 I 8% 40.59/9.76 21.50/5.55

like protein putative oligopeptide

86 transporter

CaN-198 56784228 I 5% 58.52/9.33 56.99/5.72

Hypothetical protein 44 CaN-212 15293147 I 1% 94.74/6.72 75.11/5.72

At I g55040-zf-RanBP

ATRRAN2 131 CaN-347 1668706 4 19% 25.02/6.65 34.24/6.79

Adenine nucleotide 243 CaN-560 2780194 4 10% 42.13/9.75 31.70110.0

translocator

Unknown Hypothetical protein

45 CaN-14 26449957 I 3% 32.36/8.76 23.33/5.16 At4g02550/TIOPII 16

Hypothetical protein 46 CaN-21 42408955 I 10% 16.54/10.4 7 31.20/4.87

OSJNBbOOII Hl5.25

Brain protein 44-like 46 CaN-27 37806192 I 7% 12.07/8.71 37.15/5.09

Putative polyprotein 40 CaN-30 23266291 I 2% 71.19/7.55 47.36/5.03

OSJNBaOOII F23.7 47 CaN-37 38346401 I 1% 49.05/6.00 72.05/4.80

protein

Brain protein 44-like 44 CaN-57 37806192

I 7% 12.07/8.71 98.0/5.02

At2g39730/T517.3 119 CaN-74 23308421 2 3% 51.97/5.69 59.96/5.18

Hypothetical protein 58 CaN-96 14532624 2 2% 85.94/5.48 97.0/5.21

Atlg62750

Protein At4g02550 45 CaN-115 7269015 I 3% 37.65/8.72 38.61/5.50


Hypothetical protein 41 CaN-145 53792900 2 12% 15.91/9.61 86.61/5.48

P0031A09.11 Hypothetical protein

45 CaN-154 52353656

2 2% 64.81/7.57 94.75/5.40 P0033D06.7





VFU14956 NID 68 CaN-288 729479 2 4% 40.55/8.70 41.24/6.20

OSJNBa0049H08.9 41 CaN-307 38347658 2 2% 93.70/5.87 62.09/6.18

protein hypothetical protein

41 CaN-337 5080794 2 4% 55.38/8.31 21.07/5.66 F7AI9.27

Hypothetical protein 44

CaN-86438763 I 3% 26.50/5.51 15.67/5.52

(Fragment) 339a protein F21J9.20

42 CaN-348 9743340 2 4% 61.01/9.10 34.25/6.50 [imported]

hypothetical protein 77 CaN-412 49388388 I 10% 23.98/7.79 29.81/7.14

ATU32176 NID 74 CaN-480 17065518 I 5% 18.42/9.11 11.00/4.88

Brain protein 44-like 42 CaN-491 37806192 1 7% 12.07/8.71 33.10/9.20

Brain protein 44-like 41 CaN-511 37806192 1 7% 12.07/8.71 11.50/9.29

DRSCPRBCB NID 48 CaN-551 1352767 2 4% 51.64/6.46 25.10/9.90

hypothetical protein 23.90/10.0

unknown protein 28.60/10.90

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 2.4.

b) Gene identification number as in GenBank.

2.3.4 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 Arabidopsis (Bae et al., 2003) and rice

nucleus (Khan and Komatsu, 2004) was attempted (Table 2.2 and Table 2.3). As

expected, proteins involved in signaling and gene regulation were found to be the

most abundant across the three nuclear proteomes. It is interesting to note that only

eight proteins were found to be identical in the nucleus of Arabidopsis, rice and

chickpea though the number of common proteins between any two systems was

generally higher (Figure 6). This difference in 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 (Skovgaard et

al., 2001 ). A total of thirty proteins, including the eight proteins present ubiquitously,

were common between Arabidopsis and chickpea and these proteins cover all the

functional categories. Chickpea and rice have exclusively thirteen common proteins

. between them while Arabidopsis and rice share only six 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 75% 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 amongst the exclusive 127 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.

