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JXB Advance Access published online on October 4, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm207
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots

Yuanqing Jiang1, Bo Yang2, Neil S. Harris1 and Michael K. Deyholos1,*

1Department of Biological Sciences, University of Alberta, Edmonton, Canada T6G 2E9
2Department of Agricultural, Forestry and Nutritional Science, University of Alberta, Edmonton, Canada T6G 2E9

* To whom correspondence should be addressed. E-mail: deyholos{at}ualberta.ca

Received 10 May 2007; Revised 25 July 2007 Accepted 6 August 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Conclusion
 Supplementary material
 References
 
NaCl stress is a major abiotic stress limiting the productivity and the geographical distribution of many plant species. Roots are the primary site of salinity perception. To understand better NaCl stress responses in Arabidopsis roots, a comparative proteomic analysis of roots that had been exposed to 150 mM NaCl for either 6 h or 48 h was conducted. Changes in the abundance of protein species within roots were examined using two-dimensional electrophoresis. Among the >1000 protein spots reproducibly detected on each gel, the abundance of 112 protein spots decreased and 103 increased, at one or both time points, in response to NaCl treatment. Through liquid-chromatography–tandem mass spectrometry, identity was assigned to 86 of the differentially abundant spots. The proteins identified included many previously characterized stress-responsive proteins and others related to processes including scavenging for reactive oxygen species; signal transduction; translation, cell wall biosynthesis, protein translation, processing and degradation; and metabolism of energy, amino acids, and hormones. At the resolution of individual genes and proteins, poor statistical correlation (6 h, r= –0.13; 48 h, r=0.11) of these protein expression data with previous microarray results was detected, supporting the concept that post-transcriptional regulation plays an important role in stress-responsive gene expression, and highlighting the need for combined transcriptomic and proteomic analyses.

Key words: Arabidopsis, 2-DGE, LC-MS/MS, NaCl stress, proteome, root


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Conclusion
 Supplementary material
 References
 
Soil salinity is a prevalent abiotic stress that limits the productivity and geographical distribution of plants. Natural phenomena and human practices such as irrigation can cause salts to accumulate in soil (Wiebe et al., 2007). Excess NaCl in the soil solution interferes with mineral nutrition and water uptake, and leads to accumulation of toxic ions in plants. To reduce these detrimental effects, plants use several strategies, including the regulated expression of specific proteins, which leads to the re-establishment of proper cellular ion and osmotic homeostasis with other concomitant processes of repair and detoxification (Chinnusamy et al., 2005). Roots are a site of perception and injury for several types of stress, including salinity, nutrient deficiency, and heavy metals. In many circumstances, it is the stress sensitivity of the root that limits the productivity of the entire plant (Atkin et al., 1973; Steppuhn and Raney, 2005). An improved understanding of molecular responses of roots to NaCl treatment may therefore facilitate the development of crops with increased tolerance to NaCl and other stresses.

To build a useful description of the molecular mechanisms active in the response of roots to NaCl treatment, it is necessary to characterize the components of these mechanisms, including proteins. Proteomic profiles have been produced for various stresses and species, including NaCl-treated roots of pea (Pisum sativum L.), rice (Oryza sativa L.), and wheat (Triticum aestivum L.), as well as drought-treated poplar (Populus trichocarpa Torr. & A.Gray), and cadmium- or arsenic-treated maize (Zea mays L.), rice, and Arabidopsis thaliana (L.) Heynh. (Majoul et al., 2000; Kav et al., 2004; Requejo and Tena, 2005, Yan et al., 2005; Aina et al., 2006; Plomion et al., 2006; Roth et al., 2006). Recently, a technology based on two-dimensional gel electrophoresis (2-DGE) was employed to identify NaCl- and osmotic-responsive proteins in Arabidopsis cell suspension and root microsomal fraction (Lee et al., 2004; Ndimba et al., 2005). In the present study, a moderate NaCl stress was applied to hydroponic-cultured Arabidopsis roots to identify proteins that are responsive to NaCl treatment.

Transcriptome profiling, a widely used technique to identify NaCl-responsive genes, has contributed to our understanding of salinity stress in species including Arabidopsis and rice (Kawasaki et al., 2001; Kreps et al., 2002; Seki et al., 2002; Rabbani et al., 2003; Chao et al., 2005; Jiang and Deyholos, 2006). However, transcriptome profiling has some limitations because mRNA levels are not always correlated to those of corresponding proteins, due in part to post-transcriptional regulation. Only poor or moderate correlation between changes in the levels of specific mRNAs and their corresponding proteins has been reported previously in studies involving yeast (Saccharomyces cerevisiae), animals, or Arabidopsis (Gygi et al., 1999; Tian et al., 2004; Mooney et al., 2006). Furthermore, post-translational modifications, such as phosphorylation and glycosylation, can result in a dramatic increase in proteome complexity without a concomitant increase in gene expression (Jensen, 2004; Rose et al., 2004). These biological realities motivated us to perform an analysis of NaCl stress responses at the proteome level, and to compare these results with previous, microarray-based studies of similarly treated tissues (Jiang and Deyholos, 2006).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Conclusion
 Supplementary material
 References
 
Plant materials and stress treatment
Arabidopsis plants (wild-type, ecotype Col-0) were cultured hydroponically as described previously (Jiang and Deyholos, 2006). At 18 d post-germination (dpg), the hydroponic solution was changed to fresh, half-strength Murashige and Skoog (MS) medium, either with or without 150 mM NaCl, and was maintained for 6 h or 48 h, with roots harvested from control or NaCl-treated plants in parallel at each time point, flash frozen in liquid nitrogen, and stored at –80 °C. Three biologically independent replicates were prepared at separate times.

Physiological analyses
For root elongation assays, 5 dpg, wild-type Arabidopsis seedlings, grown vertically on half-strength MS medium supplemented with 1% sucrose and 0.8% Phytablend (Caisson), were transferred onto half-strength MS plates supplemented with 1% sucrose and 0, 50, 100, 150, 200, or 250 mM NaCl in square Petri dishes. Root lengths were measured after 7 d. For ion concentration determination, hydroponic-cultured Arabidopsis roots and shoots (18 dpg) were dried for 2 d in an oven at 65 °C. Dry root or leaf samples (100–500 mg) were digested according to the EPA 3050B method (http://www.epa.gov/SW-846/pdfs/3050b.pdf) with modifications. Na and K concentrations were determined by flame emission spectroscopy (AAnalyst700; PerkinElmer). For the relative electrolyte leakage (REL) assay, ~150 mg of hydroponic-cultured seedlings, 18 dpg old, were rinsed with ddH2O, placed in test tubes containing 10 ml of ddH2O, and incubated at room temperature for 2 h, with the electrical conductivity of the solution (C1) measured using a conductivity meter (Orion 115Aplus; ThermoElectron). 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 by the formula C1/C2x100% (Cao et al., 2007).

