Comparative Proteomics of Salt Tolerance in Arabidopsis thaliana and Thellungiella halophila Qiuying Pang,†,‡ Sixue Chen,‡,§ Shaojun Dai,† Yazhou Chen,† Yang Wang,† and Xiufeng Yan*,† College of Life Sciences/Key Laboratory of Forest Tree Genetic Improvement and Biotechnology (Ministry of Education), Northeast Forestry University, Harbin 150040, China, Department of Biology, UF Genetics Institute, and Plant Molecular & Cellular Biology Program, University of Florida, Gainesville, Florida 32611, and Proteomics Facility, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida 32610 Received January 15, 2010
Salinity is a major abiotic stress affecting plant cultivation and productivity. Thellungiella halophila is a halophyte and has been used as a model for studying plant salt tolerance. Understanding the molecular mechanisms of salinity tolerance will facilitate the generation of salt tolerant crops. Here we report comparative leaf proteomics of Arabidopsis, a glycophyte, and its close relative Thellungiella, a halophyte, under different salt stress conditions. Proteins from control and NaCl treated Arabidopsis and Thellungiella leaf samples were extracted and separated by two-dimensional gel electrophoresis. A total of 88 protein spots from Arabidopsis gels and 37 protein spots from Thellungiella gels showed significant changes. Out of these spots, a total of 79 and 32 proteins were identified by mass spectrometry in Arabidopsis and Thellungiella, respectively. Most of the identified proteins were involved in photosynthesis, energy metabolism, and stress response in Arabidopsis and Thellungiella. As a complementary approach, isobaric tag for relative and absolute quantification (iTRAQ) LC-MS was used to identify crude microsomal proteins. A total of 31 and 32 differentially expressed proteins were identified in Arabidopsis and Thellungiella under salt treatment, respectively. Overall, there were more proteins changed in abundance in Arabidopsis than in Thellungiella. Distinct patterns of protein changes in the two species were observed. Collectively, this work represents the most extensive proteomic description of salinity responses of Arabidopsis and Thellungiella and has improved our knowledge of salt tolerance in glycophytes and halophytes. Keywords: salt tolerance • Arabidopsis thaliana • Thellungiella halophila • 2D gel • 8-plex iTRAQ • LC-MS/MS
Introduction Soil salinity is a major environmental stress that adversely affects crop productivity and quality in the world.1,2 Plant response and tolerance to salt stress has been a focus of plant biology research.3-8 In general, high salt concentration can cause ion imbalance, hyperosmotic stress and oxidative damage.9 Most plants under salt stress conditions exhibit slow growth, wilting or even death.10-13 To survive the stress, plants respond and adapt with complex mechanisms that include developmental, morphological, physiological and biochemical strategies.14 Many genes involved in membrane transport, signal transduction, redox reaction and other processes have been identified.15,16 However, the detailed molecular mechanisms underlying plant tolerance to salt stress remain unclear. * To whom correspondence should be addressed. Xiufeng Yan, Ph.D., College of Life Sciences, Northeast Forestry University, Harbin 150040, China. Tel: (352) 273-8103. Fax: (352) 273-8284. E-mail:
[email protected]. † Northeast Forestry University. ‡ Department of Biology, UF Genetics Institute, and Plant Molecular & Cellular Biology Program, University of Florida. § Interdisciplinary Center for Biotechnology Research, University of Florida.