2.4 Discussion

The present study is directed towards 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 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

57

Table 2.2. Comparison of Nuclear Proteomes of Chickpea, Arabidopsis and Rice

Functional Category Chickpea' Arabidopsisb Ricec

Signaling and Gene regulation centromere protein homolog subsp. indica dispersed

centromeric repeat tinnily.

tandem centromeric repeat family RCS2.

unknown protein( contains F-box (NM _114761) F-box protein

domain)

calcium ion binding (NM_II6567) putative calmodulin cal reticulin

(NM_I02304) putative calmodulin caln;ticulin

hypothetical calcium-binding protein

(NM_I 04960) calmodulin. putative

F I K23 .18 (lipase/hydrolase) (A Y08843 71 lipase/hydrolase,

putative

H+-transp01ting two-sector A TPase H+-transpotting two-sector (EC 3.6.3.14) beta chain ATPase

H+-transpotting two-sector A TPase ( EC 3.6.3.14) gamma chain OSAI mRNA for H-ATPase

precursor

H+-transpmting two-sector A TPase (EC 3.6.3.14) gamma chain

precursor

cysteine synthase tEC 4.2.99.8) 3A cysteine synthase ( rcs3)

mRNA mRNA for serine

serine carboxypeptidase-like protein carboxypeptidase-like protein

serine cbp3 gene t()r carboxypeptidase Ill

AP2 EREBP transctiption l~tctor (NM

-147879) similar to AP2

BABY BOOM domain transcription factor.

putative

phosphoglycerate kinase ( EC (N M _I 06603) phosphoglycerate 2.7.2.3) precursor kinase, putative

Cytosolic phosphoglycerate kinase (NM _I 12114) phosphoglycerate

kinase

( N M _I 121 14) phosphoglycerate kinase

phosphoglycerate kinase ( EC 2 723)0BP44

Receptor kinase 6 receptor kinase-like protein

gene, family member E

Plastidic aldolase (Fragment 1 mRNA loraldolaseC-1

ti·uctose-bisphosphate aldolase ( EC 4.1.2.13) precursor

tiuctose-bisphosphate aldolase ( EC 4.1.2.13) precursor( ti<.tgment)

Fructose-bisphosphate aldolase ( EC 4.1.2.13) '

Serine-threonine kinase (NM_IOII03) putative serine/threonine kinase

serine/threonine-specific protein kinase ARA.KIN homolog

TI5FI6J

2'-hydroxyisotlavone reductase ( EC (Al 161501) putative protein 1.3.1.45) (transcriptional repressor) (transcriptional repressor)

FllSO 14.30 (similar to isotlavone reductase)

putative calcium-dependent protein calmodulin-kinase

mRNA f(>r calcium-kina>e CPKI adapter protein 2 dependent protein-kinase

mRNA liH· protein cdc2 kinase

Protein kinase 2 (ACO I 181 0) putative protein kinase

At I g64300iFI5H21_13 {protein protein kinase homolog TI3E15.16

kinase)

At I g64300iFI5H21_13 (protein kinase)

malate dehydrogenase ( EC I .1.1.37) putative malate dehydrogenase mRNA f()r glyoxysomal malate dehydrogenase

Putative mitochondrial NAD-dependent malate dehydrogenase

Putmive malate dehydrogenase

Cytosolic malate dehydrogenase (EC 1.1.1.37)

14-3-3-like protein 14-3-3 protein homolog GF 14 chi

chain

(ABOII479) 14-3-3 protein GFI4-

glycine hydroxymethyltransferasc glycine hydrnxymethyltransfcrasc (EC 2.1.2.1 I like protein

Putative glycine h) drox ymethyltransferase

glycine hydroxymethyltransfcrasc (EC 2.12 I)

LEA 76 homologue type2 group 3 LEA protein (lea)

gene

glycine dehydrogenase (NM_I29089) glycine

(dccarboxylating) (EC 1.4.4.21 component P precursor

decarboxylase complex H-protein

NBS-LRR type disease resistance NBS-LRR type resistance protein Rps 1-k-1 protein ( r3) gene

NBS-LRR type resistance protein ( r2 1 gene

NBS-LRR type resistance protein (rll) gene

Centrin (BT000613) T5I7-ATPase families mRNA for cyc07

OsRS2 mRNA for FCA protein( fragment) (NM _122070) gennin-like protein transcription factor rough

sheath 2

unknown protein auxin-regulated protein MAP kinase homolog

(MAPKI) mRNA

FCA protein (Fragment) (ACOI 1810) DNA binding protein (var. IR36) PIR7a and PIR7b

GT-1 genes.

Transketolase (Fragment) nuclear receptor binding factor-like transcription activator REB

protein (Reb) gene.

