Proteomics Reveals the Effects of Salicylic Acid on Growth and

Article pubs.acs.org/jpr

Proteomics Reveals the Effects of Salicylic Acid on Growth and Tolerance to Subsequent Drought Stress in Wheat Guozhang Kang,* Gezi Li, Wei Xu, Xiaoqi Peng, Qiaoxia Han, Yunji Zhu, and Tiancai Guo* The National Engineering Research Centre for Wheat, the Key Laboratory of Physiology, Ecology and Genetic Improvement of Food Crops in Henan Province, Henan Agricultural University, Zhengzhou, 450002, China S Supporting Information *

ABSTRACT: Pretreatment with 0.5 mM salicylic acid (SA) for 3 days significantly enhanced the growth and tolerance to subsequent drought stress (PEG-6000, 15%) in wheat seedlings, manifesting as increased shoot and root dry weights, and decreased lipid peroxidation. Total proteins from wheat leaves exposed to (i) 0.5 mM SA pretreatment, (ii) drought stress, and (iii) 0.5 mM SA treatment plus drought-stress treatments were analyzed using a proteomics method. Eighty-two stress-responsive protein spots showed significant changes, of which 76 were successfully identified by MALDI-TOF-TOF. Analysis of protein expression patterns revealed that proteins associated with signal transduction, stress defense, photosynthesis, carbohydrate metabolism, protein metabolism, and energy production could by involved in SA-induced growth and drought tolerance in wheat seedlings. Furthermore, the SA-responsive protein interaction network revealed 35 key proteins, suggesting that these proteins are critical for SA-induced tolerance. KEYWORDS: salicylic acid, proteome, wheat, drought tolerance, growth

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INTRODUCTION Salicylic acid (SA) is a naturally occurring phenolic compound that plays an important role in the regulation of plant growth, development, ripening, and defense responses.1 The role of SA in the plant−pathogen relationship has been investigated extensively.2,3 SA significantly enhances plant tolerance to cold, heat, salt, drought, UV, and other abiotic stresses, suggesting that it has great agronomic potential in terms of improving the stress tolerance of agriculturally important crops.4,5 Much effort has focused on elucidating the mechanism of action of SA at morphological and physiological levels, such as seed germination, growth rate, stomatal closure, ion uptake and transport, photosynthesis, and antioxidant enzyme production.4−6 Exogenous SA may enhance antioxidant capacity in plants by altering the activity of antioxidative enzymes, although the data supporting this idea are somewhat inconsistent.5,6 However, little information is available about the molecular mechanisms of SA-induced abiotic tolerances, although a few SA-responsive genes, including late-embryogenesis abundant protein (LEA), heat-shock protein (HSP), and osmotin genes, have been identified in plants.4 Proteomics is becoming an increasingly important tool because proteins are directly linked to cellular functions. This technique has been used to explore the mechanisms of SA-induced pathogen tolerance, and many novel proteins have been identified.7,8 In the last few years, proteomic analysis was found to be a powerful tool to understand plant tolerance mechanisms to abiotic stress conditions mediated by signaling molecules.9,10 However, the © 2012 American Chemical Society

mechanisms underlying drought tolerance induced by exogenous SA at the proteomic level have not been elucidated. Wheat is one of the most important crops globally, and its growth and yield are seriously influenced by drought stress. Various wheat growth stages, including the seedling, stem elongation, booting, anthesis, and grain formation stages, are sensitive to drought stress.11,12 Young seedlings are susceptible to water deficit due to their low biomass, undeveloped protective structure, and water requirement for expansion growth, suggesting that younger stages are critical for the growth and subsequent development and yield of wheat under drought conditions.13,14 Exogenous SA pretreatment enhances the growth and tolerance to subsequent drought stress in some plants;2,4 however, the precise mechanism mediated by SA has remained obscure. We investigated the proteome pattern of SApretreated wheat seedlings under drought stress to further explore the molecular mechanisms underlying SA-induced drought tolerance.

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MATERIALS AND METHODS

Plant Materials

Seeds of the common wheat (Triticum aestivum L.) cultivar “Yumai 34” were sterilized with 0.01% HgCl2 followed by thorough washing with distilled water. Sterilized seeds were grown hydroponically in full-strength Hoagland’s solution in Received: August 6, 2012 Published: October 29, 2012 6066

dx.doi.org/10.1021/pr300728y | J. Proteome Res. 2012, 11, 6066−6079

Journal of Proteome Research

Article

were collected and used as protein extracts. Protein concentrations were determined using the Bradford assay with a BSA standard.

glass dishes (15-cm diameter) in an FPG-300C-30D illumination incubator (Ningbo Laifu Technology Co., Ltd., Beijing, China) under a 14-h photoperiod, 25/15 °C day/night temperatures, light intensity of 250 μmol m−2 s−1, and relative humidities of 60/75% (day/night). Each dish contained ∼60 seedlings. Two-week-old seedlings with two fully expanded leaves and almost the same heights (10.5 ± 0.2 cm) were used in this study.

2-DE Separation and Image Analysis

Protein samples (1.2 mg) were loaded onto an Immobiline DryStrip (GE Healthcare, 24 cm, pH 4−7) and rehydrated passively with 450 μL of protein solution for 16−18 h at 20 °C by an Ettan IPGphor II (GE Healthcare). Isoelectric focusing was conducted in seven steps: 100 V for 1 h; 300 V for 1 h; 500 V for 1 h; 1, 000 V for 1 h; 8000 V for 2 h, and 10 h at 8, 000 V with a total of ∼84 kV h and a constant 500 V for the final 12 h. Focused strips were equilibrated twice in equilibration solution for 15 min. The first equilibration was performed in 0.375 M Tris-HCl, pH 8.8, 6 M urea, 20% (v/v) glycerol, 2% (w/v) SDS, 0.01% bromophenol blue, and 2% DTT. The second equilibration was performed in 0.375 M Tris-HCl, pH 8.8, 6 M urea, 20% (v/v) glycerol, 2% (w/v) SDS, 0.01% bromophenol blue, and 2.5% (w/v) iodoacetamide. Then the strips were transferred to 12% vertical SDS-PAGE gels for seconddimension electrophoresis using an Ettan DALT Six Large Vertical System (GE Healthcare). SDS-PAGE was run at 1 W/ gel for 1 h, followed by 10 W/gel until completion. Protein spot images were visualized using Coomassie Brilliant Blue (CBB) and scanned at 300 dpi with a UMAX Power Look 2, 100 XL scanner (Maximum Tech Inc., Taiwan, China). Image analysis was performed with PDQuest software, version 8.0 (Bio-Rad, Hercules, CA, USA). To compare spot quantities between gels accurately, the spot volumes were normalized as a percentage of the total volume of all of the spots in the gel. The normalized percentage volumes (volume %) of protein spots from triplicate biological samples were subjected to statistical analysis using SPSS (version 17.0). Quantitative image analysis was conducted to reveal protein spots with reproducible and significant differences in abundance (volume% > 2.0-fold; p < 0.05).

