Journal of Plant Physiology 174 (2015) 166–176

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Molecular Biology

Alterations in root proteome of salt-sensitive and tolerant barley lines under salt stress conditions Agnieszka Mostek a,∗ , Andreas Börner b , Anna Badowiec a , Stanisław Weidner a a Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Oczapowskiego Street 1a, 10-957 Olsztyn, Poland b Leibniz Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, 06466 Gatersleben, Germany

a r t i c l e

i n f o

Article history: Received 10 June 2014 Received in revised form 8 August 2014 Accepted 17 August 2014 Available online 20 October 2014 Keywords: Salt stress Hordeum vulgare Root proteome

a b s t r a c t Salinity is one of the most important abiotic stresses causing a significant reduction of crop plants yield. To gain a better understanding of salinity tolerance mechanisms in barley (Hordeum vulgare), we investigated the changes in root proteome of salt-sensitive (DH14) and tolerant (DH187) lines in response to salt-stress. The seeds of both barley lines were germinating in water or in 100 mM NaCl for 6 days. The root proteins were separated by two-dimensional gel electrophoresis. To identify proteins regulated in response to salt stress, MALDI-TOF/TOF mass spectrometry was applied. It was demonstrated that the sensitive and tolerant barley lines respond differently to salt stress. Some of the identified proteins are well-documented as markers of salinity resistance, but several proteins have not been detected in response to salt stress earlier, although they are known to be associated with other abiotic stresses. The most significant differences concerned the proteins that are involved in signal transduction (annexin, translationally-controlled tumor protein homolog, lipoxygenases), detoxification (osmotin, vacuolar ATP-ase), protein folding processes (protein disulfide isomerase) and cell wall metabolism (UDP-glucuronic acid decarboxylase, ␤-d-glucan exohydrolase, UDP-glucose pyrophosphorylase). The results suggest that the enhanced salinity tolerance of DH187 line results mainly from an increased activity of signal transduction mechanisms eventually leading to the accumulation of stress protective proteins and cell wall structure changes. © 2014 Elsevier GmbH. All rights reserved.

Introduction Crop plants are often exposed to various biotic and abiotic stresses, greatly reducing the productivity of the crop worldwide. Soil salinity is among the main abiotic stresses and affects more than 800 million hectares of land, equivalent to more than 6% of the total global area of the Earth (Munns and Tester, 2008). This is

Abbreviations: 2DE, two-dimensional electrophoretic protein separation; ABA, abscisic acid; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; DOC, sodium deoxycholate; DTT, dithiothreitol; IEF, isoelectric focusing; HSC 70, heat shock cognate 70 kDa; HSP 90, heat shock protein 90-kDa; MALDI-TOF/TOF, matrix assisted laser desorption ionisation time-of-flight/timeof-flight; MeS, methionine synthase; PFP, pyrophosphate-fructose-6-phosphate-1phosphotransferase; SAH-hydrolase, S-adenosyl-l-homocysteine hydrolase; SHMT, serine-hydroxymethyltransferase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PDI, protein disulfide isomerase-like protein; PMF, peptide mass fingerprinting; TCA, trichloroacetic acid; TCTP, translationally-controlled tumor protein homolog; UGD, UDP-glucuronate decarboxylase; vATP, vacuolar ATPase. ∗ Corresponding author. Tel.: +48 89 523 35 20. E-mail address: [email protected] (A. Mostek). http://dx.doi.org/10.1016/j.jplph.2014.08.020 0176-1617/© 2014 Elsevier GmbH. All rights reserved.

a serious problem, considering the growing global population and, consequently, the increased demand for food. Therefore, improving salt tolerance of crop plants is one of the current issues of global breeding program. Plants differ considerably in their tolerance to salinity. Cereals exhibit low salinity tolerance, but significant differences in salt sensitivity can be observed within this group of plants. Among the cereals, rice (Oryza sativa) is considered the most salinity-sensitive and barley (Hordeum vulgare) the most tolerant species (Munns and Tester, 2008). Plants’ response to salt stress occurs in two phases. Initially, the harmful effect of salinity is associated with low water potential of the root medium, resulting in osmotic stress. During this phase abscisic acid (ABA) is accumulated and a range of metabolic processes is inhibited, e.g. cell expansion, cell wall synthesis, protein synthesis, stomatal conductance and photosynthetic activity (Barkla et al., 2013). In the second phase, salt is transported via the xylem to the shoot. Na+ and Cl− ions accumulate in shoot cells to a toxic extent, resulting in ionic stress. Subsequently, ion accumulation enhances the production of reactive oxygen species (ROS). By the additional

