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Proteomics Uncovers a Role for Redox in Drought Tolerance in Wheat§ Mohsen Hajheidari,†,# Alireza Eivazi,†,# Bob B. Buchanan,‡ Joshua H. Wong,‡ Islam Majidi,† and Ghasem Hosseini Salekdeh*,† Department of Physiology and Proteomics, Agricultural Biotechnology Research, Institute of Iran, Karaj, Iran, and Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, California 94720 Received October 30, 2006

Proteomic analysis offers a new approach to identify a broad spectrum of genes that are expressed in living systems. We applied a proteomic approach to study changes in wheat grain in response to drought, a major environmental parameter adversely affecting development and crop yield. Three wheat genotypes differing in genetic background were cultivated in field under well-watered and drought conditions by following a randomized complete block design with four replications. The overall effect of drought was highly significant as determined by grain yield and total dry matter. About 650 spots were reproducibly detected and analyzed on 2-DE gels. Of these, 121 proteins showed significant change under drought condition in at least one of the genotypes. Mass spectrometry analysis using MALDITOF/TOF led to the identification of 57 proteins. Two-thirds of identified proteins were thioredoxin (Trx) targets, in accordance with the link between drought and oxidative stress. Further, because of contrasting changes in the tolerant and susceptible genotypes studied, several proteins emerge as key participants in the drought response. In addition to providing new information on the response to water deprivation, the present study offers opportunities to pursue the breeding of wheat with enhanced drought tolerance using identified candidate genetic markers. The 2-DE database of wheat seed proteins is available for public access at http://www.proteome.ir. Keywords: proteomics • redox proteins • drought • wheat • seed • oxidative stress • thioredoxin h • drought tolerance

Introduction The advent of proteomics has made it possible to identify a broad spectrum of proteins in living systems. This capability is especially useful for cereals, as it may give clues not only about nutritional value, but also about yield and how these factors are affected by adverse conditions. Among the cereals, wheat has received attention because of its economic and nutritional importance. Accordingly, proteins of whole grain1,2 and its different compartments have been analyzed, that is, amyloplasts, the organelles that synthesize and store copious amounts of starch3,4 and the parent starchy endosperm that is the source of flour.5 The results have given insight into the broad metabolic capability of these systems and, in the case of endosperm, how metabolism changes during development. * Corresponding author. Dr. Ghasem Hosseini Salekdeh, Agricultural Biotechnology Research Institute of Iran, P.O. Box 31535-1897, Karaj, Iran. E-mail, [email protected]; fax, +98-261-2704539. § Dedicated to the memory of Karoly K. Kobrehel who was a true visionary in linking redox to grain proteins. † Agricultural Biotechnology Research Institute of Iran. ‡ University of California. # These two authors contributed equally to this paper. 10.1021/pr060570j CCC: $37.00

 2007 American Chemical Society

As a result of its relation to grain quality and yield, elevated temperature has also been investigated with the wheat proteome.1,2 These studies have shown that a number of grain proteins, 37 in one case1 and 38 in the other,2 are changed and thus could potentially serve as markers of temperature stress. Although studied in leaves of Arabidopsis6, sugar beet,7 rice,8,9 and oak,10 Elymus elongatum11, the effect of another major environmental parameter adversely affecting development and crop yieldsnotably, droughtshas, however, not been studied in grain. To fill this gap, we have analyzed the grain proteomes of three wheat genotypes under normal and drought conditions. The results, summarized below, show that many proteins are differentially expressed in response to water stress in these genetically distinct lines. Standing out are redox-linked proteins, including h-type thioredoxins (Trxs), that, as they seem to correlate with drought, could serve as genetic markers for this type of stress.

Experimental Procedures Plant Materials. Three spring wheat genotypes (Arvand, Khazar-1, and Kelk Afghani) differing in origin were grown in experimental fields of West Azarbayjan Agricultural Research Center (36° 58′ N, 46° 6′ E) in the 2002-2003 growing season. Journal of Proteome Research 2007, 6, 1451-1460