Protein extraction and quantification
Total protein extracts were prepared essentially according to the method described by Tsugita and Kamo (1999) with modifications. In brief, ~1 g of control and NaCl-treated roots were homogenized separately to a fine powder in liquid nitrogen, and were transferred into three 2 ml tubes. One millilitre of 10% (w/v) trichloroacetic acid/0.07% dithiothreitol (DTT) in acetone was added to each tube and incubated at –20 °C for 1 h. Afterwards, tubes were centrifuged at 18 000 g for 15 min and the supernatants discarded. The pellets were washed by resuspension in ice-cold acetone containing 0.07% DTT and centrifuged as described above. This wash was repeated three times, with the pellets dried at room temperature in a SpeedVac for 15 min and resuspended in 300 µl of lysis buffer (30 mM TRIS-HCl, 7 M urea, 2 M thiourea, 4% CHAPS, pH 8.5). The samples were mixed vigorously, incubated overnight at 4 °C, and centrifuged at room temperature for 15 min at 18 000 g, with the supernatants collected into fresh tubes. The protein extracts were cleaned up using 2-D Cleanup kit (GE Healthcare), dissolved in 200 µl of Destreak rehydration buffer containing 2% pH 3–10 immobilized pH gradient (IPG) buffer (GE Healthcare), and quantified by using a 2-D Quant kit (GE Healthcare) using bovine serum albumin as the standard.

Isoelectric focusing (IEF) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)
Two-dimensional electrophoresis of protein extracts was performed using a GE Healthcare 2-DGE system according to the manufacturer's manuals. Briefly, IPG strips (pH 3–10 NL, 24 cm) were rehydrated in 450 µl of Destreak rehydration buffer (containing 2% pH 3–10 IPG buffer) overnight (~15 h). IEF was performed using an IGPhor isoelectric focusing unit with 400 µg of protein samples loaded by cup-loading. The voltage and duration used were as follows: first step and hold: 300 V, 3 h; second gradient: 1000 V, 6 h; third gradient: 8000 V, 3 h; fourth step and hold: 8000 V, 4 h 40 min. Prior to second dimension separation, the strips were incubated first in an equilibration buffer (6 M urea, 30% v/v glycerol, and 2% SDS in 0.05 M TRIS-HCl, pH 8.8) containing 15 mM DTT for 15 min, then in an equilibration buffer containing 2.5% iodoacetamide (GE Healthcare) for another 15 min, followed by brief equilibration in 1x SDS–TRIS–glycine running buffer for 5 min. The second dimension separation of proteins was performed on SDS–PAGE gel (12.5% acrylamide, Bio-Rad) using the Ettan Dalt Six apparatus (GE Healthcare) with protein markers (Cat#SM0661, Fermentas) loaded at the left-most side. The electrophoresis was carried out at ~25 °C and 2.5 w/gel for 30 min and then 17 w/gel for ~5 h 40 min until the bromophenol blue dye front arrived at the bottom of the gels. Following SDS–PAGE, gels were washed in ddH2O three times for 15 min and proteins were detected by a modified collodial Coomassie brilliant blue staining-blue silver protocol, which was assessed to have sensitivity comparable with silver staining (Candiano et al., 2004). After three washes in ddH2O, the 2-D gels were scanned immediately using a Fuji FLA-5000 scanner (Fujifilm) with a resolution of 100 µm and 16 bit greyscale pixel depth. A total of 12 gels were analysed, i.e. three gels (biologically independent replicates) for each of two treatments (0 mM or 150 mM NaCl) at each of two time points (6 h or 48 h). This experimental design was balanced with respect to all conditions, and the use of three gels per condition is consistent with recommendations based on previous statistical analyses of protein gel electrophoresis (Hunt et al., 2005). For isoelectric point and molecular weight calibrations, the 2-D internal standards (Cat#161-0320; Bio-Rad) were used with IEF, SDS–PAGE, staining, and scanning performed as above.

Image and statistical analysis
2-D gel images were analysed using ImageMaster 2-D Platinum 6.0 (GE Healthcare). To verify the autodetected results, all spots were manually inspected and edited as necessary. After spot detection, quantification, and background subtraction, each gel analysed was matched individually to the reference gel, and matched spots were grouped into subclasses. To compensate for subtle differences in sample loading, gel staining, and destaining, the volume of each spot (i.e. spot abundance) was normalized as relative volume. This normalization method, provided by ImageMaster 2D Platinum 6.0 software, divides each spot volume value by the sum of total spot volume values to obtain individual relative spot volumes. Class reports were generated for spots of interest. The differences in expression between control and treatment were analysed by Student's t-test with P ≤0.05 considered significant. The molecular masses of proteins on gels were determined by co-electrophoresis of standard protein markers (Fermentas) and internal 2-D internal standards (Bio-Rad) according to the software manual.

For the figures shown, spot IDs were renumbered, using the annotation tool in ImageMaster Platinum 6.0, and image brightness and contrast were adjusted, and the protein marker sizes were added using Photoshop CS (Adobe).

In-gel digestion
Protein spots showing at least a 1.5-fold difference in abundance between control and treatment at one or both time points with P <0.05 were selected and excised manually into 1.5 ml microtubes. The selection of a 1.5-fold change as an arbitrary threshold allowed us to focus on the most responsive proteins for subsequent characterization, and is consistent with thresholds used previously in other microarray studies (Jiang and Deyholos, 2006, and references therein). Gel piece treatment and in-gel digestion of protein spots were performed following Jensen et al. (1999) with modifications. Briefly, gel pieces were first washed with 150 µl of HPLC-grade water (Fisher Scientific), dehydrated with 50 µl of 100% acetonitrile (ACN), then destained with 100 µl of 50 mM NH4HCO3/50% ACN for 2 h or longer until colourless. After dehydration with ACN again, gel pieces were reduced in 30 µl of 10 mM DTT/0.1 M NH4HCO3 at 56 °C for 30 min, dehydrated, and alkylated in 30 µl of 55 mM iodoacetamide/0.1 M NH4HCO3 at room temperature for 20 min in the dark, followed by rinsing with 200 µl of 0.1 M NH4HCO3, dehydration with ACN, and drying in a SpeedVac for 10 min. Afterwards, 20 µl of trypsin solution 0.02 µg µl–1 Trypsin Gold (Promega) in 40 mM NH4HCO3/10% ACN was added and incubated on ice for 1 h, then at 37 °C overnight (~14 h). Finally, 3 µl of 2% formic acid (FA) was added to stop the digestion reactions, and the supernatant was collected into fresh tubes, followed by two extractions of peptides with 15 µl of 50% ACN/0.1% FA with the extracts (~50 µl) mixed well and stored at –20 °C before use.