Effects of SA Treatments on Wheat Seedling Growth

SA solution was prepared with full-strength Hoagland’s nutrient solution and adjusted to pH 6.3 with 1 N NaOH.3 Wheat seedlings were transferred to different culture systems containing hydroponic full-strength Hoagland’s nutrient solution (pH 6.3) supplemented with 0 (control), 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, or 3.0 mM SA, respectively, for 3 days. Fresh, full-strength Hoagland’s nutrient solutions (100 mL) containing the above SA concentrations were replaced daily. Effects of SA Treatment on Drought Tolerance of Wheat Seedlings

Preliminary experiments showed, among the applied concentrations, that 0.5 mM SA was also an optimal concentration of SA to improve the tolerance to subsequent drought stress (Supplemental Figure S1 and Table S1, Supporting Information). Control seedlings were transferred to fresh Hoagland’s medium supplemented with PEG-6000 (15%) solution for 3 days (drought treatment).15 Seedlings pretreated with 0.5 mM SA were rinsed with distilled water to remove residual SA and transferred to Hoagland’s solution supplemented with PEG6000 (15%) solution for 3 days (SA + drought treatment). Samples whose uppermost leaves were fully expanded in every treatment 3 days after initiation of drought stress were collected, immediately frozen in liquid nitrogen, and stored at −80 °C to measure physiological parameters and protein extractions. Hoagland’s + PEG-6000 (15%) solution (100 mL) was exchanged daily.

In-Gel Digestion and MALDI-TOF-TOF MS Analysis

Growth Parameters

Selected protein spots were manually excised from the gel and digested with sequencing-grade trypsin. Gel slices were destained with 100 mM NH4HCO3 in 30% acetonitrile (ACN) and lyophilized prior to digestion at 37 °C overnight (20 h) with 20 μL of 50 mM NH4HCO3 containing 0.01 mg/ mL sequencing-grade modified trypsin (Promega, Madison, WI, USA). After a brief centrifugation, peptides were collected from supernatants, and the remaining gel pieces were further sonicated for 15 min in 100 μL of 60% ACN/0.1% TFA to collect the remaining peptides. The peptides from one protein spot were combined. MS and MS/MS data for protein identification were obtained using a MALDI-TOF-TOF instrument (4800 Proteomics Analyzer; Applied Biosystems). Instrument parameters were set using the 4000 Series Explorer software (Applied Biosystems). The MS spectra were recorded in reflector mode in a mass range of 800−4000 with a focus mass of 2000. MS used a CalMix5 standard to calibrate the instrument (ABI 4700 Calibration Mixture). MS spectra were obtained with 1600 laser shots per spectrum, and MS/MS spectra were acquired with 2500 laser shots per fragmentation spectrum. Up to eight of the most intense ion signals were selected as precursors for MS/MS acquisition, excluding the trypsin autolysis peaks and the matrix ion signals. Combined peptide mass fingerprinting (PMF) and MS/MS queries were performed using the MASCOT search engine, version 2.2

Growth parameters (plant height, fresh weight [FW], and dry weight [DW]) were recorded 3 days after SA pretreatment or drought stress. Absolute water content (AWC) was calculated using the following formula: AWC (%) = (FW − DW)/FW × 100. Lipid Peroxidation

Lipid peroxidation was determined by estimating malondialdehyde (MDA) concentration using the method described by Zheng et al.16 Protein Extraction

Proteins from leaf samples were extracted using the trichloroacetic acid (TCA)/acetone method with minor modifications.17,18 Triplicate leaf samples (1.5 g) were ground to powder in liquid nitrogen with a mortar and pestle. Next, 10 volumes of ice-cold 10% (w/v) TCA in acetone with 0.07% 2mercaptoethanol were added, followed by incubation at −20 °C for 2 h. After centrifugation at 14000g for 30 min at 4 °C, the supernatants were discarded and the pellets were washed three times with ice-cold acetone containing 0.07% 2mercaptoethanol. The final pellet was vacuum-dried and resuspended in lysis buffer consisting of 8 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG buffer (pH 4−7), and 1 mM fresh DTT, and incubated at 25 °C for 1−2 h. The suspension was centrifuged at 14000g for 30 min at 25 °C. Supernatants 6067



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Figure 1. Phenotypic changes in wheat seedlings following exogenous application of different concentrations of SA (0, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM, 2.0 mM and 3.0 mM) for 3 days.

Table 1. Effect of Different Concentrations of Exogenous SA on Growth Characteristics of Wheat Seedlinga plant height (cm) treatments control 0.1 mM 0.25 mM 0.5 mM 0.75 mM 1.0 mM 2.0 mM 3.0 mM a

M ± SE

% change

± ± ± ± ± ± ± ±

100 96.8 102.2 110.7 101.3 91.2 78.4 61.1

20.02 19.38 20.45 22.17 20.27 18.26 15.69 12.24

0.12b 0.25c 0.21b 0.15a 0.08b 0.15d 0.05e 0.42f

total FW (g plant−1) M ± SE 0.527 0.465 0.470 0.608 0.459 0.392 0.341 0.256

± ± ± ± ± ± ± ±

total DW (g plant−1) M ± SE

% change

0.0152b 0.0184c 0.0078c 0.0132a 0.0208c 0.0085d 0.0032e 0.0023f

100 88.2 89.2 115.4 87.1 74.3 64.7 48.6

0.069 0.063 0.067 0.076 0.066 0.061 0.054 0.044

± ± ± ± ± ± ± ±

% change

0.0021b 0.0019cd 0.0028c 0.0012a 0.0025c 0.0018d 0.0017e 0.0017f

100 91.3 97.1 110.4 95.7 88.4 78.6 63.8

AWC (%) M ± SE 87.19 84.75 85.21 87.47 86.67 84.43 83.38 82.81

± ± ± ± ± ± ± ±

0.1869a 0.0247c 0.0543c 0.0621a 0.1245b 0.3124cd 0.1212d 0.2151e

% change 100 97.2 97.7 100.3 99.4 96.8 96.2 94.9

Values were mean ± SE. Data with different letters in the same column indicate a significant difference at P < 0.05 level.

Statistical Analysis

(Matrix Science, Ltd.), embedded in the GPS-Explorer Software, version 3.6 (Applied Biosystems), on the NCBI database (900091 release, Viridiplantae proteins). The search variables were as follows: Viridiplantae, trypsin cleavage (one missed cleavage allowed), carbamidomethylation as a fixed modification, methionine oxidation as a variable modification, peptide mass tolerance of 100 ppm, and MS/MS fragment mass tolerance of 0.4 Da. A peptide charge of 1+ was considered significant. Proteins with a protein score CI% and total ion score CI% greater than 95% were identified as credible results for the MS/MS.

All experiments were repeated three times independently. Spot intensities of differential proteins in a 2D gel were calculated from three spots in three replicate gels. Growth and physiological parameters, as well as spot intensities, were statistically analyzed using a one-way analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) to determine significant differences among group means. Significant differences from control values were determined at the P < 0.05 level.

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SA-Responsive Protein Interaction Network Analysis

Further analyses were carried out using Pathway Studio software (Ariadne Genomics) according to a method described previously.3 Briefly, the protein list was blasted against the green plant database, which comprises the functional relationships of proteins supported by the scientific literature. The filters applied included “all shortest paths between selected entities.” The information received was narrowed down to our proteins of interest. Each link was built based on evidence from at least three publications. Then the interactions between the imported proteins and all proteins stored in the database were identified. Protein entities that belonged to different functional groups were represented as different shapes, according to the default settings of the software and as shown in the legend.