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production of ROS in cells, the balance between production and removal of ROS is disrupted, which eventually leads to oxidative stress (Fatehi et al., 2012). Salt tolerance is a multigenic trait demonstrating the complexity at both the genetic and physiological levels. Hence, a profound analysis of the proteome is essential to understand the fundamentals of the physiology of salinity stress. The results may finally contribute to the development of effective plant breeding strategies for salt-affected areas (Shavrukov et al., 2010). In recent years proteome analyses have gained popularity in plant science and have mostly relied on two-dimensional electrophoretic protein separation (2DE) (Barkla et al., 2013). Significant improvements in 2DE technique and mass spectrometry analysis, including MALDI-TOF/TOF enable deeper and more accurate analysis of the proteome. Salinity tolerance mechanisms are not yet fully explained, especially for an early stage of barley development as majority of the research describing salinity-induced root proteome changes in barley was conducted on 3-leaf or subsequent stages of barley development (Witzel et al., 2009; Fatehi et al., 2012). Moreover, in the previous proteomic studies, salt stress was implicated at seedling (Witzel et al., 2009, 2014) or 4-leaf stage of development (Fatehi et al., 2012). In the present study, we investigate the root proteome changes of 6-day old barley seedlings. The salt stress is applied directly on barley grains to mimic the germination conditions in saline soil where the plant is exposed to salinity from the very beginning. Salt stress conditions applied at seed stage allows to gain the knowledge about the molecular defense mechanisms activated during the germination under salinity conditions. To date, there is lack of studies describing the proteome changes under seeds-applied salt stress in the roots of two barley lines contrasting in salinity tolerance. Materials and methods Germinating conditions Seeds of salt-tolerant (DH187) and salt-sensitive genotype (DH14) were obtained from Leibniz Institute of Plant Genetics and Crop Plant Research (Gatersleben, Germany). Salinity sensitive and tolerant lines of barley belong to the Steptoe/Morex mapping population, created in terms of salinity tolerance (Sadeghil et al., 2013). Seeds germination was conducted in a growth chamber at the constant temperature of 20 ◦ C for 6 days on the tissue-paper placed in glass cylinders. The seeds that constituted the control samples of sensitive (Cs) and tolerant (Ct) barley lines were germinated in Mili-Q water. Salt stressed samples of sensitive (Ss) and tolerant (St) barley lines were germinated in 100 mM NaCl solution. One hundred seeds were sown per every biological replication. After the appointed time the roots were cut and immediately frozen in liquid nitrogen. All experiments were performed in three biological replicates (n = 3). Fresh and dry weight content measurement For the measurement of the fresh and dry weight content, approximately 100 roots from at least three independent biological replicates of each sample were examined. For the fresh weight content measurement, roots were weighed immediately after cutting. For the dry weight content measurement, roots were weighted, dried at 105 ◦ C for 24 h and weighted again. The obtained data were analyzed with Statistica 10.0 (StatSoft, Poland), using Student’s ttest and reported as means ± SD. Differences between the control samples and stress samples were considered statistically significant at a significance level p ≤ 0.05.

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Protein extraction Harvested roots were grounded in liquid nitrogen using mortar and pestle. Proteins were extracted with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% IPG buffer pH 3–11 NL (GE Healthcare), 120 mM dithiothreitol, protease inhibitors cocktail (Sigma). The protein extraction was carried out for 1 h on the laboratory shaker at 4 ◦ C. After the incubation time samples were centrifuged (14,000 × g, 10 min, 4 ◦ C) and protein extracts were purified using DOC/TCA precipitation (Gómez-Vidal et al., 2008) with some modifications described below. One ml of the protein extract was mixed with 100 ␮L of 0.2% sodium deoxycholate (DOC). The mixture was incubated on ice for 15 min. Then 340 ␮L of 24% trichloroacetic acid (TCA) was added and the entire mixture was incubated on ice for 1 h. After the appointed time the samples were centrifuged (14,000 × g, 10 min, 4 ◦ C). The precipitates were subsequently washed twice with ice-cold acetone supplemented with 0.07% [w/v] DTT. Air-dried pellets were dissolved in rehydration buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer pH 3–11 NL (GE Healthcare), 80 mM DTT and 0.002% bromophenol blue. The protein concentration was quantified using 2D-PAGE adapted Bradford assay (Ramagli and Rodriguez, 1985) with BSA dissolved in rehydration buffer as a protein standard. The BSA standards were prepared in the volume of 50 ␮L to cover the range of 0–12 ␮g ml−1 . BSA dilutions and the examined samples were acidified with 10 ␮L of 0.1 M HCl. Afterwards, 940 ␮L of Bradford reagent was added. The measurements were carried out at a wavelength of 595 nm using Infinite M200 PRO multimode microplate reader (Tecan). Two-dimensional electrophoresis Extracted proteins were separated using IEF/SDS-PAGE (Görg et al., 2004). Each sample, containing 400 ␮g of protein in rehydration buffer, was loaded onto 24 cm Immobiline DryStrip Gels (GE Healthcare) with the non-linear pH gradient 3–11. Isoelectric focusing was performed in the Ettan IPGphor 3 system (GE Healthcare). The running conditions were as follows: 30 V/10 h (active rehydration at 20 ◦ C), 500 V/1 h (step and hold), 1000 V/1 h (gradient), 8000 V/3 h (gradient), 8000 V/4:30 h (step and hold). After the isoelectric focusing, the IPG strips were equilibrated twice for 15 min in 15 ml of equilibration solution. The first equilibration solution contained 75 mM Tris–HCl buffer (pH 8.8), 6 M urea, 29.3% (v/v) glycerol, 2% (w/v) SDS, 0.002% bromophenol blue and 65 mM DDT. The second equilibration solution was modified by replacing DDT with 135 mM iodoacetamide. The second dimension (SDS-PAGE) was performed using 12.5% SDS polyacrylamide gels and was carried out in the Ettan DALTsix electrophoretic unit (GE Healthcare). Electrophoresis was performed at 30 ◦ C (2.5 W/gel for 30 min, 17 W/gel for 4:30 h). The protein spots were stained with colloidal CBB G-250. Each treatment was replicated thrice. Gels were scanned at 300 dpi using Image-Scanner III (GE Healthcare). Gel analysis was performed with Image Master 2D Platinum 7.0 software (GE Healthcare). The volume of each spot from each replicate was normalized against total spot volume and quantified. In order to investigate the salt-responsive proteins of both lines, the control samples and salt-stressed samples of both lines were matched. Proteins exhibiting at least 1.75-fold reproducible abundance changes between compared samples were subjected to statistical analysis using the t test (p ≤ 0.05). Only the spots that were present on all three replicate gels were quantified. The proteins which abundance changed significantly were designated to mass spectrometry for identification.

