Comparative proteomic analysis of the effect of temperature and fertilizer on gliadin and glutenin accumulation in the developing endosperm and flour from Triticum aestivum L. cv. Butte 86
© Hurkman et al.; licensee BioMed Central Ltd. 2013
Received: 27 September 2012
Accepted: 31 January 2013
Published: 22 February 2013
Flour quality is largely determined by the gluten proteins, a complex mixture of proteins consisting of high molecular weight-glutenin subunits (HMW-GS), low molecular weight-glutenin subunits (LMW-GS), and α-, γ-, and ω-gliadins. Detailed proteomic analyses of the effects of fertilizer and high temperature on individual gliadin and glutenin protein levels are needed to determine how these environmental factors influence flour quality.
Wheat plants (Triticum aestivum L. cv. Butte 86) were grown in greenhouses under moderate and high temperature regimens with and without post-anthesis fertilizer. Quantitative two-dimensional gel electrophoresis was used to construct accumulation profiles in developing endosperm for the entire complement of gluten proteins identified previously by tandem mass spectrometry. Amounts of individual gliadins and glutenins were also determined in flour produced under each of the regimens. Under all environmental regimens, most HMW-GS, LMW-GS, γ- and ω-gliadins accumulated rapidly during early stages of grain development and leveled off during middle stages of development. A subset of LMW-GS showed a second distinct profile, accumulating throughout development, while α-gliadins showed a variety of accumulation profiles. In flour, fourteen distinct gluten proteins responded similarly to fertilizer, high temperature, and high temperature plus fertilizer. The majority of HMW-GS and ω-gliadins and some α-gliadins increased while two LMW-GS and a minor γ-gliadin decreased. Fertilizer did not influence gluten protein accumulation under high temperature conditions. Additionally, the effects of fertilizer and high temperature were not additive; very few changes were observed when plants that received fertilizer were subjected to high temperature.
Although post-anthesis temperature and fertilizer have very different effects on grain development and yield, the two treatments elicit surprisingly similar effects on the accumulation of gluten proteins. The similarity of the responses to the different treatments is likely due to source-sink activities of nitrogen reserves in the wheat plant. Because each protein that showed a response in this study is linked to a gene sequence, the work sets the stage for transgenic studies that will better elucidate the roles of specific proteins in flour quality and in the response to the environment.
KeywordsEndosperm Fertilizer Flour Gliadins Glutenins Proteome Temperature Wheat
Wheat flour is used worldwide as an ingredient in a variety of baked products due to its unique protein properties. When flour is mixed with water, the proteins interact to form gluten, a continuous protein network that is responsible for the viscoelastic properties and gas holding capacity of flour dough. Gluten is a complex mixture of several hundred proteins with molecular weights ranging from 30,000 to 88,000 Da. The gluten proteins are synthesized in the endosperm of the developing wheat grain and comprise 60–80% of flour protein. Based on extraction and solubility properties, the gluten proteins can be separated into two major groups: the alcohol-soluble gliadins and the alcohol-insoluble glutenins . The gliadins contribute mainly to the viscosity and extensibility of wheat dough and the glutenins to elasticity and dough strength. The monomeric gliadins are classified as α-, γ-, and ω-gliadins based on their mobility in acid PAGE gels. The glutenins are classified as high molecular weight-glutenin subunits (HMW-GS) and low molecular weight-glutenin subunits (LMW-GS), which link together via intermolecular disulfide bonds to form large insoluble polymers. Variations in flour quality are related to genetic differences in gliadin and glutenin composition among wheat cultivars and to effects of environmental conditions on their relative proportions [2, 3].
Since consistent flour quality is important to end users, an increased understanding of the effects of environment on individual gliadins and glutenins and their relationship to quality is needed. Due to the difficulties in separating individual gluten proteins by chromatographic methods , quantitative two-dimensional gel electrophoresis (2-DE) has become a method of choice for identifying proteins that respond to environmental cues. To date, a number of studies have used 2-DE to discover gluten protein responses to high temperature, fertilizer, or drought [5–11] and have reported changes in as few as three  to as many as 107 2-DE spots . Mass spectrometry (MS) was used to identify some of the proteins that showed responses to environmental cues. However, in most studies, identifications were based on very few peptides and it was generally not possible to distinguish individual gluten proteins within the major classes.
