Journal of Plant Physiology 169 (2012) 117–126
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Identification of differentially expressed salt-responsive proteins in roots of two perennial grass species contrasting in salinity tolerance Yiming Liu a , Hongmei Du a , Xiaoxia He a , Bingru Huang b , Zhaolong Wang a,∗ a b
School of Agricultural and Biological Sciences, Shanghai Jiao Tong University, 800 DongChuan Road, MinHang District, Shanghai, 200240, PR China Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, United States
a r t i c l e
i n f o
Article history: Received 4 March 2011 Received in revised form 12 August 2011 Accepted 25 August 2011 Keywords: Salt tolerance Warm-season turfgrass Salt-responsive proteins Protein changes
a b s t r a c t This study was designed to identify physiological responses and differential proteomic responses to salinity stress in roots of a salt-tolerant grass species, seashore paspalum (Paspalum vaginatum), and a salt-sensitive grass species, centipedegrass (Eremochloa ophiuroides). Plants of both species were exposed to salinity stress by watering the soil with 300 mM NaCl solution for 20 d in a growth chamber. The 2DE analysis revealed that the abundance of 8 protein spots significantly increased and 14 significantly decreased in seashore paspalum, while 19 and 16 protein spots exhibited increase and decrease in abundance in centipedegrass, respectively. Eight protein spots that exhibited enhanced abundance in seashore paspalum under salinity stress were subjected to mass spectrometry analysis. Seven protein spots were successfully identified, they are peroxidase (POD, 2.36-fold), cytoplasmic malate dehydrogenase (cMDH, 5.84-fold), asorbate peroxidase (APX, 4.03-fold), two mitochondrial ATPS␦ chain (2.26-fold and 4.78fold), hypothetical protein LOC100274119 (5.01-fold) and flavoprotein wrbA (2.20-fold), respectively. Immunblotting analysis indicated that POD and ATPS␦ chain were significantly up-regulated in seashore paspalum at 20 d of salinity treatment while almost no expression in both control and salt treatment of centipedegrass. These results indicated that the superior salinity tolerance in seashore paspalum, compared to centipedegrass, could be associated with a high abundance of proteins involved in ROS detoxification and energy metabolism. © 2011 Elsevier GmbH. All rights reserved.
Introduction Salinity is a major abiotic stress that affects plant growth and development. Salt stress causes water deficit, ion toxicity, nutrient imbalance, and oxidative stress, leading to cellular damage and growth reduction, and even plant death (Tester and Davenport, 2003; Munns, 2005; Sahar et al., 2007). Salinity tolerance of plant is a complex phenomenon that involves physiological, biochemical, and molecular processes (Greenway and Munns, 1980; Munns and Tester, 2008). Roots are the first organ of a plant to experience the salt stress when Na+ and Cl− are present in the soils. The toxic ions are transported into the plant along with the water stream, which moves from soil to the vascular system of the root by different pathways including symplastic and apoplastic pathways (Tester
Abbreviations: RWC, relative water content; REL, root electrolyte leakage; 2DE, two-dimensional electrophoresis; POD, peroxidase; APX, asorbate peroxidase; MDA, monodehydroascorbate; cMDH, cytoplasmic malate dehydrogenase; ATPS␦, ATP synthase subunit Delta; LSD, least significant difference; ROS, reactive oxygen species; CBB, coomassie brilliant blue; DTT, dithiothreitol; Fv/Fm, variable fluorescence to maximal fluorescence; IEF, isoelectric focusing. ∗ Corresponding author. E-mail address:
[email protected] (Z. Wang). 0176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2011.08.019
and Davenport, 2003). The ability of plants to maintain low cytosolic salt concentrations is controlled by their ability for selective ion uptake, ion exclusion (Shi et al., 2000) and compartmentalization, particularly of toxic levels of Na+ in vacuoles (Blumwald and Poole, 1987; Apse et al., 1999). Several root proteins, such as plasma membrane and vacuolar H+ -ATPase and Na+ /H+ antiporters, play an essential role in ion uptake and transport (Munns, 2002; Aharon et al., 2003). In addition, antioxidant enzymes and soluble proteins in the cytoplasm have an important function in protecting cells from salt-induced oxidative stress (Gossett et al., 1994; Marcum and Murdoch, 1994). Proteomic analysis is an effective method for studying protein responses to salt stress (Parker et al., 2006). Separation of proteins by two-dimensional electrophoresis (2-DE) allows for analysis of the expression of stress-induced proteins in plants, and may help to identify the roles of these proteins under a variety of physiological and environmental conditions (Komatsu et al., 2003). Previous studies have revealed salt-related proteins by 2-DE in roots of various plant species, such as Arabidopsis (Arabidopsis thaliana) (Jiang et al., 2007), rice (Oryza sativa) (Yan et al., 2005), wheat (Triticum aestivum) (Majoul et al., 2000), pea (Pisum sativum) (Kav et al., 2004), cucumber (Cucumis sativus) (Du et al., 2010) and sugar beet (Beta vulgaris L.) (Wakeel et al., 2011). These studies demonstrated