Liquid chromatography–tandem mass spectrometry (LC-MS/MS)
LC-MS/MS analysis of digested peptide mixtures was performed using an Agilent 1100 LC/MSD Trap XCT (Agilent Technologies). Briefly, an autosampler was used to inject 20 µl of each tryptic digest onto the first of two C-18 columns. This short 5 µm enrichment column, Zorbax 300SB-C18, 5 µm, 5x0.3 mm, served to trap and concentrate the samples. Next, the sample was eluted onto the next C-18 column (Zorbax 300SB-C18, 5 µm, 150x0.3 mm), which was used in conjunction with a solvent gradient to separate the peptides. The peptide-separation gradient started at 85% solvent A (0.1% FA in H2O) and ended at 55% solvent B (0.1% FA, 5% H2O in ACN) over a 42 min span. This was followed by a 10 min period of 90% solvent B to cleanse the columns before returning to 97% solvent A for the next sample. The ion trap mass spectrometer collected information by first running an MS 300–2200 m/z scan and followed that with an MS/MS analysis of the most intense ions. In addition to the most intense ion for each scan, the software was set to exclude this ion after two spectra and gather MS/MS information on the next most intense ion(s). Raw spectral data were processed into Mascot Generic File (.mgf) format using the default method in the ChemStation Data Analysis module. The MS/MS ion search was performed using MASCOT (http://www.matrixscience.com) searching the NCBInr database and taking Arabidopsis as the taxonomy. The parameters for searching were: an MS/MS tolerance of ±0.8 Da, one missed cleavage site, enzyme of trypsin, fixed modifications of carbamidomethyl, peptide tolerance of ±2 Da, peptide charge of 1+ 2+ 3+, monoisotopic, and ESI-TRAP instrument. Only significant hits, as defined by the MASCOT probability analysis (P <0.05), were accepted. In case multiple significant hits were found for a protein, only the highest scoring hit was listed in Table 1, with all significant hits listed in Supplementary Table S2 available at JXB online.


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  		Table 1. Differentially expressed proteins identified by LC-MS/MS

 

   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Conclusion
 Supplementary material
 References
 
Plant growth response to NaCl stress
Arabidopsis thaliana is a glycophyte and is sensitive to NaCl exposure. To find an appropriate concentration of NaCl to use for treatment of plants prior to proteomic profiling, a root elongation dose response assay was performed. The present results demonstrated that 100 mM and 150 mM NaCl inhibited root elongation by 53% and 78%, respectively (Fig. 1A). However, concentrations higher than 200 mM NaCl almost completely inhibited root growth and led to death of almost all seedlings. In a post-stress recovery assay, 18 dpg plants were treated by 150 mM NaCl for 6, 24, or 48 h, and were transferred into fresh media to recover for 1 week. Almost all of the NaCl-treated plants recovered and resumed normal growth (Fig. 1C, D). Therefore, 150 mM was selected as the treatment concentration to be used in the present study, because it induced visible signs of stress including retarded growth rate and loss of turgor. This concentration of NaCl has been used in several previous gene expression studies, because it induces a moderate stress response and is not acutely lethal (Jiang and Deyholos, 2006; Ma et al., 2006). Higher concentrations of NaCl appear to lead to plasmolysis and lethality (Munns, 2005), although other previous Arabidopsis studies have used an NaCl treatment concentration of 250, 300, or even 600 mM (Seki et al., 2002; Ndimba et al., 2005).


Figure 1
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  		Fig. 1. Physiological responses induced by NaCl treatment. (A) Effect of increasing [NaCl] on root elongation of seedlings (n=9). (B) Relative electrolyte leakage from roots of control (filled circles) or NaCl-treated (open circles) roots following 6, 24, or 48 h exposure to 150 mM NaCl (n=5). Vertical bars indicate standard deviation (SD). (C, D) Post-stress recovery of NaCl-treated plants. Plants were treated with 150 mM NaCl for 6 h (C) or 48 h (D), then returned to normal growth medium for 1 week before being photographed (scale bar=1 cm, diameter of rafts=13.4 cm).

 
REL is an indicator of membrane damage caused by NaCl stress. Stress-induced changes were measured in plants treated with 150 mM NaCl. As shown in Fig. 1B, the REL of seedlings treated by 150 mM NaCl for 6 h was 8.6%. After 24 h treatment, the REL increased to 18% and further increased to 26% after 48 h of NaCl treatment. When compared with the control plants, the REL values of NaCl-treated seedlings were 1.7, 2.8, and 4.4 times higher at 6, 24, and 48 h, respectively, which indicated that stress-induced cellular damage accumulated throughout the duration of the experiment.

To establish further the physiological status of plants that were to be subjected to proteome analysis, next the concentrations of K and Na were quantified in both leaves and roots of control and 150 mM-treated plants (Fig. 2). Na and K concentrations significantly increased and decreased, respectively, in both leaves and roots under 150 mM NaCl treatment. Moreover, the Na concentrations in roots treated by 150 mM NaCl for 6, 24, and 48 h were 115-, 167-, and 132-fold higher than those of the control roots at comparable time points. The concentration of Na in roots reached a maximum after 24 h (Fig. 2A); however, leaves continued to accumulate more Na after the 24 h time point (Fig. 2B), presumably due to transpiration-driven flux. The K:Na ratios in both leaves and roots decreased as the NaCl treatment progressed. Part of the basis of Na+ toxicity in plants is that high Na+ concentrations in the cells increase the Na:K ratio, which is adverse for most metabolic processes (Chinnusamy et al., 2005). Thus, the quantitative ion analysis was consistent with the REL data in describing the progressive accumulation of stress symptoms throughout the 48 h duration of the treatments.