OSJNBa0042F21.1 3 protein RRM-containing protein mRN A for Dof zinc finger

protein

syringolide-induced protein (MYB) putative methyltransferase domain, DNA for repetitive sequence DUF248, putative ankyrin protein RGS

(AJ251 087) ABI3-interacting mRNA for polypeptide

putative diacylglycerol kinase protein 2, AIP2

chain-binding, partial sequence

auxin binding protein (ABP44); putative pectate-lyase mRNA for OsNAC3 protein

isovaleryl-CoA Dehydrogenase

mitochondrial nad9 gene for DNA-binding protein-like putative methyltransferase NADH dehydrogenase

subunit9

transcription factor/ zinc ion mpa22_p_30 (putative stellar K+

DNA forphyBI gene binding

outward rectifying channel, (SKOR) protein)

Hypothetical protein At2g43170 (AC069273) deoxyguanosine OsiFI-1 mRNA for FIFO-

kinase, putative A TPase inhibitor protein

putative ZF-HD homeobox protein (NM_l27384) syntaxin SYPII2 Sgtl (Sgtl) mRNA

G5bf protein homeobox protein knotted-! like 2

mRNA forPib (KNAT2) (ATKI)

glycerate dehydrogenase (EC nuclear receptor binding factor-like lipoxygenase (CM-LOX2) 1.1.1.29) protein mRNA

maturase K (AB052241) MYB transcription NADP-specific isocitrate

factor Atmyb2 dehydrogenase mRNA

maturase dioxygenase-like protein MADS box protein MADS!

gene, promoter

(AL021636) putative protein RFTI gene for IT-like

putative OsD305 (similarity to HSR201 protein, Nicotiana tabacum)

protein

AY045676 NID (WRKY-DNA (NM_I04470) serine lipoxygenase (CM-LOXI) binding domain) acetyl transferase mRNA

Homeobox-leucine zipper protein, (U75205) germin-like protein

endogenous double-stranded putative RNA encoding polyprotein

DNA cytosine methyltransferase (U75205) germin-like protein

hypothetical protein, fertilin Zmet3, putative alpha subunit

mitochondrion cox3 gene for (U75205) germin-like protein cytochrome oxidase subunit

3

(NM_l22070) germin-like protein G II A protein

TAT A-binding protein-associated SEC31 mRNA

phosphoprotein Dr!

(U75205) germin-like protein embryonic flower !-like

protein mRNA

(AC023628) putative GTP-binding mRNA for OsNAC4 protein.

protein

(NM _11253 7) translationally abscisic acid-and stress-

controlled tumor protein-like inducible protein (Asrl)

protein

TAT A-binding protein-associated phosphoprotein Dr! protein mRNA for RPRI

homolog

hypothetical protein (plant mRNA for allergenic protein, transposase (Ptta/En/Spm family)) clone RA14c

(NM_l25904) MADS box pRRD3 gene promoter transcription factors-like protein region

(NM_ll2768) GTP-binding protein, putative

(NM_ll2480) GTP-binding protein, putative

(AJ250184) NlMIN-1 protein

(NM_l26660) En/Spm-like transposon protein

(NM _121 041) bZIP transcription factor, OBF4

CHP-rich zinc finger protein, putative

(AF488587) putative bHLH transcription factor

(NM _112341 ) hypothetical protein; (zinc finger BED type profile)

(AF439975) SGT!a (TPR repeat, SGS domain)

probable transcription factor MYB34

putative bHLH transcription factor similar to the MYC

(NM _I 02535) putative clathrin-coat assembly protein

germin-like protein

MADS-box protein like

histone acetyltransferase HA T1

putative protein [contained serine-rich region]

putative protein (contained serine-rich region)

glyceraklehydc-3-piH>sphatc glyccraldchydc-3-phosphatc DNA replication and dehydrogenase (NADP) gl yccra luchydc-3-phosphatc Jehyurogenase ( GAPDH)

transcription I phosphmylating) (EC 1.2.1.13) B Jehyurogcnasc ( EC 1.2.1.12) mRNA precursor

g I ycera luehyde-3 -phosphate glyccra!Jchyuc 3-phosphatc

Jchyurogenasc (phospho1ylating) (EC 12.112)

dehydrogenase

g.lyceraldehyde-3-phosphate dehydrogenase (NADP) glyccraldehydc-3-phosphate

(phosph01ylating) (EC 12.113) A dehydrogenase precursor

glyceraldehyde-3-phosphate dehydrogenase (NADP)

(phosph<llylating) (EC 1.2.1 13) A precursor

glyceraldehyde-3 -phosphate dehydrogenase (NADP)

(phosph01ylating) (EC 1.2.1.13) A precursor

glyceraldehydc-3-ph<>sphate dehydrogenase ( N A Dl')