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research articles Plots of 2.4 m2 were sowed at a density of 250 seeds per plot. The experimental design was a randomized complete block design with four replications. Plots were uniformly irrigated from emergence until the booting stage12 using a system based on evaporation from Class A Pan. Control and water deficittreatments were irrigated at 75 ( 5 and 150 ( 5 mm evaporation, respectively. At the end of the growing season, calculations of total dry weight and grain yield were performed based on four central rows (1 m2/plot). The mature seeds of the main ears from each plot were collected for proteome analysis. The whole grain, 20 g, was ground in a UDY mill (Model MS UDY Cyclone Sample Mill, UDY CO., Fort Collins, CO). The samples obtained were analyzed for protein by near-infrared reflectance spectroscopy13 using an NIR System 8100 (NIR System, Inc., Silver Springs, MD). Data were analyzed using of Mstat-c software based on Steel and Torrie14 procedure. Protein Extraction and Two-Dimensional Gel Electrophoresis (2-DE). Protein was extracted from the ground meal according to Finnie et al.15 with minor modifications. The seeds of each of the main ears collected from different plots (replications) were ground in liquid nitrogen with a mortal and pestle. Approximately, 4 g of ground grain was suspended in 20 mL of extraction buffer consisting of 5 mM Tris, pH 7.5, 1 mM CaCl2, and 20 mM DTT. The soluble proteins were extracted by shaking 30 min at 4 °C and then clarified by centrifugation (30 min at 15 000g). The supernatant fraction was applied to analytical 2-D gels. Protein was quantified according to Bradford16 using reagents from Bio-Rad (Hercules, CA) and bovine serum albumin as the standard. For preparing gliadins, 10 g of mill flour was mixed with about 8 mL of distilled water, centrifuged for 5 min at 10 000g, and washed with water. The residue (gliadins and glutenins) were separated by centrifugation at 10 000g, and the gliadin fraction was dried at room temperature and weighed. The precipitate was solubilized in buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, 1% (v/v) pH 3-10 ampholyte, and 35 mM Tris base. For IEF, 18 cm IPG strips with a linear gradient (pH 4-7) were rehydrated using 350 µL of rehydration buffer (8 M urea, 2% (w/v) CHAPS, 20 mM DTT, 0.5% (v/v) IPG buffer 4-7, and 0.01% (w/v) bromophenol blue) in a reswelling tray (Amersham Pharmacia Biotech, Uppsala, Sweden) at room temperature for 16 h. For analytical and preparative gels, 120 µg and 1.5 mg of protein, respectively, were added to rehydration buffer. IEF was performed using Multiphor II and a DryStrip Kit (Amersham Pharmacia Biotech). Gels were run at 500 V for 2 h followed by 1000 V for 2 h and finally 3500 V for 14 h. The focused IPG strips were equilibrated twice for 15 min in 10 mL of equilibration solution. The first equilibration was performed in a solution containing 50 mM Tris-HCl buffer, pH 8.8, 6 M urea, 30%(v/v) glycerol, 2% (w/v) SDS, and 1% (w/v) DTT, with a few grains of bromophenol blue. The second equilibration was as the first except that DTT was replaced with 2.5% (w/v) iodoacetamide. The second dimension was developed with a 12% SDS-polyacrylamide gel using a Protean II Multi Cell (BioRad). Protein spots were visualized in analytical gels by staining with silver nitrate.17 Preparative gels were stained with colloidal Commassie Brilliant Blue G-250 (CBB).18-19 Image and Data Analysis. Wet silver-stained gels were scanned with a GS-800 calibrated densitometer (Bio-Rad, Hercules, CA) at a resolution of 600 dots and 12-bit per inch and analyzed using Melanie-3 software (GeneBio, Geneva, Switzerland) according to the user’s manual. After scanning, 1452

