Proteomics Approach to Identify Dehydration Responsive Nuclear Proteins from Chickpea (Cicer arietinum L.)*S

Dehydration or water-deficit is one of the most important environmental stress factors that greatly influences plant growth and development and limits crop productivity. Plants respond and adapt to such stress by altering their cellular metabolism and activating various defense machineries. Mechanisms that operate signal perception, transduction, and downstream regulatory events provide valuable information about the underlying pathways involved in environmental stress responses. The nuclear proteins constitute a highly organized, complex network that plays diverse roles during cellular development and other physiological processes. To gain a better understanding of dehydration response in plants, we have developed a comparative nuclear proteome in a food legume, chickpea (Cicer arietinum L.). Three-week-old chickpea seedlings were subjected to progressive dehydration by withdrawing water and the changes in the nuclear proteome were examined using two-dimensional gel electrophoresis. Approximately 205 protein spots were found to be differentially regulated under dehydration. Mass spectrometry analysis allowed the identification of 147 differentially expressed proteins, presumably involved in a variety of functions including gene transcription and replication, molecular chaperones, cell signaling, and chromatin remodeling. The dehydration responsive nuclear proteome of chickpea revealed a coordinated response, which involves both the regulatory as well as the functional proteins. This study, for the first time, provides an insight into the complex metabolic network operating in the nucleus during dehydration.

Environmental stress limits growth, development, and crop productivity (1,2) and plays a major role in determining the geographic distribution of plant species. Several environmental stresses are united by the fact that at least part of their detrimental effect on plant performance is caused by disruption of plant water status. Periods of little or no rainfall can lead to a meteorological condition called drought. However, water deficit or dehydration, can also occur at conditions in which water is not limiting, through altered ion content and water uptake caused by high salinity or by formation of extracellular ice during freezing. Desiccation is the extreme form of dehydration wherein most of the protoplasmic "free" water is lost and the cell survival relies on the 'bound' water associated with the cell matrix. Dehydration is one of the most common environmental stresses to which plants are exposed, and in many regions it is the bottleneck of agricultural development (3). There is hardly a physiological process in plants that is not impaired by water deficit or dehydration. However, very few plants have been subjected to biochemical and molecular studies to analyze the mechanisms of dehydration stress tolerance. It appears that the intrinsic ability of plant to tolerate stress is a result of different biochemical and molecular mechanisms, and the elucidation of the nature of these mechanisms would be an interesting area of research. The stress perception and signal transduction to switch on adaptive responses are critical steps in determining the survival of plants exposed to adverse environments. Based on the presence of general and specific stress tolerance mechanisms (4), it is logical to expect plants to have multiple stress perception and signal transduction pathways, which may cross-talk at various steps. The alteration of protein synthesis or degradation is one of the fundamental metabolic processes that may influence dehydration tolerance (5,6). Both quantitative and qualitative changes of proteins have been detected during dehydration stress (7). Increasing evidence indicates a relationship between the accumulation of dehydration-induced proteins and physiological adaptations to water limitation (7,8).
Most studies on dehydration to date have mainly focused on the changes in gene expression, while there is far less information available on their functional products. The changes in gene expression are regulated by a number of different, and potentially overlapping, signal transduction pathways (9,10). However, the level of mRNA does not always correlate well with the level of protein, the key player in the cell (11,12). It is thus insufficient to predict protein expression level from quantitative mRNA data. Proteome studies aim at the complete set of proteins encoded by the genome and thus complement the transcriptome studies. Nevertheless, the resolution of protein spots on a two-dimensional gel is limited by several factors such as protein abundance, size, hydrophobicity, and other electrophoretic properties (13). Therefore, the complete proteome is not recommended but is rather fractionated into subproteomes, to improve sensitivity and resolution and to reduce the overall complexity. Further, the compartment specific proteome is preferred because fractionated subsets of proteins provide the suitable information in which they exert their particular function (14 -16). The nucleus is the subcellular organelle that contains nearly all the genetic information required for the regulated expression 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. The nuclear proteome is dynamic, changing its composition in response to intracellular and environmental stimuli.