(phosphorylating) ( EC 12. 113) B precursor

Glyceraldehyde-3-phosphate dehydrogenase 8 subunit

Retrotransposon protein, putative, reverse transcriptasc gen.: of Ty-1 copia subclass Copia-like rctrotransposon

Putative glycine-rich RNA-binding DNA-directed RNA polymerase I transgene, right border of protein 2 190K chain-like protein integrated DNA

Putative glycine-rich RNA-binding putative Helicase C contained in LINE retrotransposon,

protein 2 DEAD/DEAH box endonuclease region of

RILNI4

Putative glycine-rich RNA binding (NM_l22996) replication protein transgene, left border of protein I Al-like integrated DNA

Putative glycine-rich RNA-binding (NM_lll360) DNA-damage- Plai (Ngam) ACS mRNA for protein 2 repair/toleration protein DRTI02 ACC synthase

Putative glycine-rich RNA-binding RNA-binding protein homolog DNA for repetitive sequence protein I Fl8A5.250 RG2

Putative glycine-rich RNA-binding putative protein; contains similarity

protein 2 to DNA mismatch repair protein MutS2 from Synechocystis sp.

Putative glycine-rich RNA-binding expressed protein (serine-rich protein 2 region profile, S I domain)

Putative glycine-rich RNA-binding putative U2 small nuclear

protein 2 ribonucleoprotein A (U2 SNRNP-

A)

Putative glycine-rich RNA-binding chloroplast RNA-binding protein protein 2 cp33-homolog, putative

Putative glycine-rich RNA binding chloroplast RNA-binding protein protein I cp33-homolog, putative

Putative glycine-rich RNA-binding chloroplast RNA-binding protein protein 2 cp33-homolog, putative

Putative glycine-rich RNA binding protein I

Putative glycine-rich RNA-binding protein 2





Aspartate carbamoyltransferase





AAA A TPase, central region; Homeodomain-like

S 19 self-incompatibility ribonuclease

Metabolism beta-1.3-glucanase endo-1. 3-beta-glucanase

mRNA

beta-1 ,3-glucanase

Glycolate oxidase like protein glycolate oxidase, putative

(Fragment)

Glycolate oxidase like protein glycolate oxidase

(Fragment)

Ribulose-! ,5-bisphosphate beta-glucosidase beta-amylase gene.

carboxylase/oxygenase large subunit (Fragment)

Ribulose-! ,5-bisphosphate OSamy-c gene for alpha-

carboxylase large subunit beta-glucosidase homolog-precursor (Fragment) amylase

Ribulose I ,5-bisphosphate RAmy3A gene for alpha-

carboxylase small subunit precursor beta-glucosidase (EC 4.1.1.39)

amylase

Methionine synthase beta-glucosidase alpha-amylase mRNA, clone

pOSI37

probable alanine-glyoxylate transaminase (EC 2.6.1.44) Enolase

[imported]

mRNA for cytosolic pyruvate

Protein degradation AF041258 NID ( ATP dt·pendcnt (NM_129557) 265 protcasomc

265 proteasome regulatOJy subunit) regulato1y subunit

(AY062556) 265 proteasomc regulatory subunit

(NM _115865) E3 ubiquitin ligase Protein disulfide isomerase

Putative FtsH-like protein Pftf SCF complex subunit SKPI/ASKl (At5), putative

precursor

Cyclin-like F-box; Agenet DegP protease

Hypothetical protein (kelch repeat-(NM_ll3709) DegP protease

containing F-box family protein)

(NM_l28260) 20S proteasome alpha subunit G (PAGI)

(NM_I28260) 20S proteasome alpha subunit G (PAGJ)

(NM _128260) 20S proteasome alpha subunit G (PAG I)

multicatalytic endopeptidase complex, alpha subunit

multicatalytic endopeptidase complex, alpha subunit

(NM_II9701) cysteine protease XCPl

Protein folding dnaK-type molecular chaperone (NM_I20328) dnaK-typc molecular dnaK-lype molecular CS5 I precursor c hapcrone hsc 70. I chaperone F20DI0.30

Chaperon in 60 alpha subunit CHAPERONIN H5P60

MITOCHONDRIAL

probable chaperonin 60 beta chain Chaperonin 60

probable chapcronin 60 beta chain

Prohibitin, putative HSP70-like protein Prunus dulcis heat shock

PHB2 hypothetical protein, heat shock

simulans heat shock transcription factor HSF8

HSP90-like protein

Structure Actin actin

Actin (Fragment) (NM - 115235) actin (ACTJ)

(NM _112764) actin 2

(AF486849) tubulin t<>lding alpha-tubulin (TubA I) gene, cofactor B promoter and complete cds.