Recently, we developed improved methods for tandem mass spectrometry (MS/MS) identification of the gluten proteins. Proteins in 2-DE spots were digested individually with chymotrypsin, thermolysin, and trypsin to increase sequence coverage. In addition, search strategies were improved and databases that included cultivar-specific gluten protein sequences were constructed for analysis of spectral data . This methodology made it possible to develop a comprehensive proteome map of flour from the US wheat Butte 86 in which 233 individual 2-DE spots were assigned to specific gene sequences . Gluten proteins identified included 5 HMW-GS, 22 LMW-GS, 13 γ-gliadins, 23 α-gliadins and 4 ω-gliadins, many of which had sequence coverage greater than 50%. Among the proteins identified in this study were α-, γ- and ω-gliadins that contain extra cysteine residues and may be linked into the glutenin polymer. In addition, α-gliadins containing specific epitopes involved in celiac disease were distinguished from those that do not contain these sequences. The proteome map made possible a detailed analysis of the effects of fertilizer on gliadin and glutenin levels in flour . One-hundred twenty-two gluten protein spots representing 19 gluten protein gene sequences responded significantly to fertilizer: five HMW-GS, six α-gliadins, and four ω-gliadins increased and three LMW-GS and one γ-gliadin decreased.
In this paper, we compare the effects of high temperature and fertilizer singly and in combination on the entire complement of gliadins and glutenins in developing endosperm and in flour from mature grain. The study extends earlier studies in which we analyzed the effects of high temperature and fertilizer on a limited number of gliadins and glutenins in developing endosperm by 2-DE and evaluated flour composition by RP-HPLC [5, 6]. Because the Butte 86 proteome map links gluten proteins to gene sequences, the goal of this study was to identify genes encoding proteins that respond to fertilizer and high temperature that can be used in transgenic approaches to determine the roles of specific proteins in flour quality and in the response of the grain to environmental cues.
High temperature and fertilizer alter fresh weight and gluten protein levels during grain development
Key to plant growth regimens and expected results
Differences due to fertilizer under moderate temperature.
Differences due to high temperature.
Differences due to high temperature plus fertilizer.
Differences due to high temperature in the presence of fertilizer.
Differences due to fertilizer under high temperature.
Principal component analysis reveals equivalent time points between the 24/17°C and 37/28°C time courses
Comparison of the 2-D gel patterns of proteins in the developing endosperm provided a partial explanation for the temporal distribution of the clusters generated by PCA (Figures 2B-E). Under the 24/17°C regimen at 8 dpa, the most abundant proteins were ATP synthase A (AtpA), calreticulin (CRT), 70 kD heat shock protein (Hsp70), protein disulfide isomerase (PDI), and stress responsive protein R40 (R40) (Figure 2B). PDI and Hsp70 were identified previously [13, 19] and ATP synthase A (AtpA), calreticulin (CRT), and stress responsive protein R40 (R40) were identified in this study (Additional file 2 contains the MS/MS identification data). In keeping with the onset of gluten protein biosynthesis, CRT, Hsp70, and PDI, which reside in the ER, facilitate the folding and maturation of proteins. CRT also modulates calcium homeostasis and Hsp70 assists in transport of precursor proteins into organelles and targets damaged proteins for degradation . PDI regulates the formation, reduction, and isomerization of disulfide bonds associated with protein folding and AtpA is a key enzyme in bioenergetics. The specific role of the R40 proteins remains unclear. It has been proposed that these ABA- and osmotic stress-responsive proteins [21, 22] have both protein- and carbohydrate-binding domains enabling them to cross-link proteins and glycoconjugates . The non-gluten proteins appear to decrease throughout development, but this is due to a concomitant large increase in gluten protein accumulation that masks the presence of most non-gluten proteins. In an earlier study , a fraction in which gluten proteins were depleted was essential for analysis of non-gluten protein accumulation during Butte 86 grain development. Under the 24/17°C regimens, gluten proteins were present at very low levels at 8 dpa (Figure 2B), but comprised a major proportion of proteins from 13 to 37 dpa (Figure 2D and E, Additional file 1). Under the 37/28°C regimens, gluten protein levels were more abundant at 6 dpa (Figure 2C) than at 8 dpa under the 24/17°C regimens and comprised a major proportion of proteins from 8 to 21 dpa (Additional file 1).