Legumes are valuable agricultural and commercial crops that serve as important nutrient sources for human diet and animal feed. The characteristic feature of legumes is the biological nitrogen fixation. Chickpea (Cicer arietinum L.) is one of the most important grain legumes comprising high protein content, 25.3-28.9% (17). The bulk of chickpea is produced and consumed in South Asia and increasingly in Middle East and some Mediterranean countries. India is the largest producer of chickpea in the world. It is relatively dehydration tolerant (3) and is traditionally cultivated as a second crop mostly during the dry season. Its growth is limited mainly by the content of the remaining soil moisture, and dehydration tolerance is thus the most important ecophysiological trait for determining the occurrence of reliable harvests. In a previous study, we developed the nuclear proteome map of chickpea (16). Here, we have reported the nucleus-specific comparative proteome of chickpea to identify novel components involved in dehydration tolerance with a wider aim to use them in future crop improvement program. The quantitative image analysis revealed 205 protein spots that changed their intensities significantly for more than 2.5-fold, at least, at one time point during dehydration. A total of 147 differentially expressed nuclear proteins were identified during the course of dehydration using classical two-dimensional electrophoresis coupled with LC-MS/ MS. The comparison of dehydration responsive nuclear proteome in chickpea reveals predicted and unexpected components indicating their possible role in dehydration tolerance.

EXPERIMENTAL PROCEDURES
Plant Growth, Maintenance, and Dehydration Treatment-Seeds of chickpea (Cicer arietinum L. var. JG-62) were obtained from ICRISAT (International Crops Research Institute for the Semi-Arid Tropics), Hyderabad, India and grown in pots containing a mixture of soil and soilrite (2:1, w/w; 10 plants/1.5 L capacity pots with 18 cm diameter) in an environmentally controlled growth room. The seedlings were maintained at 25 Ϯ 2°C, 50 Ϯ 5% relative humidity under 16 h photoperiod (270 mol m Ϫ2 s Ϫ1 light intensity). The pots were provided with 100 ml of water everyday that maintained the soil moisture content to ϳ30%. A gradual dehydration condition was applied on the 3-week-old seedlings by withdrawing water and tissues were harvested at every 24 h up to 144 h. The unstressed and the stressed plants were kept in parallel in the same growth room. The samples from the unstressed (control) plants were collected at each time point during dehydration and were finally pooled to normalize the growth and developmental effects. The harvested unstressed and stressed tissues were instantly frozen in liquid nitrogen and stored at Ϫ80°C.
Isolation of Pure Nuclei-The nuclei were isolated as described earlier (16). The integrity of the isolated nuclei was analyzed by staining with 4Ј,6Ј-diamidino-2-phenylindole hydrochloride (DAPI) 1 . The nuclear fraction was stained for 15 min with 0.1 g/ml DAPI in 0.1 M potassium phosphate buffer (pH 7.4) and then washed twice with phosphate-buffered saline. For microscopy, a small volume of suspension was placed on a slide, covered with a cover glass and the images were captured with or without UV filter.
Extraction of Nuclear Protein and Two-dimensional Electrophoresis-The nuclear proteins were prepared from the nuclei-enriched pellet using TriPure Reagent (Roche) according to the manufacturer's instructions. The 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). The enrichment of nuclear proteins was evaluated by immunoblot analysis for two nuclear resident proteins, fibrillarin and histone core. IEF was carried out with 150 g of protein.
Image Acquisition and Data Analysis-Gel images were digitized with a Bio-Rad FluorS equipped with a 12-bit camera. The PD Quest version 7.2.0 (Bio-Rad) was used to assemble first level matchset (master image) from three replicate two-dimensional electrophoresis gels. Experimental molecular mass and pI were calculated from digitized two-dimensional electrophoresis images using standard molecular mass marker proteins. Each spot included on the standard gel met several criteria: it was present in at least two of the three gels and was qualitatively consistent in size and shape in the replicate gels. We defined "low-quality" spots as those with a quality score Ͻ30; these spots were eliminated from further analysis. The remaining highquality spot quantities were used to calculate the mean value for a given spot, and this value was used as the spot quantity on the standard gel. The first level matchset spot densities were normalized against the total density in the gel image. The replicate gels used for making the first level matchset had, at least, correlation coefficient value of 0.8. After obtaining the first level matchsets, a second level matchset that allowed a comparison of the standard gels from each of the time points was obtained. A second normalization was done with a set of three unaltered spots identified from across the time points. From this matchset, the filtered spot quantities from the standard gels were assembled into a data matrix of high-quality spots from the seven time points for further analysis.