Kinesin-rdated protein (NM_ll6758) kinesin-like protein

A

Beta-conglutin myrosinase binding like protein alpha-expansin (EXP16)

gene

Histone H3 (Fragment) myrosinase binding like protein mRNlA for prolamin, strain: lambda RMI.

Putative MAR binding filament-like (AF054906) myrosinase-binding beta-expansion (EXPB2) protein I protein homolog mRNA

myrosinase binding like protein cellulose synthase-like protein OsCsiF4 gene. .. myrosinase-associated protein, cellulose synthase-like

putative protein OsCsiA6 gene.

(NM_ll2517) putative lectin subsp. japonica RSWI-like cellulose synthase catalytic

(A Y088397) putative lectin

putative lectin, (AC001645) jasmonate inducible protein isolog

putative lectin

F-actin capping protein alpha chain

(NM_123567) luminal binding protein

putative fibrillin (contained PAP _fibrillin domain)

Translation putative translation elongation

translational elongation Translational elongation !actor Tu !actor EF-T u precursor I ike

factor Tu (tufA) gene. homolog

putative tmnslation elongation Putative elongation J:tctor !-gamma litctor EF-Tu precursor like

hotnolog

Elongation tactor !-gamma

TufA

Translation elongation !actor EF-Tu precursor. chlnroplast

Elongation Etctor-1 alpha-:~

tAF0~527lJ) hypothetical EIF-2- el F4A-2 gene li>r cuk<nyotic Alpha initiation tact or 4A

40S tibosomal protein S5 40S ribosomal protein SA tlaminin

receptor homolog)

putative 40S ribosomal protein SA ( laminin receptor-like protein)

putative 40S ribosomal protein SA ( laminin receptor-like protein)

40S ribosomal protein SA (p40) ( laminin receptor homolog)

40S tibosomal protein SA (p40) ilaminin receptor homolog)

tNM 12S766) 40S ribosomal -proteinS 12

(NM 12X766) 40S ribosomal -proteinS 12

Ribosomal protein subunit 2 ribosomal protein L4

rps I 0 gene tor mitocondtial (Fragment) ribosomal protein S I 0

A Y085926 NID 60S acidic ribosomal protein P2 mitocondrial rrnl8 gene for ISS rRNA, partial sequence.

(A Y08 7 50 I) 60S acidic ribosomal protein P2

50S ribosomal protein l21-homolog

(X90855) L1 protein

(X90855) L1 protein

(X90855) L1 protein

30S ribosomal protein S5

putative 60S acidic ribosomal protein PO

50S ribosomal protein l21-homolog

60S ribosomal protein L12

60S ribosomal protein L12

60S ribosomal protein-like protein

Miscellaneous A TP synthase beta chain A TP synthase beta chain.

(Fragment) mitochondrial precursor

AtpB (Fragment) A TP synthase beta chain

A TPase beta subunit

oxygen-evolving complex protein I mitochondrial atp6 gene

precursor

Carbonic anhydrase (EC 4.2.1.1) Halobacterium sp.NRC-1

Carbonic anhydrase (EC 4.2.1.1) Sulfolobus solfataricus

Formate dehydrogenase protein phosphate I mRNA

Ferredoxin-binding subunit

protochlorophyllide reductase (EC 1.3 .1.33) precursor

putative phytochelatin synthetase

L-ascorbate peroxidase

2-cys peroxiredoxin-like protein

NADPH oxidase

Glyoxalase/bleomycin resistance protein/ dioxygenase

Chlorophyll alb binding protein precursor

Superoxide dismutase (EC 1.15.1.1) (Mn) I

Transport ATRRAN2 (A YOX4274) Rab-type small GTP- rab 16B gene. rab 16B-j allele.

binding protein-like par1ial.

Hypothetical protein Atlg55040-zf-RanBP

Putative sorbitol transporter

Amino acid transporter-like protein

Putative oligopeptide transporter

Aenine nucleotide translocator

Unknown Brain protein 44-like

mRNA J(,r brain specific protein (S94 gc·ne)

Brain protein 44-like


Brain prokin 44-like






At2g39730ff517 .3 (NM 106632) unknown protein genomic DNA, chromosome

I, PAC clone:P0004A09

genomic DNA, chromosome Hypothetical protein Atlg62750 (NM_ll3293) hypothetical protein 6, BAC

clone:OSJNBa0007020

A TU32176 NID mudrA-Iike protein ( transposon- mRNA EN77, partial

like) sequence.