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that salt-responsive proteins are involved in a variety of metabolic processes, such as scavenging for ROS, signal transduction, transcription and translation, transporting, chaperones, cell wall biosynthesis, processing and degradation, metabolism of energy, amino acids, and hormones (Taylor et al., 2009). Changes in proteins and other cellular components for plant acclimation to salt stress largely depend on the salt concentration, salt treatment duration and plant species. In addition, it is still unclear which proteins or metabolic processes are associated with salinity tolerance. Perennial turfgrass species exhibit a wide range of salinity tolerance (Peacock and Dudeck, 1985; Marcum and Murdoch, 1994; Marcum et al., 1998; Lee et al., 2004). Seashore paspalum is often found growing around brackish ponds and estuaries, and is among the most salinity-tolerant warm-season turfgrass species. Our recent research found that the salinity tolerance thresholds for seashore paspalum and centipedegrass were 474.0 mM and 222.4 mM, respectively (Liu et al., 2009). Seashore paspalum exhibited superior salt tolerance to centipedegrass, which has previously been attributed to its ability to maintain greater levels of photosynthesis, osmotic adjustment, shoot growth rates and tissue water content (Peacock and Dudeck, 1985; Marcum and Murdoch, 1994; Marcum et al., 1998; Lee et al., 2004). However, little information is available about interspecific variations in proteomic responses to long-term salt stress in perennial grass species. The identification of these variations will provide further insight into the molecular mechanisms of salt tolerance in C4 perennial grass species. Salt-tolerant species such as seashore paspalum may possess some unique proteins, up-regulated stress defensive proteins, or proteins maintained to a greater extent than in salt sensitive species, which would allow better plant survival of severe salt stress in the tolerant plants. The objective of this study was to identify saltresponsive proteins in roots of seashore paspalum that differ from centipedegrass species, which may be associated with superior salinity tolerance using 2-DE and MS technologies.
Materials and methods Plant materials and treatments ‘Salam’ seashore paspalum and ‘Civil’ centipedegrass sod pieces were collected from 3-year-old field plots in the Turfgrass Experiment Farm at Shanghai Jiao Tong University, Shanghai, China. They were transferred into plastic pots (17 cm diam., 20 cm height, with 4 holes at the bottom for drainage) filled with washed sand (0.25–1.0 mm diam.). Plants were maintained in a greenhouse for 2 months and then moved into a growth chamber. The chamber was set at 30/25 ◦ C (day/night temperature), 75% relative humidity, 14 h photoperiod, and 400 mol m−2 s−1 of photosynthetically active radiation. Plants were fertilized weekly with full-strength Hoagland solution (Hoagland and Arnon, 1950). Plants were watered 3 times a week until drainage was observed at the bottom of the pot. Plants were allowed to acclimate to growth chamber conditions for 2 weeks before salinity treatment was imposed. All plants were trimmed to the canopy height of 5 cm before salt stress treatment was imposed to start with a uniform turf canopy. Plants of both species were irrigated daily with deionized water (control) or salt solution to induce salinity stress for 20 d. The salinity level was gradually increased by adding NaCl solution of 6, 12 and 18 dS m−1 until the EC of the leachate reached 26 dS m−1 (300 mM) in the soil and then by watering soil with 300 mM NaCl solution daily for 20 d. Each treatment had 4 replicates. Exposure of plants to increasing salt concentration allows gradual acclimation of plants to salinity conditions to avoid sudden death of plants at high salt concentration. The solution with 300 mM NaCl was slowly watered into the soil. Care was taken to avoid salt solution on leaves,
minimizing potential salt-induced leaf burning. Plants were treated for 20 d and root sample were collected every 4 d during the whole experiment. At the end of the salinity treatment, all roots from each container were washed free of sand, harvested and immediately frozen in liquid nitrogen, and then stored at −80 ◦ C prior to analysis. Measurements of physiological parameters Physiological responses to salinity stress were evaluated by measuring root electrolyte leakage (REL), root osmotic potential and root viability during the treatment period. All measurements were taken on 4 replicates in each treatment during the experimental period. Root electrolyte leakage (REL) was measured by the method of Radoglou et al. (2007) with some modifications. Roots were excised and cut into 2 cm segments. After being rinsed 3 times with deionized water, 0.2 g root segments