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Protein digestion and MALDI-TOF/TOF analysis The spots containing the proteins of interest were manually cut out from the gels with a scalpel blade. Prior to digestion, the proteins were washed and destained (Shevchenko et al., 1996). Proteins were dried in a vacuum centrifuge and digested using a trypsin solution (Promega) containing 15 ng ␮l−1 trypsin in 25 mM ammonium bicarbonate. The digestion was carried out overnight at 37 ◦ C. Samples were subsequently sonicated for 5 min and briefly centrifuged. A 1 ␮l aliquot of each peptide mixture was applied onto the ground steel MALDI target plate, previously covered by an equal volume of a freshly prepared 5 mg ml−1 solution of ␣cyano-4-hydroxy-cinnamic acid (CHCA) (Sigma) dissolved in 50% acetonitrile with 0.1% trifluoroacetic acid. Another layer (1 ␮l) of CHCA was applied as the peptide mixtures dried up. Peptide mass spectra were acquired using MALDI-TOF/TOF Autoflex III SmartBeam mass spectrometer (Bruker Daltonics). Up to 4 of the most intense ions were selected for MS/MS spectra acquisition. The MS together with MS/MS spectra were searched against the National Center for Biotechnology Information database (NCBI). MASCOT search engine (http://www.matrixscience.com) was used for PMF search. Statistical probability of the predicted protein was calculated by MASCOT, including PMF and ion scores. Scores above 72 (p < 0.05) were considered significant. The following parameters were applied: trypsin cleavage, taxonomy Viridiplantae, monoisotopic masses MH+ , up to ±0.2 Da mass tolerance, one missed cleavage site allowed, carbamidomethylation of cysteine as fixed modification and oxidation of methionine as variable modification.

Results Fresh and dry weight measurement

Fig. 1. Changes fresh (A) and dry weight (B) of barley roots isolated from sensitive (s) and tolerant (t) lines germinating under optimal (Control) or salt stress conditions (Stress). Results are presented as means ± SD. The statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001) between control and stress samples were marked with an asterisk.

The effect of salinity on barley seedlings growth was investigated in terms of differences in root fresh and dry weight content. The applied salinity stress affected fresh and dry weight of the roots in both barley lines (Figs. 1 and 2). However, the average root fresh weight of the tolerant line (84 mg) was substantially higher than that of the sensitive line (40 mg) under salt stress. The root fresh weight in the tolerant line was reduced by about 23% (Fig. 1A) while in the sensitive line it was reduced to a greater extent, by about 59%. Similar trend was also observed for root dry weight (Fig. 1B). The dry weight in tolerant and sensitive lines was decreased by about 21% and 52%, respectively, under salt stress conditions (Fig. 1B).

Identification of salt-responsive proteins The aim of this study was to investigate changes in protein patterns, occurring under salt stress in the roots of salt-sensitive and salt-tolerant barley lines. To separate and identify differentially expressed salt-responsive proteins, IEF/SDS-PAGE, coupled with MALDI TOF/TOF mass spectrometry were used. Among the controls and salt-treated samples of the two barley lines, more than 1800 reproducible protein spots were detected on gel replicates of which more than 1000 could be matched in the compared gels. Among the matched spots of both barley lines, the abundance levels (% vol) of 72 protein spots were changed more than 1.75-fold (p < 0.05), 47 spots were successfully identified by spectrometric analysis (Fig. 3). The identified proteins are summarized in Table 1. To demonstrate the quality of spots matching, relative abundances of selected spots of sensitive and tolerant barley lines under control and saline conditions were shown (Fig. 4A and B). Also protein spot abundance, given by the normalized spot

Fig. 2. Phenotypes of 6-days old barley seedlings of salt-sensitive and tolerant lines germinating under control and salt-stress conditions. Cs, control sample sensitive line; Ss, stress sample of sensitive line; Ct, control sample of tolerant line; St, stress sample of tolerant line. Detailed description of the germination conditions can be found in the text.

volume as determined by the image analysis software was demonstrated (Fig. 4 C and D). The protein profile of the tolerant barley line displayed high similarity to that of the sensitive line under non-salt conditions, but the profiles differed significantly under salt stress. Under control conditions only 9 proteins of both lines shown significant changes (data not shown) which is a small number compared to 72

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Fig. 3. The 2-DE analysis of root proteins from salt sensitive (s) and tolerant (t) barley lines under control (Cs, Ct) and salt stress conditions (Ss, St). Proteins of 400 ␮g were separated by 2-DE and visualized by Coomassie G-250 staining. Proteins were separated in the range of pH 3–11 (24 cm, nonlinear) in the first dimension and 12.5% SDS-PAGE in the second dimension. The marked spots exhibiting significant abundance changes (1.75-fold change, p ≤ 0.05) between control and salt-stressed samples in both barley lines were analyzed by MALDI TOF/TOF MS. Protein information is listed in Table 1.

Fig. 4. Change in the abundance of spots 976 and 26 in the sensitive and tolerant barley lines under control and saline conditions. (A and B) Sections of 2D gel for close-up view of the spots 976 and 26, respectively. (C and D) Protein spot abundance of the spots 976 and 26, respectively, given by the normalized spot volume as determined by the image analysis software.

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Table 1 Salt stress-responsive proteins identified in roots of salt-sensitive and tolerant barley lines. Spot no.a

Protein name

Function

Exp. pI/MWd

Calc. pI/MWe

19 8 17 12 11 9

5.5/51 6.0/72.3 6.5/54.8 6.6/55.3 4.8/85.3 8.4/14

5.8/49.9 5.5/40.3 9.51/77.2 8.2/56.2 5.09/75.9 8.75/22.5

133 123 93 129

11 13 6 16

5.2/78 6.2/49 9.5/22 6.1/61

5.1/82.9 8.5/58.8 9.57/23.5 6.18/61.4

gi|474166845

226

25

6.5/41.7

7.16/39.2

T. urartu

gi|473887274

259

23

5.9/94.2

6.04/93.8

H. vulgare H. vulgare T. aestivum H. vulgare T. urartu H. vulgare H. vulgare H. vulgare H. vulgare T. urartu H. vulgare H. vulgare B. distachyon

gi|68655500 gi|50897038 gi|461744058 gi|473781647 gi|474071954 gi|532572 gi|2429087 gi|2429087 gi|56682582 gi|474139853 gi|1203832 gi|11527563 gi|357135971

383 164 139 193 166 196 217 181 112 105 85 92 334

18 16 14 6 16 16 25 12 9 6 8 8 14

6/85.1 5.6/90 5.3/51.2 5.85/43.6 6.9/43.1 5.7/94 6.1/94 6.2/92 6.9/19 6.5/35 7/79.7 5.1/81.2 5/61

5.97/84.7 5.68/84.7 5.49/48.5 5.9/45.2 7.1/39.2 5.73/96.4 6.25/96.8 6.34/96.6 6.04/25.8 6.15/35 7.96/67.8 5.23/68.7 5.95/59

Accession no.