Protein Identification and Expression Clustering-Protein samples were excised mechanically using pipette tips, destained, in-gel digested with trypsin and peptides extracted according to standard techniques (18). Peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (LC/MS/TOF) using an Ultimate 3000 HPLC system (Dionex) coupled to a Q-Trap 4000 mass spectrometer (Applied Biosystems). Tryptic peptides were loaded onto a C18PepMap100, 3 m (LC Packings) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). The MS/MS data were extracted using Analyst Software v.1.4.1 (Applied Biosystems). Peptides were identified by searching the peak-list against the MSDB 20060831 (3239079 sequences; 1079594700 residues) database using the MASCOT v.2.1 (http://www.matrixsciences.com) search engine. Because the chickpea genome sequence is not known, a homology based search was performed. The database search criteria were: taxonomy, Viridiplantae (Green Plants, 247882 sequences); peptide tolerance, Ϯ1.2 Da; fragment mass tolerance, Ϯ0.6 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 Ͻ 0.05 is set by Mascot algorithm and is based on the size of the database used in the search. The details regarding the precursor ion mass, expected molecular weight, theoretical molecular weight, delta, score, rank, charge, number of missed cleavages, p value, and the peptide sequence for proteins identified with a single peptide are mentioned in supplemental document 1. Further, the fragment spectra for these proteins are provided in supplemental document 2. Wherein there were more than one accession numbers for the same peptide, the match was considered in terms of putative function. In the case of same protein being identified in multiple spots where several peptides were found to be shared by the isoforms, differential expression pattern was observed for each of the candidate and thus the proteins were listed as independent entities. The function of each of the identified protein has been analyzed in view of the metabolic role of the candidate protein in the nucleus. The protein functions were assigned using a protein function database Pfam or Inter-Pro. As functional annotation is based on pFam and Interpro, the functional redundancy, if any, is thus greatly minimized.
Self-organizing tree algorithm (SOTA) clustering was performed on the log transformed fold induction expression values across seven time points by using Multi Experiment Viewer software (TIGR). The clustering was done with Pearson correlation as distance with 10 cycles and maximum cell diversity of 0.8 (19).
Immunoblot Analysis-Immunoblotting was done by resolving nuclear protein on a uniform 12.5% SDS-PAGE and then electrotransferring onto nitrocellulose membrane at 150 mA for 2 h. The membranes were subsequently blocked with 5% (w/v) nonfat milk for 1 h and incubated with respective primary antibodies for 2 h. Further, the blots were incubated with alkaline phosphatase conjugated secondary antibody for 1 h and the signal was detected using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate method.
Enzyme Assays-The isolated nuclei pellet was suspended and homogenized in 100 mM triethanolamine (Tea, pH 7.4) for superoxide dismutase (SOD), 50 mM of KPO 4 Ϫ buffer (pH 7.0) for ascorbate peroxidase (APx), and 57 mM KPO 4 Ϫ buffer (pH 7.5) for nitrate reductase (NR), respectively. The homogenate was centrifuged at 16,000 ϫ g for 20 min at 4 C. The supernatant was transferred into fresh tube and was used for the assay of SOD, APx, and NR. SOD activity was determined by spectrophotometric method based on the inhibition of superoxide-driven NADH oxidation (20). The assay mixture contained 100 mM triethanolamine (pH 7.4), 100 mM/50 mM EDTA/MnCl 2 , 7.5 mM NADH, and 10 mM mercaptoethanol in a total volume of 1.0 ml. The oxidation of NADH was followed at 340 nm (an absorbance coefficient of 6.2 mM Ϫ1 cm Ϫ1 ). The oxidation rates were initially low, then increased progressively (usually 2-4 min after mercaptoethanol addition) to yield a linear kinetics (12-15 min), which were used for calculation. APx was assayed from the decrease in absorbance at 290 nm (an absorbance coefficient of 2.8 mM Ϫ1 cm Ϫ1 ) as ascorbate is oxidized by its activity (21). The reaction mixture for the peroxidase contained 50 mM potassium phosphate (pH 7.0), 0.5 mM ascorbate, and 0.1 mM H 2 O 2 in a total volume of 1.0 ml. The reaction was initiated by adding H 2 O 2 , and the absorbance was recorded 30 s after the addition. Correction was done for the low, nonenzymatic oxidation of ascorbate by H 2 O 2 . On the other hand, the activity of NR was determined in the reaction mixture containing 57 mM potassium phosphate (pH 7.5), 0.005 mM FAD, 0.2 mM NADPH, and 10 mM potassium nitrate. The oxidation of NADH was followed at 340 nm (an absorbance coefficient of 6.2 mM Ϫ1 cm Ϫ1 ) for ϳ5 min and the maximum linear rate was used to calculate the activity (22).