were placed in a test tube containing 20 ml deionized water. Test tubes were agitated on a shaker for about 24 h, and the solution conductivity (C1) was measured with a conductivity meter (DDS-320, Shanghai Kangyi Co., Ltd., Shanghai, China). Samples then were killed in an autoclave at 100 ◦ C for 20 min, and the conductivity of the solution containing killed tissue was measured after tubes cooled down to room temperature (C2). The REL was calculated as (C1/C2) × 100. Root osmotic potential was measured with a calibrated vapor pressure osmometer (Vapro 5520, Wescor, Logan, UT). Fresh roots (0.5 g) were obtained and soaked into deionized water for 6–8 h, surface water was removed, then roots were placed in a 1.5 ml centrifuge tube and immediately frozen in liquid nitrogen for 1 h. Tubes were thawed and cell sap was pressed from roots, which was subsequently analyzed for osmolarity (C) (mmol kg−1 ). Osmolarity of cell sap was converted from mmol kg−1 to osmotic potential (Mpa) using the formula: Mpa = −C × 2.58 × 10−3 (Blum and Sullivan, 1986; Blum, 1989). Root viability was determined by measuring respiratory activity with triphenyltetrazolium chloride (TTC) (Steponkus and Lanphear, 1967). About 0.5 g root tips were isolated and immersed completely in a 1/1 mixture of 4% 2,3,5-triphenyl tetrazolium chloride (TTC) and phosphate buffer. The mixtures were placed in the dark at 37 ◦ C for 2 h before 2 ml 1 M H2 SO4 was added to end the reaction. Roots were taken out and grinded with ethyl acetate. Reduced red tetrazolium was extracted and the absorbance read at 485 nm. Protein extraction and labeling At 20 d of treatment, roots were washed free of sand, harvested and immediately frozen in liquid nitrogen and kept at −80 ◦ C. Three independent samples were harvested from each treatment and analyzed by electrophoresis. About 1 g of root samples were homogenized and incubated with 5 ml 10% w/v trichloroacetic acid (TCA) in acetone containing 0.3% dithiothreitol (DTT) for 12 h at −20 ◦ C. The precipitated proteins were pelleted and washed with ice-cold acetone containing 0.3% DTT until the supernatant was colorless. The pellet was dried and resuspended in lysis buffer [7 M urea + 2 M thiourea + 65 mM DTT + 4% CHAPS + 1% cocktail (protease Inhibitor Cocktail Set III, Merck, Cat. No. 539134-1ml)], and vortexed for 1 h to extract proteins. Insoluble tissue was removed by centrifugation at 14,000 g for 60 min. The protein content was determined using the 2D-Quant Kit (Amersham Biosciences, San Francisco, CA, USA). Two-dimensional electrophoresis An Ettan IPGPhor3 apparatus (GE Healthcare, Piscataway, NJ, USA) was used for isoelectric focusing (IEF) with immobilized pH gradient (IPG) strips (pH 3–10, NL gradient, 13 cm). The IPG
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strips were rehydrated for 12 h at 20 ◦ C with 250 l of rehydration buffer (8 M urea + 2% CHAPS + 15 mM DTT + 0.002% bromophenol blue + 0.5% IPG buffer pH3-10 NL) containing 300 g proteins. The voltage settings for IEF were 100 V for 3 h, gradient 200 V for 3 h, 1000 V for 1 h, gradient 8000 V for 3 h and 8000 V for 64,000 V h to a total 18 h. Following IEF, the protein in the strips was denatured with equilibration buffer (50 mM Tris–HCl pH 8.8 + 6 M urea + 30% glycerol + 2% SDS + 1% DTT + 0.002% bromophenol blue) and then incubated with the same buffer containing 2.5% iodoacetamide (IAA) instead of DTT for 20 min. The second dimension electrophoresis was performed on a 12.5% gel using a Hoefer SE 600 vertical chambers (GE Healthcare) with standard protein markers (Fermentas) loaded at the left-most side. The gels were run at 10 mA per gel for the first 30 min and followed by 20 mA per gel. After SDS-PAGE, gels were washed in ddH2 O 3 times for 15 min and stained with colloidal Coomassie Blue G-250 (Neuhoff et al., 1988). Image and statistical analysis The 2-D gels were scanned using Image scanner (Amersham Biosciences Company, Sweden). The scanner control software used was LabScan version 5.0 (Amersham Biosciences Corp., Piscataway, NJ). The scanned gels were saved as TIFF images for subsequent analysis. To correct the variability because of staining, 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 accumulated proteins. Spot detection, spot intensity measurement, background subtraction and spot matching were performed specifically after CBB staining of the gels using Image Master 2D Platinum 6.0 software (GE Healthcare, Uppsala, Sweden). Following automatic spot detection, gel images were carefully edited. Before spot matching, one of the gel images was selected as the reference gel. The amount of a protein spot was expressed as the volume of spot, which was defined as the sum of the intensities of all the pixels that make up the spot. In order to correct the variability due to CBB staining and to reflect the quantitative variations in intensity of protein spots, the spot volumes were normalized as a percentage of the total volume of all spots present in the gel. The molecular masses of proteins on gels