H. vulgare H. vulgare T. urartu T. monococcum T. aestivum T. aestivum

gi|68655456 gi|51592190 gi|474154142 gi|115589736 gi|82582811 gi|471270462

378 144 141 122 105 152

T. urartu T. urartu T. urartu B. distachyon

gi|473983421 gi|474326909 gi|473808028 gi|357124641

T. urartu

Peptides no.c

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Proteins up-regulated under salt stress in both sensitive and tolerant barley lines Amino acid metabolism S-adenosyl-l-homocysteine hydrolase 783 Nucleotide pyrophosphatase Cell wall 907 Amino acid metabolism Serine-hydroxymethyltransferase 228 Serine-hydroxymethyltransferase Amino acid metabolism 748 966 Chaperone Heat shock protein 90 Cyclophilin A, chain A Chaperone 26 Proteins up-regulated under salt stress only in the sensitive barley line 909 Heat shock cognate 70 kDa Chaperone Citrate synthase Carbohydrate and energy metabolism 678 Other Stem 28 kDa glycoprotein 148 Pyrophosphate-fructose-6-phosphateCarbohydrate and energy metabolism 846 1-phosphotransferase 548 Pyruvate dehydrogenase E1 Carbohydrate and energy metabolism component subunit alpha, mitochondrial Aconitate hydratase, putative Carbohydrate and energy metabolism 942 Proteins up-regulated under salt stress only in the tolerant barley line Amino acid metabolism Methionine synthase 2 959 Methionine synthase Amino acid metabolism 976 Enolase Carbohydrate and energy metabolism 770 Phosphoglycerate kinase Carbohydrate and energy metabolism 544 Cell Wall UDP-glucuronic acid decarboxylase 519 Linolate 9S-lipoxygenase 1 Signal transduction 988 979 Lipoxygenase 2 Signal transduction Lipoxygenase 2 Signal transduction 980 Detoxification Thaumatin-like protein (osmotin) 126 Annexin Signal transduction 402 ␤-d-Glucan exohydrolase Cell Wall 912 Detoxification Vacuolar proton ATP-ase 943 832 ATP-synthase subunit beta, Carbohydrate and energy metabolism mitochondrial-like

Scoreb

Species

812 677 90

a b c d e

H. vulgare B. distachyon H. vulgare

gi|6136111 gi|357151587 gi|20140865

192 206 233

15 11 11

5.05/56.5 4.7/48.3 4.05/18

5.2/51.7 5.3/42 4.53/18.9

O. sativa T. urartu M. sinensis

gi|108710846 gi|474416088 gi|379054892

107 111 230

8 13 11

4.9/49 5.9/96 5.2/65

4.89/46.5 5.96/97.3 6.3/43

T. aestivum

gi|74048999

139

11

5.5/16.2

5.7/17.5

T. aestivum

gi|299469378

131

12

5.9/40

6.17/40.5

H. vulgare

gi|326526063

105

8

5.5/79

6.07/81.2

T. uratu H. vulgare H. vulgare H. vulgare

gi|474012573 gi|357134819 gi|326519550 gi|3688398

166 94 129 181

8 10 14 14

4.92/45.1 5.7/38 4.6/43 5.6/22.5

5.07/71.4 6.02/38.2 4.83/40.4 5.85/27.5

O. sativa T. aestivum

gi|15808779 gi|74048999

156 142

14 9

4.8/23.5 5.9/17

5.1/27.9 5.7/17.5

H. vulgare

gi|120668

122

12

6/24.9

6.2/33.4

T. aestivum H. vulgare H. vulgare

gi|194425591 gi|30025164 gi|326493760

103 180 185

9 22 16

8/31.2 5.85/37 5.1/25

8.59/32.9 6.29/40.6 5.43/40.3

H. vulgare H. vulgare H. vulgare

gi|50659026 gi|326494746 gi|326511695

82 126 96

9 9 9

5.2/22 5.4/39 6.2/34

7.1/39 5.66/38.3 9.97/35.1

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UDP-glucose pyrophosphorylase Cell Wall Predicted actin-1-like Other Signal transduction Translationally-controlled tumor protein homolog 723 Tubulin alpha-1 chain Other Elongation factor 2 Translation 994 Translation Eukaryotic translation initiation factor 780 4A-3-like protein, partial 46 Eukaryotic translation initiation factor Translation 5A1 Chaperones Protein disulfide isomerase-like 532 protein, putative Predicted protein Unknown 936 Proteins down-regulated under salt stress in both sensitive and tolerant barley line Heat shock cognate 70 kDa Chaperones 625 Glutelin type-A1-like, predicted Other 464 598 Predicted protein Unknown Ascorbate peroxidase Detoxification 116 Proteins down-regulated under salt stress only in the sensitive barley line l-Ascorbate peroxidase Detoxification 150 62 Eucaryotic translation initiation factor Translation 5A1 248 Glyceraldehyde-3-phosphate Carbohydrate and energy metabolism dehydrogenase 2 Root peroxidase Detoxification 357 Lipoxygenase 1 Signal transduction 469 281 Predicted protein Unknown Proteins down-regulated under salt stress only in the tolerant barley line UDP-d-glucuronate decarboxylase Cell wall 188 Predicted protein Unknown 464 Predicted protein Unknown 355

Spot ID numbers correspond to the spots in Fig. 3. Statistical probability of the predicted protein calculated by MASCOT including PMF and ion scores; scores above 72 (p < 0.05) are considered significant. The number of matched peptides. Experimental values of isoelectric point and molecular mass of the protein. Theoretical values of isoelectric point and molecular mass of the protein.