2584 Journal of Proteome Research 2010, 9, 2584–2599 Published on Web 04/08/2010
Arabidopsis thaliana is an excellent model with rich genetic resources for modern plant biology research. There have been many reports on salt tolerance in Arabidopsis.4,7,8,17-22 However, the outcome of such work is rather limited because of the fact that Arabidopsis is a true glycophyte. Studies using Arabidopsis reveal little information on salt tolerance, and certain novel adaptive responses may be overlooked when using a glycophyte as the exclusive model. Halophytes are plants that can tolerate saline soils and can complete their life cycle in soils with salinity above 200 mM NaCl. Some species require high salt concentrations for optimal growth. Recently, a halophytic plant species Thellungiella halophila has been proposed as an ideal model for studying molecular mechanisms of salt tolerance in plants because of its “extremophile” characteristics manifested by extreme tolerance to high salinity.5,20,23-26 As a model experimental system, Thellungiella shares many of the advantages of Arabidopsis. It has a small genome (less than twice the size of the Arabidopsis genome), a short life cycle, high seed yield and ease of being transformed.5,20 Moreover, Thellungiella is a close relative of Arabidopsis. Analysis of ESTs revealed 90-95% nucleotide identity in transcripts of well-known housekeeping genes in both spe10.1021/pr100034f
2010 American Chemical Society
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cies. A small number of sequences with lower identity were also found, suggesting the presence of paralogous genes.15 Despite the close genetic relationship and a similar morphological appearance, the two species differ considerably in their biochemistry and physiology in salt stress response and adaptation. To date, there have been studies on plant osmotic adjustment,27 ion distribution,24 membrane salt overly sensitive 1 (SOS1) and H+-ATPase activity,28 gene transcription regulation,15 and transcriptomics16 using Thellungiella and Arabidopsis. Research using Thellungiella is primarily focused on salt-tolerant genes and eco-physiological aspects of salt tolerance.29-31 Although Thellungiella plants are salt tolerant and can grow in 500 mM NaCl medium, they do not have salt glands or other morphological alterations. It was hypothesized that the mechanisms of salt tolerance in halophytes may be similar to those in glycophyte and that subtle differences in regulation result in large differences in tolerance or sensitivity.5 The gene expression profiling of Thellungiella obtained using full-length Arabidopsis cDNA microarrays showed that only a few genes were induced by 250 mM NaCl treatment, in strong contrast to in Arabidopsis. Notably, a large number of stress inducible genes, including Fe-superoxide dismutase (Fe-SOD), pyrroline 5-carboxylase synthase (P5CS), plant defensin 1.2 (PDF1.2), 9-cis-epoxycarotenoid dioxygenase (AtNCED), Pprotein, β-glucosidase and SOS1 were expressed at high levels in Thellungiella even in the absence of stress.14 None of the studies mentioned above offered insights into the quantity and quality of the final gene products, that is, proteins. Proteomic applications provide a powerful tool for the study of plant response to salt stress.32-34 The technology has been particularly useful in analyzing and comparing synthesis, turnover and modification of proteins during plant growth, development or response to environmental changes.35 Comparative proteomics has been successfully applied for systematic scrutiny of proteins in several plant species under a wide range of abiotic challenges, including salt stress,36-38 drought,39-41 high or low temperature,42-44 ultraviolet radiation,45,46 heavy metals47 and herbicides.48,49 In the present study, a systematic proteomic analysis was conducted to investigate proteins responsive to salt stress in Arabidopsis and Thellungiella during vegetative growth. Proteins from control and salt-treated Arabidopsis and Thellungiella samples were extracted and separated by two-dimensional gel electrophoresis (2-DE). Differentially expressed proteins were identified by nanoflow liquid chromatography (LC) coupled to Q-Trap MS/MS and Mascot database searching. In addition, multiplex isobaric tag for relative and absolute quantification (iTRAQ) and two-dimensional LC-MS was employed to identify and quantify differentially expressed microsomal proteins involved in salinity responses in Arabidopsis and Thellungiella. A comprehensive inventory of salinity responsive proteins was established.