were determined by coelectrophoresis of standard protein markers (Fermentas) and pI of the proteins determined by the migration of the protein spots on 12 cm IPG (pH 3–10, linear strips. In gel tryptic digestion Eight up-regulated protein spots that showed a change of at least 1.5-fold (mean of the 6 gel replicates) between control and treated samples in seashore paspalum were selected for digestion and identification by mass spectrometry (MS) analysis. Protein spots were excised from the preparative gels, destained with 100 mM NH4 HCO3 in 30% v/v acetonitrile (ACN). After removing the destaining buffer, the gel pieces were lyophilized and rehydrated in 30 l of 50 mM NH4 HCO3 containing 50 ng trypsin (sequencing grade; Promega, Madison, WI, USA). After overnight digestion at 37 ◦ C, the peptides were extracted 3 times with 0.1% TFA in 60% ACN. Extracts were pooled together and lyophilized. The resulting lyophilized tryptic peptides were kept at −80 ◦ C until mass spectrometric analysis. A protein-free gel piece was treated as above and used for a control to identify autoproteolysis products derived from trypsin. MALDI-TOF/TOF MS analysis and database searching All mass spectra were acquired on MALDI-TOF/TOF mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA). Data were analyzed using GPS Explorer software 3.6 (Applied Biosystem) and MASCOT software 2.2 (Matrix
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Science, London, UK). NCBInr and rice was selected as the database and taxonomy, respectively. National Center for Biotechnology non-redundant (NCBInr) database (updated on May 5, 2009), with Green Plant as taxonomy was searched against. The other parameters for searching were enzyme of trypsin; one missed cleavage; fixed modifications of carbamidomethyl (C); variable modifications of oxidation (Met). Peptide tolerance of 100 ppm; fragment mass tolerance of ±0.5 Da; peptide charge of 1+ were selected. Only significant hits, as defined by the MASCOT probability analysis CI% protein score >95%, were accepted. Protein blot analysis Methods used for immunoblotting were based on a modification from Cunningham and Volenec (1996). Proteins from SDS-PAGE gels were transferred to a polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA) with a VE-186 electrophoresis transfer unit (Tian Neng, Shanghai, China) using a continuous buffer system [0.39 M glycine, 0.48 M Tris, 20% methanol (v/v)] at 100 V for 1 h. The membranes were then blocked in a blocking solution (PA106-01, TIANGEN, Beijing) overnight at 4 ◦ C. After blocking, the membranes were incubated with a 1:2000 dilution of rabbit anti-POD polyclonal antibody (P1419, Uscnlife Science & Technology Company, Wuhan, China) and anti-ATP synthase polyclonal antibody (AS08370, Agrisera, Vaennaes, Sweden) in Tris-buffered saline Tween-20 (TBST) [0.05% (v/v) Tween-20 in TBS (0.015 M NaCl, 0.01 M Tris–HCl, pH 7.5)] for 1 h, and then kept at 4 ◦ C overnight. After primary antibody incubation, membranes were washed with TBST for 3 times, each time for 15 min and then immersed in TBST containing the secondary goat anti rabbit IgG antibody (dilution 1:4000) conjugated to alkaline phosphatase (L5904rB, Uscnlife Science & Technology Company, Wuhan, China) for 1.5 h. Secondary antibodies were detected using 5-bromo-4-chromo-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) substrate solution after washed in TBST for 45 min (Blake et al., 1984). The intensity of protein bands was analyzed for optical intensity using a protein imaging software (Tanon 2500, Tanon Science and Technology Co., Ltd., Shanghai, China). Activities of antioxidant enzymes and MDA content For enzyme extracts and assays, 0.5 g fresh root were frozen in liquid nitrogen and then ground in 4 ml solution containing 50 mM phosphate buffer (pH 7.0) and 1% (w/v) polyvinylpolypyrrolidone. The homogenate was centrifuged at 15,000 × g for 30 min, and the supernatant was collected for enzyme assays. The activity of POD was measured by following the change of absorption at 470 nm due to guaiacol oxidation. The activity was assayed for 1 min in a reaction solution (3 ml final volume) composed of 100 mM potassium phosphate buffer (pH 7.0), 20 mM guaiacol, 10 mM H2 O2 and 0.15 ml enzyme extract (Polle et al., 1994). The activity of APX was measured as a decrease in absorbance at 290 nm for 1 min (Nakano and Asada, 1981). The assay mixture consisted of 0.5 mM AsA, 0.1 mM H2 O2 , 0.1 mM EDTA, 50 mM sodium phosphate buffer (pH 7.0), and 0.15 ml enzyme extract. Lipid peroxidation was measured in term of MDA content (Dhindsa et al., 1981). A 1 ml aliquot of supernatant was mixed with 4 ml of 20% trichloroacetic acid containing 0.5% thiobarbituric acid. The mixture was heated at 100 ◦ C for 30 min, quickly cooled, and then centrifuged at 10,000 × g for 10 min. The absorbance of the supernatant was read at 532 nm. The unspecific turbidity was corrected by A600 subtracting from A532. The concentration of MDA was calculated using the molar extinction coefficient of 155 mM−1 cm−1 .