VFU14956 NID (AY069906) F4Pl2_50 genomic DNA, chromosome I, BAC clone:OJIIII G12.

DRSCPRBCB NID (NM_ll3031) unknown protein mitocondrial DNA for ORF

155,strain IR 62829 B

chromosome 10 clone hypothetical protein unknown protein OSJNBa0079B05, complete

sequence.

hypothetical protein (A Y088216) unknown genomic DNA, chromosome

6, 08007 RFLP marker.

genomic DNA, chromos unknown protein (NM _11194 7) expressed protein Borne l,BAC

clone:Bl003B09.

Hypothetical protein (AC009322) hypothetical protein

genomic DNA, chromosome

At4g02550/TIOPII.l6 6,BAC

clone:OSJNBa0038F22.

Hypothetical protein chromosome I 0 clone

OSJNBbOOIIHI5.25 (NM_l18310) hypothetical protein OSJNBa0079B05, complete

sequence.

Putative polyprotein (NM I 03636) expressed protein genomic DNA, chromosome

I, clone:P0503G09.

OSJNBaOOIIF23.7 protein (NM_l28973) hypothetical protein genomic DNA, chromosome

I, PAC clone:P0707DIO.

Protein At4g02550 (NM_l29149) hypothetical protein random single-copy DNA

fragment 09RG136F.

Hypothetical protein P0031 A09.11 (AP000737) genomic DNA, chromosome

gene id:LA522.1-unknown protein 6, BAC clone:JNBa0033

Hypothetical protein P0033D06.7 (AC013354) Fl5H18.22 chromosome I 0 BAC clone

OSJNBa0057L21

OSJNBa0049H08.9 protein (NM 119322) hypothetical protein mRNA EN320, partial

sequence.

hypothetical protein F7 A 19.27 (NM 102665) expressed protein mRNA EN205, partial

sequence.

Hypothetical protein (Fragment) (A Y039905)F17 A22.12 Genomic sequence for Oryza

sativa, Nipponbare Strain,

protein F2119.20 [imported] hypothetical protein F18F4.130 Hd3a gene,

cultivar:Nipponbare.

(A Y085177) unknown mRNA ENIIO, partial

sequence.

(NM 118545) putative protein

(A Y065463) unknown protein

hypothetical protein T7M24.5

(NM 112128) expressed protein

putative protein

(NM_II9726) putative protein

(NM_II3243) expressed protein

hypothetical protein T2711.15

(AF367319)nunn10_180

hypothetical protein

expressed protein

(A Y080851) unknown protein

(AYI36403) unknown protein

hypothetical protein

expressed protein

a) Proteins identified in this study. b) Proteins identified in Arabidopsis nucleus (Bae eta!., 2003 ). c) Proteins identified in Rice nucleus (Khan and Komatsu, 2004). d) The matched proteins are indicated in pink.

C. arietinum

A. thaliana 0. sativa

Figure 2.6. Comparative analyses of plant nuclear proteome. Venn diagram depicting overlaps within Arabidopsis thaliana, Oryza sativa and Cicer arietinum nuclear proteomes. The numbers signify the unique and/or orthologous proteins among the organisms studied.

Table 2.3. Comparison of Nuclear Proteins Identified in Chickpea, Arabidopsis and Rice.

Functional Identification Cicer Arabidopsis Oryza sativae catej!ory arietinum0 thalianah

Signaling and gene Centromere protein - 15233570 M AF078903 regulation homolog AF058902

F-box protein CaN-282 15229075 M -

Ca-binding protein CaN-140 115480 M T05703 15236276 M T05703 15221593 M 11135591 M

Lipase/hydrolase CaN-283 21594055 -

H+ ATPase CaN-135 - S17916 CaN-298 D10207 CaN-353

Cysteine synthase - 2118307 M AF073697 Serine carboxypeptidase CaN-310 - D17587

Dl0985 AP2 transcription factor CaN-331 22326940 M -Phosphoglycerate kinase CaN-180 15230595 M -