Materials and Methods Plant Material and NaCl Treatment. Seeds of Arabidopsis (ecotype Col-0) were obtained from the Arabidopsis Biological Resource Center. Seeds of Thellungiella (ecotype Shandong) were kindly provided by Dr. Hui Zhang at Shandong Normal University (Jinan city, China). Arabidopsis and Thellungiella seeds were surface sterilized and maintained at 4 °C for 3 and 10 days, respectively, to break seed dormancy. Seeds were sown in small pots (5 cm diameter, 6 cm deep) filled with soil and
research articles vermiculite mixture (1:1). Seedlings were grown in a growth chamber with an 8 h/16 h light/dark cycle, 25 °C/20 °C day/ night temperature, 150 µmol m-2 s-1 light intensity and a relative humidity of 60%. The plants were irrigated with water every three days and were free from attack by pests. Four-weekold Arabidopsis and 6-week-old Thellungiella (similar in size and developmental stage) were irrigated with water (control) and 50 mM and 150 mM NaCl. Plant materials were harvested directly into liquid nitrogen after 5 days of salt treatment and stored at -80 °C freezer. Three independent biological replicates were prepared for each sample. The NaCl concentrations were chosen according to previous reports showing that 150 mM NaCl inhibited Arabidopsis root elongation up to 78%. Concentrations higher than 200 mM NaCl can completely inhibit root growth and led to death of the seedlings.21 Our own data demonstrated that after 150 mM NaCl treatment for 5 days Arabidopsis plants exhibited notable salt stress phenotypes but could survive the treatment (data not shown). Tissue Water Content and Relative Electrolyte Leakage Analysis. Plant fresh weight (FW) was measured immediately after harvesting. Dry weight (DW) was determined after drying for 72 h in an oven at 60 °C. Relative tissue water content (TWC) was calculated as follows: TWC ) [(FW - DW)/FW] × 100. For the relative electrolyte leakage (REL) assay, leaf materials were rinsed with ddH2O, placed in test tubes containing 10 mL of ddH2O, and incubated at room temperature for 2 h. The electrical conductivity of the solution (C1) was measured using a conductivity meter (Orion 115Aplus; Thermo Electron). Then, the tubes were boiled for 15 min and cooled to room temperature, and the electrical conductivity (C2) was measured again. The REL was calculated using the formula C1/C2 × 100%.50 Preparation of Microsomal Membranes. Microsomal membranes were isolated as previously described.51 Approximately 10 g fresh leaves of each sample were harvested from control and 150 mM NaCl treated samples and ground into fine powder in liquid nitrogen. Ground tissues were suspended in 50 mL ice-cold homogenization medium containing 50 mM Tris-HCl, 2 mM EDTA, 10 mM β-mercaptoethanol, 250 mM sucrose, pH 7.5. The resulting slurry was filtered through two layers of cheesecloth and centrifuged at 8000× g for 15 min at 4 °C. The supernatant was recovered and centrifuged again to remove debris. The resulting supernatant was centrifuged at 100 000× g for 1 h at 4 °C in a Beckman ultracentrifuge (Beckman Coulter, USA). The pellet was resuspended in 1 mL ice-cold 100 mM sodium carbonate and homogenate with a dounce homogenizer. The homogenate was centrifuged at 100 000× g for 1 h at 4 °C. The pellet was washed with 80% cold acetone and stored at -80 °C freezer. 2-DE and Image Analysis. Total protein extraction and quantification were conducted according to previous methods.52 Each sample containing 1.3 mg total protein in 450 µL rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer (pH 4-7), 0.04 M DTT, and 0.002% Bromophenol Blue) was loaded onto a 24 cm, pH 4 to 7 linear gradient IPG strip (GE Healthcare, USA). Isoelectric focusing was performed using an Ettan IPGphor isoelectric focusing system according to the manufacturer’s instruction. Briefly, active rehydration was carried out at 30 V for 8 h, followed by 50 V for 4 h, 100 V for 1 h, 300 V for 1 h, 600 V for 1 h, 1000 V for 1 h, a linear increase of voltage to 8000 V for 12 h at 20 °C. After IEF, the strips were equilibrated with an equilibration solution (50 mM Tris pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue) containing 1% DTT, followed by 2.5% iodoacJournal of Proteome Research • Vol. 9, No. 5, 2010 2585