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Experimental design and statistical analysis The experiment was a 2 × 2 factorial combination (2 turfgrass species: seashore paspalum and centipedegrass, and 2 salinity levels: 0, 300 mM NaCl treatment) in a complete block design (1 treatment of 1 species served as the block) with 4 replications. All data were subjected to analysis of variance (SAS 8.1, SAS Institute Inc., Cary, NC). Treatment means were separated using the least significant difference (LSD) test at P ≤ 0.05. Results Effect of salinity on physiological parameters The REL of salt treated centipedegrass was significantly higher than the control level beginning at 4 d and reached 24.18% (2.30fold of the control), however, the REL of seashore paspalum was significantly affected under the salt treatment until 12 d and reached 18.42% (3.22-fold of the control) (Fig. 1A). The REL increased with the duration of salinity treatment in both turfgrass species, but the seashore paspalum increased to a lesser extent than centipedegrass. At the end of treatment (20 d), the REL of centipedegrass increased to 54.35% (3.82-fold of the control), and the REL of seashore paspalum only reached 32.25% (2.96-fold of the control). Root osmotic potential declined in both species with increasing stress duration, but the rate of decline in centipedegrass was much greater than seashore paspalum (Fig. 1B). Root osmotic potential of centipedegrass decreased significantly below the control level beginning at 4 d under the salt treatment and decreased to −0.93 Mpa (the control level is −0.45 Mpa). However, root osmotic potential of seashore paspalum decreased significantly below the control level beginning at 8 d under the salt treatment and decreased to −1.254 Mpa (the control level is −0.875 Mpa). At the end of salt treatment (20 d), root osmotic potential of centipedegrass decreased to −1.656 Mpa (the control level is −0.401 Mpa), while seashore paspalum decreased to −1.455 Mpa (the control level is −0.906 Mpa). Root viability declined in both species with increasing stress duration, but the decline in centipedegrass was much greater than seashore paspalum (Fig. 1C). Root viability of centipedegrass decreased significantly below the control level beginning at 8 d under the salt treatment and decreased to 64.5 g g−1 h−1 (73.24% of the control level). Root viability of seashore paspalum decreased significantly below the control level beginning at 12 d under the salt treatment and decreased to 72.3 g g−1 h−1 (68.51% of the control level). At the end of salt treatment (20 d), root viability of centipedegrass decreased to 21.8 g g−1 h−1 (25.37% of the control level), while root viability of seashore paspalum decreased only to 62.1 g g−1 h−1 (63.31% of the control level). 2-DE analysis of root proteins in seashore paspalum and centipedegrass In order to investigate the changes of root proteome in response to salt stress, 2-DE analysis of the total proteins at 20 d of salt treatment in seashore paspalum and centipedegrass was carried out. Fig. 2 shows the 2-DE maps obtained from control plants and salt-treated plants. Each treatment has three replications. Approximately 250 protein spots were detected on seashore paspalum stained gels and about 300 protein spots were detected on centipedegrass stained gels. Salt-responsive proteins of seashore paspalam and centipedegrass Quantitative image analysis revealed the protein spots that changed their abundance (vol.%) significantly (P < 0.05) by 1.5-fold
Fig. 1. Effects of the salt stress on (A) root electrolyte leakage (root EL), (B) root osmotic potential and (C) root viability for seashore paspalum and centipedegrass. Vertical bars indicate LSD value (P ≤ 0.05).
or higher. 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). In our research, 8 spots (Fig. 2A, spots 1–8) exhibited significant increases in abundance (up-regulated) and 14 spots had a decline in abundance (Fig. 2A, spots 9–22) (down-regulated) in seashore paspalum. Nineteen spots (Fig. 2B, spots1–19) were significantly up-regulated and 16 (Fig. 2B, spots 20–35) were significantly down-regulated in centipedegrass. Seven up-regulated proteins in seashore paspalum roots were successfully identified by MALDI TOF/TOF (Table 1; see Supplementary data). The examination of up-regulated proteins, particularly in salinity-tolerant paspalum plants may give us specific mechanisms of stress adaption or tolerance. Spots 1–4 and spots 6–8 (Fig. 2A) were successfully identified. These proteins were POD (spot 1), hypothetical protein LOC100274119 (spot 2), cMDH (spot 3), APX (spot 4), mitochondrial ATPS␦ chain (spots 6 and 7) and putative flavoprotein wrbA (spot 8), respectively. Most of
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Fig. 2. Coomassie-stained 2-DE gel of separated proteins from seashore paspalum (A) and centipedegrass (B) root (control and NaCl-treated). Proteins were separated in the first dimension on an IPG strip (pH 3.0–10.0) and in the second dimension on a 12.5% gel. Marked proteins were salt-responsive proteins of seashore paspalam and centipedegrass (up and down regulated, >1.5-fold, n = 3).
these proteins belong to the ROS scavenging category and energy metabolism. The POD was shown to increase 2.3-fold expression abundance in seashore paspalum roots after 20 d of treatment. The cMDH and APX were strongly up-regulated, which were 5.6-fold and 3.7-fold more abundant in seashore paspalum roots after 20 d salinity treatment compared to the control. Mitochondrial ATPS␦ chain increased 2.3-fold and 4.7-fold in seashore paspalum roots, respectively, under salinity stress (Table 2). The seven successfully identical proteins were confirmed in the corresponding protein spots (protein spot at the same position with the same values of molecular mass and pI in treated and untreated protein 2D gels) of centipedegrass. Three corresponding protein spots were not expressed in centipedegrass (POD, APX, flavoprotein wrbA), while the other 4 corresponding protein spots (cMDH, 2 ATPS␦ chain, LOC100274119) were expressed in centipedegrass (Figs. 3 and 4).
Validation of spot expression by immunoblotting analysis In order to confirm the protein expression revealed by 2-D gel electrophoresis, Protein blot analysis was performed on POD and
ATPS␦ chain. A higher level of expression of POD and ATPS␦ chain was detected at 20 d of salt treatment for seashore paspalum compared to control signals obtained from immunoblotting (Fig. 5). While centipedegrass had a very low level of expression of POD and ATPS␦ chains in both control and salt treatments.