Figure 2
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  		Fig. 2. Changes in Na and K ion concentrations following NaCl exposure. Na (closed symbols) and K (open symbols) concentrations (mg g–1 dry weight) were determined by flame emission spectroscopy in roots (A) or rosette leaves (B) of hydroponically grown Arabidopsis plants, following 6, 24, or 48 h of exposure to media supplemented with either 0 mM NaCl (control, circles) or 150 mM NaCl (treated, triangles). Na concentrations in control samples are very low (<0.4 mg g–1). Data are from three biological replicates, and vertical bars indicate ±SD.

 
2-DGE analysis of NaCl-responsive proteins in Arabidopsis roots
To investigate the temporal changes of protein profiles during NaCl stress, 2-DGE analysis of the total proteins in Arabidopsis roots from three biologically independent replicate experiments was carried out. A representative gel is shown in Fig. 3. Approximately 1000 protein spots were detected on Coomassie brilliant blue-stained gels and about 600 protein spots were matched between six control gels and six treatment gels. Quantitative image analysis revealed a total of 215 protein spots that changed their abundance (vol%) significantly (P ≤0.05) by <1.5-fold at one or two time points. A 1.5-fold threshold value was selected in order to focus protein identification efforts on the most responsive proteins and for consistency with previous microarray experiments (Jiang and Deyholos, 2006). Essentially arbitrary threshold values ranging from 1.3- to 2.0-fold have been used in previous proteomics studies (Casati et al., 2005; Amme et al., 2006; Parker et al., 2006; Yan et al., 2006). It was noted that some protein spots also demonstrated qualitative changes in intensity. For example, spots 21, 24, 28, and 75 were absent in the NaCl-treated gels at one or more time points while spots 26, 62, 82, and 85 were absent in control gels at one or more time points.


Figure 3
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  		Fig. 3. Representative 2-DE gels of Arabidopsis root proteins. Eighty-six of the spots showing at least a 1.5-fold change under NaCl treatment at least at one time point with P <0.05 were analysed by LC-MS/MS.

 
LC-MS/MS identification and classification of NaCl-responsive proteins
Eighty-nine of the 215 differentially expressed spots described above were arbitrarily selected and excised for tryptic digestion and analysis by LC-MS/MS. From 89 gel plugs excised, 86 proteins were successfully identified (Table 1 and Supplementary Table S1 available at JXB online), of which 81 were unique. Some NaCl-responsive spots were not excised because of their low abundance. It was found that at least 23 (26%) of these spots contained peptides that matched proteins from unrelated families (Table 1 and Supplementary Table S2 available at JXB online), indicating the presence of multiple proteins in some spots. Conversely, it was found that four proteins were identified in more than one spot, although they were excised from the same gel (Table 1). For example, L-ascorbate peroxidase (APX1) was identified from three spots (35, 36, and 61), glutathione S-transferase (AtGSTF2) was identified in two spots (41 and 42), glycosyl hydrolase family 17 protein was identified in two spots (26 and 62), and glycosyl hydrolase family 1 protein was identified twice (65 and 86). Further examination of electrophoresis patterns indicated that the inferred mass or isoelectric point values of these spots differed, due perhaps to post-translational modification or degradation. Post-translational modifications such as glycosylation, phosphorylation, etc. can change the molecular weight and/or charge of proteins. Alternatively, proteins that were present in multiple spots could result from being translated from alternatively spliced mRNAs (Ishikawa et al., 1997). This phenomenon was also reported previously (Holmes-Davis et al., 2005; Ndimba et al., 2005). Proteomic studies have also shown that some proteins may be degraded during abiotic stress. For example, the Rubisco large subunit was detected as 19 different fragments plus the intact protein in NaCl-treated rice roots (Yan et al., 2006). Similar phenomena have also been reported in pea mitochondrial proteome under chilling stress (Taylor et al., 2005). It is possible that reactive oxygen species (ROS) may also contribute to the degradation of proteins under stress conditions (Desimone et al., 1996; Kingston-Smith and Foyer, 2000).

The proteins identified were classified into 11 categories similar to the convention used by Ndimba et al. (2005) (Fig. 4). Proteins implicated in energy metabolism (e.g. glycolysis, citrate cycle, electron transport), ROS scavenging and defence, and protein metabolism (e.g. translation, processing, and degradation) comprised 52% of the proteins identified. Further examination showed that after 6 h of stress, the abundance of most NaCl-responsive proteins had decreased. By contrast, after 48 h of treatment, it was observed that the number of proteins that had increased in abundance was approximately equal to the number of proteins that had decreased in abundance (Fig. 4). This suggests that during the initial (6 h) phase of NaCl stress, the synthesis of many proteins was inhibited and/or their degradation increased, while after 48 h, plant roots began to adapt to water deficit and ionic accumulation by synthesizing selected stress-response proteins. This is generally consistent with previous observations of NaCl-treated roots, in which the abundance of transcripts for almost all ribosomal proteins decreased after 6 h, while transcripts for >30 peptidases increased at the same time point (Jiang and Deyholos, 2006). However, in the previous microarray analysis, transcripts for ribosomal proteins remained at decreased levels between 6 h and 48 h, suggesting that the increased expression of selected stress proteins reported here may involve specialized mechanisms of translation that are not reflected in the bulk transcript level of all cellular ribosomal proteins.


Figure 4
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  		Fig. 4. Functional classification of NaCl-responsive proteins. The number of proteins that either (A) increased or (B) decreased by at least 1.5-fold in 11 functional categories is shown for roots exposed to 150 mM NaCl for 6 h (grey bars) or 48 h (black bars). Functional categories are named according to the major metabolic substrate, except for the categories representing processes of signal transduction, transcriptional regulation (transcription), and unclassified or unknown (unclass. and unk.) proteins.