171

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Fig. 5. Pie chart showing the functional distribution of salt-responsive proteins in the root of salt sensitive and tolerant barley lines. (A) Proteins up-regulated in sensitive line; (B) proteins up-regulated in tolerant line; (C) proteins down regulated in sensitive line; (D) proteins down-regulated in tolerant line.

significantly changed salt-responsive proteins. The total number of salt-responsive proteins was higher in the tolerant (35 proteins) than in the sensitive line (22 proteins). Most of the salt-responsive proteins in the tolerant line were up-regulated (28 proteins). The number of up- and down-regulated proteins in the sensitive line was comparable, amounting 12 and 10 proteins, respectively. Functional distribution of salt-responsive proteins The identified proteins were classified according to their function into several groups such as: proteins involved in carbohydrate and energy metabolism, amino acid metabolism, signal transduction, detoxification processes, translation, cell wall metabolism, chaperones and other functions as well as hypothetical or putative proteins with unknown functions. The up-regulated proteins in the sensitive line were assigned to four functional groups: carbohydrate and energy metabolism (37%), amino acid metabolism (27%) chaperones (27%) and cell wall (9%) (Fig. 5A). The tolerant line showed accumulation of proteins associated with nine functional groups: amino acid metabolism (19%), signal transduction (15%), cell wall metabolism (12%), translation (12%), chaperones (11%) carbohydrate and energy metabolism (11%), detoxification (8%), other (8%) and unknown (4%) (Fig. 5B). It should be emphasized that the up-regulation of proteins involved in signal transduction, translation and detoxification was observed only in tolerant line. Most of the proteins showing down-regulation in the sensitive line are associated with detoxification processes (Fig. 5C), whereas the majority of down-regulated proteins in the tolerant line are assigned as unknown (Fig. 5D). Comparative proteomic analysis of salt-sensitive and tolerant barley lines under salt stress Although some proteins were regulated similarly in both lines under salt-stress conditions, the majority of salt-responsive proteins were line-specific. We identified 10 proteins regulated similarly in both barley lines, 12 proteins specifically regulated in

the sensitive line and 25 proteins regulated only in the tolerant line under salt stress conditions (Table 1). Among the 10 spots exhibiting similar patterns in both lines, 6 were up-regulated: spot 783 (S-adenosyl-l-homocysteine hydrolase), spot 907 (nucleotide pyrophosphatase), spot 228 and 748 (serine hydroxymethyltransferase, spot 966 (heat shock protein 90), spot 26 (cyclophilin A, chain A). Four other spots were downregulated: spot 625 (heat shock cognate 70 kDa), spot 464 (glutelin type-A1-like, predicted), spot 598 (predicted protein), spot 116 (ascorbate peroxidase). Out of the 12 spots specifically regulated in the sensitive line, 6 were up-regulated: spot 909 (heat shock cognate 70 kDa), spot 678 (citrate synthase), spot 148 (stem 28 kDa glycoprotein), spot 846 (pyrophosphate-fructose-6-phosphate1-phosphotransferase), spot 548 (pyruvate dehydrogenase E1 component subunit alpha, mitochondrial), spots 942 (aconitate hydratase, putative). The other 6 spots demonstrating downregulation pattern were: spot 150 (l-ascorbate peroxidase), spot 62 (eucaryotic translation initiation factor 5A1), spot 248 (glyceraldehyde-3-phosphate dehydrogenase 2), spot 357 (root peroxidase), spot 469 (lipoxygenase 1), spot 281 (predicted protein). Among the 25 salt-responsive spots regulated exclusively in tolerant line, 22 were up-regulated: spots 959 and 976 (methionine synthase), spot 770 (enolase), spot 544 (phosphoglycerate kinase), spot 519 (UDP-glucuronic acid decarboxylase), spot 988 (linolate 9S-lipoxygenase 1), spot 979 and 980 (lipoxygenase 2), spot 126 (thaumatin-like protein), spot 402 (annexin), spot 912 (␤-d-glucan exohydrolase), spot 943 (vacuolar proton ATP-ase), spot 832 (ATP-synthase subunit beta, mitochondrial-like), spot 812 (UDP-glucose pyrophosphorylase), spot 677 (predicted actin-1like), spot 90 (translationally-controlled tumor protein homolog), spot 723 (tubulin alpha-1 chain), spot 994 (elongation factor 2), spot 780 (eucaryotic translation initiation factor 4A-3-like protein, partial), spot 46 (eucaryotic translation initiation factor 5A1), spot 532 (protein disulfide isomerase-like protein, putative), spot 936 (predicted protein). Three spots revealed down-regulation under salt stress: spot 188 (UDP-d-glucuronate decarboxylase), spots 464 and 355 (predicted proteins).

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The significance and possible biological functions of the identified proteins in salt stress-induced molecular pathways are discussed in the next section. Discussion In this work we investigated a morphological and proteomic changes of two barley lines contrasting in salinity tolerance under control and salt stress conditions. The effect of salinity on barley seedlings growth was investigated in terms of differences in root fresh and dry weight. The roots fresh and dry weight content was significantly higher in the tolerant barley line under saline conditions than in the sensitive one (Figs. 1 and 2), which may result from more effective salinity-defense mechanisms applied by the tolerant line. Comparative proteomic analysis allows to indicate the proteins conferring salinity resistance in plants. Changes in gene expression and protein accumulation are among the first plant defensive responses to salt stress. To provide new information underlying salt stress tolerance in crops, it is important to investigate differences between the varieties contrasting in salinity tolerance. Forty-seven spots exhibiting abundance changes under salt stress were successfully identified. Among the identified spots several proteins already known to be linked to salt stress response were found, e.g. subunits of the V-ATPase, the vacuolar proton pump which plays a key role in the sequestration of Na+ into the vacuole; but also proteins such as annexin, lipoxygenase, PDI-like protein, which salt-protective function is known but poorly documented. The obtained results indicate that the tolerant line activates greater spectrum of metabolic processes in response to salt stress than the sensitive line (Fig. 5A and B). The most noticeable differences concern proteins involved in signal transduction, translation and detoxification which are accumulated only in the tolerant line. Carbohydrate and energy metabolism-involved proteins The diversified resistance to salt stress may be a result of different defense strategies developed by the sensitive and tolerant barley lines, such as diverse regulation of proteins associated with carbohydrate and energy metabolism. In the tolerant line we found the up-regulation trend in glycolysis-involved enzymes, such as enolase and phosphoglycerate kinase. On the other hand, in the sensitive line we observed the down-regulation of glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase and up-regulation of enzymes involved in link reaction and TCA cycle: citrate synthase, pyruvate dehydrogenase and aconitase. Overall, the results indicate changes in carbon metabolism in response to reduced photosynthesis and a higher demand for osmotic adjustment in salt-stressed tissue. Perhaps the accumulation of glycolysis-related proteins in the tolerant line plays a role in acclimation of barley seedlings to anaerobic conditions caused by oxidative stress (Rasoulnia et al., 2011). The proteomic study performed on halophyte plant Aeluropus lagopoides also revealed the up-regulation pattern of glycolysis-related enzymes (Sobhanian et al., 2010). The literature is ambiguous regarding salt stress impacts on the TCA cycle at the protein level. Based on proteome and metabolome studies, the TCA cycle is supported in A. lagopoides under salt stress (Sobhanian et al., 2010). The up-regulation of the TCA cycle-related proteins might reflect the action of the plant for detoxification processes. Among the cellular respiration-involved proteins we identified beta subunit of ATP synthase which was strongly up-regulated under salt stress in the tolerant line. Mitochondrial ATP synthase is the key enzymatic complex of energy metabolism, supplying ATP