RESULTS

Effects of Exogenous SA Pretreatment on Phenotypes and Growth Parameters of Wheat Seedlings

Wheat seedlings were pretreated with 0.1−3.0 mM SA for 3 days. We found that 0.5 mM SA accelerated seedling growth, and that other concentrations had adverse effects, particularly high concentrations (2.0 and 3.0 mM) (Figure 1). These phenotypic results were confirmed by quantitative analysis (Table 1). For example, the heights, FWs, and DWs of wheat seedlings pretreated with 0.5 mM SA for 3 days were significantly higher than those of the control, by 10.7%, 15.4%, and 10.4%, respectively. However, the difference in AWC between the control and 0.5 mM SA pretreatment was not significant. In contrast, these parameters in seedlings 6068



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treatment groups. The difference in wilting between the drought and SA + drought-treatment groups indicates that drought stress leads to substantial water loss from seedlings, which was alleviated by pretreatment with 0.5 mM SA. The inhibitory effect of drought stress on wheat seedling growth was significantly abrogated by pretreatment with 0.5 mM SA. The AWC content and total FW and DW in the SA + drought-treatment group were 4.8%, 35.4%, and 15.7% higher than those in the drought-treatment group, respectively. In contrast, the MDA concentration in the SA + droughttreatment group was 29.9% lower than that in the droughttreatment group (Figure 4).

pretreated with 3.0 mM SA were significantly lower than those of the control by 38.9%, 51.4%, 36.2%, and 5.1%, respectively (Table 1). No significant difference in MDA concentrations between control and 0.5 mM SA pretreatment was found; however, MDA concentrations after other SA pretreatments, particularly 2.0 and 3.0 mM SA, were notably higher than those of the control and 0.5 mM SA pretreatment (Figure 2).

Leaf Protein Expression Patterns in the SA-Pretreatment, Drought, and SA + Drought-Treatment Groups

To elucidate the proteomic response of wheat seedlings to SA treatment during drought stress, the leaf proteomes of the control, 0.5 mM SA pretreatment, drought, and 0.5 mM SA + drought-treatment groups were compared. Representative 2DE gels are shown in Figure 5. More than 800 protein spots were detected from wheat leaves in the SA-pretreatment, drought, and SA + drought-treatment groups, of which 82 were differentially expressed. These were analyzed by mass spectrometry, resulting in the identification of 76 of them. The identified proteins were annotated based on the NCBI databases using BLASTp analyses (Table 2). A number of the identified proteins are enlarged in Figure 6 to illustrate the changes in differentially expressed proteins among the groups. Some spots were identical, despite being excised from the same gel. For example, spots 9 and 10 were the ferredoxinNADP(H) oxidoreductase, as were 25 and 59 (triosephosphate isomerase), 49 and 71 (glyceraldehyde-3-phosphate dehydrogenase B), and 57 and 58 (ascorbate peroxidase). This may be due to post-translational modifications, different isoforms, or degradation.19 On the basis of their putative physiological functions, the identified proteins were classified into eight categories: signal transduction (3 spots, 3.9%), stress and defense (7 spots, 9.2%), photosynthesis (20 spots, 26.3%), carbohydrate metabolism (18 spots, 23.7%), protein metabolism (12 spots, 15.8%), energy production (6 spots, 7.9%), toxin metabolism (2 spots, 2.6%), and unknown function (8 spots, 10.5%) (Figure 7 and Table 2).

Figure 2. Effects of different concentrations of SA on MDA concentrations in wheat seedling leaves. Different letters indicate significant differences at P < 0.05. Bars represent standard errors of triplicate experiments.

Effect of SA Pretreatment on Wheat Seedling Drought Tolerance

To demonstrate the effect of SA pretreatment on drought tolerance, wheat seedlings of both the control and pretreatment with 0.5 mM SA groups were exposed to PEG-induced drought stress (PEG-6000, 15%) for 3 days. Drought stress resulted in a dramatic morphological change in wheat seedlings without SA pretreatment, as indicated by marked wilting; in contrast, 0.5 mM SA-pretreated seedlings exhibited less wilting (Figure 3). The phenotypic results were confirmed by physiological measurements. As shown in Figure 4, drought stress greatly decreased the total FW, DW, and AWC, but increased MDA concentrations, in both the drought and SA + drought-

Figure 3. Phenotypic changes of wheat seedlings in drought and SA + drought treatments 3 days after 15% PEG 6000 application. 6069



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Figure 4. Effects of exogenous 0.5 mM SA pretreatment on total fresh weight (A), total dry weight (B) absolute water content (AWC) (C) and MDA concentrations (D) of wheat seedlings under drought stress (PEG) conditions. Different letters indicate significant differences at P < 0.05. Bars represent standard errors of triplicate experiments. CK, control; SA, SA pretreatment; PEG, drought treatment; SA + PEG, SA + drought treatment.

Proteins Differentially Expressed by SA Pretreatment, Drought, and SA + Drought Treatment

Interaction Network Analysis of SA-Responsive Proteins

Proteins in a living cell do not act as single entities, but rather work together in the context of networks.3 Using the Pathway Studio software, we analyzed all protein regulation network pathways, and generated protein interaction maps connected to each of the identified proteins in the green plant molecular networks database. Thirty-five key proteins were induced by SA, which were associated mainly with carbohydrate metabolism (11 proteins), protein metabolism (11 proteins), stressdefense (5 proteins), photosynthesis (4 proteins), energy (2 proteins), signal transduction (1 protein), and unknown function (1 protein) (Figure 8). These proteins included glutathione S-transferase, dehydroascorbate reductase, ascorbate peroxidase, cys peroxiredoxin, ribulose 1,5-bisphosphate carboxylase activase, carbonic anhydrase, isocitrate dehydrogenase, malate dehydrogenase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, S-adenosylmethionine synthase, glutamine synthetase, and ATP synthase, etc.

The identified proteins were further classified into four groups according to the conditions under which they were differentially expressed (Table 2): (1) Thirty-seven protein spots (48.7%) in the SA-pretreatment group were upregulated significantly compared to the control. Under drought stress, the abundance of these proteins in the SA + drought-treatment group was significantly higher than that in the drought-stress group. These spots included 2, 3, 5, 9−14, 18−20, 22, 26, 32− 35, 37, 38, 44, 46, 50, 54, 55, 57, 58, 60, 62, 63, 66−69, 72, 75, and 81. (2) Twenty-one protein spots (27.6%) in the SApretreatment group were downregulated compared to the control. Under drought stress, SA addition either significantly upregulated their abundance or alleviated the decline thereof. The abundance of these protein spots in the SA + droughttreatment group was markedly higher than that in the droughtstress group. These spots included 15, 16, 25, 36, 42, 43, 45, 47−49, 52, 53, 59, 71, 73, 76−80, and 82. (3) Thirteen protein spots (17.1%) in the SA-pretreatment group were significantly upregulated compared to the control. Under drought stress, the abundance of these protein spots was decreased or upregulated. However, their abundance in the SA + drought-treatment group was significantly higher than in the drought-treatment group. These spots included 1, 4, 7, 8, 17, 23, 24, 40, 41, 56, 64, 65, and 74. (4) Five protein spots (6.6%) in SA treatment were significantly downregulated compared to the control. The abundance of these was significantly lower than in the droughttreatment group. These spots included 6, 27, 29, 39, and 70.