Two-dimensional Electrophoresis of Nuclear Proteins of Chickpea-
The primary objective of this study was to characterize global protein expression in nucleus of chickpea during dehydration. Chickpea seedlings were subjected to gradual dehydration over 144 h. There were no visible changes in the seedlings during 24 h of dehydration. The leaflets rolled after 36 h of dehydration, and the damage aggravated further during 48 -144 h. The nuclei were isolated from the seedlings 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 DAPI staining (Fig. 1A). The nuclear FIG. 1. Analysis of isolated chickpea nuclear fraction. A, the purified nuclear fraction was stained with DAPI and visualized by confocal microscopy. Phase contrast micrograph of the nuclei is shown in the left panel, while the DAPI-stained nuclei are shown in the right panel. B, immunoblot analysis of extracted nuclear proteins with anti-histone core and anti-fibrillarin antibodies. Aliquots of 100 g 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 one-dimensional gel was electroblotted onto Hybond-C membrane, and histones and fibrillarin were detected using alkaline phosphatase conjugated secondary antibodies.
proteins were prepared from the purified nuclei using TriPure reagent (Roche Diagnostics) 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, fibrillarin and histone core (Fig. 1B). Isolated nuclear proteins from control and dehydrated chickpea seedlings were resolved and detected using high-resolution two-dimensional electrophoresis followed by silver staining as detailed in "Experimental Procedures." For each time point, three replicate two-dimensional electrophoresis gels were run, which were then computationally combined into a representative standard gel (Fig. 2). The only spots that survived several stringent criteria (classified as "high-quality" spots) were used to estimate spot quantities; otherwise, a large number of protein spots were included in the matchset. For example, 321 spots were detected in the control gel, but 316 were classified as high quality (Table I). The spot densities at the lower level were normalized against the total density present in the respective gel to overcome the experimental errors introduced due to differential staining. To make comparison between the time points, a second level matchset was created (Fig. 3). The intensity of spots was normalized to that of landmark proteins used for internal standardization. From the higher level matchset, the filtered spot quantities were assembled into a data matrix that consisted of 501 unique spots indicating change in intensity for each spot during dehydration. The data reveal that nearly 95% of the spots on the standard gels were of high quality reflecting the reproducibility of the experimental replicates (Table I).
More than 400 protein spots were reproducibly detected on silver-stained gels. Quantitative image analysis revealed a total of 205 protein spots that changed their intensities significantly by more than 2.5-fold at least at one time point. While most spots showed quantitative changes, some spots showed qualitative changes also. Six typical gel regions are enlarged as shown in Fig. 4. Of the 205 dehydration responsive proteins (DRPs), 80 proteins were clearly up-regulated and 46 were down-regulated, while 79 proteins showed a mixed pattern of time-dependent expression.
Identification of the Differentially Expressed Proteins-MS/MS analysis was carried out for 147 DRPs resulting in 105 proteins with a significant match, representing an identification success rate of ϳ70%. As an advantage of using classical two-dimensional electrophoresis, we could detect different isoelectric species of various proteins. For example, five isoelectric species were detected for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is suggestive of posttranslational modification(s). This type of information cannot be acquired using transcriptomic approaches. Although adept at resolving isoelectric species, the technique of two-dimensional electrophoresis is somewhat restricted at quantifying low abundance proteins. This might account for the absence of many of the transcription factors in differential proteome of chickpea nucleus. Despite the known limitation of current two-dimensional electrophoresis methodology at detecting underrepresented proteins, the differential expression pattern of 105 nuclear proteins would nevertheless help in elucidating the complex regulatory network involved in dehydration tolerance.
The identified DRPs might play a variety of functions during cellular adaptation against dehydration, e.g. gene regulation,  signaling, proteolysis, and chaperone type activities. All the identified DRPs were grouped into 10 functional classes based on their respective roles in dehydration response ( Fig.   5 and Table II). The proteins for which no known function could be assigned were grouped under the unknown category that accounted for 24% of the dehydration responsive nuclear proteome. Gene transcription and replication (17%) were found to be the most abundant class of proteins, reflecting the role of nucleus in gene expression and regulation. This was closely followed by the molecular chaperones (16%), which might help in protecting and proper functioning of the cellular machinery following dehydration stress. The cell signaling, chromatin remodeling, protein degradation, and the reactive oxygen species (ROS) pathway related proteins were other major classes identified. Some of these DRPs are likely to be breakdown products as is evident from the gel-based size of these proteins and have been discussed in that light.
Dynamics of Protein Networks during Dehydration-To summarize the information contained in Table II and to cluster the proteins showing similar expression profiles during dehydration response in chickpea, self-organizing tree algorithm clustering was applied to the DRPs. The data were taken in terms of fold expression with respect to the control expression value. Further, the data sets were log transformed to the base two in order to level the scale of expression and to reduce the noise. The analysis yielded 11 expression clusters and only the clusters with n Ն 10 were taken to study the coexpression patterns for functionally similar proteins (Fig. 6). Detailed information on proteins within each cluster can be found in supplemental Fig. 1.