research articles etamide in the equilibration solution, each for 15 min. The second dimension was performed on 12.5% polyacrylamide gels using an Ettan DALT Six Electrophoresis Unit (GE Healthcare, USA) according to the manufacturer’s instructions. The 2-DE experiments were repeated three times using protein samples prepared independently from Arabidopsis and Thellungiella materials, respectively. Proteins were visualized by Coomassie brilliant blue R250 staining, and gel images were acquired using an ImageScanner (GE Healthcare, USA). Image analysis was performed with ImageMaster 2D Platinum Software Version 5.0 (Amersham Biosciences). Experimental Mr (kDa) of each protein was estimated by comparison with the Mr markers, and experimental pI was determined by its migration on the IPG strip. The abundance of each protein spot was estimated by the percentage volume (% Vol). Only those with significant and reproducible changes were considered to be differentially expressed proteins. In-Gel Digestion and Protein Identification. Protein in-gel tryptic digestion and nanoESI MS/MS analysis were carried out on a QSTAR XL MS/MS system (Applied Biosystems/MDS Sciex, USA) as previously described.52 For MALDI-TOF/TOF MS analysis, tryptic peptides were desalted with C18 Ziptips (Millipore) and spotted onto a MALDI plate by mixing 1:1 with the matrix solution (10 mg/mL CHCA in 60% ACN and 0.1% TFA). MS/MS spectra were acquired using a 4700 MALDI-TOF/ TOF mass spectrometer (Applied Biosystems/MDS Sciex, USA). The peptide MS/MS spectra were searched against NCBI nonredundant fasta database (8 224 370 entries, downloaded on April 14, 2009) using MASCOT search engine (http:// www.matrixscience.com). Mascot was set up to search green plants only, assume trypsin digestion and one allowed miscleavage. The mass tolerance for both parent ion and fragment ion mass was set to be 0.2 Da. Iodoacetamide derivatization of Cys, deamidation of Asn and Gln, and oxidation of Met are specified as variable modifications. Unambiguous identification was judged by the number of peptides, sequence coverage, MASCOT mowse score and the quality of MS/MS spectra (Supplemental Figure 1, Supporting Information).52 iTRAQ Labeling and 2D LC-MS/MS. The acetone pellets from four control samples and four 150 mM NaCl treated microsomal protein samples (each of 100 µg protein) from Arabidopsis and Thellungiella were dissolved in 1% SDS, 100 mM triethylammonium bicarbonate, pH 8.5. The samples were reduced, alkylated with methylmethanethiosulfate, trypsindigested and labeled using the iTRAQ Reagents 8-plex kit according to the manufacturer’s instructions (Applied Biosystems, USA). The control replicates were labeled with iTRAQ tags 113, 114, 115 and 116, and the 150 mM NaCl replicates were labeled with tags 117, 118, 119 and 121, respectively. After labeling, the two groups of microsomal protein samples were mixed and lyophilized dry before dissolving in strong cation exchange (SCX) solvent A (25% v/v acetonitrile, 10 mM ammonium formate, pH 2.8). The peptides were fractionated on an Agilent HPLC system 1100 using a polysulfethyl A column (2.1 × 100 mm, 5 µm, 300 Å, PolyLC, Columbia, MD). Peptides were eluted at a flow rate of 200 µL/min with a linear gradient of 0-20% solvent B (25% v/v acetonitrile, 500 mM ammonium formate) over 50 min, followed by ramping up to 100% solvent B in 5 min and holding for 10 min. The absorbance at 214 nm was monitored, and a total of 19 fractions were collected. The fractions were analyzed on a nanoflow LC-MS system as previously described.53 2586
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Pang et al. iTRAQ Data Analysis. The iTRAQ MS/MS Data were processed by a thorough search considering biological modification and amino acid substitution against the NCBI nonredundant database using the Paragon algorithm54 of ProteinPilot v3.0 software (Applied Biosystems, USA). Plant species, fixed modification of methylmethanethiosulfatelabeled cysteine, fixed iTRAQ modification of free amine in the N-terminus and lysine, and two miscleavages of trypsin digestion were considered. The mass tolerance for both MS and MS/MS was 0.2 Da. The raw peptide identification results from the Paragon algorithm were further processed by the ProGroup algorithm. The ProteinPilot cutoff score used was 1.3, which corresponds to a confidence limit of 95%. For protein relative quantification, only MS/MS spectra unique to a particular protein and where the sum of the signal-to-noise ratio for all of the peak pairs greater than nine were used for quantification (software default settings). The mean, standard deviation, and p-values to estimate statistical significance of the protein changes were calculated by ProGroup. Proteins were selected for further analysis based on the following criteria: at least three confident MS/MS spectra (allowing generation of a p-value), a p-value smaller than 0.05, and a fold change of at least two in more than one independent experiment.