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for the cell. Subunits of this enzyme are over-expressed under stress conditions, providing additional energy needed for cell homeostasis maintenance (Moghadam et al., 2012). Huseynova et al. (2007) also found that beta-subunit of the ATP-synthase complex accumulated less in drought-sensitive wheat cultivar than in drought-tolerant one under water stress, which is consistent with our results. Grater accumulation of ATP synthesis-associated proteins was also observed in wheat leaf under salt stress (Gao et al., 2011). Accumulation of pyrophosphate-fructose-6-phosphate-1phosphotransferase (PFP) observed in the tolerant line may also contribute to improved salt stress-resistance. The enzyme catalyzes the conversion of fructose-6-phosphate to fructose-1,6bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis/gluconeogenesis. PFP uses inorganic phosphate as its phosphoryl donor in place of ATP during fructose-6phosphate phosphorylation which consequently gives an energetic advantage to the plant cell (Mertens et al., 1990). Lim et al. (2009) demonstrated that the altered expression of PFP affects the growth of transgenic Arabidopsis plants. The PFP-overexpressing lines displayed faster growth than wild type plants. The data suggest that the enzyme is important in CO2 assimilation and carbohydrate metabolism. Thus, PFP can be considered as one of the possible markers of salinity tolerance. Amino acid metabolism related proteins The synthesis of various amino acids such as glutamine, methionine and cysteine may be affected by salt stress (Guo et al., 2012). We observed up-regulation of several enzymes involved in methionine cycle: S-adenosyl-l-homocysteine hydrolase (SAH-hydrolase), serine-hydroxymethyltransferase (SHMT) and methionine synthase (MeS). Increased abundance levels of SAH-hydrolase and SHMT were detected in both lines under salt stress, while up-regulation of MeS was found only in the tolerant line. A similar expression profile was observed in the roots of salt-sensitive and tolerant wheat varieties (Guo et al., 2012). In the methionine cycle, methionine is converted into S-adenosyl methionine that serves as a methyl group donor in transmethylation of proteins, nucleic acids, cell wall components and secondary metabolites. Disturbances in methylation processes may have serious consequences for plant organism (Roeder et al., 2009). Due to retain homeostasis under stress conditions, sufficient concentrations of methionine and S-adenosyl-l-methionine must be maintained. Thus, the up-regulation of enzymes involved in the methionine cycle is not surprising. On the other hand, the responsiveness of amino acid-related proteins might reflect the start of the synthesis of compatible osmolites. Translation associated proteins The modulation of gene expression, mainly involving regulation of translation, is a very important strategy that allows plants to survive under the unfavorable environment conditions ˜ et al., 2013). Translation elongation factor 2 (Echevarría-Zomeno (EF-2) and eukaryotic translation initiation factor 4A-3-like (eIF4A) displayed similar up-regulation patterns in both barley lines. It was previously shown that eIF4A confers salt tolerance when overexpressed in tobacco (Sanan-Mishra et al., 2005). On the other hand, Guo et al. (2012) observed its down-regulation in salt tolerant wheat cultivar under salt stress. In our study, translation initiation factor 5A1 (eIF5A1) was differently regulated, showing down-regulation in sensitive line and up-regulation in tolerant line. It was previously demonstrated that amino acid starvation induces the expression of translation initiation factor eIF5 which regulates

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general translation by altering start codon selection (Jennings and Pavitt, 2013).

Increased accumulation of lipoxygenases in the tolerant line can contribute to a better adaptation to salt stress conditions by affecting stress-signal transduction.