Cluster 1 and 2 both were found to be early dehydration responsive; however, no clear clustering pattern was detected for either of them. Almost all the functional categories are represented in these two clusters. The proteins in Cluster 5 first decreased in relative expression followed by a gradual increase during late dehydration. This cluster is enriched in the ROS pathway-related proteins. Cluster 6, with 17 members, showed maximum co-clustering for the proteins involved in gene transcription and replication. Nonhomogeneous expression patterns were observed for different subunits in protein complexes. For example, dnaK and dnaJ were present in Clusters 5 and 4, respectively, which might underline the complexity of such responses. The unknown category of proteins comprised a major fraction in all the clusters. The fact that the unknown proteins were identified as dehydration responsive gives the first functional annotation to these genes. Identification of these proteins can provide valuable insight into the kinetics of dehydration tolerance mechanisms.
Immunoblot Analysis-Several proteins were identified as the same protein having the same molecular weight but different pI values that were expressed differentially during dehydration, including GAPDH and malate dehydrogenase (MDH). Previously, we showed that some other isoforms of the same protein are also a part of the chickpea nuclear proteome (16) even though these were not found to be differentially expressed under dehydration. Changes in protein levels alone do not imply changes in function as many proteins are known to be regulated by post-translational modifications. Proteins can resolve into multiple spots due to proteolytic processing, multiple isoforms, or changes in charge state resulting from posttranslational modifications, including phosphorylation, deamidation, acetylation, glycation, and glutathionylation (23)(24)(25)(26). It is possible that post-translational modifications may affect in-gel migration of a protein such that a protein classified as repressed may be present elsewhere on the gel or that a putative de novo protein may be found in a new place in the gel. Because some proteins migrated in multiple spots, we have used one-dimensional Western blot analysis to determine whether changes in individual isoelectric variants were representative of the entire protein population. GAPDH and MDH along with 14-3-3 and dnaK were subjected to immunoblot analysis in an attempt to address this discrepancy. 14-3-3 and dnaK both followed exactly the same profile as detected on the two-dimensional electrophoresis gel (Fig. 7 and Table II). GAPDH, which was represented by nine spots (5 differentially expressed and 4 with constitutive expression) on the two-dimensional electrophoresis gel, showed two bands on the one-dimensional Western blot, each showing a different profile. MDH, which had three spots (1 differentially expressed and 2 with constitutive expression) on the two-dimensional electrophoresis gel, showed an up-regulation toward the later time points (Fig. 7).
Effect of Dehydration on SOD, APx, and NR-The functional   TABLE II-continued a Spot number as given on the two-dimensional 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 Fig. 3.

Dehydration Responsive Nuclear Proteome of Chickpea
b Gene identification number as in GenBank. c Protein expression profile represents the average change in spot density at various time points C (Control), 24 h, 48 h, 72 h, 96 h, 120 h and 144 h. The data were taken in terms of fold expression with respect to the control value and were log transformed to the base two in order to level the scale of expression and to reduce the noise. aspect of an enzymatic protein is dependent on its activity rather than on its presence in the proteome. Post-translational modifications are known to affect the activity and also the fact that few of the proteins were expected to be degraded forms owing to their low molecular weight, compelled us to study the enzyme activity for few of the proteins. It is likely that dehydration stress may give rise to increased ROS levels, particularly superoxide radical (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ) (27,28). The SOD catalyzes the dismutation of O 2 Ϫ to molecular oxygen and H 2 O 2 , thus playing a key role in cell defense and rescue mechanism (29). Subsequently, H 2 O 2 can be decomposed by catalase or through a series of oxi-FIG. 6. Clustering analysis of expression profiles of nucleus-specific DRPs in chickpea. The differentially expressed 105 proteins were grouped into 11 clusters based on their expression profiles. The self-organizing tree algorithm cluster tree is shown at the top and the expression profile in self-organizing tree algorithm clusters are shown below. The expression profile of each individual protein in the cluster is depicted by gray lines, whereas the mean expression profile is marked in pink for each cluster. The number of proteins in each cluster is given in left upper corner and the cluster number in the right lower corner. Detailed information on proteins within each cluster can be found in supplemental Fig. 1. doreduction reactions involving ascorbate peroxidase (APx) and glutathione peroxidase (GPx) using ascorbate or reduced glutathione, respectively. Because oxidative stress pathway appeared to play a very important role in the dehydration responsive chickpea nuclear proteome, we investigated the behavior of the antioxidant enzymes, SOD and APx, under dehydration. The enzyme assay showed that both SOD and APx had maximum activity during early stress perception (48 h), whereas APx also showed a late induction at 120 h of dehydration stress. Furthermore, SOD displayed ϳ3-fold increase in activity, whereas APx showed a maximum increase of less than 2-fold (Fig. 8).
