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• W2160492411 abstract "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. 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 (1Araus J.L. Slafer G.A. Reynolds M.P. Royo C. Plant breeding and drought in C-3 cereals: what should we breed for?.Ann. Bot. 2002; 89: 925-940Crossref PubMed Scopus (883) Google Scholar, 2Boyer J.S. Plant productivity and environment.Science. 1982; 218: 443-448Crossref PubMed Scopus (2860) Google Scholar) 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 (3McKersie B.D. Leshem Y.Y. Desiccation.in: McKcKersie B.D. Leshem Y.Y. Stress and Stress Coping in Cultivated Plants. Kluwer, Dordrechet, The Netherlands1994: 132-144Crossref Google Scholar). 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 (4Shinozaki K. Yamaguchi-Shinozaki K. Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways.Curr. Opin. Plant Biol. 2000; 3: 217-223Crossref PubMed Google Scholar), 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 (5Chandler P.M. Robertson M. Gene expression regulated by abscisic acid and its relation to stress tolerance.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1994; 45: 113-141Crossref Scopus (461) Google Scholar, 6Ouvrard O. Cellier F. Ferrare K. Tousch D. Lamaze T. Dupuis J.M. Casse-Delbart F. Identification and expression of water stress- and abscisic acid-regulated genes in a drought-tolerant sunflower genotype.Plant Mol. Biol. 1996; 31: 819-829Crossref PubMed Scopus (116) Google Scholar). Both quantitative and qualitative changes of proteins have been detected during dehydration stress (7Riccardi F. Gazeau P. Vienne D.V. Zivy M. Protein changes in responses to progressive water deficit in maize.Plant Physiol. 1998; 117: 1253-1263Crossref PubMed Scopus (337) Google Scholar). Increasing evidence indicates a relationship between the accumulation of dehydration-induced proteins and physiological adaptations to water limitation (7Riccardi F. Gazeau P. Vienne D.V. Zivy M. Protein changes in responses to progressive water deficit in maize.Plant Physiol. 1998; 117: 1253-1263Crossref PubMed Scopus (337) Google Scholar, 8Bray E.A. Molecular responses to water deficit.Plant Physiol. 1993; 103: 1035-1040Crossref PubMed Scopus (660) Google Scholar). 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 (9Seki M. Narusaka M. Ishida J. Nanjo T. Fujita M. Oono Y. Kamiya A. Nakajima M. Enju A. Sakurai T. Satou M. Akiyama K. Taji T. Yamaguchi-Shinozaki K. Carninci P. Kawai J. Hayashizaki Y. Shinozaki K. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray.Plant J. 2002; 31: 279-292Crossref PubMed Scopus (1634) Google Scholar, 10Shinozaki K. Yamaguchi-Shinozaki K. Gene expression and signal transduction in water-stress response.Plant Physiol. 1997; 115: 327-334Crossref PubMed Scopus (901) Google Scholar). However, the level of mRNA does not always correlate well with the level of protein, the key player in the cell (11Tian Q. Stepaniants S.B. Mao M. Weng L. Feetham M.C. Doyle M.J. Yi E.C. Dai H.Y. Thorsson V. Eng J. Goodlett D. Berger J.P. Gunter B. Linseley P.S. Stoughton R.B. Aebersold R. Collins S.J. Hanlon W.A. Hood L.E. Integrated genomic and proteomic analyses of gene expression in mammalian cells.Mol. Cell. Proteomics. 2004; 3: 960-969Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar, 12Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Crossref PubMed Scopus (3187) Google Scholar). 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 (13Westbrook J.A. Wheeler J.X. Wait R. Welson S.Y. Dunn M.J. The human heat proteome: 2D maps using narrow range immobilised pH gradients.Electrophoresis. 2006; 27: 1547-1555Crossref PubMed Scopus (40) Google Scholar). 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 (14Dreger M. Proteome analysis at the level of subcellular structures.Eur. J. Biochem. 2003; 270: 589-603Crossref PubMed Scopus (103) Google Scholar, 15Bhushan D. Pandey A. Chattopadhyay A. Choudhary M.K. Chakraborty S. Datta A. Chakraborty N. Extracellular matrix proteome of chickpea (Cicer arietinum L.) illustrates pathway abundance, novel protein functions and evolutionary perspect.J. Proteome Res. 2006; 5: 1711-1720Crossref PubMed Scopus (57) Google Scholar, 16Pandey A. Choudhary M.K. Bhushan D. Chatttopadhyaya A. Chakraborty S. Datta A. Chakraborty N. The nuclear proteome of chickpea reveals predicted and unexpected proteins.J. Prot. Res. 2006; 5: 3301-3311Crossref PubMed Scopus (79) Google Scholar). 