Results and Discussion Morphological and Physiological Changes Under Salt Stress. Exposure of Arabidopsis seedlings to 5-day salt stress led to chlorosis in young leaves under 50 and 150 mM NaCl. Apparently, 150 mM NaCl treatment was more effective (Figure 1A). Despite the physical deterioration, the plants could survive the 5-day treatment. In contrast, Thellungiella did not exhibit obvious chlorosis under the above conditions (Figure 1B). It was clearly more tolerant to salt treatment than Arabidopsis. We then measured tissue water content and relative electrolyte leakage. NaCl treatment caused a significant decrease in tissue water content in Arabidopsis, whereas it only caused a slight decrease in Thellungiella (Figure 1C). This suggests that under high salt conditions water uptake was impaired in Arabidopsis, which could affect a series of physiological processes. Relative electrolyte leakage (REL) is an indicator of membrane damage. After a 5-day salt treatment, REL increased in both Arabidopsis and Thellungiella (Figure 1D). However, the increase in Arabidopsis was much higher than that in Thellungiella. Interestingly, Thellungiella also showed significant electrolyte leakage as previously reported.55 The salt tolerance capability of Thellungiella may be closely related to the maintenance of ion homeostasis and membrane recovery under salt stress.56 Identification of Salt Stress-Responsive Proteins by 2-DE and MS. Comparative proteomic analysis was used to investigate the changes of protein profiles under different salt stress conditions. Leaves from control and treated plants were harvested, and total protein was extracted and separated by 2-DE. After image analysis, more than 1100 protein spots were reproducibly detected and matched between all the gels. Representative 2D gel maps are shown in Figures 2 and 3. Proteins were well separated in both dimensions. The isoelectric points (pI) of the spots ranged from 4.2 to 7 and the molecular mass ranged from 10 to 130 kDa. Such high quality 2D gel maps are essential for detailed quantitative analysis and protein identification (Figures 2 and 3). Protein spots from all the replicate gels were compared and quantified using the ImageMaster 2D Platinum Software Version 5.0 (Amersham Biosciences). The normalized percentage volumes (% Vol) of
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Figure 1. Morphology and physiological changes of Arabidopsis and Thellungiella growing under different NaCl concentrations. (A) Arabidopsis and (B) Thellungiella were grown in soil and vermiculite mixture (1:1) and irrigated with water containing 0, 50, and 150 mM NaCl for 5 days. (C) Effect of salt stress on water content and (D) relative electrolyte leakage were measured.
Figure 2. 2D gel analysis of proteins extracted from Arabidopsis leaves. A total of 1300 µg of protein was loaded on each IPG strip (pH 4-7). After isoelectric focusing, 12.5% SDS-PAGE gels were used for second dimension separation. Protein spots were visualized using coomassie brilliant blue staining. 2D gels from samples treated with (A) 0, (B) 50, and (C) 150 mM NaCl. Ninety spots that showed significant volume changes under salt stress are labeled in (A).
protein spots from triplicate biological samples were subjected to statistical analysis using means ( standard errors. Only the protein spots that exhibited reproducible and significant changes (>1.5 fold and p-value < 0.05) under different NaCl treatments were included for further analysis. A total of 88 protein spots from Arabidopsis gels and 37 protein spots from Thellungiella gels showed significant changes in NaCl-treated samples compared to control samples (Figures 2 and 3; Supplemental Tables 1 and 2, Supporting Information).