Dehydration is known to cause increased NR protein turnover (30). NR was identified as a degraded product with a lower molecular weight than expected (Table II). The activity graph of NR showed that the activity goes down until 72 h and then tries to recover a little (Fig. 8), which indicates that NR is also present as an intact protein in the chickpea nuclear proteome. However, under dehydration stress, the NR protein might undergo degradation even though a direct relationship between the accumulation of degraded product and the activity was not observed. This might be the result of other regulatory mechanisms for this protein. It has been documented that the regulation of maize NR is dependent on both gene expression as well as protein phosphorylation (31). DISCUSSION Within the larger goal of profiling protein expression in plant under dehydration, the aim of this study was to characterize the regulatory and functional pathways operating in chickpea nucleus. A model representing the regulatory and functional network activated under dehydration is depicted in Fig. 9. Proteins of the regulatory network include transcription factors, RNA-binding proteins, protein kinases, and phosphatases (4,(32)(33). The functional network comprises proteins whose activity enables the plant to survive stress conditions and includes proteins involved in ion homoeostasis (34), water channels (35), enzymes involved in scavenging of ROS (36), enzymes involved in the synthesis of osmolytes and compatible solutes (37), and protective proteins (38).

Dehydration Responsive Regulatory Network in Nucleus-
The nucleus is the site where the genetic material is transcribed into mRNAs, which carry the blueprint to synthesize proteins. Because the protein synthesis is a very complicated and highly regulated process, many different proteins are needed to ensure that a gene is transcribed at the right time in the life of a cell. This indicates that a lot of molecules have to get in and out of the nucleus at any given time. The major regulator in this transport is an intracellular signaling protein called Ran (CaN-347), which stands for Ras-related protein in the nucleus. The small Ras-like GTPase Ran was highly upregulated under dehydration (Table II). This protein is known to play a crucial role in nucleocytoplasmic transport in that it FIG. 7. Immunoblot analysis for few of the representative DRPs. The nuclear expression profile for 14-3-3, dnaK, GAPDH, and MDH was detected using one-dimensional Western blot analysis; 100 g of nuclear protein for all seven time points was separated by 12.5% SDS-PAGE and blotted onto Hybond-C membrane. The blots were probed with respective primary antibodies and signal detected. provides the identity of the two compartments and thus ensures directionality of transport (39). The cytoplasmic fibrils of the nuclear pore consist of a very large molecule called RanBP (CaN-212), for Ran-Binding protein. It is thought that the function of RanBP is to watch out for Ran-cargo complexes coming out of the nuclear pore (40). 14-3-3 protein (CaN-8) is also involved in the nucleocytoplasmic transport (41); however, we have included this protein as a part of signal transduction because it is known to have a number of interacting protein partners.
Several proteins involved in early stress responsive signaling were identified in this study. Dehydration is known to induce abscisic acid (ABA) in the stressed cells. It has been documented that ABA can cause an increased generation of O 2 Ϫ (42) and H 2 O 2 (43,44), and induce the expression and activity of antioxidant genes (42,45,46). FCA (CaN-1, 236,  711) is a known ABA receptor in nucleus of higher plants (47), which might be involved in this signaling network even though it may correspond to a degradation product as is evident from its gel-based molecular weight. However, there is not much information on the downstream elements of this pathway. Signaling partners like 14-3-3 (CaN-8), protein kinase (CaN-229), serine-threonine kinase (CaN-305), histidine kinase (CaN-640), receptor-like protein kinase (CaN-693) and tyrosine phosphatase (CaN-623) might be involved in relaying this signal to the heterochromatin region so as to regulate gene expression. While all of these signaling partners were early dehydration responsive (up-regulated as early as 24 h), serine-threonine kinase was up-regulated only after 96 h (Table  II). Recently, it has been shown that tobacco cells transformed with an antisense construct of 14-3-3 no longer accumulate ROS (48) suggesting 14-3-3 protein as a very important component in this signaling pathway. NR (CaN-710) is known to catalyze a one electron transfer from NADPH to nitrite to produce nitric oxide (NO) (49,50), a potential signaling intermediate (51). Dehydration stress causes increased NR protein turnover and accelerated mRNA turnover (30, 52) and 14-3-3 proteins are instrumental in bringing about this degradation. The availability of 14-3-3s for binding to phosphorylated-NR controls the stability of NR via proteolysis (53), which explains the concomitant rise in degraded NR levels along with the rise in 14-3-3 protein levels (Table II).