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% (17Hulse, J. H. (1991) Nature, composition and utilization of grain legumes. p. 11–27. In: Uses of tropical Legumes: Proceedings of a Consultants’ Meeting, 27–30 March 1989, ICRISAT Center. ICRISAT, Patancheru, A.P. 502 324, India.Google Scholar). 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 (3McKersie B.D. Leshem Y.Y. Desiccation.in: McKcKersie B.D. Leshem Y.Y. Stress and Stress Coping in Cultivated Plants. Kluwer, Dordrechet, The Netherlands1994: 132-144Crossref Google Scholar) 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 (16Pandey A. Choudhary M.K. Bhushan D. Chatttopadhyaya A. Chakraborty S. Datta A. Chakraborty N. The nuclear proteome of chickpea reveals predicted and unexpected proteins.J. Prot. Res. 2006; 5: 3301-3311Crossref PubMed Scopus (79) Google Scholar). 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. 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 ± 2 °C, 50 ± 5% relative humidity under 16 h photoperiod (270 μmol m−2 s−1 light intensity). The pots were provided with 100 ml of water everyday that maintained the soil moisture content to ∼30%. 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 −80 °C. The nuclei were isolated as described earlier (16Pandey A. Choudhary M.K. Bhushan D. Chatttopadhyaya A. Chakraborty S. Datta A. Chakraborty N. The nuclear proteome of chickpea reveals predicted and unexpected proteins.J. Prot. Res. 2006; 5: 3301-3311Crossref PubMed Scopus (79) Google Scholar). The integrity of the isolated nuclei was analyzed by staining with 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) 1The abbreviations used are: DAPI, 4′,6′-diamidino-2-phenylindole hydrochloride; SOD, superoxide dismutase; APx, ascorbate peroxidase; NR, nitrate reductase; DRPs, dehydration responsive proteins; HDAC, histone deacetylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MDH, malate dehydrogenase; GRPs, glycine-rich proteins; ROS, reactive oxygen species; ABA, abscisic acid. . The nuclear fraction was stained for 15 min with 0.1 μg/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. 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 μg of protein. Aliquots of proteins were diluted with two-dimensional rehydration buffer [8 M urea, 2 m thiourea, 2% (w/v) CHAPS, 20 mm dithiothreitol, 0.5% (v/v) pharmalyte (pH 4–7), and 0.05% (w/v) bromphenol blue], and 250 μl of solution was used to rehydrate immobilized pH gradient strips (13 cm; pH 4–7). Protein was loaded by in-gel rehydration method onto IEF strips, and electrofocusing was performed using the IPGphor system (Amersham Biosciences, Bucks, United Kingdom) at 20 °C for 30,000 Vh. The focused strips were subjected to reduction with 1% (w/v) dithiothreitol 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). 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 <30; these spots were eliminated from further analysis. The remaining high-quality 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 samples were excised mechanically using pipette tips, destained, in-gel digested with trypsin and peptides extracted according to standard techniques (18Casey T.M. Arthur P.G. Bogoyevitch M.A. Proteomic analysis reveals different protein changes during endothelin-1- or leukemic inhibitory factor-induced hypertrophy of cardiomyocytes in vitro.Mol. Cell. Proteomics. 2005; 4: 651-661Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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.matrixscience.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, ±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. 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 (19Romijin E.P. Christis C. Wieffer M. Gouw W.J. Fullaondo A. Sluijs P. Braakman I. Heck A.J.R. Expression clustering reveals detailed co-expression patterns of functionally related proteins during B cell differentiation.Mol. Cell. Proteomics. 