 
Energy metabolism
Under NaCl stress, plants decrease energy metabolism rates to conserve energy and limit further generation of ROS (Moller, 2001). Previously it had been reported that the transcript abundance of components of the glycolytic, citrate cycle, mitochondrial respiration, and pentose phosphate pathways generally decreased in NaCl-treated Arabidopsis roots (Jiang and Deyholos, 2006). Therefore, it was not surprising to observe in the present study that the abundance of 11 proteins involved in glycolysis, citrate circle, pentose phosphate pathways, and electron transport decreased at one or both time points after NaCl treatment (Table 1). For example, aconitase (At2g05710), isocitrate dehydrogenase (At1g65930), fumarase (At2g47510), and malate dehydrogenase (At1g04410) are four enzymes of the citrate cycle that each also decreased in abundance following NaCl treatment. By contrast, the abundance of nucleoside diphosphate kinase 1 (NDPK1, At4g09320), an enzyme converting GTP to ATP, and a mitochondrial malate dehydrogenase (At1g53240) increased at the early phase (6 h) and late phase (48 h) following NaCl treatment, respectively. Differential regulation of structurally related transcripts and proteins has been reported previously in NaCl-treated roots, and may reflect sub-functionalization of related enzymes for optimal activity in different cells or cellular microenvironments (Jiang and Deyholos, 2006). It was noted that phosphopyruvate hydratase/enolase (LOS2, At2g36530), which catalyses the formation of high-energy phosphoenol pyruvate from 2-phosphoglycerate in the glycolytic pathway, decreased after both 6 h and 48 h of stress. Previous reports have shown that mutation in the LOS2 locus results in repression of cold-responsive genes and therefore it acts as a positive regulator of cold-responsive genes (Lee et al., 2002). The decreased abundance of LOS2 protein that was observed indicates that NaCl-induced perturbation of metabolic flux in glycolysis may be different from that of cold stress. Other energy-related proteins, including three proton-transporting ATPases, two vacuolar ATP synthases (At1g78900 and At4g11150), and a mitochondrial ATP synthase delta chain (At5g47030) were found to be responsive to NaCl stimulus. Vacuolar H+-ATPase can generate a proton electrochemical gradient, which is the driving force utilized by the tonoplast Na+/H+ antiporter, AtNHX1, to compartmentalize Na+ into the vacuole (Chinnusamy et al., 2005). Vacuolar sequestration of Na+ is an important and cost-effective strategy for osmotic adjustment that also reduces the Na+ concentration in the cytosol in plants.

ROS scavenging and detoxifying enzymes
Abiotic stresses induce the production of ROS, which, on one hand can cause damage to cellular components, and on the other hand, can act as signalling molecules for stress responses (Apel and Hirt, 2004). Plants can regulate the ROS level through complex mechanisms such as scavenging them with ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferase (GST), and superoxide dismutase (SOD), of which nine proteins were identified in this study (Table 1). In general, the abundance of these nine identified proteins was increased upon NaCl treatment. It is proposed that some members of APX, GPX, GST, and SOD families are part of the antioxidant system employed by plants (Apel and Hirt, 2004) and, previous microarray results demonstrated that members of them are responsive to various stresses including NaCl, osmotica, drought, and cold (Kreps et al., 2002; Seki et al., 2002; Jiang and Deyholos, 2006). The increased abundance of GST, peroxidase, and SOD proteins following NaCl is consistent with the presence of oxidative stress in NaCl-stressed roots. The alleviation of oxidative damage and increased resistance to environmental stresses is correlated with an efficient anti-oxidative system (Smirnoff, 1998). Overexpression of some SOD, APX, and GST genes has been shown to improve oxidative stress tolerance in transgenic plants (Allen, 1995; Roxas et al., 1997; Mittler, 2002).

GSTs are abundant proteins which are encoded by a highly divergent, ancient gene family and have protective functions such as detoxification of herbicides, and the reduction of organic hydroperoxides formed during oxidative stress. Recent studies have also implicated GSTs as components of ultraviolet-inducible cell signalling pathways and as potential regulators of apoptosis (Dixon et al., 2002). Previous microarray analysis indicated that transcripts for at least 19 GST genes increased in abundance in NaCl-treated Arabidopsis roots (Jiang and Deyholos, 2006). Here, proteins of four plant-specific GSTs—AtGST1/GSTF6 (At1g02930), AtGSTF2/GST4 (At4g02520), AtGST6/GSTF8 (AT2g47730), and AtGST11/GSTF7 (AT1g02920)—were identified, which all ultimately increased in abundance after 48 h of NaCl treatment, although several of them slightly decreased in abundance at the initial time point sampled (6 h). Previous studies showed that AtGST1 was up-regulated by a variety of treatments, while AtGSTF2 and AtGST6 each showed a selective spectrum of inducibility to different stresses, indicating that regulation of gene expression in this super-family is controlled by multiple mechanisms (Wagner et al., 2002).

APX and GPX can directly detoxify H2O2 to H2O, and previous studies showed that APX1 is a central component of the reactive oxygen gene network of Arabidopsis (Davletova et al., 2005). In Arabidopsis, a family of seven related proteins named AtGPX1–AtGPX7 was identified and several AtGPX genes were up-regulated coordinately in response to abiotic stresses (Milla et al., 2003). AtGPX6 possibly encodes mitochondrial and cytosolic isoforms by alternative initiation, and AtGPX6 transcript showed the strongest responses under most abiotic stresses tested, thus supporting an important role for it in protection against oxidative damage (Milla et al., 2003).

In the present study, an NaCl-dependent increase in protein abundance for two class III plant peroxidases—PER22 (At2g38380) and PER23 (At2g38390)—was observed. Class III peroxidases are plant-specific oxidoreductases that are implicated in various physiological processes such as H2O2 detoxification, auxin catabolism, liginfication, suberization, stress response (wounding, pathogen attack, NaCl), and senescence (Hiraga et al., 2001; Passardi et al., 2005). Previously it had been reported that the majority of class III peroxidases are responsive, at the transcript level, to NaCl treatment in Arabidopsis roots (Jiang and Deyholos, 2006). The diverse functions of class III peroxidases are, in part, due to two possible catalytic cycles, peroxidative and hydroxylic, involving the consumption or release of H2O2 and ROS (Passardi et al., 2005). Although some functions of these peroxidases appear to be paradoxical, the whole process is probably regulated by a fine-tuning that has yet to be elucidated (Passardi et al., 2005).

SODs catalyse the dismutation of superoxide into oxygen and H2O2, and constitute the first line of defence against ROS within a cell (Alscher et al., 2002). Surprisingly, in our previous microarray analyses, transcripts for all three detectable SOD genes decreased in response to NaCl treatment (Jiang and Deyholos, 2006). Among these was manganese superoxide dismutase, MSD1 (At3g10920), a protein which was observed here to increase in abundance by 3.7-fold at 48 h post-NaCl treatment (Table 1). The contrast in protein and transcript abundance in similarly treated tissues for MSD1 highlights the importance of an integrated proteomic and transcriptomic analysis of gene expression.