High temperature and fertilizer alter individual gliadin and glutenin accumulation profiles during grain development
Effect of temperature and fertilizer on accumulation of individual gluten proteins in flour
Predominant protein 1
Spot numbers 5
Spot volume × 10 –5 (6)
% Change 11
12, 16, 17, 18, 19, 23, 24, 28
20, 21, 25, 26, 27, 29, 33, 302
3, 5, 7, 10, 14, 36, 307, 538
40, 47, 52, 349, 350, 351, 358, 360
42, 48, 51, 333, 424, 514
119, 119a, 120, 131, 132, 161, 237, 310, 316
140‡, 141, 141a
144, 145‡, 472
314, 317, 318, 322
GenBank: AAB48469 (i-type)
328, 329, 387, 524, 525
190, 206, 331, 551
166, 169, 337
134, 320, 323, 324
335, 346, 347
Bu-1 or Bu-83
Gli-B3 type (ω-5)†
60, 63, 69, 71, 73, 74
Gli-D3 type (ω-1, 2)†
Bu-D5 (ω-1, 2)†
Cys type TC-262770 (ω-1, 2)†3
107, 113, 115, 116
Under the 24/17°C regimen without fertilizer, the gluten proteins exhibited four developmental profiles (Figure 4, MN). The HMW-GS and α-gliadins reached maximum levels at 18 dpa and remained at these levels until 37 dpa. The ω-gliadins attained maximum levels at 13–18 dpa and declined slightly by 37 dpa. The majority of LMW-GS (Figure 4, LMW-GSA) had profiles similar to the γ-gliadins and attained maximum levels at 28 dpa and declined slightly by 37 dpa. A small group of LMW-GS (spots 140, 145, 153, 170, 315) increased gradually throughout development and attained maximum levels at 37 dpa (Figure 4, LMW-GSB, indicated by ‡ in the Spot Numbers column in Table 2). These spots were identified as the m-type LMW-GS Bu-1, Bu-7, Bu-11 and Bu-18 and the i-type LMW-GS Bu-4 . Under the 24/17°C regimen with fertilizer, the gluten proteins exhibited only two developmental profiles (Figure 4, MF). The HMW-GS, LMW-GSA, and the α-, γ-, and ω-gliadins peaked at 28 and the LMW-GSB were maximal at 37 dpa. Compared to the 24/17°C regimen without fertilizer, the HMW-GS, α-gliadins, and ω-gliadins were higher from 28–37 dpa, the LMW-GSA were lower from 28–37 dpa, the LMW-GSB were lower from 18–37 dpa, and the γ-gliadins were somewhat lower at 33 dpa (Figure 4, compare MN and MF). At 37 dpa, the most notable changes in gluten protein levels were increases in the α- and ω-gliadins and decreases in the LMW-GSB.
Under the 37/28°C regimens, the gluten proteins exhibited three developmental profiles without and with post-anthesis fertilizer (Figure 4, HN and HF). The HMW-GS and LMW-GSA attained maximum levels at 13 dpa and remained at these levels until 21 dpa. The α-, γ-, and ω-gliadins also peaked at 13 dpa, but declined slightly by 21 dpa. The LMW-GSB increased gradually throughout development and attained maximum levels at 21 dpa. The most notable changes in gluten protein levels at 21 dpa under the two high temperature regimens were increases in the ω-gliadins.