Signal transduction associated proteins

Detoxification-associated proteins

Salt stress response is usually initiated in plant cells after sensing and transferring stress signals through signal transduction networks. Plants can adapt to stress conditions through activation of several signaling pathways that regulate the expression levels of specific functional proteins (Guo et al., 2012). The best known salt-stress signaling pathway is the Overly Sensitive Pathway which includes proteins such as SOS3, SOS2 and SOS1. SOS3 is a Ca+ binding protein that senses the Ca+ change triggered by salt stress. The protein activates SOS2 which is a Ser/Thr protein kinase. The SOS3–SOS2 kinase complex regulates the expression and activity of ion transporters such as SOS1, a plasma membrane Na+ /H+ exchanger, eventually removing Na+ from the cytosol (Mahajan et al., 2008). Annexin, translationally-controlled tumor protein homolog and lipoxygenases, which are probably involved in salt stress-signal transduction, were significantly up-regulated under salt stress in the tolerant line. Annexins are calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction. Lee et al. (2004) confirmed the salt-stress protective role of annexins in Arabidopsis thaliana. It was suggested that annexins comprise a novel class of Ca+ binding proteins that play an important role in ABA-mediated stress response in plants. The abundance of annexins significantly increased upon NaCl treatment in the root microsomal proteome of Arabidopsis. It is speculated that annexin senses the Ca+ signal induced by ABA and transmits it to downstream signaling pathways via protein degradation and translocation to the membrane (Lee et al., 2004). The up-regulation of annexins was also observed during recovery after chilling stress in the roots of germinating Phaseolus vulgaris (Badowiec and Weidner, 2013). Translationally-controlled tumor protein homologue (TCTP), which was also found to be up-regulated under salt stress in tolerant line, is Ca+ binding protein that performs important functions in different cellular processes such as protection against stress and apoptosis, cell growth and cell cycle progression. Although TCTP is a well-known animal protein, little is known about the protein function in plants. Previous study, conducted by Hoepflinger et al. (2013) showed cytoprotective effects of plant TCTP. In A. thaliana TCTP binds Ca2+ and reduces its cytosolic levels in the same way as described for animals. Additionally, TCTP interacts with other cytosolic or membrane-bound proteins of the cell death machinery, thereby inhibiting cell death progression. Protein levels of plant TCTPs were shown to be altered by various stress conditions such as cold, salt, draught, aluminum, and pathogen infection (Hoepflinger et al., 2013). We observed a considerable dissimilarity in the regulation of lipoxygenases in the examined barley lines. The tolerant line displayed an increased accumulation of linolate 9S-lipoxygenase 1 and lipoxygenase 2 under salt stress, whereas the sensitive line showed down-regulation of the latter. Lipoxygenases play an important role in the biosynthesis of oxylipins by catalyzing the oxidation of fatty acids, mainly linolenic and linoleic. In addition to the role in development of fertile flowers, oxylipins are an important class of signaling molecules related to plant stress responses and primary immunity. The best-characterized oxylipins are jasmonic acid and 12-oxo-phytodienoic acid, which are accumulated in the roots upon osmotic and drought stress (Reinbothe et al., 2009). Oxylipins are probably involved in stress signaling and root-to-shoot communication by supporting the regulation of stomatal closure (Singh et al., 1987).

As plant resistance to stress is usually associated with accumulation of various defense proteins, detection of specifically regulated defense proteins in the tolerant line was expected. We observed a significant up-regulation of thaumatin-like protein under salt stress in the tolerant line, whereas the protein abundance in the sensitive line remained unchanged. Thaumatin-like proteins, more commonly known as osmotins, are multifunctional proteins involved in plants osmotolerance (Singh et al., 1987; Aghaei et al., 2008). Osmotins belong to a group of Pathogenesis Related proteins (PR-5) that are induced in response to various biotic and abiotic stresses. It was demonstrated that over-expression of osmotins induces accumulation of proline which increases tolerance to osmotic stress in transgenic tobacco (Barthakur et al., 2001). Osmotins probably act as a modulator of transcription factors or metabolic signaling, eventually leading to the accumulation of osmoprotectants (Abdin et al., 2001). This makes it very likely that the early up-regulation of this protein is related to osmotic adjustment. In addition to its osmoprotectant role, osmotins are shown to exhibit antifungal activity against a broad range of fungal pathogens (Yun et al., 1998). In order to survive under saline conditions, plants have to maintain ion homeostasis. The cytotoxic ions are compartmentalized into the vacuole and further used as osmotic solutes. Vacuolar ATPase (V-ATPase), which was up-regulated specifically in the tolerant line, is of prime importance in sodium sequestration into the central vacuole. Impaired ion homeostasis, in particular elevated Na+ levels, inhibits enzyme activity resulting in disturbances of essential metabolic processes. V-ATPase is well known for its responsiveness to salt stress, manifested in its overexpression and increased activity under salinity treatment (Golldack and Dietz, 2001). It is widely known that salt-tolerance in plants correlates with the induction of antioxidant enzymes (Rasoulnia et al., 2011; Guo et al., 2012). Interestingly, we observed down-regulation of peroxidases in both barley lines. Moreover, other antioxidant enzymes such as catalase and superoxide dismutase showed no significant abundance differences under salt stress. The down-regulation of three different peroxidases was observed in the sensitive line. Similar results were obtained by Sairam and Srivastava (2002) who showed that most antioxidant enzymes isolated from susceptible wheat genotype exhibited decreased activity under salt stress conditions. Down-regulation of peroxidase in the tolerant line can be explained by the occurrence of distinct defense mechanisms. Sobhanian et al. (2010) revealed that exposure of halophyte plant A. lagopoides to salt stress also results in a decreased activity of catalase and ascorbate peroxidase, which may indicate the involvement of other defense mechanisms in plants resistant to salinity. However, in most proteomic studies up-regulation of antioxidant enzymes were reported. It is possible that antioxidant proteins were among the unidentified spots. Chaperones The misfolded proteins or peptides may accumulate in plant cells subjected to severe biotic and abiotic stresses. Plants may engage chaperone proteins to prevent and rearrange incorrect interactions between proteins and to facilitate correct protein folding (Ranford et al., 2000). Several identified stress-responsive proteins have been shown to act as molecular chaperones which are responsible for protein