Ascorbate is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase is crucial for ascorbate regeneration and essential for maintaining a reduced pool of ascorbate. Surprisingly, it was found that the abundance of one monodehydroascorbate reductase (AtMDAR2, At5g03630) was down-regulated by NaCl stress, suggesting that although plants require reduced ascorbate to remove free radicals, the fine tuning of the levels of various antioxidants is also an important consideration in stress responses (Lisenbee et al., 2005).

Protein translation, processing, and degradation
Regulation of gene expression is achieved at several levels, i.e. transcriptional, post-transcriptional, translational, and post-translational. Thirteen proteins implicated in protein translation, processing, and degradation were identified in the present study. A decrease in bulk de novo protein synthesis following NaCl treatment has been detected in Arabidopsis (Ndimba et al., 2005), and previous microarray data also demonstrated down-regulation of the majority of transcripts for almost all cytosolic and plastidic ribosomal proteins (Jiang and Deyholos, 2006). Similarly, microarray profiling of Arabidopsis seedlings under hypoxia indicated a repression of bulk protein synthesis followed by selective translation of specific transcripts (Branco-Price et al., 2005). Under dehydration conditions, >90% of the Arabidopsis mRNAs showing a strong decrease in abundance as detected by microarray displayed reduced polysomal association, indicating a decreased translation of those transcripts (Kawaguchi et al., 2004).

Consistent with these observations, three ribosomal proteins (At1g15930, At3g09200, and At3g53870), whose abundance decreased following NaCl treatment, were identified, suggesting that short-term NaCl stress represses protein synthesis in vivo (Table 1). The expression level of a eukaryotic translation initiation factor 3 subunit protein (eIF3I1, At2g46280), which is a homologue of mammalian TGF-beta receptor-interacting protein, was also found to have decreased in this study. Previous studies showed that eIF3I1/TGF-beta receptor-interacting protein 1 is involved in brassinosteroid-regulated plant growth and development, thereby revealing a putative link between a developmental signalling pathway and the control of protein translation (Jiang and Clouse, 2001). Several proteins that promote the proper folding of proteins and/or prevent the aggregation of nascent or damaged proteins were detected. Two protein disulphide isomerase-like (PDIL) proteins (PDIL1-1, At1g21750; PDIL1-2, At1g77510), putative nascent polypeptide-associated complex alpha chain protein (At3g12390), and mitochondrial HSC70-2 (70 kDa heat-shock cognate, At5g09590) all ultimately decreased in protein abundance in NaCl-treated tissues as compared with untreated controls after 48 h, although At3g12390 showed transient increases in abundance after 6 h of NaCl treatment. Members of HSC70 proteins are often involved in assisting the folding of de novo synthesized polypeptides and the import/translocation of precursor proteins (Wang et al., 2004). It was proposed that HSC70 might be used as a motor for transporting the precursor protein through the membranes by interacting with the signal peptides (Zhang and Glaser, 2002). The decreased abundance of MtHSC70-2 after NaCl treatment suggests a decrease in the transportation of newly synthesized peptides into mitochondria, due partly to a decreased de novo protein synthesis under saline conditions.

Cell wall-related proteins
Moderate NaCl stress reduces water availability and leads to the inhibition of plant growth by increasing the threshold pressure for wall yielding in expanding cells or inducing hydraulic limitations to water uptake (Neumann et al., 1994; Steudle, 2000). In the current study, four glycosyl hydrolase (GH) family proteins, three of which belong to GH family 1 (At1g47600, At1g66280, At3g09260) and one to GH family 17 (At4g16260), were identified (Table 1). GH1 and GH17 protein families include ß-glucosidases and ß-1,3-glucanases, respectively, which play important roles in many physiological processes in plants, including cell wall remodelling (Bray, 2004; Xu et al., 2004). Each of the four GH proteins identified had a distinctive temporal expression pattern: two of the GH1s decreased in abundance after 6 h, while the third GH1, At1g47600, increased at 6 h; however, after 48 h, At3g09260 (spots 65 and 86) showed increased abundance compared with controls. The two GH17 protein spots identified (spots 26 and 62) were more abundant in treated tissues than controls at both time points. The diversity of expression patterns suggested that several different physiological processes were represented by these results. Further experiments on the substrate specificity, localization of the enzymes with respect to potential substrates, and the activities of the substrates and hydrolysis products are required to determine the roles of these enzymes in root responses to NaCl.

Glycine-rich proteins (GRPs) containing >60% glycine have been found in the cell walls of many higher plants and form a group of structural protein components of the wall in addition to extensins and proline-rich proteins (Ringli et al., 2001). GRPs play roles in post-transcriptional regulation of gene expression in plants under various stress conditions and, in most cases, they are accumulated in the vascular tissues and their synthesis is part of the plant's defence mechanism (Mousavi and Hotta, 2005). It was observed that salinity caused a transient increase in the abundance of AtGRP7 (At2g21660) (Table 1). Previous studies showed that AtGRP7 transcript level was repressed by ABA, high NaCl, and mannitol (Cao et al., 2006). More recent studies suggest that AtGRP7 exhibits RNA chaperone activity and can promote the cold adaptation process in Escherichia coli (Kim et al., 2007b).

Also identified were two germin-like proteins (GLPs): GLP9 (At4g14630) and oxalate oxidase-like protein (At5g38940), whose abundance increased in Arabidopsis roots subjected to NaCl treatment (Table 1). GLPs exhibit sequence and structural similarity to cereal germins and may be associated with the cell wall (Membre et al., 2000). Although GLPs mostly lack oxalate oxidase activity, some GLPs have SOD activity. GLPs are thought to play a significant role both during embryogenesis and in biotic and abiotic stress conditions. For example, GLP expression was detected in barley roots after exposure to NaCl (Hurkman et al., 1994). A reversibly glycosylated polypeptide (RGP1, At3g02230) was also found to be induced by NaCl treatment. RGP1 is possibly involved in plant cell wall synthesis (Dhugga et al., 1997). Cell wall rigidification, the formation of a physical barrier, and a process of class III peroxidase-mediated cross-linking of several compounds (Passardi et al., 2004), would protect plant roots from further dehydration under water deficit.