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DISCUSSION

Morphological and Physiological Response of Wheat Seedlings to Exogenous SA

The effect of exogenous SA on the growth and stress tolerance of plants depends on not only the applied concentrations and the mode of application, but also the overall states of the plants, and the determination of an optimum concentration is a prerequisite because SA applied beyond a certain range might be detrimental.4,20 For instance, pretreatment with 0.25 mM SA and 0.5 mM acetyl salicylic acid (ASA) provides optimal protection against drought stress in barley and muskmelon seedlings, respectively.21,22 6070



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Figure 5. 2-DE analysis of leaves proteins from leaves of control, SA pretreatment, drought and SA + drought treatments. Total of proteins (1.2 mg) were loaded on a 24 cm IPG strip with linear gradient (pH 4−7) and SDS-PAGE was performed on a 12% gel. Proteins were stained with CBB G250. Proteins indicated by arrow were differentially regulated according to treatment and identified by MS and MS/MS.

total FW and DW, and decreased MDA concentrations (Figures 3 and 4). These results are consistent with previous reports in barley and Phillyrea angustifolia.23,24 The morphological and physiological responses of wheat seedlings suggest that pretreatment with optimum SA concentrations could modify the physiological metabolic rate and modulate regulatory pathways, resulting in not only improved growth but also enhanced drought tolerance. To further investigate the molecular mechanism underlying the regulation of wheat seedling growth and drought-stress tolerance by SA, differential proteomic analyses were performed on control, SA pretreatment, drought treatment, and SA + drought treatment plants (Figure 5 and Table 2).

Exogenous SA application can cause oxidative stress in plants. At relatively low concentrations it acts as a moderate stress, having an effect on the oxidative status of the plant similar to that of stress-acclimating processes, whereas higher concentrations of SA cause serious oxidative stress, which greatly damages plants.3,4,6 In the present study, the phenotype and growth parameter data suggested that exogenous SA induced significant changes in the growth of wheat seedlings in a dose-dependent manner. Concentration of 0.5 mM SA might acts as a moderate stress, and it effectively increased plant height, total FW and DW, and decreased MDA concentrations. This implies that, among the applied concentrations, 0.5 mM may be the optimum concentration of SA. Higher SA concentrations (>2.0 mM) and other lower concentrations (0.1 and 0.25 mM) had significant inhibitory effects, manifested by decreased plant height and root and shoot DW, and increased MDA concentrations (Figure 1, Table 1). Such concentrations induce oxidative stress that may result in plant cell death.3

Proteins Associated with SA Mediate Increased Growth and Drought-stress Tolerance

Seventy-six of the identified proteins could be subdivided into four groups. Those in the first group were upregulated in both the SA pretreatment and SA + drought-treatment groups. Their abundances in the SA pretreatment and SA + droughttreatment groups were significantly higher than those in the control and drought-treatment groups, respectively. Presumably, these proteins participated in both the growth and drought-stress tolerance induced by SA. In the second group, the abundance of identified proteins in the SA-pretreatment group was lower than that in the control, but that in the SA + drought-treatment group was markedly higher that in the drought-treatment group. This implies that these proteins are associated mainly with the improved drought tolerance.

Effect of SA Pretreatment on Drought Tolerance as Indicated by Morphological and Physiological Changes

Under PEG-induced drought stress, the growth of wheat seedlings was severely inhibited, as indicated by the marked wilting and significantly decreased AWC content, total FW, DW, and increased MDA concentrations (Figures 3 and 4). However, pretreatment with 0.5 mM SA restored growth and significantly counteracted the effects of subsequent drought stress on wheat seedlings, manifested in less wilting, increased 6071



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Table 2. Identification of Differentially Expressed Proteins in Leaves of Wheat Seedlings Treated with PEG and SA fold changee accession nob

protein PI/ MWc

coverage (%)

prot. score/ ion scored

pep. count

Signal transduction 56 14−3−3 protein

gi|40781605

4.83/29.38

60

600/432

17

68

gi|4582456

7.04/21.71

62

78/

10

spot noa

75

protein name

SET-domain transcriptional regulator protein agamous-like 26

Stress defense 22 glutathione S-transferase 1 27

dehydroascorbate reductase DHAR3

37

gi| 334187967

8.10/29.20

39

73/

gi|232196

5.29/25.93

24

218/172

10

7

8.68/29.01

68

191/139

8

glutathione S-transferase

gi| 357124703 gi|20067415

6.35/25.09

49

462/346

14

40

putative ascorbate peroxidase APX4

gi|31980500

8.97/32.10

42

135/129

2

57 58 62

ascorbate peroxidase ascorbate peroxidase 2-cys peroxiredoxin BAS1

gi|15808779 gi|15808779 gi| 357163385

5.10/27.96 5.10/27.96 5.98/28.36

44 48 56

588/517 502/431 789/718

9 9 9

5.76/39.01

44

489/363

15

8.29/39.18

39

503/413

13

Photosynthesis 2 phosphoglycolate phosphatase-like 9

ferredoxin-NADP(H) oxidoreductase

gi| 357164381 gi|20302471

10

ferredoxin-NADP(H) oxidoreductase

gi|20302471

8.29/39.18

39

419/331

13

11

gi|167096

8.62/47.34

36

662/577

15

18

ribulose 1,5-bisphosphate carboxylase activase isoform ribulose1,5-bisphosphate carboxylase/ oxygenase large subunit ribulose 1,5-bisphosphate carboxylase/ oxygenase large subunit ribulose 1,5-bisphosphate carboxylase/ oxygenase large subunit thylakoid lumenal 29 kDa protein

26

thylakoid lumenal 19 kDa protein

36 38

ribulose 1,5-bisphosphate carboxylase/ oxygenase large subunit putative carbonic anhydrase

42

oxygen-evolving enhancer protein 1

47

putative inner envelope protein

14 15 16

52

photosystem II stability/assembly factor HCF136 53 ribulose 1,5-bisphosphate carboxylase activase isoform Photosynthesis 63 rubisco large subunit-binding protein subunit alpha 72 ribulose-1,5-bisphosphate carboxylase activase 76 ferredoxin-NADP(H)oxidoreductase 79

rubisco large subunit-binding protein subunit beta 80 ribulose-1,5-bisphosphatecarboxylase/ oxygenase large subunit Carbohydrate metabolism 1 fibrillin-like protein 4 fructose-bisphosphate aldolase cytoplasmic isozyme 1-like 5 isocitrate dehydrogenase [NAD] catalytic subunit 5

S

D

S+D

(S + D)/D

Triticum aestivum Arabidopsis thaliana Arabidopsis thaliana

1.66f

5.06f

4.88f

0.96

2.38f

2.17f

3.20f

1.47

f

f

f

1.90

Triticum aestivum Brachypodium distachyon Triticum aestivum Arabidopsis thaliana Hordeum vulgare Hordeum vulgare Brachypodium distachyon