More than twice as many proteins in Arabidopsis changed in their abundance levels as those in Thellungiella under salt stress. This is an interesting observation, considering Arabidopsis as a glycophyte is more sensitive to NaCl exposure. Thellungiella can tolerate much higher salt levels than Arabidopsis.24 Our treatment is relatively mild to Thellungiella, which is a halophyte with stress-tolerant genes.14 Protein spots containing differentially expressed proteins were excised, trypsin digested, and analyzed by MS. Proteins Journal of Proteome Research • Vol. 9, No. 5, 2010 2587
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Figure 3. 2D gel analysis of proteins extracted from Thellungiella leaves. A total of 1300 µg protein was loaded on each IPG strip (pH 4-7). After isoelectric focusing, 12.5% SDS-PAGE gels were used for second dimension separation. Protein spots were visualized using coomassie brilliant blue staining. 2D gels from samples treated with (A) 0, (B) 50, and (C) 150 mM NaCl, respectively. Thirtyseven spots that showed significant volume changes under salt stress are labeled in (A).
in all the protein spots were successfully identified by MS and MASCOT database searching with high confidence (Supplemental Tables 1 and 2, Supporting Information). The identified protein spots corresponded to 90 unique proteins in Arabidopsis and 40 in Thellungiella. Since the 2-DE method has a disadvantage in distinguishing proteins with similar isoelectric points and molecular weights, 9 protein spots in Arabidopsis and 5 protein spots in Thellungiella were identified to contain two proteins (Supplemental Tables 1 and 2, Supporting Information). These proteins were excluded from quantitative analysis (Tables 1 and 2). It was interesting to note that 7 proteins were identified in two spots in Arabidopsis, that is, putative alanine aminotransferase (spots 143 and 689), oxygenevolving enhancer protein 2 (spots 532 and 824), Rubisco activase (spots 181 and 710), chlorophyll a/b binding protein (spots 478 and 812), thiazole requiring (THI1) (spots 396 and 422), CLpC ATP-Dependent protease (spots 12 and 632), ATP synthase CF1 alpha subunit (spots 135 and 675) and two proteins were identified in two spots in Thellungiella, that is, phosphoglycerate kinase (spots 441 and 513) and ascorbate peroxidase (spots 125 and 613) (Tables 1 and 2; Supplemental Tables 1 and 2, Supporting Information). Further inspection of the gel patterns revealed that the experimental mass and/ or isoelectric point values of the spots differed from their theoretical values. This could be due to post-translational modifications such as glycosylation, phosphorylation and cleavage that can alter protein molecular weight and/or charge. Alternatively, proteins present in multiple spots could result from translation from alternatively spliced mRNAs.21,57-59 Five proteins, chloroplast PSI type III chlorophyll a/b-binding protein, phosphoglycerate kinase, ribulose bisphosphate carboxylase, carbonic anhydrase (CA1) and pathogenesis-related gene 5 (PR5) were identified in both Arabidopsis and Thellungiella. Interestingly, some proteins showed differential responses to salt stress in Arabidopsis and Thellungiella. As an example, PR5 was increased in expression in Arabidopsis, but clearly decreased in expression in Thellungiella. Because the salt stress responsive proteins varied in the two species, we postulated that glycophyte and halophyte took different strategies facing NaCl stress. 2588
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To visualize the coordinately regulated proteins listed in Tables 1 and 2, hierarchical clustering was conducted. Two major clusters were produced, one included 50 proteins whose abundance increased and the other contained 29 proteins with decreased levels in Arabidopsis (Figure 4). Most of the changes were observed in samples treated with 150 mM NaCl, with 42 and 19 proteins up- and down-regulated, respectively, compared to control samples (Table 1, Figure 5). Comparison of protein abundance differences among different samples revealed de novo synthesis of unique proteins. In the increased protein cluster, one of the subclusters comprised 14 spots (spots 27, 35, 74, 92, 135, 157, 215, 233, 268, 272, 315, 374, 685 and 689) that were only visible in the treated samples (Table 1, Figure 4A). Two protein spots (233 and 685) were detectable only in the 150 mM NaCl treated samples, suggesting that they were induced by high salinity. Although the number of differentially expressed protein spots in Thellungiella was smaller than in Arabidopsis, many proteins were found to exhibit differential patterns after salt treatment (Figure 