Hormone-related proteins
Ethylene and jasmonic acid (JA) are hormones whose activity has been correlated previously with environmental stress (Chen et al., 2005; Devoto and Turner, 2005). The majority of ethylene- and JA-related transcripts detected in previous microarray experiments were responsive to NaCl treatment (Jiang and Deyholos, 2006). 1-Aminocyclopropane-1-carboxylic acid oxidase (ACO) catalyses the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene. ACOs are encoded by a small gene family. It was found that ACO2 (At1g62380) increased in abundance at the early (6 h) time point but later decreased in abundance (Table 1). S-Adenosylmethionine synthetase catalyses the production of S-adenosyl-L-methionine (SAM) from L-methionine and ATP. SAM serves as a methyl group donor in numerous transmethylation reactions and is the precursor for the biosynthesis of polyamines and ethylene among other metabolites. In plants, SAM synthetases are encoded by small gene families that contain members that are differentially regulated by NaCl stress (Espartero et al., 1994). Here SAM1 (At1g02500) decreased in abundance following NaCl treatment, which is consistent with previously reported microarray results (Jiang and Deyholos, 2006).

JA is involved in a wide range of stress, defence, and developmental processes (Devoto and Turner, 2005). One enzyme implicated in JA biosynthesis, allene oxide cyclase 2 (AOC2, At3g25780), whose abundance increased upon NaCl challenge, was identified, indicating that increased JA biosynthesis may also be associated with NaCl responses in Arabidopsis roots. One jacalin lectin family protein (JR1, At3g16470), which is similar to myrosinase-binding protein, was found to be positively regulated by NaCl. JR1 transcript was strongly induced by wounding and JA (Leon et al., 1998), supporting the concept of cross-talk between various abiotic stresses.

Signal transduction network involved in NaCl stress responses
An increase in NaCl concentration in the extracellular space can be perceived by putative sensors in the cell membrane of Arabidopsis and transmitted to the cellular machinery to regulate gene expression (Chinnusamy et al., 2005). Some proteins involved in signal transduction were identified in this study (Table 1), i.e. two calcium ion-binding proteins (CRT1, At1g56340; CRT2, At1g09210), a vacuolar calcium-binding protein-related (At1g62480), and a small Ras-like GTP-binding protein (Ran-1, At5g20010). In plant cells, Ca2+ is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Modulation of intracellular Ca2+ levels is partly regulated by calcium-binding proteins, which, after activation, induce specific kinases. Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Plants have three CRT isoform groups (CRT1, CRT2, and CRT3) and Arabidopsis has 18 CRT proteins, and members of the different isoform groups respond differently to applied external stimuli (Persson et al., 2003). The CRT1 and CRT2 identified in this study support that they are the major isoforms, possibly due to an enhanced Ca2+-binding efficiency, and play important roles in Ca2+ homeostasis under osmotic stress. Ran is an evolutionarily conserved eukaryotic GTPase, which is likely to be involved in nuclear translocation of proteins and cell cycle progression (Yang, 2002). However, little is known about the function of Ran in plant response to stresses. It was found that the abundance of Ran-1 increased after 48 h NaCl treatment, suggesting that Ran could also play a specific role under saline conditions.

Amino acid metabolism
The amount of Pro and certain other amino acids is reported to increase following NaCl treatment (Fougère et al., 1991; Di Martino et al., 2003). It was observed that the abundance of four amino acid biosynthesis-related enzymes was influenced by NaCl (Table 1). 3-Isopropylmalate dehydrogenase (AtIMD1, At5g14200), which is involved in Leu biosynthesis, and cobalamine-independent methionine synthase (ATCIMS/AtMetE, At5g17920) decreased in abundance following NaCl treatment. However, glutamate dehydrogenase 2 (GDH2, At5g07440) and glutamine synthetase (GS, At1g66200) both decreased in abundance at the 6 h time point, but increased at the 48 h time point. GS functions as the major assimilatory enzyme for ammonia, and GDH works as a link between carbon and nitrogen metabolism as it can aminate 2-oxoglutarate into glutamate (biosynthetic reaction) or deaminate glutamate into ammonium and 2-oxoglutarate (catabolic reaction). GS and GDH, together with a number of other enzymes, play key roles in maintaining the balance of carbon and nitrogen (Miflin and Habash, 2002). A recent study showed that a salinity-generated ROS signal induces {alpha}-GDH subunit expression, and the anionic iso-GDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis (Skopelitis et al, 2006).

Cytoskeleton
Actin and tubulin dynamics have important functions in cellular homeostasis. The cytoskeleton is rapidly remodelled by various endogenous and external stimuli such as hormones, low temperature, aluminium, and NaCl (Abdrakhamanova et al., 2003; Dhonukshe et al., 2003; Sivaguru et al., 2003). For example, the transverse orientation of cortical microtubule arrays in tobacco BY-2 cells was remodelled to a more random arrangement after treatment with 150 mM NaCl for 15 min (Dhonukshe et al., 2003). More recent research suggests that NaCl stress compromises the organization of cortical microtubule arrays, in which SPR1 is involved, and inhibits anisotropic growth (Shoji et al., 2006). It was found that one actin protein, ACT8 (At1g49240), and one tubulin ß-chain (At5g62690) decreased in abundance following NaCl treatment, while tubulin {alpha}-6 chain (TUA6) was induced by NaCl (Table 1). These observations are consistent with previously reported microarray results (Seki et al., 2002; Jiang and Deyholos, 2006) and, although their mechanistic significance is not fully clear, the stress-responsiveness of these common, cytoskeletal proteins calls into question their designation sometimes as housekeeping genes.

Transcription-related proteins
Transcriptional control of the expression of stress-responsive genes is a crucial part of the plant response to various abiotic and biotic stresses. Nascent polypeptide-associated complex (NAC) is a heterodimeric complex ({alpha}- and ß-NAC) that can reversibly bind to eukaryotic ribosomes. Rospert et al. (2002) suggested that NAC is a negative regulator of translocation into the endoplasmic reticulum and a positive regulator of translocation into the mitochondria. Previous studies found that the {alpha} chain of NAC in osteoblasts functions as a transcriptional coactivator (Yotov et al., 1998). Here, two NAC domain-containing proteins (At1g17880 and At1g73230) were identified, which are similar to human transcription factor BTF3 (RNA polymerase B transcription factor 3) and whose abundance increased following NaCl treatment (Table 1). Interestingly, transcripts for these proteins significantly decreased in abundance in the previous microarray study of Jiang and Deyholos (2006). Furthermore, proteomic evidence showed that a rice {alpha}-NAC was down-regulated by NaCl and cold stresses (Yan et al., 2005, 2006). Based on previous microarray results, it is likely that hundreds of other transcription factors changed in abundance in the tissues examined, but that these proteins fell below the sensitivity threshold of analysis, and were therefore not detected in the protein gels (Seki et al., 2002; Jiang and Deyholos, 2006).