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spot detection, protein quantification, and spot pairing were carried out based on Melanie-3 default settings. Then, spot pairs were investigated visually, and the scatter plots of each data point between gels were displayed to estimate gel similarity and experimental errors. The molecular mass of proteins in the gels was estimated by co-electrophoresis with standard protein markers (Amersham Pharmacia Biotech); pI was determined by measuring spot migration on 18 cm IPG (pH 4-7, linear) strips. Since there were two treatments for each genotype (well-watered and droughted), treatment combinations were analyzed by two-way analysis of variance (ANOVA). Spots were concluded to be significantly up- or down-regulated when P < 0.05. Protein Identification and Database Search. Protein spots were excised from CBB-stained gels and analyzed using an Applied Biosystems 4700 Proteomics Analyzer at the Protein and Proteomics Center, University of Singapore (Mass Spectrometry Services, Department of Biological Sciences). Protein digestion, desalting, and concentration of samples were carried out using Montage In-Gel Digestion Kits (Millipore and Applied Biosystems, Foster City, CA). The samples were dissolved in solvent consisting of 0.1% trifluroacetate and 50% acetonitrile (ACN) in MilliQ Water. Then 0.5 µL of sample solution was mixed with 0.5 µL of matrix solution (5 mg/mL R-cyano-4hydroxycinnamic acid dissolved in the above solvent), applied to a MALDI sample target plate, and dried in air. Before each analysis, the instrument was calibrated with the Applied Biosystems 4700 Proteomics Analyzer Calibration Mixture. Data interpretation was carried out using the GPS Explorer Software (Applied Biosystems), and an automated database search was carried out using the MASCOT program (Matrix Science, Ltd., London, U.K.). Combined MS-MS/MS searches were conducted with the selection of the following criteria: MSDB 20040710 (1501893 sequences; 480537664 residues), all entries, parent ion mass tolerance at 50 ppm, MS/MS mass tolerance of 0.2 Da, carbamidomethylation of cysteine (fixed modification) and methionine oxidation (variable modification). According to MASCOT probability analysis (P < 0.05), only significant hits were accepted. Western Blot Analysis. Flour protein, 100 mg, was extracted with 1.0 mL of 0.5 N NaCl by shaking for 1 h at ambient temperature. The supernatant fraction was recovered by centrifugation (10 min at 15 800g) at 4 °C. Western blot analysis was performed on clarified salt-soluble protein extracts prepared from the indicated cultivars and subjected to SDS-PAGE on a pre-cast 10-20% linear gradient Criterion Tris-HCl gel (Bio-Rad, Hercules, CA) at pH 8.5 at a constant voltage of 150 V for 75 min. Several concentrations of protein (5-20 µg) were dissolved in 1× Laemmli sample buffer, boiled for 5 min, clarified, and loaded onto the above gel. Proteins were transferred to nitrocellulose at a constant voltage of 50 V for 1 h at 4 °C using a Criterion Blotter Cell Assembly (Bio-Rad, Hercules, CA). Nitrocellulose membrane was blocked with 5% powdered milk in buffer (20 mM Tris-HCl, pH 7.5, supplemented with 0.15 M NaCl) for 2 × 30 min at 25 °C and incubated in primary antibody overnight at 4 °C and in secondary antibody for 1 h at 25 °C. Primary antibody was wheat anti-Trx h II20 diluted 1:1000; secondary antibody was goat anti-rabbit IgG-HRP Conjugate (Bio-Rad, Hercules, CA) diluted 1:3000. Blots were developed in TMB Substrate Kit for Peroxidase (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions.

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Redox Role in Drought Tolerance

Figure 1. The effect of drought stress on grain yield and dry weight of three genotypes.

Protein Determination, Blot Scanning, and Analysis. The blots were scanned using a UMAX PowerLook II00 scanner fitted with Photoshop 6. Images were transferred to Quantity One Quantitation Software (Bio-Rad, Hercules, CA), version 4.0, and the volume (intensity × area) of the immunoreactive bands was measured.

Results and Discussion On the basis of the analysis of variance, the overall effect of drought was highly significant (P < 0.01) as determined by grain yield (Figure 1, left panel) and total dry matter (Figure 1, right panel). Ears per m2 and grains per spike were also affected with respective drought-induced decreases of 35 and 33%. In all cases, the tolerant genotype (Khazar-1) was significantly less affected than its susceptible counterparts (Afghani and Arvand). There were also protein differences. One particularly striking change was observed with the gliadin storage proteins which showed an increase of 9-fold in drought-tolerant Khazar-1 versus a 3-fold increase in susceptible Arvand and Afghani. Interestingly, while total protein increased in all cases during drought stress, the extent was relatively modest: Khazar-1 (3%) < Afghani (7%) < Arvand (9%). These changes may reflect the effect of stress as drought tends to increase protein content during grain filling because the accumulation of starch is more severely affected than that of nitrogen.21 Overall, it appears that, while effecting only a small increase in total grain protein, drought elicits a major redirection of resources to the synthesis of gliadins. Further, this redirection appears to be more pronounced in drought-tolerant genotypes. Identification of Drought-Responsive Proteins. We applied 2-DE to analyze the seed proteomes of the three wheat genotypes in response to prolonged drought stress (Supporting Information Figure 1). Drought-responsive proteins were excised from preparative gels and examined by MALDI-TOF/TOF. A spot that consistently showed a particular position in the different gels was considered to be the same protein. About 650 spots were reproducibly detected in four replications of each genotype, that is, Khazar-1 (Figure 2), Arvand, and Afghani. Of these, 121 proteins showed significant change under drought condition in at least one of the genotypes (Figure 2 and Supporting Information Table 1). Mass spectrometry analysis using MALDI-TOF/TOF led to the identification of 57 proteins (Supporting Information Table 2) that were classified according to function (Table 1). Some of the proteins in Table 1, for example, Trx h, serpin, R-amylase inhibitor, and mitochondrial aldehyde dehydrogenase, have functions other than the one indicated.