Effect of salinity on activities of antioxidant enzymes and MDA content Immunoblotting analysis showed that a higher level of POD expression was detected at 20 d of salt treatment for seashore paspalum compared to the control while centipedegrass had very low levels of expression of POD in both control and salt treatments. So, activities of antioxidant enzymes and MDA content were measured for the purpose of comparing ROS detoxification abilities between seashore paspalum and centipedegrass. The MDA content of centipedegrass and seashore paspalum were significantly higher than the control level at 4d and reached 30.2 nmol g−1 Fw (2.19-fold of the control) and 26.3 nmol g−1 Fw (1.67-fold of the control), respectively (Fig. 6A). With the duration of salinity treatment, the MDA content increased in both turfgrass
Table 1 Up-regulated protein in seashore paspalum roots identified by MALDI TOF/TOF. Spot numbers correspond to Fig. 2A. The assigned protein that best matched was given with the species in which it had been identified and its accession number. T MW/PI, theoretical molecular weight (kD)/isoelectrical point(pI); O MW/PI, observed molecular weight (kD)/isoelectrical point (pI); fold change, salt/control; PS, protein score; PM, the number of unique peptides matched. Coverage percent (%). Spot no.
Protein name [species]
T MW/PI
O MW/PI
Fold change
Accession no.
PS
PM
Coverage percent (%)
1 2 3 4 5 6 7 8
Peroxidase [Zea mays] Hypothetical protein LOC100274119 [Zea mays] Malate ehydrogenase, cytoplasmic [Zea mays] Asorbate peroxidase [Pennisetum glaucum] No detected ATPS␦ chain, mitochondrial [Zea mays] ATPS␦ chain, mitochondrial [Zea mays] flavoprotein wrbA, putative [Ricinus communis]
51/6.22 50/6.01 43/5.92 32/5.67
55.8/7.59 48.1/6.9 45.9/5.77 37.6/5.69
2.36 5.01 5.84 4.03
gi|226493663 gi|226492249 gi|162464321 gi|145388991
232 316 646 410
7 15 13 8
29.27 32.09 57.83 44.4
26/5.10 26/5.17 18/6.10
29.915/5.19 29.915/5.19 21.581/5.83
2.26 4.78 2.20
gi|226507194 gi|226507194 gi|255555109
224 271 178
4 6 3
31.76 41.18 21.67
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Table 2 Up-regulated proteins in seashore paspalum roots identified by MALDI TOF/TOF. Spot numbers correspond to Fig. 2A. The assigned protein that best matched was given with the species in which it had been identified and its sequence. IS, ion score. Spot no.
Protein name [species]
Sequence
Calculated mass
Observed mass
IS
Modification
1
Peroxidase [Zea mays] Hypothetical protein LOC100274119 [Zea mays]
3
Malate ehydrogenase, cytoplasmic [Zea mays]
1218.5474 1730.886 1024.557 1300.596 1374.716 1373.7423 1679.7806 1807.8756 2000.1361 2016.1310 1343.6631 1803.9097 2071.0237 1415.6914 1571.7925 1857.8442 1985.9392 1415.6914 1571.7925 1857.8442 1985.9392 1240.5779 2572.3293
1218.485 1730.7915 1024.5602 1300.5974 1374.7229 1373.7704 1679.7863 1807.8846 2000.142 2016.135 1343.5946 1803.8413 2070.9458 1415.6859 1571.7931 1857.8534 1985.9464 1415.6868 1571.7974 1857.8575 1985.9473 1240.5779 2572.3391
56 137 49 60 96 105 130 161 143 41 85 159 106 55 26 61 62 61 37 76 63 80 79
Carbamidomethyl (C) [6]
2
THFHDCFVR DSVGVIGGPFWSVPTGR LFADFQKR TFTYYHPESR IAAVQALSGTGACR IVQGLPIDEFSR FSSALSAASSACKHIR KFSSALSAASSACDHIR VLVTGAAGQIGYALVPMIAR VLVTGAAGQIGYALVPMIAR NPLVFDNSYFK NPAEQAHGANAGLDIAVR QMGLSDQDIVALSGGHTLGR AFDDVNHQLQTK RAFDDVNHQLQTK FSQEPQPIDWEYYR FSQEPQPIDWEYYRK AFDDVNHQLQTK RAFDDVNHQLQTK FSQEPQPIDWEYYR FSQEPQPIDWEYYRK AFMDATGGLWR SDVPIITPNELAEADGLLF
4
Asorbate peroxidase [Pennisetum glaucum]
6
ATPS␦ chain, mitochondrial [Zea mays]
7
ATPS␦ chain, mitochondrial [Zea mays]
8
Flavoprotein wrbA, putative [Ricinus communis]
Carbamidomethyl (C) [13] Carbamidomethyl (C) [12] Carbamidomethyl (C) [13] Oxidation (M) [17]
Oxidation (M) [2]
Oxidation (M) [3]
Fig. 3. Magnified views of 7 proteins up-regulated in the roots of seashore paspalam and centipedegrass under well-watered (control) and salt treatmemt. ND, no detected. Spot numbers of seashore paspalam correspond to Fig. 2A. Spot numbers of centipedegrass correspond to Fig. 2B.