1.15f

1.86f

2.96f

1.59

f

f

f

0.81

species

Brachypodium distachyon Triticum aestivum Triticum aestivum Hordeum vulgare

8.35/28.51

54

524/405

13

6.08/34.72

57

756/593

17

Triticum aestivum Hordeum comosum Triticum aestivum Brachypodium distachyon Brachypodium distachyon Ilex cochinchinensis Triticum turgidum Leymus chinensis

4.85/26.74

29

448/365

11

Hordeum vulgare

gi| 118430396 gi|31087879

6.22/53.67

49

766/440

25

6.22/53.74

45

817/539

24

gi|32966580

6.22/53.44

47

650/361

24

8.98/39.29

43

210/179

7

5.76/26.52

64

236/208

5

5.52/19.21

55

280/230

6

1.41

0.83

1.49

2.30

1.20

2.12f

3.76f

5.23f

1.39

1.09

3.58f

3.38f

0.94

1.14f 1.04 1.62f

1.85f 1.62f 1.66f

2.07f 2.09f 1.96f

1.12 1.29 1.18

1.14f

1.21f

1.32f

1.09

1.64f

2.71f

2.78f

1.03

1.28f

1.11f

1.40f

1.26

1.77f

0.53f

1.17f

2.21

f

f

f

1.25

2.75

2.21

2.76

0.86f

1.42f

3.26f

2.30

0.62f

1.82f

3.54f

1.95

f

f

f

1.03

gi| 357165619 gi| 357148370 gi| 331704149 gi| 290875537 gi| 147945622 gi| 159885648 gi|75252730

9.02/45.50

32

488/411

10

Oryza sativa

0.90

gi|167096

8.62/47.34

44

790/675

17

Hordeum vulgare

gi|134102

4.83/57.66

23

375/344

7

gi| 115392208 gi|20302473

6.52/40.26

26

153/123

7

6.92/40.49

47

576/469

13

gi|2493650

4.88/53.72

29

175/114

11

Triticum aestivum Triticum aestivum Triticum aestivum Secale cereale

gi| 339305387

5.82/26.34

50

740/596

10

gi|29367475 gi| 357123886 gi| 357148997

5.04/33.92 6.86/37.98

49 44

294/262 201/156

6 8

6.33/39.97

52

583/504

12

6072

1.21

1.38

4.07

4.20

1.12f

1.37f

2.26f

1.65

0.59f

1.34f

2.12f

1.58

f

1.24

f

f

1.26

0.68f

0.91

1.02

0.65f

0.74f

1.42f

1.92

f

f

1.12

f

1.44

0.71f

0.65f

0.96

1.48

1.32f

0.93

1.61f

1.73

1.86f

1.53f

2.06f

1.35

0.98

0.56f

0.69f

1.23

0.85f

0.39f

0.58f

1.49

Elymus hystrix

0.62f

2.07f

3.74f

1.81

Oryza sativa Brachypodium distachyon Brachypodium distachyon

1.12f 1.64f

4.46f 2.70f

3.92f 2.34f

0.88 0.87

1.26f

2.12f

2.56f

1.21

1.14

0.78

1.56

1.12



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Table 2. continued fold changee spot noa

protein name

8

malate dehydrogenase 1

12

cytosolic malate dehydrogenase

23

soluble inorganic pyrophosphatase

25 35 41

triosephosphate isomerase 6-phosphogluconate dehydrogenase decarboxylating-like isoform 1 triosephosphat isomerase

46

isocitrate dehydrogenase

48

glyceraldehyde-3-phosphate dehydrogenase B glyceraldehyde-3-phosphate dehydrogenase B triosephosphate isomerase transketolase

49 59 64 70

77

6-phosphogluconate dehydrogenase, decarboxylating-like isoform 1 glyceraldehyde-3-phosphate dehydrogenase B fructose-1,6-bisphosphatase

78

quinone oxidoreductase-like protein

71

Protein metabolism 3 putative PDI-like protein 24

ATP-dependent Clp protease proteolytic

32

putative mitochondrial cysteine synthase

33

S-adenosylmethionine synthase 3

34

S-adenosylmethionine synthase 1

Protein metabolism 45 glutamine synthetase 2 50

glutamine synthetase isoform GS1c

54

serine-type peptidase

65 66

DEAD-box ATP-dependent RNA helicas 56-like glutamine decarboxylase 1-like

81

aspartic proteinase

82

30S ribosomal protein 2

Energy production 13 ATP synthase subunit 39

vacuolar proton ATPase subunit E

43

ATP synthase beta subunit

44

ATP synthase CF1 beta subunit

67

ATP1

69

ATP synthase CF1 alpha subunit

accession nob

protein PI/ MWc

coverage (%)


pep. count

gi| 357132456 gi| 229358240 gi| 226499836 gi|1174745 gi| 357110692 gi|11124572

8.54/35.60

32

356/313

7

5.75/35.83

66

660/525

15

5.45/24.37

33

230/195

6

gi| 211906502 gi| 108705994 gi| 357114230 gi|1174745 gi| 357110873 gi| 357110692 gi| 357114230 gi| 300681469 gi| 357147655

6.00/31.96 5.61/52.86

70 48

749/607 1030/881

15 24

5.38/27.01

66

637/501

14

S

D

S+D

(S + D)/D

Brachypodium distachyon Triticum aestivum Zea mays

1.61f

2.98f

2.25f

0.76

1.32f

1.19f

1.89f

1.59

1.02

2.27f

1.78f

0.78

Secale cereale Brachypodium distachyon Triticum aestivum Gossypium hirsutum Oryza sativa

f

0.87 1.14f

f

0.64 1.27f

0.92 1.56f

1.44 1.23

1.05

1.53f

1.42f

0.93

f

4.68

f

1.14

0.89f

0.78f

1.03

1.32

Brachypodium distachyon Secale cereale Brachypodium distachyon Brachypodium distachyon Brachypodium distachyon Triticum aestivum Brachypodium distachyon

0.82f

1.15f

1.35f

1.17

0.95 1.27f

0.58f 2.03f

0.98 1.89f

1.70 0.93

0.93

1.21f

1.12f

0.93

0.80f

0.79f

1.36f

1.72

0.93

0.51f

0.87f

1.71

0.93

1.02

1.26f

1.19

1.26f

2.12f

2.54f

1.20

f

f

f

0.96

species

6.29/46.41

50

537/428

15

4.99/34.03

40

272/248

5

6.03/47.68

35

351/305

9

6.00/31.96 5.93/80.01

69 33

813/659 302/227

16 14

5.61/52.86

34

589/497

14

6.03/47.68

42

289/244

9

5.38/37.85

50

562/436

15

8.29/39.84

40

439/355

12

gi| 299469378 gi| 357150338 gi| 213958273 gi| 122220777 gi| 223635282

6.17/40.58

38

556/480

11

5.61/43.25

72

803/547

16

Triticum monococcum

1.41

gi| 251832993 gi|71361904

5.75/47.00

40

760/681

11

Triticum aestivum Triticum aestivum Medicago truncatula Brachypodium distachyon Brachypodium distachyon Triticum aestivum Brachypodium distachyon