Both the total number of drought-responsive proteins and the number of up-regulated proteins (P < 0.05) were higher in the tolerant genotype, Khazar-1, than in the susceptible genotypes, Arvand and Afghani (Figure 3). Additionally, the response patterns of the proteins generally differed among the three genotypes. Most of the proteins up-regulated during drought in Khazar-1 were either up-regulated to a lesser extent, down-regulated, or showed no change in Arvand and Afghani. Conversely, a small number of proteins up-regulated in the susceptible genotypes either showed no change or were downregulated in the resistant counterpart. Function of Drought-Responsive Proteins. The proteins function in fundamental processes, including stress/defense, protein synthesis/assembly, metabolism, and storage. Four of the proteins were unknown, and two had no known function. Of the 57 proteins of known function, 38 (designated with an asterisk, *) have been identified as either a confirmed or candidate Trx target, thus, highlighting a link of drought tolerance to redox.22-24 The individual proteins are discussed below in relation to their function and link to redox. The change in each protein in response to drought is given in Table 1. Corresponding changes in the different processes are summarized in Figure 4. The majority of the proteins identified functioned in stress/defense, protein synthesis/assembly, and metabolism. A small number were storage proteins and proteins with unknown function. Proteins Responding in Three Genotypes. Because of the production of reactive oxygen species (ROS) in multiple types of stress, systems have been developed to minimize their deleterious effects during seed development and germination.25,26 In particular, desiccation and resumption of respiration following the hydration of dry seeds give rise to ROS that should be removed or neutralized. The appearance of antioxidant enzymes at the onset of the maturation-drying phase is in accord with this function and acquisition of desiccation tolerance.27 In view of these events, it is not surprising that the largest number of drought-related proteins in cereal grain are redox-related and are known to undergo a pronounced change in sulfhydryl status during development and germination.23 Four enzymes functional in stress/defense, 1-Cys peroxiredoxin, glutathione S-transferase, and two forms of Trx h (spots 37 and 43), showed a decisive response in the drought-tolerant genotype (Kharzar-1) and both susceptible counterparts. 1. Thioredoxin h. A member of a family of small proteins that appear to be ubiquitous, Trx h functions in scavenging, but perhaps more importantly, the protein regulates a number of fundamental seed processes.23,28-30 All Trxs share a conserved Journal of Proteome Research • Vol. 6, No. 4, 2007 1453

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Figure 2. 2-D gel analysis of proteins extracted from grain of Khazar-1 genotype harvested under well-watered conditions. In the first dimension (IEF), 120 µg of protein was loaded on an 18 cm IPG strip with a linear gradient of pH 4-7. In the second dimension, 12% SDS-PAGE gels were used, with a well for molecular weight standards. Proteins were visualized using silver staining. Arrows represent drought-responsive spots of which 57 have been identified by MS (Table 1).