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Fig. 4. Relative volume of 7 proteins up-regulated in the roots of seashore paspalam and centipedegrass under well-watered (control) and salt treatmemt. sp, seashore paspalum; cg, centipedegrass; ND, no detected. Spot numbers of seashore paspalam correspond to Fig. 2A. Spot numbers of centipedegrass correspond to Fig. 2B.
species, but the seashore paspalum increased to a lesser extent than centipedegrass. At 20 d of the salt treatment, the MDA content of centipedegrass and seashore paspalum increased to 64.3 nmol g−1 Fw (4.3-fold of the control) and 42.2 nmol g−1 Fw (2.6-fold of the control). Root POD and APX activities of seashore paspalum were markedly increased under salinity treatments, which were significantly higher than the control level, beginning at 4 d and reached 144.5 U min−1 g−1 Fw (1.30-fold of the control) and 82.3 mol g−1 Fw (1.26-fold of the control), respectively (Fig. 6B and C). But root POD and APX activities of centipedegrass almost had no signifcant increase at the first 12 d of salt treatment. With the duration of salt treatment, root POD and APX activities of seashore paspalum had a decline, but they were still maintained significantly higher than the control level even at 20 d of treatment. Root POD and APX activity of centipedegrass decreased throughout the treatment, while at the end of the experiment, root POD and APX of centipedegrass declined to 84.6 U min−1 g−1 Fw (83.1% of the control) and 26.4 mol g−1 Fw (51.8% of the control). Discussion Salinity stress caused cellular damage and oxidative stress in roots of both species as indicated by the increases in REL and
MDA content, which may have led to the reduction in root viability, caused cellular membrane damage and growth inhibition. In this study it became clear that the kinetics of salinity treatment in seashore paspalum and centipedegrass is different in the first 4 days. The physiological data show that the first 4 days are important for survival under salt treatment. At 4 d the salt-sensitive centipedegrass suffers, the REL and root osmotic potential reached significantly higher and lower levels than the control, respectively, whereas the tolerant species seashore paspalum were unchanged. This was also visible in the root viability in days 0–8. Root viability of seashore paspalum decreased significantly below the control level beginning at 12 d while centipedegrass decreased beginning at 8 d. This indicated that seashore paspalum has the greatest ability to withstand initial salt stress than centipedegrass in the first days. Seashore paspalum also had relative lower REL and root osmotic potential and higher root viability than centipedegrass at 20 d of salt treatment. The MDA content of both species were significant increased in the first 4 days, this showed that the two species had suffered oxidative damage at the beginning of the salt treatment. The POD and APX activities of two species were significantly increased in the first 4 days, but the increases of seashore paspalum were significant higher than centipedegrass. With the duration of salinity treatment, POD and APX activities of two species were both declined,
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Fig. 5. Estimated protein abundance of POD based on 2-D gel electrophoresis and Protein blot analysis. sp, seashore paspalum; cg, centipedegrass.
but the POD and APX activities of salt treated seashore paspalum were maintained higher than the control level during the whole experiment. That indicated that seashore paspalum had higher ROS scavenging abilities than centipedegrass. Our results of root responses to salinity stress are consistent with previous studies that evaluated shoot responses to salt stress. In these studies, the two warm-season turfgrass species differed in physiological responses to salt stress, seashore paspalum exhibiting long-term salt tolerance than centipedegrass with higher leaf RCW, Fv/Fm, chlorophyll content, soluble proteins, lower leaf EL, etc. (Peacock and Dudeck, 1985; Marcum and Murdoch, 1994; Marcum et al., 1998; Lee et al., 2004). The differential physiological responses to salt stress between the two warm-season turfgrass species could be related to the changes at the protein level. There are two basic effects that could lead to salt acclimation. First is the regulation of ion homeostasis by regulation of channels such as SOS pathway, and this has been confirmed in Arabidopsis (Mehlmer et al., 2010; Monneuse et al., 2011). Second is the metabolic acclimation such as compatible solutes, ROS scavenging, energy metabolism (Taylor et al., 2009). Previous studies showed that the root proteins identified were classified into 11 categories similar to the convention used by Ndimba et al. (2005). Proteins involved in ROS scavenging and defense, energy metabolism (e.g. glycolysis, citrate cycle, electron transport) and protein metabolism (e.g. translation, processing, and degradation) comprised 52% of the proteins identified (Jiang et al., 2007). For example, the initial reaction of the proteome and phosphoproteome of maize after adjustment to saline conditions reveals members of sugar signaling and cell signaling pathways such as calmodulin, and gave hint to a transduction chain which is involved in NaCl-induced signaling (Zorb et al., 2010). In our research, the up-regulated salt-responsive proteins of seashore paspalum roots were found mostly to be related to energy metabolism and ROS scavenging, which might work cooperatively to re-establish cellular homeostasis under water deficiency and ionic toxicity. Our results confirmed that general principles in the biochemical acclimation to salt stress appears to be similar in diverse species like Arabidopsis, rice, or these turfgrasses (Hernandez et al., 2000; Salekdeh et al., 2002; Flagella et al., 2006; Jiang et al., 2007).