0.98

gi| 357495999 gi| 357135175 gi| 357117563 gi|73912433

6.24/32.48

31

276/233

7

5.36/22.58

67

741/654

3

5.51/43.14

55

624/434

16

5.41/39.4

53

477/359

13

6.79/45.95

40

311/287

5

5.46/49.02

22

292/252

7

5.30/56.32

20

277/230

10

5.14/54.96

31

472/403

11

gi| 357157795

9.01/26.44

55

232/202

6

gi| 285014508 gi|82502214

8.18/40.04

31

412/313

14

6.38/26.27

61

612/489

16

5.08/51.04

57

977/824

17

5.06/53.88

46

906/762

18

6.01/53.98

49

882/700

21

6.11/55.32

52

140/859

28

gi| 110915668 gi|14017579 gi| 166165274 gi|14017569

Triticum aestivum Brachypodium distachyon Aegilops speltoides Hordeum vulgare

4.03

3.27

f

4.12

2.50

2.39

1.15f

0.86f

1.40f

1.63

2.32f

2.86f

3.21f

1.12

f

f

f

1.14

0.48f

0.86f

1.79

f

f

1.32

1.12

f

1.72

1.48

1.96

1.96

1.05

1.42f

1.68f

1.18

2.32f

3.83f

2.85f

0.74

f

f

f

2.36

1.12

1.21

2.85

1.26f

0.77f

1.98f

2.57

0.47f

0.59f

0.85f

1.44

2.43f

3.37f

4.52f

1.34

0.61f

1.62f

1.37f

0.85

0.84f

0.34f

0.52f

1.53

Triticum aestivum Secale strictum

1.03

0.46f

1.58f

3.43

1.04

1.31f

1.51f

1.15

Triticum aestivum

1.72f

0.38f

0.95

2.50

Triticum aestivum Triticum aestivum Mibora minima

Toxin metabolism 6073



Article

Table 2. continued fold changee spot noa

protein name

accession nob

protein PI/ MWc

coverage (%)


pep. count

S

D

S+D

(S + D)/D

Triticum aestivum Triticum aestivum

1.41f

1.87f

2.24f

1.20

1.51f

1.69f

2.09f

1.24

species

19

Ptr ToxA-binding protein 1

gi|38570261

9.01/32.42

29

286/252

5

20

Ptr ToxA-binding protein 1

gi|38570261

9.01/32.42

18

161/143

4

gi| 326517467 gi| 326517467 gi| 297809989 gi| 326533176 gi| 226533870 gi| 326528955 gi| 357111159 gi| 326533372

5.71/35.76

44

299/220

11

Hordeum vulgare

0.38f

3.23f

1.62f

0.50

5.71/35.76

41

120/51

10

Hordeum vulgare

1.07

2.21f

1.42*

0.64

9.15/31.87

8

90/83

2

Arabidopsis lyrata Hordeum vulgare

1.38f

1.92f

1.75f

0.91

f

f

0.98

0.88

Unknown 6 predicted protein 7

predicted protein

17 29

hypothetical protein ARALYDRAFT_912061 predicted protein

55

Cp31BHv

60

predicted protein

73

uncharacterized protein At3g63140

74

predicted protein

8.54/41.93

57

1100/935

18

4.85/18.99

67

636/549

9

4.85/29.38

37

479/414

8

8.54/41.30

33

393/329

10

5.45/74.03

18

128/91

9

Triticum aestivum Hordeum vulgare Brachypodium distachyon Hordeum vulgare

0.90

1.11

2.10f

2.91f

4.64f

1.59

1.21f

1.02

1.63*

1.60

0.53f

0.32f

0.78f

2.43

3.21f

2.86f

2.05f

0.72

a

Spot numbers correspond with 2-D gel as shown in Figure 5. bAccession number in NCBI database. cProtein pI/MW (kDa): pI of predicted protein/molecular mass of predicted protein. dProtein score was based on combined MS and MS/MS spectra. The proteins that had a statistically significant (p < 0.05) protein score of 55 or more were considered successfully identified. eFold changes were from three biologically independent experiments of 2-DE, compared with the control; S, SA pretreatment; D, drought treatment; S + D, SA + drought treatment; (S + D)/D, (SA + drought treatment)/drought treatment. fIndicates significant difference between control and treatments at the p < 0.05 level.

Figure 6. Magnified views of some differentially expressed protein spots in Figure 5. The identified proteins by MS/MS (see Table 2) were labeled using squares.

Similarly, in the third group, the abundances of identified proteins in the SA-pretreatment group were higher than that in the control, but abundances in the SA + drought-treatment group were markedly lower than in the drought-treatment group. In the fourth group, the abundances of the five identified proteins in the SA or SA + drought-treatment groups were significantly lower than in the control or drought-treatment groups, respectively. Thus these proteins may be SA- or drought-responsive proteins. The role of these proteins in the SA-enhanced growth and drought tolerance needs to be further explored. The upregulated proteins might play important roles in hormone-induced growth or abiotic tolerance of plants.8,25 The great majority (93.4%) of identified proteins belonged to the first, second, and third groups, in which they were predicted to

be associated with SA-induced growth and drought tolerance. These upregulated proteins are associated with signal transduction, stress defense, photosynthesis, carbohydrate metabolism, protein metabolism, energy production, and toxin metabolism, suggesting more effective defense systems, more efficient photosynthesis, more active anabolism, and more abundant energy supply in the SA treatment and SA + droughttreatment groups. These three groups are discussed further below. Signal Transduction. The involvement of proteins responsible for signal transduction is of profound importance to SA-mediated growth and abiotic tolerance.26 In the present study, three proteins (spots 56, 68, and 75) involved in signal transduction were markedly upregulated in both the SA pretreatment and SA + drought-treatment groups. 6074



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signal transduction pathways, apoptosis, and cellular growth and survival.27 Our data suggest that the 14−3−3 protein was induced by SA or drought, whereas there was no significant difference between drought and SA + drought treatments, indicating its involvement mainly in SA-induced growth. SET domain proteins have been found in chromatinassociated complexes, which play a role in the transcriptional regulation of gene expression by histone methylation in animals.28,29 However, its function in higher plants remains unclear. Two proteins containing an SET domain, CLF (CURLY LEAF) and MEA (MEDEA), have been identified by the developmental phenotype associated with loss-offunction mutations in Arabidopsis.29−31 In this study, an SET domain transcriptional regulator (spot 68) was rapidly upregulated by SA pretreatment, and its abundance in the SA + drought-treatment group was also higher than that in the drought-treatment group. Thus it may function as an element of a signaling pathway and have immense implications for the initiation of growth and drought tolerance in wheat seedlings. Agamous, a member of the MADS-domain family of regulatory factors, integrates floral inductive pathways, controls

Figure 7. Functional classification and distribution of all 76 identified proteins based on analysis of sequence homology as listed in Table 2. Eight protein groups were categorized based on the putative functions of identified proteins and the percentages of each protein group were indicated. Different color codes represented different functional groups.