active-site motif, [-Trp-Cys-Gly(Pro)-Pro-Cys-]. In their reduced state, Trxs reduce regulatory disulphide bridges of numerous target proteins. Subsequently, the oxidized Trxs formed are reduced by either of two mechanisms: one linked to ferredoxin (chloroplasts and cyanobacteria) and the other to NADPH (heterotrophic cells and cell compartments). Plant Trxs reduced by NADPH, via NADP-thioredoxin reductases (NTR), are designated the h-type and have been described for the cytosol, mitochondria, and ER.23 Trx h is also involved in a range of biochemical processes. These include the mobilization of protein and starch in germinating cereal seeds,31 self-incompatibility,32,33 and cellular protection against oxidative stress, particularly during seed desiccation and germination.34 Trxs h reduce a variety of target proteins that contain disulfide bonds, including storage proteins, such as glutenins and gliadins in wheat35,36 and hordein and glutelin in barley,37 and proteins related to oxidative stress such as peroxiredoxin.23 In the current study, we identified three isoforms of Trx h that showed contrasting response patterns in the tolerant and susceptible genotypes. One form (spot 43) increased in the tolerant genotype and decreased in the susceptible counterparts, and another (spot 42) decreased in all three genotypes (both of these changes were statistically significant). A third spot, 37, significantly increased in the tolerant genotype and decreased in the susceptible ones, although these changes were not statistically significant. 1454

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Because of its pronounced change in drought stress, we applied an immunological approach to confirm the proteomic results for Trx h. As seen in Figure 5, Western blot analysis showed that the amount of Trx h differed in the three genotypes subjected to drought, that is, the level decreased in the sensitive genotypes (Arvand and Afghani) and increased in the tolerant counterpart (Khazar-1). This differential change was observed with both the major and minor bands, two well-known isoforms of Trx h (major and minor bands are designated with a dot and a star in Figure 5).38 Further, the proteomic and Western blot analyses were complementary in showing an increase in Trx h in Khazar-1 and a decrease in both Arvand and Afghani under drought stress (Figure 6). Thus, the level of Trx h in whole grain decreased in sensitive and increased in tolerant genotypes based on two lines of evidence. In view of its apparent dual improving effect, it becomes of interest to relate the abundance of Trx not only to drought tolerance, but also to dough quality.39 It is known that drought leads to a decrease in the ability of flour produced acceptable products.40 The question, therefore, arises as to whether Trx will help minimize this deleterious effect. The three wheat Trx h spots37,42 are closest to Arabidopsis Trx h5 (81% similarity). They are, however, also quite close to Trx h3 (79% and 78% similarity). Trx h5 and h3 are characterized by a relatively common but atypical active site, CPPC. The three isoforms of Trxs in our study could, in fact, be the wheat counterpart of Arabiopsis Trx h3 and/or Trx h5. Both Trxs are

Redox Role in Drought Tolerance

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Table 1. Drought Responsive Proteins of Wheat Grain Identified Using MALDI TOF-TOF MS/MS and Their Corresponding Induction Factor (Percent Volume of Spot in Stress Condition/Percent Volume of Spot in Well-Watered Condition)a

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Table 1 (Continued)

a According to Mascot probability analysis (P < 0.05), only significant hits were accepted; (*) thioredoxin target (22,23); (**) change statistically significant in at least one variety in response to drought stress compared to well-watered. Spots were concluded to be significantly up- or down-regulated when P < 0.05. Dark gray highlights, change statistically significant in two varieties in response to drought stress compared to well-watered;10 light gray highlights, change statistically significant in three varieties in response to drought stress compared to well-watered.9 (§) Change differs between tolerant and at least one susceptible variety in response to drought stress compared to well-watered.11

desiccation tolerance during late stages of seed development have been elucidated.45 These authors suggested that 1-Cysperoxiredoxin may help maintain dormancy. In reaching a similar conclusion, Haselkas et al.46 suggested that the antioxidant function of 1-Cys Prx is to sense harsh environments, thereby preventing germination under unfavorable conditions. In our study, the change in 1-Cys-peroxiredoxin was pronounced but was inconsistent in the three genotypes. Proteins Responding in Two Genotypes. Three types of protein in the stress/defense category showed significant opposing changes in Khazar-1 and one of the susceptible genotypes (R-amylase inhibitor, a cold-regulated protein and dehydroascorbate reductase). Figure 3. The number of grain proteins differing significantly in abundance in drought-stressed and re-watered plants of wheat cultivars compared with well-watered controls. Solid bars, proteins more abundant in stressed plants; open bars, proteins less abundant in stressed plants.