Correlation analysis of mRNA and proteins levels
The relationship between gene expression measured at the mRNA level and the corresponding protein level has not been well characterized in plant roots under abiotic stresses. To evaluate the correlation between mRNA and the corresponding protein levels, the differentially expressed protein levels were compared with previous oligonucleotide microarray data (Jiang and Deyholos, 2006). Tissues for both types of analyses were grown and treated under nearly identical conditions to minimize the effects of experimental variation on the results. The oligonucleotide probes for two proteins identified in the proteomic study were not represented in the microarray: ACO2 (At1g62380) and putative tubulin ß-2/ß-3 chain (At5g62690). Another 32 genes were filtered out from the microarray data as their signal intensities did not pass the threshold background intensity and the statistical analysis. Altogether, 54 genes/proteins were obtained for the comparison (Fig. 5). For the proteins whose abundance decreased at one or more time points following NaCl treatment, mRNA of most genes (85%, 22 out of 26) also decreased. However, for the 28 proteins that increased in abundance after NaCl treatment, only 10 genes (36%) also increased at the mRNA level (Fig. 5; the other 22 spots showed different expression patterns between mRNA and protein at least at one time point). Interestingly, a similar pattern was reported in a comparison of 2-DGE and qRT-PCR data for chilling stress in rice: 88% (15 out of 17) of proteins and their corresponding transcripts decreased in parallel following stress, while only 19% (5 of 27) of proteins that increased in abundance following stress had cognate transcripts that also increased in abundance (Yan et al., 2006). Together, these studies suggest that transcript abundance may be more directly relevant to expression of genes that are down-regulated following stress than those genes that are up-regulated. However, further study is required to demonstrate the generality of this pattern across a larger sample of proteins, treatments, and measurement techniques.


Figure 5
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  		Fig. 5. Comparison of NaCl-induced changes in mRNA and cognate protein abundance. The relative change in abundance (treated/untreated) is shown in log2 scale for proteins extracted from roots treated for (A) 6 h or (B) 48 h, as compared with previously reported (Jiang and Deyholos, 2006) changes in mRNA abundance levels in tissues grown under identical conditions.

 
In quantitative terms, the correlation between the expression ratios (i.e. treated/control) observed for proteins in the present experiment and the transcript in previous microarray experiments was low at both 6 h (r= –0.13) and 48 h (r=0.11). These results support the conclusion of other authors that, in statistical terms, measurements of mRNA are not well correlated with protein abundance (Gygi et al., 1999; Tian et al., 2004; Noir et al., 2005; Mooney et al. 2006; Yan et al., 2006).


   Conclusion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
Supplementary material
 References
 
In this study, a proteomic analysis of Arabidopsis roots subjected to non-lethal NaCl treatment for 6 h or 48 h, with physiologically defined responses, was performed (Fig. 1). Symptoms of stress, such as electrolyte leakage and Na concentration, continued to increase until at least 48 h, even though considerable remodelling of the proteome had apparently occurred before this time point (Figs 1, 2). Eighty-one different NaCl-responsive proteins were identified by LC-MS/MS (Table 1). The proteins identified were implicated in a wide range of physiological processes, i.e. energy metabolism, ROS scavenging and detoxification, protein translation, processing, and degradation, signal transduction, hormone and amino acid metabolism, cell wall modifications, as well as cytoskeleton remodelling, which might work cooperatively to re-establish cellular homeostasis under water deficiency and ionic toxicity. Some of the proteins identified here were also identified in previous microarray profiling of Arabidopsis response to NaCl stress as well as in 2-DGE analysis of Arabidopsis cell suspension cultures (Kreps et al., 2002; Seki et al., 2002; Ndimba et al., 2005; Jiang and Deyholos, 2006). The proteins identified in this study represent only a small part of the Arabidopsis proteome responsive to NaCl treatment, and many other NaCl-responsive proteins still need to be identified. Considering the limitations of a proteomic study based on 2-D gels, i.e. inability to resolve membrane proteins and detect low-abundant proteins, complementary strategies at the transcript, protein, and metabolite levels should be used to gain more insight into the intricate network of plant response to high salinity. Such approaches will include use of two-dimensional high-performance liquid chromatography, sub-proteomics study, or other alternative approaches (Lee et al., 2004; Peck, 2005; Baginsky and Gruissem, 2006; Kim et al., 2007a). The identification of novel NaCl-responsive proteins provides not only new insights into NaCl stress responses but also a good starting point for further dissection of their functions using genetic and other approaches.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 Supplementary material
References
 
Table S1. Peptide sequence data for the 86 Arabidopsis root proteins identified by LC-MS/MS.

Table S2. Significant hits to protein spots as analysed by MASCOT with a Mowse score of more than 100 and number of matched peptides more than 3.


   Acknowledgements
 
We are grateful to Dr Anthony Cornish in the Molecular Biology Service Unit (MBSU), University of Alberta for help in 2-DGE facility and Agilent 1100 system use, and to Manjeet Kumari, Naomi Hotte, and Matt Bryman for providing methods or technical assistance. We also thank anonymous reviewers for constructive comments. The project was funded by an NSERC (Natural Sciences and Engineering Research Council) Discovery grant to MKD.


   Abbreviations
 
ACN, acetonitrile; ACO, ACC oxidase; APX, ascorbate peroxidase; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate; CRT, calreticulin; 2-DGE, two-dimensional gel electrophoresis; dpg, days post-germination; DTT, dithiothreitol; FA, formic acid; GDH, glutamate dehydrogenase; GLP, germin-like protein; GPX, glutathione peroxidase; GRP, glycine-rich protein; GS, glutamine synthetase; GST, glutathione S-transferase; IEF, isoelectric focusing; IPG, immobilized pH gradient; JA, jasmonic acid; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; NAC, nascent polypeptide-associated complex; REL, relative electrolyte leakage; ROS, reactive oxygen species; SAM, S-adenosyl-L-methionine; SOD, superoxide dismutase; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis.


   References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 Supplementary material
 References
 
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