2005; 4: 1297-1310Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). 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. The isolated nuclei pellet was suspended and homogenized in 100 mm triethanolamine (Tea, pH 7.4) for superoxide dismutase (SOD), 50 mm of KPO4− buffer (pH 7.0) for ascorbate peroxidase (APx), and 57 mm KPO4− buffer (pH 7.5) for nitrate reductase (NR), respectively. The homogenate was centrifuged at 16,000 × g for 20 min at 4ο 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 (20Paoletti F. Aldinucci D. Mocali A. Caparrini A. A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts.Anal. Biochem. 1986; 154: 538-541Crossref Scopus (521) Google Scholar). The assay mixture contained 100 mm triethanolamine (pH 7.4), 100 mm/50 mm EDTA/MnCl2, 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−1 cm−1). 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−1 cm−1) as ascorbate is oxidized by its activity (21Nakano Y. Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts.Plant Cell Physiol. 1981; 22: 867-880Google Scholar). The reaction mixture for the peroxidase contained 50 mm potassium phosphate (pH 7.0), 0.5 mm ascorbate, and 0.1 mm H2O2 in a total volume of 1.0 ml. The reaction was initiated by adding H2O2, and the absorbance was recorded 30 s after the addition. Correction was done for the low, nonenzymatic oxidation of ascorbate by H2O2. 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−1 cm−1) for ∼5 min and the maximum linear rate was used to calculate the activity (22Gilliam M.B. Sherman M.P. Griscavage J.M. Ignarro L.J. A spectrophotometric assay for nitrate using NADPH oxidation by aspergillus nitrate reductase.Anal. Biochem. 1993; 212: 359-365Crossref PubMed Scopus (207) Google Scholar). 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 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).Fig. 2Dehydration responsive comparative proteome of chickpea nucleus and the representative two-dimensional gel electrophoresis. Three-week-old chickpea seedlings were subjected to dehydration and tissue harvested every 24 h until 144 h. The nuclear proteins were isolated from the control and the stressed tissue. An equal amount (150 μg) of protein from each time point was separated by two-dimensional gel electrophoresis. Three replicate gels for each time point (A) were computationally combined using PDQuest software to generate the standard gels (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IReproducibility of 2-dimensional gelsTimeAverage no. of spotsaAverage number of spots present in three replicate gels of each time point.High quality spotsbSpots having quality score more than 30 assigned by PDQuest (Ver.7.2.0).ReproducibilityControl (h)32131698.44%2431028792.584834433095.937230527991.489631229895.5112025824896.1214429929197.65Total2149204995.34a Average number of spots present in three replicate gels of each time point.b Spots having quality score more than 30 assigned by PDQuest (Ver.7.2.0). Open table in a new tab Fig. 3Higher level matchset of protein spots detected by two-dimensional gel electrophoresis. The matchset was created in silico from seven standard gels for each of the time point as depicted in Fig. 2. The boxed areas marked with dotted lines represent the zoomed in gel sections in Fig. 4. The numbers correspond with the spot ID mentioned in Table II.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. MS/MS analysis was carried out for 147 DRPs resulting in 105 proteins with a significant match, representing an identification success rate of ∼70%. As an advantage of using classical two-dim" @default.
• W2160492411 created "2016-06-24" @default.
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• W2160492411 date "2008-01-01" @default.
• W2160492411 modified "2023-10-09" @default.
• W2160492411 title "Proteomics Approach to Identify Dehydration Responsive Nuclear Proteins from Chickpea (Cicer arietinum L.)" @default.
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• W2160492411 doi "https://doi.org/10.1074/mcp.m700314-mcp200" @default.
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