4B). In 150 mM NaCl treated Thellungiella, 9 proteins were increased and 10 were decreased. Interestingly, there were more proteins, that is, 10 were increased and 13 were decreased, in 50 mM NaCl treated samples (Table 2, Figure 5). The results are consistent with the results of transcriptomics,60 suggesting that tolerant plants respond quickly and better equip themselves even with a milder stress. Identification of Salt-Responsive Microsomal Proteins using iTRAQ LC-MS. Here we focused our analysis on microsomal proteins, which could play major roles in salt tolerance. Due to the physicochemical properties of membrane proteins, 2-DE is not appropriate for comprehensive profiling of membrane proteins.35 iTRAQ LC-MS is a recent technology for protein quantification, and is advantageous in quantitative analysis of hydrophobic proteins. Two independent iTRAQ experiments were conducted using four-week-old Arabidopsis and six-week-old Thellungiella plants irrigated with water and 150 mM NaCl, respectively. Of the 677 proteins in Arabidopsis and 764 in Thellungiella identified, 152 proteins in Arabidopsis and 93 in Thellungiella could be quantified with at least three different peptide MS/MS spectra
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Table 1. Protein Identities and Their Relative Changes under Salt Stress in Arabidopsis from 2D-Gel Analysis
a Assigned spot number as indicated in Figure 2. b Database accession numbers according to NCBInr. c Theoretical (c) and experimental (d) mass (kDa) and pI of identified proteins. Experimental values were calculated using Image Master 2D Platinum Software. Theoretical values were retrieved from the protein database. d Theoretical (c) and experimental (d) mass (kDa) and pI of identified proteins. Experimental values were calculated using Image Master 2D Platinum Software. Theoretical values were retrieved from the protein database. e Mascot score reported after searching against the NCBInr database. f Sequence coverage. g Number of peptides sequenced. h Mean of relative protein abundance and standard error. Three treatments including control (0, 50, and 150 mM NaCl for 5 days) were performed.
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Table 2. Protein Identities and Their Relative Changes under Salt Stress in Thellungiella from 2D-Gel Analysis
Figure 4. Hierarchical clustering analysis of the differentially expressed proteins in (A) Arabidopsis and (B) Thellungiella. The three columns represent 0, 50, and 150 mM NaCl treatment for 5 days, respectively. The rows represent individual proteins. The up- and down-regulated proteins are indicated in red and green, respectively. Proteins not detected on control gels or 50 mM gels are indicated in gray. The intensity of the colors increases as the expression differences increase, as shown in the bar at the top.
Figure 5. Number of protein spots significantly (A) downregulated or (B) up-regulated under different salt treatment conditions. Solid bars represent salt-responsive proteins in Arabidopsis, and open bars represent salt-responsive proteins in Thellungiella.
a Assigned spot number as indicated in Figure 2. b Database accession numbers according to NCBInr. c Theoretical (c) and experimental (d) mass (kDa) and pI of identified proteins. Experimental values were calculated using Image Master 2D Platinum Software. Theoretical values were retrieved from the protein database. d Theoretical (c) and experimental (d) mass (kDa) and pI of identified proteins. Experimental values were calculated using Image Master 2D Platinum Software. Theoretical values were retrieved from the protein database. e Mascot score reported after searching against the NCBInr database. f Sequence coverage. g Number of peptides sequenced. h Mean of relative protein abundance and standard error. Three treatments including control (0, 50, and 150 mM NaCl for 5 days) were performed.
and a p-value smaller than 0.05 in at least one of the experiments (Supplemental Tables 3 and 4, Supporting Information). 2590
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To determine a significance threshold, ratios of replicate samples were plotted against the p-values. Replicates of the same sample types, that is, identical iTRAQ samples showed similar overall quantification results, while comparison between control and NaCl treated samples revealed differentially expressed proteins (Supplemental Figure 2, Supporting Information). Based on this analysis, a protein has to meet the following criteria before it can be considered differentially expressed, that is, being quantified with at least three peptide spectra (allowing generation of a p-value), a p-value 1.5 or