Stress-mediated effects on chromatin remodeling are well established in yeast. Histone deacetylase (HDAC; CaN-603) interacts with the 14-3-3 family of proteins that are known to bind specifically to conserved phosphoserine-containing motifs. 14-3-3 proteins negatively regulate HDAC by preventing its nuclear localization and thereby uncovering a novel regulatory mechanism for HDACs (54). Alternatively, association with 14-3-3 proteins may stimulate the nuclear export of HDAC and might account for the decrease in HDAC protein level after 48 h and subsequent increase after 120 h when 14-3-3 levels go down (Table II). DNA cytosine methyltransferase Zmet3 (CaN-240) was found to be consistently upregulated following dehydration stress. DNA methylation and histone deacetylation work in combination to regulate gene transcription (55,56). High methylcytosine contents and low histone acetylation are frequently associated with silent genes. Matrix or scaffold attachment regions (MARs or SARs) are involved in looping of open chromatin fibers. The interaction of chromatin with the nuclear matrix via MARs on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. MARs have a positive effect on gene expression (57,58) and MAR binding filament-like protein 1 (MFP1; CaN-230) with a filament protein-like structure is a good candidate for a MAR binding constituent of the nuclear filaments (59). H1 histone has long been known to be induced by waterdeficit (60). We found that histones H3 (CaN-574) and H2B (CaN-575) were continuously induced after 48 h of dehydration. The up-regulation of histones might be involved in controlling downstream gene expression during dehydration stress. Actin (CaN-72) is not only a major cytoskeletal component in all eukaryotic cells but also a nuclear protein that plays a role in gene transcription (61). Actin's functional complexity makes it a likely target of oxidative stress. 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with actin-depolymerizing factor protein (62). Kinesin (CaN-103), a microtubule-dependent ATPase, is also thought to be a part of this machinery and was found to be differentially regulated by dehydration stress.
The differential proteome of chickpea nucleus also revealed the presence of a number of glycolytic pathway enzymes. All of these proteins are known to have nonglycolytic function in the nucleus. GAPDH (CaN-242, 269, 363, 398, 682) has been proposed to act as a tRNA-binding protein and may participate in RNA export (63). All five isoforms of this protein showed a different expression profile. Aldolase (CaN-299), on the other hand, was initially identified as a DNA-binding protein when it was purified from SEWA sarcoma cells of vertebrate species (64). The phosphorylation of fructose-bisphosphatase (FBPase) (CaN-628) might be the signal inducing the movement of the enzyme to the nucleus where it might induce the expression of its own gene, although a possibility that FBPase influences the expression of other genes cannot be ruled out (65). Transketolase (CaN-251), another enzyme of the same pathway, was also found to be differentially regulated under dehydration stress. Retrotransposons have been shown to be specifically activated by environmental stress signaling (66), which was again corroborated by our results showing a constant increase in the protein levels as early as after 24 h of dehydration.
Numerous studies have shown that transcription factors are important in regulating plant responses to environmental stress. Different members of transcription factors regulate the gene expression in response to dehydration stress. However, many of these would have escaped detection as the expression level for transcription factors is generally not very high. The AP2/ EREBP transcription factor BABY BOOM (CaN-331), involved in plant growth and development (67), was found to be completely shutdown under dehydration. Other transcription factors identified in this study are homeobox-leucine zipper protein (CaN-233), homeobox protein HB3 (CaN-713), putative transcription factor (CaN-18), and WRKY-DNA binding domain protein (CaN-338), all of which showed an up-regulation. The exact function of these proteins can only be predicted after extensive studies, and might be interesting for future investigations.