CaN-183 15230595 M 2129669 M 15219412 M

Receptor kinase CaN-221 - U72724 Aldolase CaN-119 - D50301

CaN-184 CaN-244 CaN-299

Ser/Thr kinase CaN-305 15221284 M -

CaN-450 Transcriptional repressor CaN-369 7267238 M -

CaN-291 Ca dependent protein CaN-5 15221781 M Dl3436

kinase D64036 Protein kinase CaN-137 9958055 -

CaN-148 7488270 M CaN-229

Malate dehydrogenase CaN-293 15219721 M D85763 CaN-296 CaN-359 CaN-439

14-3-3 protein CaN-8 1361987 -9759623

Glycine CaN-447 15235745 M -

hydroxymethyltransferase CaN-446 CaN-517

LEA protein - 15232660 M AF046884 Glycine decarboxylase CaN-402 15226973 M -

NBS-LRR type resistance CaN-238 AF032689 protein AF032698

AF032690 DNA replication Glyceraldehyde-3- CaN-242 81622 M AF010582 and transcription phosphate dehydrogenase CaN-269 16974416

CaN-297 15222848 M CaN-358 CaN-363 CaN-366 CaN-398

Copia-like CaN-208 - Z75500 retrotransposon

Metabolism 13-1 ,3-glucanase CaN-139 - AF337174 CaN-473

Glycolate oxidase CaN-503 15231850 M -

CaN-541 15229497 M Protein degradation 26S proteasome CaN-370 15225611 M -

regulatory subunit 17064960 M Protein folding DnaK-type molecular CaN-44 15241849 M T05618

chaperone Chaperonin 60 CaN-38 - P80502

CaN-82 AF489695 CaN-133

Structure Actin CaN-72 71633 M -

CaN-118 18409908 M 15230191 M

Tubulin - 20514259 AF182523 Kines in CaN-103 15234638 M -

Translation EF-Tu CaN-130 23397095 AF327413 CaN-195 23397095 CaN-257 CaN-279 CaN-304 CaN-535

ElF - 4588003 AB046415 40S ribosomal protein CaN-550 11467967 M -

15218458 M 15218458 M 11467967 M 11467967 M 15225180 M 15225180 M

Ribosomal protein CaN-558 21537296 AB035347 subunit

Miscellaneous A TP synthase CaN-81 - Q01859 CaN-105 Q07233 CaN-235

Transport RAN binding protein CaN-307 21536533 AF333275 CaN-347

Unknown Brain protein CaN-27 - Dl6140 CaN-57 CaN-116 CaN-157 CaN-188 CaN-511 CaN-172 CaN-228 CaN-491

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 (Bae et al., 2003). c) Accession numbers according to Rice pro teo me database (Khan and Komatsu,

2004).

processes (Raikhel and Coruzzi, 2003). 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 170 proteins are 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 non-classical proteins were identified in

the chickpea nucleus, which have never been associated with this compartment.

The proteins involved in signaling and gene regulation (32%) were found to be

the most abundant. Spots 1 and 236 were identified to be a RNA-binding protein,

FCA. It is a plant specific nuclear protein that is involved in flowering time control

and was touted as an ABA receptor (Razem et al., 2006). However, this conclusion

was later retracted as it was found that FCA does not bind ABA (Risk et al., 2008).

The role of this protein in dehydration response would be of great interest. 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 (Anderson et al., 1995). Aldolase (spots 119, 184, 244 and 299) was

included in this category as it was identified as a DNA-binding protein (Ronai et al.,

1992). Apparently, aldolase is also located in the perinuclear space and functions as a

nuclear protein in plants (Anderson et al., 1995). A few kinases were found to be a

part of nuclear proteome of chickpea (spots 137, 148, 229, 305 and 450). A 14-3-3-

like protein (spot 8) was also identified in the nuclear proteome. Although the

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 (Brunet et al., 2002). Proteins

involved in calcium signaling were also observed in chickpea proteome, particularly

calcium dependent protein kinase CPK1 adapter protein 2 (spot 5) and calcium ion

binding protein (spot 140). Spot 331 is an AP2/EREBP transcription factor, BABY

BOOM, which functions mainly as a developmental regulator of cell/organ identity

and fate (Boutilier et al., 2002). Quite a few other transcription factors were identified

in the nuclear fraction. Another protein, DNA cytosine methyltransferase Zmet3

(CaN-240) was also present, which is known to regulate gene transcription by

methylating cytosine residues in the DNA (Loidl, 2004). It is interesting to note that

proteins involved in signaling and gene regulation dominated other categories,

reflecting the role of nucleus in gene expressiOn and regulation. While, in other

58

organelles, like chloroplast and mitochondria, the largest percentage of proteins were

reported to be involved in energy production, either in electron transport or in A TP

production (Millar et al., 2001).