High temperature and fertilizer alter levels of specific gliadins and glutenins in flour
Since flour quality depends on gluten protein composition, the effect of fertilizer and high temperature on gluten protein levels was examined in flour milled from mature grain harvested from plants used for the developmental studies (original data in Additional file 4). Because the same protein sequences were often associated with multiple 2-D spots, the volumes of spots containing proteins with the same identifications were summed for this analysis as was done by Altenbach et al. . This resulted in 42 different gluten proteins (Table 2). The percent change in volume relative to the 24/17°C regimen without fertilizer was calculated to determine specific gluten protein responses to fertilizer, high temperature, and high temperature plus fertilizer. In a previous study, 19 gluten proteins showed statistically significant changes of 20% or more in response to fertilizer under a 24/17°C regimen (Table 2, indicated by † in the Predominant Protein column) . In this study, it is notable that 14 proteins responded not only to fertilizer (MNvMF), but also to high temperature (MNvHN) and high temperature plus fertilizer (MNvHF). These included three HMW-GS (Ax2*, Dx5, By9), one m-type LMW-GS (Bu-11), five α-gliadins (Bu-5, Bu-11, Bu-12, Bu-14, Bu-BQ807130), one γ-gliadin (Bu-1) and one ω-5 gliadin and three ω-1,2 gliadins.
Effect of fertilizer and high temperature on developmental profiles of gluten proteins
Quantitative 2-DE was used to determine the effects of post-anthesis fertilizer and high temperature on the accumulation of individual gliadins and glutenins in the developing endosperm and in flour milled from the mature grain. To distinguish responses to temperature from those of fertilizer, plants were grown under 24/17°C and 37/28°C regimens without and with post-anthesis fertilizer. 2-DE spots identified as HMW-GS, ω-gliadins or γ-gliadins generally showed expression profiles characteristic of each protein group while spots identified as α-gliadins showed a variety of expression profiles. Individual LMW-GS spots exhibited two distinctly different developmental profiles. One group (LMW-GSA) had expression profiles similar to the γ-gliadins and another group (LMW-GSB) increased gradually throughout development. Of the five proteins comprising LMW-GSB, four were identified by MS/MS as m-type LMW-GS (Bu-1, Bu-7, Bu-11, Bu-18) and one as an i-type LMW-GS (Bu-4) (Table 2). These proteins had the same MS/MS identities as other proteins in the LMW-GSA group, but had different pIs and Mrs. Although MS/MS sequence coverage for these LMW-GS was quite high, generally about 50%, differences in the expression profiles suggest that very similar proteins in the LMW-GSA and LMW-GSB groups are encoded by different genes. Juhász et al.  recently found two expression profiles for LMW-GS genes. However, they reported that s-type and i-type LMW-GS had expression profiles similar to the LMW-GSB in the present study. Liu et al.  also reported multiple expression profiles for LMW-GS genes in a proteomic analysis of three Chinese wheat cultivars.
The developmental profiles demonstrate that gluten protein accumulation is a complex process that is subject to spatial and temporal regulation as well as environmental signaling. Individual proteins within each gluten protein class accumulated to different levels, suggesting that the corresponding genes had different basal levels of expression. Ultimately, the proportions of ω-gliadins and HMW-GS and a few α-gliadins and LMW-GS changed in flour in response to both post-anthesis fertilizer and high temperature while those of most α- and γ-gliadins and LMW-GS were unchanged. The data suggest that there are differences in the promoter sequences of the gluten protein genes, both between and within the major gene families. Promoter elements responsible for tissue specificity of the gluten proteins are the endosperm box, which is comprised of the prolamin box and a GCN4-like motif (GLM), and the AACA motif [26, 27]. GLM, also known as the N motif, is thought to play a role in the response of the gluten protein genes to nitrogen [28, 29]. Recently, Juhász et al.  compared regulatory elements in the 5′ non-coding regions of LMW-GS genes available in public databases and developed a model for their transcriptional regulation based on an in silico analysis of expression profiles. Clearly, it will be important to confirm this model using experimental data and extend the model to include other classes of gluten protein genes. Comparisons of promoter regions from genes encoding gluten proteins characterized in this study should prove valuable for this type of analysis.