Dehydration Responsive Functional Network in Nucleus-Whether ABA acts upstream of ROS or vice-versa is controversial as gene expression via ROS-independent ABA signaling is also common in plants. NADPH oxidase (CaN-158), a so-called "respiratory burst oxidase homologue" mediating ABA induced ROS (68), are known nuclear membrane proteins that may produce O 2 Ϫ in the vicinity of membrane ion channels (69). The resulting oxidative species, in turn, act as secondary messengers to control a variety of physiological responses. It is clear that this "respiratory burst" must have an adequate supply of NADPH for ROS generation, which suggests that a transient increase in NADP(H) is a requirement for this signaling pathway (70). The required NADPH for this reaction might be generated by MDH (CaN-293), making it an important member of the ROS pathway (71). Both NADPH oxidase and MDH were down-regulated under dehydration, but their accumulation showed a slight increase around 120 h. Although ROS is necessary for signaling and stress-induced gene expression, excess ROS can cause damage to proteins, DNA, and lipids in the cell. Thus, removal of excess free radicals from the cell is necessary through specific detoxification mechanisms. SOD (CaN-344) constitutes the first line of defense against ROS, thereby converting O 2 Ϫ to H 2 O 2 . APx (CaN-231) and GPx (CaN-644) in turn act in parallel to convert the peroxide ion into molecular oxygen and water. Even though APx and GPx have not been reported in plant nucleus, their presence in animal nuclei (72,73) is known. Methylglyoxal has a binding site on GPx, and its high levels inhibit enzyme activity (74). Since GPx catalyzes the detoxification of H 2 O 2 , its inactivation leads to the accumulation of ROS. Glyoxalase (CaN-176) carry out catabolism of methylglyoxal and is indirectly involved in scavenging ROS (75). 2-cys peroxiredoxins (CaN-6) are a large family of peroxidases, which reduce alkyl hydroperoxides and hydrogen peroxide. Although they are known chloroplastic enzymes, PER1 is one of the first enzymatic plant antioxidants found to be localized in the nucleus (76).
The ROS-mediated proteolytic degradation might be executed by kelch repeat-containing F-box family protein (CaN-350), putative FtsH-like protein Pftf (CaN-84), and ATP-dependent 26S proteasome (CaN-370). Because intact proteins are less sensitive to oxidation than misfolded proteins (77), the protein chaperones get up-regulated in response to stresses. Different chaperones are documented to play complementary, and sometimes overlapping, roles in protection of proteins. The dnaK (CaN-644), an Hsp70-like protein chaperone together with its co-chaperones dnaJ (CaN-715) and GrpE constitutes the KJE system and prevents aggregation of misfolded proteins, thus facilitating their subsequent refolding (78). The small heat shock proteins (sHsps; CaN-641 and 654) are proposed to bind unfolding proteins, providing a reservoir of substrates available for subsequent refolding by the KJE system (79). Redox sensitive molecular targets usually contain highly conserved cysteine residues, and their oxidation, nitration, and formation of disulfide links are crucial events in oxidant/redox signaling. ROS has also been shown to cause increased gene expression of stress response genes (HSP-27, 70, and 90) and antioxidant enzymes (APx, SOD, GPx), mediated or regulated by redox-sensitive transcription factors (such as NF-B and AP-1) (80). Protein translation may represent another way of combating dehydration, which explains the initial increase in protein levels for translation elongation factor (CaN-218 and 345) even though it shows a complete down-regulation after 72 h.
Dehydrins (CaN-600), which are also known as late-embryogenesis-abundant protein group II, have been most extensively studied in relation to drought and cold stresses (81,82). Dehydrins have been proposed to have a protective function during abiotic stress via a number of different mechanisms. These include improving or protecting enzyme activity under cold or dehydration conditions (83,84); or acting as radical scavengers (85) or as membrane stabilizers (81,86). Vicilin (CaN-656) and conglutin (CaN-17) are basically storage proteins, which possess a lectin-type activity. However, there is a report that a pea nuclear protein belonging to the vicilin superfamily gets accumulated under dehydration stress (87). As the protein was shown to share homology with dehydrins, it was suggested that the function of this protein may be related to the protection of chromatin structure against desiccation. Another protein with a chaperone-like activity was the class of glycine-rich RNA binding proteins (GRPs; CaN-25, 63, 132, 166, 277, 339, 375, and 577). These proteins were identified from different parts of the two-dimensional electrophoresis gel representing different molecular weight and pI, which is fairly surprising. The GRPs are RNA chaperones, and have been shown to be responsive to environmental stresses in particular, cold stress (88).
Dehydration stress also showed differential response for a number of ATPases (CaN-105, 135, 235, 298, 353, and 660), glycine dehydrogenase protein (CaN-402), and phenylcoumarin benzylic ether reductase or isoflavone reductase (PCBER/ IFR; CaN-291, 369, and 674). PCBER/IFR is involved in secondary metabolism and plays an important role in plant defense (89). As many as 26 proteins with no known function were also identified in chickpea nucleus. The prediction of a possible function for these proteins under dehydration would make up an interesting study.
In conclusion, we have investigated the molecular responses to dehydration stress in chickpea at nuclear-specific proteome level and identified the probable DRPs that play a variety of cellular functions.