It has been reported that the 14−3−3 protein may interact with more than 200 target proteins involved in cellular organization, such as GTPase and H(+)-ATPase, and regulate

Figure 8. An interaction network of drought and SA responsive proteins in wheat seedlings. The pathways and interactions connected to all of the differentially expressed proteins analysized using the pathway studio software. The red and blue circles indicated up- and down-regulation, respectively. Protein entities which belonged to distinct functional groups were represented in different shapes according to the default settings of the software as described in the legend. ADG1, aspartic proteinase; APX1, ascorbate peroxidase; APG1, inner envelope protein; atpA, ATP synthase CF1 alpha subunit; atpB, ATP synthase CF1 beta subunit; CA1, carbonic anhydrase; ClpP, ATP-dependent Clp protease proteolytic; CMDH, cytosolic malate dehydrogenase; CS, mitochondrial cysteine synthase; CSP41A, A RAP41 homologue; Cys1, 2-Cys peroxiredoxin BAS1; DHAR3, dehydroascorbate reductase DHAR3; ERD9, glutathione S-transferase; FLC, protein agamous-like 26; FIB, fibrillin protein; FQR1, quinone oxidoreductase-like protein; F19H22.70, fructose-bisphosphate aldolase cytoplasmic isozyme 1-like; GAPB, glyceraldehyde-3-phosphate dehydrogenase B; GAD, glutamine decarboxylase 1-like; GS1, glutamine synthetase isoform GS1c; GS2, glutamine synthetase 2; GSTF6, glutathione S-transferase 1; ICDH, Isocitrate dehydrogenase [NAD] catalytic subunit 5; MAT1, S-adenosylmethionine synthase 1; MAT3, Sadenosylmethionine synthase 3; MDH1, malate dehydrogenase 1; PDIL1−1, PDI-like protein; PGD, 6-phosphogluconate dehydrogenase decarboxylating-like isoform 1; PPA, soluble inorganic pyrophosphatase; PSBQA, oxygen-evolving enhancer protein 1; rbcL, ribulose 1,5bisphosphate carboxylase activase isoform; RPS2, 30S ribosomal protein 2; SP, serine-type peptidase; TIM/TPI, triosephosphate isomerase. 6075



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reproductive organ identity, and maintains floral meristem determinacy.32 Agamous 15 transcript factor is capable of perceiving and responding to application of exogenous ethylene and interacts within the promoter regions of two cold-shock domain protein genes and alters their expression patterns.33,34 In the present study, the abundance of agamous-26 protein in the SA pretreatment and SA + drought-treatment groups was significantly higher than in the control and drought-treatment groups, respectively. Thus, it likely participates in the regulation of SA signaling. Stress Defense. Glutathione S-transferase catalyzes the conjugation of GSH to a wide range of xenobiotic and natural cytotoxic compounds with a reactive electrophilic center during plant responses to biotic and abiotic stresses.35 Ascorbate peroxidases are a specific type of antioxidant enzyme encoded by a large multigene family that contains many isoenzymes. Their function in abiotic tolerance was reported to be detoxification of H2O2 to H2O by oxidation of specific substrates such as ascorbate.36 The protein 2-Cys peroxiredoxin (2-Cys Prx) is a member of an ancient family of peroxidedetoxifying enzymes and has acquired a plant-specific function in the oxygenic environment of the chloroplast.37 In the present study, two glutathione S-transferases (spots 22 and 37), ascorbate peroxidases (spots 57 and 58, representing one protein) and a 2-Cys Prx (spot 62) were significantly upregulated by SA pretreatment for 3 days. Three days after drought stress, these proteins continued to be upregulated in both the drought and SA + drought-treatment groups, and their abundance in the SA + drought-treatment group was higher than that in the drought-treatment group. These results suggest that SA pretreatment enhanced the antioxidant defense system to decrease oxidative damage under drought stress, possibly providing a favorable environment for growth and development processes. Photosynthesis. Proteins in this group were involved in the Calvin cycle (spots 11, 14, 15, 16, 36, 38, 52, 53, 63, 72, 79, and 80), photorespiration (spot 2), electron transfer (spots 9, 10, 47, and 76), and oxygen evolution (spot 42). Of these, the most abundant involved in photosynthesis was ribulose 1,5bisphosphate carboxylase/oxygenase (Rubisco) (spots 14, 15, 16, 36, and 80) and its related enzymes (spots 11, 53, 63, 72, and 79). Rubisco is an enzyme involved in the Calvin cycle that catalyzes the first major step of carbon fixation. Previous studies have focused on changes in Rubisco content, activity, and mRNA level induced by exogenous SA under drought- and saltstress conditions.38,39 Our data confirm that exogenous SA decreased the abundance of many Rubisco proteins and related enzymes (spots 15, 16, 36, 53, and 79). This was confirmed by one previous report, in which SA pretreatment of barley seedlings decreased the carboxylating activity of Rubisco, and increased both the CO2 compensation point and stomatal resistance. These changes could in part be due to stomatal closure and a reduced CO2 supply.40 Under drought stress, however, the abundances of Rubisco proteins (spots 14, 15, 16, 36, 72, and 80) were higher in the SA + drought treatment than in the drought-treatment groups. This suggests that SA pretreatment and drought stress have a synergistic effect on Rubisco proteins and that photosynthesis may be enhanced in the SA + drought-treatment group. This is in agreement with a previous study that reported SA pretreatment-induced enhancement of photosynthesis under abiotic stress conditions.38,41Activation of Rubisco in vivo