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synthesis, maturation, targeting and degradation. Among the saltresponsive chaperone proteins we found: heat shock protein 90kDa (HSP 90), cyclophilin A, heat-shock cognate 70-kDa (HSC 70) and protein disulfide isomerase-like protein (PDI). The HSP 90 is mainly involved in protein folding but it is also engaged in signal-transduction networks, cell-cycle control, protein degradation and protein trafficking (Wang et al., 2004a). The HSP 90 was significantly up-regulated in both barley lines, which indicates that the sensitive line also possess some molecular mechanisms to counteract stress. On the other hand, HSC 70 was differentially regulated under salt stress in the examined lines. In the tolerant line HSC 70 was found to be down-regulated whereas in sensitive line it was up-regulated in spot 909 and down-regulated in spot 625. According to Wang et al. (2004a), HSC 70 are not stressresponsive, but rather constitutively expressed proteins. They are mainly involved in the folding of de novo synthesized polypeptides and the translocation of precursor proteins. Decreased quantity of HSC 70 was also reported in pea roots under chilling stress conditions (Badowiec et al., 2013). Cyclophilins are likely to catalyze the cis–trans isomerization of the amide bond between proline and preceding residues. It functions as a molecular chaperone involved in protein folding and refolding of denatured proteins (Ruan et al., 2011). We detected up-regulation of cyclophilin A in both barley lines. However, the accumulation of this protein under salt stress was greater in the tolerant than in the sensitive line- 3.5 and 2.1-fold change, respectively (data not shown). The similar up-regulation pattern of cyclophilins was also shown in wheat (Guo et al., 2012) and barley leaves (Fatehi et al., 2012). In addition, it was demonstrated that the cyclophilin OsCYP2 is responsible for salt tolerance in rice (Ruan et al., 2011). Protein disulfide isomerase-like protein (PDI) was significantly up-regulated in the tolerant line. It is suggested that PDI promotes folding of protein chains as well as formation of disulfides and plays a role similar to chaperones in folding process. PDI is known to be a multifunctional protein capable of non-specific peptide binding. This property is closely connected to its possible function as a chaperone. PDI was significantly accumulated in the seedlings of salt tolerant rice under saline conditions (Ghaffari et al., 2014). The obtained results indicate the important role of PDI in maintaining correct conformation of the proteins under salt stress conditions and therefore it can be considered as one of the potential markers of salt tolerance. Cell wall-related proteins Abundance changes of UDP-glucuronate decarboxylase (UGD) and ␤-d-glucan exohydrolase in the tolerant line may indicate some cell wall structural rearrangements that can be important for salt stress tolerance mechanism. UGD, which was down-regulated in the tolerant line, catalyzes the formation of UDP-xylose from UDP-glucuronate. UDP-xylose is then used to initiate glycosaminoglycan biosynthesis on the core protein of proteoglycans (Moriarity et al., 2002). On the other hand, ␤-d-glucan exohydrolase was significantly up-regulated under salt stress in the tolerant line. The enzyme releases single glucosyl residues from the nonreducing ends of ␤-d-glucans which leads to the degradation or modification of cell wall (Hrmova and Fincher, 2001) and may result in enhanced adaptation to salt stress conditions. In our study the up-regulation of two enzymes involved in nucleotide sugars metabolism was observed. Nucleotide pyrophosphatase was up-regulated in both barley lines, while UDP-glucose pyrophosphatase was up-regulated only in the tolerant line. UDPglucose pyrophosphorylase catalyzes the synthesis of UDP-glucose from glucose-1-phosphate and UTP, whereas nucleotide pyrophosphatase catalyzes the opposite reaction. UDP-sugars serve as the

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main precursors of cellulose, hemicellulose, pectins, glycoproteins as well as sucrose and glycolipids. It is assumed that UDP-sugars are the main precursors for biomass production in plants (Kleczkowski et al., 2011). The results here suggest the intensification of anabolic processes leading to a greater accumulation of biomass in the tolerant line.

Other proteins Significant changes in the accumulation of some storage proteins were found under salt stress. Stem 28 kDa glycoprotein, which may function as somatic storage protein during early seedling development (http://www.uniprot.org), was up-regulated under salt stress in the sensitive barley line. Glutelin type-A 1-like-protein, which belongs to seed storage proteins, was downregulated under salt stress in both lines. We also found changes in abundance of cytoskeleton-associated proteins in response to stress. Predicted actin-1-like and tubulin alpha-1 chain were up-regulated under salt stress in the tolerant barley line. It has been well established that cytoskeleton is not just involved in fundamental processes like mitosis, cytokinesis, cell polarity, and intracellular trafficking but also plays a pivotal role in modulating the response of plants to environmental factors (Wasteneys and Yang, 2004). Moreover, the role of the actin cytoskeleton in the modulation of intracellular signaling was highlighted. Wang et al. (2004b) showed that actin microfilaments regulate extracellular calcium influxes and cytosolic calcium accumulation in Arabidopsis pollen. The up-regulation of actin and tubulin, the building blocks of microfilaments and microtubules, may improve the cytoskeleton stability of barley tolerant line. Actin and tubulin levels significantly changed under salt stress in wheat (Guo et al., 2012) and chilling stress in pea (Badowiec et al., 2013).

Conclusions One of the most challenging issues in present plant research is to explain the molecular basis of plant adaptation to environmental stress and our results may greatly improve the understanding of mechanisms leading to salt tolerance in barley. In this experiment it was demonstrated that the sensitive and tolerant barley lines respond differently to salt stress. A number of salt-responsive proteins with various functions were identified. Some of the identified proteins are already known as salt stress tolerance markers. However, several of the identified proteins are already known to be associated with other abiotic stresses but have not been detected in response to salinity. The most significant differences concerned proteins involved in signal transduction (annexin, translationallycontrolled tumor protein homolog, lipoxygenases) detoxification processes (osmotin, vacuolar ATP-ase), protein folding (protein disulfide isomerase) and cell wall metabolism (UDP-glucuronic acid decarboxylase, ␤-d-glucan exohydrolase, UDP-glucose pyrophosphorylase) which were up-regulated only in the tolerant line under salt stress. Presumably, enhanced salinity tolerance of DH187 line results mainly from increased activity of signal transduction mechanisms leading to the accumulation of stress protective proteins and changes in cell wall structure, which requires further analysis. The results suggests that salinity tolerance may be highly dependent on the efficiency of salt stress signal transduction mechanisms which should be subjected to more detailed examinations in the future experiments. Since many of the identified proteins were proved to be membrane-associated, the further study includes the investigation of salinity-induced membrane proteome changes. The areas for further study also include the investigation of additional barley lines from the Steptoe/Morex population to find out

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Alterations in root proteome of salt-sensitive and tolerant barley lines under salt stress conditions.

Salinity is one of the most important abiotic stresses causing a significant reduction of crop plants yield. To gain a better understanding of salinit...
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