Effect of fertilizer and high temperature on gluten protein composition in flour
Dupont et al.  used RP-HPLC to quantify the effects of fertilizer and high temperature on the accumulation of Butte 86 flour proteins and reported changes in the levels of ω-gliadins and HMW-GS. However, changes in specific LMW-GS, α- and γ-gliadins were difficult to detect because of the complexity of these groups of proteins. In this study, 2-DE analysis revealed a number of changes in distinct gluten proteins within these classes in response to fertilizer and temperature. Of the 42 uniquely identified gluten proteins, 16 increased with fertilizer under moderate temperatures (four HMW-GS, eight α-gliadins, and four ω-gliadins) and three decreased (two LMW-GS and one γ-gliadin). The response to the 37/28°C regimen without fertilizer (MNvHN) was comparable to that of 24/17°C regimen with fertilizer (MNvMF); thirteen of the same proteins increased (four HMW-GS, five α-gliadins, and four ω-gliadins) and two of the same proteins decreased (one LMW-GS and one γ-gliadin). Most of these proteins also responded to high temperature plus fertilizer (MNvHF). It is notable that the effects of fertilizer and high temperature were not additive and that very few significant changes were observed when plants receiving fertilizer were subjected to high temperature (MFvHF). Likewise, the application of fertilizer under a high temperature regimen had little effect on gluten protein accumulation (HNvHF). As noted previously [5, 14, 30, 31], the response to high temperature and fertilizer is related to the proportion of the S-containing amino acids in the different gluten protein classes. Generally, the proportions of low S-proteins (ω-gliadins) and low to medium S-proteins (HMW-GS and α-gliadins) increased and those of the high S-proteins (LMW-GS and γ-gliadins) remained the same or decreased.
In agreement with previous studies [6, 14, 32], the ratios of gliadins to glutenins increased in response to fertilizer and high temperature. The higher ratios reflect increases in the proportions of ω-gliadins and certain α-gliadins. The ratios of the HMW-GS to LMW-GS also increased in response to both fertilizer and high temperature, largely due to increases in HMW-GS and decreases in certain LMW-GS. Of the gliadins containing an odd number of cysteines and likely to function as LMW-GS, only the cys-type ω-gliadins showed responses to fertilizer or high temperature. Others have reported that the ratio of the HMW-GS to LMW-GS in a glutenin polymer fraction of SDS unextractable protein is significantly correlated with dough strength [33–37].
Because individual proteins in this analysis were associated with specific gene sequences, changes in the immunogenic potential of the flour that result from the growth conditions of the plant can now be assessed. It is notable that the Gli-B3 ω-gliadins (also referred to as ω-5 gliadins) are associated with the food allergy wheat-dependent exercise-induced anaphylaxis. These proteins increased significantly in response to both fertilizer and temperature. Among the gluten proteins, the α-gliadins are considered most immunogenic in celiac disease. Several of the α-gliadins that increased in response to both fertilizer and temperature contain epitopes important in celiac disease (Bu-5, Bu-11, Bu-14) while others do not contain these sequences (Bu-12) (Table 2) [13, 38]. It is interesting that proteins that do not contain celiac epitopes comprised 31–32% of the total α-gliadins under all treatments, suggesting that neither fertilizer nor temperature influenced the immunogenic potential of the flour with respect to celiac disease. The accumulation of other immunogenic proteins were not assessed in this study. However, Altenbach et al.  analyzed a fraction of total SDS-extractable protein and reported changes in the proportions of other proteins with fertilizer that are likely food allergens. These included increases in serpins and decreases in lipid transfer protein (LTP), chitinase, the α-amylase/trypsin inhibitor CM3 and globulin-2. Hurkman et al.  analyzed an albumin/globulin fraction and reported that the same proteins had opposite responses to high temperature when plants were supplied with fertilizer. They observed increases in LTP, chitinase and globulin-2 and decreases in the proportions of several serpins.