requires ribulose-1,5-bisphosphate carboxylase activase and Rubisco large subunit-binding protein. Carbonic anhydrase raises the CO2 concentration within the chloroplast, resulting in an increase in the carboxylation rate of the Rubisco enzyme. Our data indicate a higher abundance of ribulose-1,5bisphosphate carboxylase activase (spots 11 and 53, representing one protein), two Rubisco large subunit-binding proteins (spots 63 and 79), and a carbonic anhydrase (spot 38) in the SA + drought-treatment group, implying that these proteins increased the Rubisco activation state and carboxylation rate under drought stress. Carbohydrate Metabolism. Carbohydrate metabolism regulates sugar synthesis and transformation as well as carbon partitioning, and drought stress disrupts carbohydrate metabolism in plants.42 Eighteen proteins in this group involved in glycolysis (spots 4, 23, 25, 35, 41, 48, 49, 59, 64, 70, 71, and 77), the tricarboxylic acid cycle (spots 5, 8, 12, 46, and 78), and formation of elastic fibers (spot 1) were differentially regulated 3 days after SA pretreatment or drought stress. Three days after SA pretreatment, 10 of the identified protein spots were upregulated and 8 were downregulated. Under drought stress, most of the identified proteins in this group (11 of 18) had higher expression abundance in the SA + drought treatment than in the drought-treatment groups. These enzymes included two isocitrate dehydrogenases (spots 5 and 46), a malate dehydrogenase (spot 12), a triosephosphate isomerase (spots 25 and 59, representing one protein), a 6-phosphogluconate dehydrogenase decarboxylating-like protein (spot 35), two glyceraldehyde-3-phosphate dehydrogenases (spots 48, 49, and 71; spots 49 and 71, representing one protein), a fructose-1,6bisphosphatase (spot 77), and a quinone oxidoreductase (spot 78), which are key enzymes in glycolysis and the tricarboxylic acid cycle. This suggests that SA pretreatment regulated carbohydrate metabolism and, under drought stress, further enhanced carbohydrate synthesis in wheat seedlings. This is supported by two previous reports, in which SA pretreatment enhanced the levels of glucose, fructose, and sucrose, and the abundance of many enzymes related to carbohydrate metabolism in sweet cherry fruits and sunflower seedlings under biotic and abiotic stresses.26,43 Protein Metabolism. An active protein quality-control system may play an important role in the production of osmotic regulators (including proline, free amino acids, and other compounds), and improved nitrogen metabolism is an important tolerance mechanism induced by SA pretreatment, as indicated by an increase in nitrate reductase (NR) activity, protein level and nitrogen contents of SA-pretreated wheat seedlings.38 In the present study, SA pretreatment for 3 days enhanced the abundance of 10 protein-metabolized spots (3, 24, 32, 33, 34, 50, 54, 65, 66 and 81), had no effect on that of one spot (45), and decreased that of another (82). Three days after drought stress, the majority of these proteins in the SA + drought-treatment group (10 of 12) had higher abundances than in the drought-treatment group. Glutamine synthetase catalyzes the ATP-dependent condensation of ammonium with glutamate to yield glutamine, which then provides nitrogen groups for the biosynthesis of all nitrogenous compounds in plants. Glutamine decarboxylase catalyzes decarboxylation of L-glutamine to γ-aminobutyrate. Overexpression of glutamine synthetase or decarboxylase in plants confers resistance to abiotic and biotic stresses.44,45 Expression of one glutamine synthetase 2 (spot 45) was relatively unaffected by SA pretreatment and greatly inhibited 6076



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by subsequent drought stress. Exogenous SA pretreatment compensated for the inhibition of this protein by drought stress. Another glutamine synthetase isoform, GS1c (spot 50), and a glutamine decarboxylase 1-like enzyme (spot 66) were upregulated by SA pretreatment or subsequent drought stress, and exhibited higher abundance in the SA + drought-treatment group (Table 2). Ribosomes are responsible for protein biosynthesis. Protein disulfide isomerase (PDI)-like proteins play a role in disulfide bond formation in the intracellular proteins of thermophilic organisms.46 A PDI-like protein (spot 3) was induced rapidly, and a ribosomal protein (spot 82) was inhibited, by SA pretreatment and drought stress, respectively. However, their abundances in the SA + drought-treatment group were significantly higher than in the drought-treatment group. The enhanced abundance of proteins in the SA pretreatment and SA + drought-treatment groups may be associated with the increased protein and nitrogen contents of SA-pretreated, drought-stressed wheat seedlings.37 Energy Production. Plants need a large quantity of ATP for sufficient energy for growth, development, and stress responses.47 ATP synthase, a 400-kDa protein complex, provides energy for a large number of fundamental biological processes.48 Energy production proteins identified in this group were uniquely involved in ATP synthesis (spots 13, 39, 43, 44, 67, and 69). SA pretreatment for 3 days increased the abundance of four protein spots (13, 44, 67, and 69). Under drought stress, SA + drought treatment accelerated the abundance of spots 13, 44, and 67 upregulated by drought alone, and compensated for the downregulation of spots 43 and 69. This suggests that energy generation processes were more activated in the SA pretreatment and SA + drought-treatment groups than in the control and drought-treatment groups, respectively, to increase growth and cope with drought stress. Toxin. Ptr ToxA, a proteinaceous host-selective toxin produced by the wheat tan-spot pathogen Pyrenophora triticirepentis, induces cell death and results in the accumulation of reactive oxygen species in toxin-sensitive wheat cultivars. ToxAbinding protein 1, a ToxA-interacting protein, is likely involved in downstream events in response to oxidative burst and ToxAinduced cell death.49 In the present study, ToxA-binding protein 1 (spots 19 and 20, representing one protein) was upregulated by SA pretreatment and subsequent drought stress, but the extent of upregulation was greater in the SA + droughttreatment group. Thus this protein may function as a common regulator of the oxidative burst produced by SA pretreatment and drought stress under biotic and abiotic stress conditions in wheat seedlings.

determine the key proteins during SA action using the pathway studio software. The results indicate that 35 key proteins in the metabolic processes are induced by SA, and they are related to some processes of physiology and metabolism, including carbohydrate metabolism, protein metabolism, stress defense, photosynthesis, energy, signal transduction (Figure 8). This implies that these proteins may be critical in the SA-responsive protein interaction network. For instance, glutathione Stransferase 1 (GSTF6) (spot 22) is associated with antioxidative reactions, significantly induced by exogenous SA under abiotic stress conditions, and overexpression of GSTF6 gene significantly increases the drought tolerance in tobacco.20,50 The changes in the expression levels of these key proteins and regulatory pathways could be important factors in SA-induced growth and drought tolerance.

■

CONCLUSION Treatments with 0.5 mM SA for 3 days significantly increase the growth of wheat seedlings and the tolerance to subsequent drought stress. These were manifested by the increased heights, AWC, DWs, FWs and the decreased lipid peroxidation level. Proteomic analysis indicated involvement of 76 proteins differentially regulated by SA pretreatment in signal transduction, stress defense, photosynthesis, carbohydrate metabolism, protein metabolism, and energy production. After these identified proteins were analyzed using Pathway Studio software, we found that 35 might play critical roles in drought tolerance induced by SA. To the best of our knowledge, this is the first study on the proteomic mechanism of the SA-induced growth and drought-stress tolerance of higher plants.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1. Phenotypes of wheat seedlings pretreated with different concentrations (0, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM, 2.0 mM, and 3.0 mM) of SA and then exposed to drought stress for 3 days. Table S2. Effect of different concentrations (0, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM, 2.0 mM, and 3.0 mM) of exogenous SA pretreatments on growth characteristics of wheat seedling exposed to drought stress for 3 days. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 371 6355 8205. Fax: +86 371 6355 8202. E-mail: [email protected]; [email protected].

Putative Mechanism of SA-Induced Growth and Drought-Stress Tolerance

Notes

This proteomic study provides evidence that 0.5 mM SA pretreatment for 3 days results in differential regulation of the expression of many proteins. The abundances of the majority of the identified proteins were higher in the SA pretreatment and SA + drought-treatment groups than in the control and drought-treatment groups, respectively. This suggests that photosynthesis, energy production, carbohydrate and protein metabolism, defense systems were increased in both the SA pretreatment and SA + drought-stress groups, which enhanced both growth and drought-stress tolerance. In the present study, we have analyzed the pathways and interactions connected to each of the identified proteins to

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Lan Zhang of Institute of Apicultural Research, Chinese Academy of Agricultural Science, for pathway studio software analysis. This study was financially supported by the open item of the State Key Laboratory of Crop Biology of China (2009KF01), the open item of the State Key Laboratory of Crop Genetics & Gerplasm Enhancement (ZW2009003) and the National Basic Research Program of China (2009CB118602). 6077



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Proteomics Reveals the Effects of Salicylic Acid on Growth and

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