ROS scavenging enzymes Oxidative stress is commonly encountered in salt-stressed plants (McCue and Hanson, 1990; Gossett et al., 1994). Salt stress is often accompanied by accumulation of ROS such as superoxide rad• ical (O2 − ), hydroxyl radical (• OH) and hydrogen peroxide (H2 O2 ) in plant cells (Mittler, 2002). These ROS cause membrane damage and attack macromolecules. Plants have developed enzymatic and nonenzymatic systems to scavenge these toxic compounds, and plant salt tolerance has been found to be positively associated with a more efficient antioxidant system (Mittler, 2002; Noreen and Ashraf, 2008). The APX and POD are the common antioxidant enzymes. The APX use ascorbate as a reducing agent to catalyze the conversion of H2 O2 to water (Apel and Hirt, 2004). In our study, APX and POD were shown to increase 5.6-fold and 2.3-fold abundance in seashore paspalum roots after 20 d of treatment, respectively. However, APX and POD of centipedegrass were not seen to increase in a similar manner. Also, the APX and POD activity of seashore paspalum increased significantly while centipedegrass decreased significantly, especially at the end of the treatment. The MDA content of centipedegrass was significantly higher than seashore paspalum. The data indicates that seashore paspalum has greater antioxidant capacity than centipedegrass under salt treatment. The APX and POD have also been found to be up-regulated by salt stress in many other plants. It was also shown that in the absence of stress, APX is 4.4-fold more abundant in roots of a salt-tolerant rice genotype (‘Pokkali’) than in roots of a salt-sensitive genotype (‘IR29’) and suggested that the greater salt tolerance of Pokkali compared with IR29 may be due to a higher constitutive level of antioxidant capacity (Salekdeh et al., 2002). In Arabidopsis roots, the expression of APX was increased by 150 mM NaCl treatment for either 6 h or 48 h (Jiang et al., 2007). The APX and POD were also strongly induced by salt stress in the pea (Hernandez et al., 2000). In cotton ovules cultured in vitro, tolerance of salt stress is associated with higher constitutive levels of APX and glutathione reductase and a greater capacity to up-regulate superoxide dismutase (Rajguru et al., 1999). It is possible that the greater salt-tolerance of seashore paspalum compared with centipedegrass is due to a higher constitutive level of antioxidant capacity.
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Fig. 6. Effects of the salt stress on root MDA (A), POD (B) and APX (C) for seashore paspalum and centipedegrass. Vertical bars indicate LSD value (P ≤ 0.05).
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(contains 5 submits, ␣33␥1␦11). The Fo portion forms a transmembrane ion channel for the translocation of protons (Echt et al., 1987). In this study, spot 6 and 7 were identified as the ATPS␦ chain, which were markedly up-regulated 2.3-fold and 4.2-fold at 20 d of salt treatment. In Arabidopsis roots, mitochondrial ATPS␦ chain increased 1.53-flod at 6 h when exposed to 150 mM NaCl treatment (Jiang et al., 2007). In rice leaves, ATP synthase  subunit (small isoform) was up-regulated 2.6- and 5-fold at 24 h and 7 d under 50 mM NaCl, respectively. It is also found that mitochondrial ATP synthases were stimulated in osmotic stressed wheat (Flagella et al., 2006). However, the reason for enhanced expression of ATP synthase after long-term salt stress is unclear. Enhanced ATPS␦ chain in salt stressed seashore paspalum may reflect the requirement for increased ATP synthesis. The proteins identified in this study represent only the upregulated proteins of the seashore paspalum proteome responsive to salt treatment, and many other salt responsive proteins such as down-regulated in centipedegrass still need to be identified. The identification of salt-responsive proteins provides not only new insights into salt stress responses but also a good starting point for further dissection of their functions using genetic and other approaches. Considering the limitations of a proteomic such as 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. These approaches will include use of two-dimensional high-performance liquid chromatography, sub-proteomics study or other approaches (Lee et al., 2004; Peck, 2005; Baginsky and Gruissem, 2006; Kim et al., 2007; Jiang et al., 2007). In summary, salinity altered protein expression in roots of two warm-season turfgrass species. Seashore paspalum exhibited superior salinity tolerance, as demonstrated by the less severe physiological damages compared to centipedegrass. Seashore paspalum had higher levels of POD and APX in roots, which is critical for antioxidant defense against oxidative stress. In addition, seashore paspalum root also had higher expression of cMDH and mitochondrial ATPS␦ chains under salt treatment, which are involved in ATP synthesis. The functions of some of these differentially expressed proteins (Fig. 2A, spot 2, hypothetical protein LOC100274119; spot 8, putative flavoprotein wrbA) and direct involvement in salinity tolerance are not yet clear and warrant further analysis in future studies. This study gives new insights into salt stress response in warm-season turfgrass roots.
Energy metabolism
Acknowledgments
Malate dehydrogenase is an enzyme in the citric acid cycle that catalyzes the conversion of malate into oxaloacetate (using NAD+ ) and vice versa, it is also involved in gluconeogenesis, the synthesis of glucose from smaller molecules (Minárik et al., 2002). Malate dehydrogenase is essential for ATP production. ATP is required for many biosynthetic pathways in plant cells, and during external stress maintenance of energy requirements may increase considerably (Dooki et al., 2006). In this study, cMDH was 5.6-fold up-regulated in seashore paspalum roots after 20 d salinity treatment. The upregulation of these enzymes has been observed under salt, drought, and cold conditions (Salekdeh et al., 2002; Imin et al., 2004; Liska et al., 2004). In Arabidopsis roots, mitochondrial malate dehydrogenase increased 1.08-fold and 1.52-fold at 6 h and 48 h under 150 mM NaCl treatment, respectively (Jiang et al., 2007). In rice young panicles and leaves, cytosolic malate dehydrogenase was up-regulated up to 23% under 75 mM NaCl treatment (Dooki et al., 2006). The ATP synthase, a large 400 kDa protein complex, consists of an integral membrane Fo portion and an extrinsic F1 portion
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