Source-sink activities and gluten protein responses to fertilizer and high temperature
The question arises as to why post-anthesis high temperature and the post-anthesis addition of fertilizer alter gluten protein composition in the same manner. The similarity in responses of the gliadins and glutenins to high temperature and fertilizer might be explained by source-sink activities in the wheat plant [39–45]. All plants were grown with fertilizer and adequate water under moderate temperatures until anthesis so the amount of nitrogen and carbon reserves that could be mobilized to the developing grain for the synthesis of protein and starch were comparable at the beginning of the treatments. The addition of post-anthesis fertilizer enhances the remobilization of reserves during grain fill and the rate of protein accumulation in the grain, resulting in increases in flour protein percentage and specific changes in gliadin and glutenin composition in flour [5, 6, 14]. On the other hand, high temperature conditions elicit premature leaf senescence and shorten the period of grain fill . Because remobilization of nitrogen reserves to the developing grain occurs over a significantly shorter period of time, the temperature treatment may essentially mimic high nitrogen conditions, resulting in changes in the same complement of gluten proteins. The lack of response to fertilizer under high temperature conditions suggests that the plants are unable to utilize fertilizer under the temperatures used in this study. This would also explain why post-anthesis fertilizer and temperature do not elicit an additive response.
This study provides insight into the manner in which specific growth conditions during wheat grain development influence the accumulation of the individual gluten proteins that determine flour quality. While many gluten proteins respond to fertilizer and high temperature, most of the changes are small in magnitude and surprisingly similar between the two treatments. It is notable that both fertilizer and high temperature increase the proportions of most HMW-GS and ω-gliadins and some of the same α-gliadins while decreasing the proportions of several LMW-GS and a minor γ-gliadin. A critical question is how the observed changes in gluten protein composition influence flour quality. Many different proteins contribute to flour quality. While the roles of HMW-GS have received considerable attention, the functions of individual LMW-GS and gliadins in flour quality remain poorly defined. The identification of genes within these classes that respond to fertilizer or temperature now makes it possible to utilize transgenic approaches to determine the importance of specific proteins in flour quality and in the response of the grain to the environment. For example, RNA interference was used recently to create transgenic Butte 86 plants in which genes encoding the ω-5 gliadins were silenced . Since these proteins show some of the largest responses to fertilizer and temperature, analyses of transgenic lines should elucidate how the proteome responds to fertilizer and high temperature in their absence and reveal the contribution of the ω-5 gliadins to flour quality.
Triticum aestivum L. cv. Butte 86 was grown in climate-controlled greenhouses as described previously [14, 47]. Seven seeds were sown per pot (25 cm diameter by 24 cm high). The pots contained Sunshine Mix Number 1 (SunGro Horticulture, Inc., Bellevue, WA, USA), a planting mix that contains 70–80% Sphagnum peat moss plus perlite, dolomitic limestone, gypsum, and wetting agent. Prior to anthesis, plants were grown at a maximum daytime temperature of 24°C and a minimum nighttime temperature of 17°C. Pots were watered by drip irrigation with 20-20-20 fertilizer (Plantex, Plant Products Co. Ltd., Brampton, Ontario, Canada). Pots received 500 mls of 0.6 g/l fertilizer or 0.06 nitrogen units per day. Pots did not receive S. S contents of flour from grain produced under the same environmental conditions indicated that S was not limiting . At anthesis, pots were divided into four groups. One group of pots was left in the 24/17°C greenhouse and irrigated with fertilizer. The second group of pots was flushed with water to remove fertilizer, returned to the 24/17°C greenhouse, and hand-watered at the same rate without fertilizer. The third group of pots was transferred to a second greenhouse that had a maximum daytime temperature of 37°C and a minimum nighttime temperature of 28°C and supplied with fertilizer. The fourth group of pots was flushed with water, transferred to the 37/28°C greenhouse, and hand-watered without fertilizer. Wheat heads were harvested throughout grain fill. Grain number per head ranged from 29–49. For the 24/17°C regimens, heads were collected at 4 to 5-day intervals from 8 to 37 dpa. Because high temperature accelerated the grain developmental program, heads were collected at 2 to 5-day intervals from 6 to 21 dpa for the 37/28°C regimens. The region of the grain containing the embryo was first excised with a razor blade. The endosperm was squeezed through the resultant opening onto a spatula, leaving most of the pericarp/testa/aleurone behind. Endosperm was transferred immediately into tubes cooled in liquid nitrogen and stored at −80°C [19, 48]. For developmental studies, the end point for each regimen was the oldest age that endosperm could be squeezed or scraped from the grain after the embryo was excised. Flour was milled from mature grain samples (100 g) with a Brabender Quadrumat Junior (South Hakensack, NJ) at the Hard Winter Wheat Quality Laboratory (US Department of Agriculture, Agricultural Research Service, Manhattan, KS). Flour protein content was determined by NIR and was 7.7 for MN, 14.0 for MF, 15.9 for HN, and 17.7 for HF . Flour was stored in sealed containers at −80°C.