of interest in this context because h5 has been associated with oxidative stress41 and h3 confers H2O2 tolerance.42 Our results support these previously assigned functions and extend the role of Trx h to drought stress. 2. Glutathione S-Transferase. A participant in ROS scavenging, glutathione S-transferase showed decisive differential expression in the tolerant and susceptible genotypes. The enzyme limits oxidative damage by removing ROS formed in stress and by detoxifying xenobiotics under normal conditions. The endogenous products of oxidative damage, for example, membrane lipid peroxides and products of oxidative DNA degradation, are highly cytotoxic. Glutathione S-transferase detoxifies these endogenously generated electrophiles by conjugating them with GSH.43 In accord with this conclusion, the enzyme increased 2-fold under drought stress compared to the well-watered counterpart in Khazar-1, whereas it was downregulated by a similar factor in Arvand and Afghani (Table 1). 3. 1-Cys-peroxiredoxin. 1-Cys-peroxiredoxin is also directly involved in scavenging ROS. In plants, the cDNA of 1-Cysperoxiredoxin was first isolated as a dormancy-related protein expressed in the embryo and aleurone layer of barley.44 The antioxidant activity of this enzyme and its contribution to 1456

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1. Dehydroascorbate Reductase. This enzyme was upregulated in response to drought stress in the tolerant genotype, Khazar-1, and down-regulated in the susceptible genotypes either markedly (Arvand) or marginally (Afghani). The enzyme is noteworthy in view of its role in the ascorbate-glutathione cycle for ROS removal. Dehydroascorbate reductase also showed the highest increase in activity during seed development among the enzymes studied.25 As there is a shortage of ascorbic acid in seeds,47 enhanced dehydroascorbate activity early in germination would help maintain the limited pool of this antioxidant.26 2. r-Amylase Inhibitor. The activity and expression level of R-amylase inhibitor is associated with starch during grain filling and seed maturation. We identified nine protein spots as isoforms of the R-amylase inhibitor. Of these, four (spots 18, 20, 31, and 543) were up-regulated to a greater extent in Khazar-1 than in the sensitive genotypes. Four (spots 32, 152, 153, and 190) did not change markedly in Khazar-1, but were down-regulated to varying degrees in Afghani, and two proteins (spots 32 and 153) were down-regulated in both Afghani and Arvand. Only one isoform (spot 36) was up-regulated in Arvand. Although a single form of R-amylase did not change in an opposing manner in the tolerant and susceptible genotypes, the observed up-regulation of individual forms in tolerant Khazar-1 and their down-regulation in the susceptible genotypes may offer protection against oxidative stress and help preserve grain starch, a component crucial for germination.

Redox Role in Drought Tolerance

Figure 4. Functional annotation of the identified droughtresponsive proteins in three genotypes classified by biological function described in Table 1. The numbers represent the percent and number of proteins in each class.

3. Cold-Regulated Protein. We identified two cold-regulated proteins, both of which were significantly up-regulated in Khazar-1. One (spot 30) was down-regulated in one of the susceptible genotypes, Arvand. The UniGene cluster of this gene (Ta.13183) represents ESTs from leaf, root, and developing