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 amino

terminal RNA-binding domain and a Gly-rich carboxy-terminal domain. These

proteins have already been reported in the Arabidopsis nucleolar proteome (Pendle et

al., 2005). GAPDH was also identified as multiple protein spots. It was earlier

detected in Arabidopsis and rice nuclear proteomes. Recent evidence suggests that

besides its glycolytic activity, GAPDH is a multifunctional protein with both

cytoplasmic and nuclear functions, one of which is an essential component of a

transcriptional activator complex regulating histone expression (Pendle et al., 2005).

Aspartate carbamoyltransferase (A TCase) 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 (Nagy et al., 1982).

Structural proteins like actin (spots 72 and 118) and kinesin (spot 103)

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 (Grzanka et al., 2004; Hu et al., 2004; Percipalle et al., 2003).

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 (MFP 1 ). The interaction of chromatin

with the nuclear matrix via matrix attachment regions (MARs) on the DNA is

important for higher-order chromatin organization and the regulation of gene

expression. The animal nuclear matrix proteins with the greatest structural similarity

to MFPI are the nuclear lamins (Harder et al., 2000).

Proteins involved in translational machinery are standard in case of any

nuclear proteome. In our study, 5% of the total identified proteins belong to this

category. Eukaryotic elongation factor (eEF-la) plays a pivotal role in protein

biosynthesis, present mainly in the cytoplasm, but a small population of eEF -1 a

molecules has been previously identified in the nucleus where it forms a complex

with zinc finger protein (Gangwani et al., 1998). Another protein in this category was

59

the ribosomal protein, which have also been identified in the nuclear proteome of

Arabidopsis (Bae et al., 2003) and Medicago (Repetto et al., 2008).

The molecular chaperones account for 4% 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 (Nallen et al., 2001). Spot 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 (Nijtmans et al., 2000).

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 (Takechi et

al., 2000). ATP-dependent 26S proteasome regulatory subunit (spot 370) was another

protein in this category.

Several metabolism-related proteins have also been identified in the chickpea

nucleus. Spot 139 and 4 73 represent f3-l, 3-glucanase and fall under this category.

These proteins are members of 0-Glycosyl hydrolases family that hydrolyse 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 (Khan and Komatsu, 2004). 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 (Bae et al., 2003). 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.

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 (Gao et al., 2003).

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 (Noiraud et al.,

2001). The other important protein identified is a RAN2 (spot 347, Ras-related protein

60

in the nucleus), an intracellular signaling protein which acts as a major regulator of

nucleocytoplasmic transport (Braunwarth et al., 2003). RanBP, Ran-Binding protein

(spot 212) works in close association with Ran during this process. It is thought that

the function of RanBP is to watch-out for Ran-cargo complexes coming out of the

nuclear pore (Vetter et al., 1999). An amino-acid transporter-like protein (spot 164), a

putative oligopeptide transporter (spot 198) and an adenine nucleotide translocator

(spot 560) make up 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 starting from A TP

synthase (spots 81 and 105) involved in energy production to a putative phytochelatin

synthetase (spot 557), a protein involved in metal-ion homeostasis (Grill et al., 1989).

On the other hand, NADPH oxidase (spot 158), glyoxalase (spot 176), ascorbate

peroxidase and superoxide dismutase (spot 344) might be involved in ROS

metabolism directly or indirectly.

For a complete understanding of cellular functions, it is important to determine

and characterize (a) the proteomes at different subcellular locations and (b) their

involvement in biosynthetic and signaling pathways. Most of the identified proteins

are verified nuclear proteins as evident by the literature. However, few of the

nonclassical proteins were identified in the chickpea nucleus that have never been

associated with this compartment. These results, in part, are in agreement with the

previous reports on plant nuclear proteomes and also show 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. In conclusion, a combined

approach of subcellular isolation followed by careful purification can significantly

enhance the systematic identification of nuclear proteins. This information, coupled

with analysis of biological processes, molecular functions, and regulatory networks,

can be used to gain insiglits into the complexity of functions controlled by the protein

machinery in the nucleus. 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 onto increasing the number of analyzed proteins with an aim to draw a

complete functional map of nuclear proteome. Further, we will focus on identifying

61

the dynamics associated with the nuclear proteome toward cells metabolic and

regulatory pathways at different physiological conditions.

62

analysis of nuclear pro teo me in...

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