research articles and dormant seed tissues. Drought, salt, and cold conditions of dormant seed and drought condition represent the highest number of ESTs. In Arabidopsis, most cold-regulated genes respond to dehydration, and conversely, most dehydrationinduced genes also respond to cold stress.48 The current work suggests that a common set of genes is responsible for both drought and cold tolerance. 4. Proteins Functional in Synthesis and Assembly. Two of the proteins identified in this category, elongation factor-1 R and HSP17, were up-regulated in Khazar-1 and down-regulated in one of the susceptible genotypes in accord with a protective function in drought response (Table 1). Others have observed that this protein family, which is expressed during seed development,49 is up-regulated in mature wheat seed under heat stress.1,2 Additionally, several studies have suggested that HSPs may function to protect cellular components during seed desiccation.50-53 In the current study, the types of HSPs identified were consistently up-regulated to a greater extent in drought-tolerant (Khazar-1) than in the susceptible genotypes (Afghani and Arvand). It is noteworthy that three HSP 70 identified (spots 170, 171, and 173) were down-regulated in the susceptible genotype, Afghani. These proteins are involved in a wide range of cellular functions, including protein folding, and the correct assembly of oligomeric proteins,54 prevention of the aggregation of denatured proteins,55 refolding of stress-denatured proteins,56 and import of proteins across membranes.57 Their downregulation would thus likely have detrimental effects on proteins at multiple levels. The changes in expression of these HSP70 proteins in the other genotypes was less clear. Three forms of protein disulfide isomerase3 precursor were identified. One (spot 119) showed behavior not observed with other proteins in this study: down-regulated in each of the tree genotypes, but less so in the two susceptible genotypes than in Khazar-1. The observation suggests that this form of the enzyme may be detrimental for drought tolerance. 5. Proteins Functional in Metabolism. Of the metabolic enzymes identified, glyceraldehyde-3-phosphate dehydrogenase showed the most striking response to drought stress: up-regulation in drought-susceptible Afghani and Arvand and down-regulation in tolerant Khazar-1. This finding suggests that glycolysis would be decreased during drought. The pattern of the other metabolic enzymes was less clear. Thus, methylmalonate semialdehyde dehydrogenase, a mitochondrial enzyme functional in the catabolism of valine and pyrimidines, was increased in one susceptible and down-regulated in the other as well as in the tolerant counterpart. Starch granule-bound starch synthase showed similar inconsistent behavior. 6. Storage Proteins. One globulin was up-regulated under stress selectively only in drought-sensitive genotypes and down-regulated in Khazar-1. The results suggest that it is advantageous to down-regulate the synthesis of this storage protein and direct available resources to minimize the effects of stress during drought. 7. Unknown. Two proteins of unknown function (spots 28 and 29) were up-regulated in the drought-tolerant genotype and down-regulated in one or both of the susceptible counterparts. Another protein, spot 99, that was down-regulated up to 2-fold in the susceptible genotypes and not significantly changed in the tolerant counterpart, was identified as a hypothetical protein from Sporobolus stapfianus (resurrection grass) with 89% identity to rice glyoxalase I (BAA36759). Glyoxalase I plays a central role in the prevention of glycation Journal of Proteome Research • Vol. 6, No. 4, 2007 1457

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Figure 5. Western blot analysis of Trx h in grain extracts from drought-susceptible and tolerant wheat genotypes grown in the field. The gel was developed with 5 µg of flour protein. Similar results were obtained with 10 and 20 µg samples. Seeds were taken from plants grown in one of the four indicated replicate field plots (R1-R4).

nontransgenic controls.61 Glyoxalase I has also been found to be one of several genes induced in drought and cold stresses in Arabidopsis.62 Further study is needed to determine how glyoxalase is linked to the drought-tolerance mechanism in plants.

Concluding Remarks

Figure 6. Comparison of Western blot and proteomic analysis for drought-induced changes in Trx h isoforms in grain of drought-susceptible and tolerant wheat varieties grown under field conditions. The amount of protein in the Western blot was assessed by estimating the volume of each band by densitometric scanning. The values shown represent averages taken from the relevant lanes designated in Figure 5. The average of significant induction factors of all Trx h isofrorms in the proteomic (2-DE) analysis are presented as indicated.

reactions by detoxifying R-oxoaldehydes such as methylglyoxal and glyoxal through conjugation with glutathione. These derivatives are further metabolized by glyoxalase II, thereby minimizing their mutagenic and cytotoxic activities that, among other effects, lead to arrested growth.58 A decrease in glyoxalase I activity in situ during aging or oxidative stress results in increased glycation and tissue damage.59 Transgenic tobacco underexpressing glyoxalase I showed an enhanced accumulation of methylglyoxal which resulted in the inhibition of seed germination.60 By contrast, overexpression of glyoxalase I resulted in improved tolerance against methylglyoxal and higher levels of resistance to salinity stress compared to 1458

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In highlighting the role of redox, the present findings add insight to our understanding of the response of wheat to drought stress. The results provide evidence that drought causes a redirection in protein synthesis to increase the formation of gliadins, interestingly, to a greater extent in drought-tolerant genotypes than in susceptible genotypes. Of the 57 drought-responsive proteins identified, two-thirds were Trx targets, highlighting the link between drought and oxidative stress that was earlier observed with leaves of Arabidopsis.6 Further, because of contrasting changes in the tolerant and susceptible genotypes studied, several proteins emerge as key participants in the drought response. Included are two proteins that are clearly selectively up-regulated in the drought-tolerant genotype, Khazar-1, notably, Trx h and glutathione S-transferase. Other possible candidates include cold-regulated protein, dehydroascorbate reductase, elongation factor-1 R, HSP17, and unknown protein (P0022BO5.25). There are also two proteins that behaved in an opposing manner, that is, downregulated in the tolerant Khazar-1 and up-regulated in the susceptible Afghani and Arvand: glyceraldehyde-3-phosphate dehydrogenase and at least one globulin storage protein. One enzyme, protein disulfide isomerase3 precursor, was consistently down-regulated, but more extensively in the tolerant than in the susceptible genotypes. In addition to providing new information on the response to water deprivation, the present study offers opportunities to pursue the breeding of wheat with enhanced drought tolerance. Specific areas worthy of further study include:

Redox Role in Drought Tolerance

• Identification of the remaining 63 proteins found to change during drought stress in the genotypes in the current investigation. This information may give further insight into droughtresponsive pathways and the genes that control them. Efforts should be made to take advantage of ongoing improvements in proteomics technology to increase sensitivity in an attempt to identify less abundant proteins such as transcription factors. • Development of additional wheat genotypes tolerant and susceptible to drought. Added analyses will provide an assessment of the extent to which present findings can be generalized and whether the redox and related proteins identified in this study undergo selective change in other genotypes. This work is also central to efforts to identify candidate markers for drought tolerance. • The effect of drought on the redox state of grain proteins in susceptible versus tolerant genotypes. A change in redox state can markedly alter the activity of certain enzymes, for example, by oxidative deactivation or glutathionylation, in addition to alerting the solubility and digestibility properties of storage proteins.23 • Analysis of the proteome in mapping grain populations. Proteome maps will help determine whether the clustering of drought-responsive proteins has a genetic basis and whether they can be applied as markers for breeding. The two-dimensional gel electrophoresis (2-DE) databases of wheat seed proteins contain clickable 2-DE gel images and descriptive textual information such as protein name, Mr/pI values, MS score, and sequence coverage. These and other information are available for public access at http:// www.proteome.ir. Abbreviations: CBB, Coomassie Brilliant Blue; BSA, bovine serum albumin; Trx, thioredoxin; ROS, reactive oxygen species.

Acknowledgment. This project was partially funded by grants from the Agricultural Biotechnology Research Institute, Iran, to G.H.S. and from the California Agricultural Experiment Station to B.B.B. We are grateful to the Iranian National Science Foundation for the financial assistance to establish 2-DE database and to Mohammad Ghareyazie and Taha Abachi for their technical assistance in creating the database. Supporting Information Available: List of drought responsive proteins and identified proteins in wheat. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Majoul, T.; Bancel, E.; Triboı¨, E.; Ben Hamida, J.; Branlard, G. Proteomics 2003, 3, 175-183. (2) Skylas, D. J.; Cordwell, S. J.; Hains, P. G.; Larsen, M. R.; Basseal, D. J.; Walsh, B. J.; Blumenthal, C.; Rathmell, W.; Copeland, L.; Wrigley, C. W. J. Cereal Sci. 2002, 35, 175-188. (3) Andon, N. L.; Hollingworth, S.; Koller, A.; Greenland, A. J.; Yates, J. R.; Haynes, P. A. Proteomics 2002, 2, 1156-1168. (4) Balmer, Y.; Vensel, W. H.; Dupont, F. M.; Buchanan, B. B.; Hurkman, W. J. J. Exp. Bot. 2006, 57, 1591-1602. (5) Vensel, W. H.; Tanaka, C. K.; Nai, N.; Wong, J. H.; Buchanan, B. B.; Hurkman, W. J. Proteomics 2005, 5, 1594-1611. (6) Rey, P.; Pruvot, G.; Becuwe, N.; Eymery, F.; Rumeau, D.; Peltier, G. Plant J. 1998, 13, 97-107. (7) Hajheidari, M.; Abdollahian-Noghabi, M.; Askari, H.; Heidari, M.; Sadeghian, S. Y.; Ober, E. S.; Salekdeh, G. H. Proteomics 2005, 5, 950-60. (8) Salekdeh, Gh. H.; Siopongco, J.; Wade, L. J.; Ghareyazie, B.; Bennett, J.; Proteomics 2002, 2, 1131-1145. (9) Ali, G. M.; Komatsu, S. J. Proteome Res. 2006, 5, 396-403. (10) Jorge, I.; Navarro, R. M.; Lenz, C.; Ariza, D.; Jorrin, J. Proteomics 2006, 6 (Suppl. 1) S207-214.

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