A KCl-insoluble fraction enriched in gliadins and glutenin subunits was prepared from endosperm collected at each time point as described previously . Fifty mg of endosperm was ground to a fine powder in liquid nitrogen using a mortar and pestle and suspended in 200 μl of cold (4°C) KCl buffer containing protease inhibitors (1 tablet per 10 ml of Mini Complete Protease Inhibitor Cocktail, Roche Applied Science, Indianapolis, IN). The suspensions were incubated on ice for 5 min with intermittent mixing and centrifuged at 14,000 g for 15 min at 4°C. The pellet or KCl-insoluble fraction was suspended in 800 μl of SDS buffer (2% SDS, 10% glycerol, 50 mM DTT, 40 mM Tris-Cl, pH 6.8), incubated for 1 h at room temperature, and insoluble material removed by centrifugation at 14,000 g for 10 min at room temperature (Eppendorf 5415C; Brinkman Instruments, Inc., Westbury, NY). Proteins were then precipitated by addition of 4 vol of cold (−20°C) acetone and incubation overnight at −20°C to remove SDS, which interferes with protein determination and prevents separation of proteins by IEF. Samples were centrifuged at 14,000 g for 10 min at room temperature, the pellet was rinsed by pipetting cold acetone onto the pellet, centrifuging at 14,000 rpm for 10 min at room temperature, and pipetting the acetone off of the pellet. The pellet was dried at room temperature and solubilized in urea buffer (9 M urea, 4% NP-40, 1% DTT, and 2% ampholytes) to a final concentration of 3 μg protein/μl. For protein determinations, triplicate 5 μl samples were removed from the SDS extracts, precipitated with acetone, and, following addition of 0.1 N NaOH, protein amount assayed by the procedure of Lowry et al.  as described by Hurkman and Tanaka .
Flour was extracted with SDS buffer (2% SDS, 10% glycerol, 50 mM DTT, 40 mM Tris-Cl, pH 6.8) as described in detail by Dupont et al. . Protein amounts from 50 mg of flour from MN, MF, HN and HF treatments were determined using the Lowry assay  and were 3.29, 5.38, 6.14 and 6.64 mg, respectively.
Separation of proteins by 2-DE
Endosperm and flour proteins were separated by 2-DE as described previously [15, 49]. Briefly, samples were suspended in urea buffer (9 M urea, 4% NP-40, 1% DTT, and 2% ampholytes) at a concentration of 3 μg protein/μl. The first dimension capillary tube gels contained 9.2 M urea, 4% (total monomer) acrylamide:BIS, 2% Nonidet P-40, 2% 3–10 Iso-Dalt Grade Servalyts (Crescent Chemical Co., Islandia, NY), 0.015% ammonium persulfate, and 0.125% TEMED. Triplicate IEF gels were loaded with 18 μg of protein and run using a Mini Protean II Tube Cell (BioRad Laboratories, Richmond, CA). Gels were extruded into microcentrifuge tubes and equilibration buffer (2.3% SDS, 10% glycerol, 0.05% dithiothreitol, and 62.5 mM Tris-Cl, pH 6.8) added. The gels were frozen by placing the tubes in crushed dry ice and then stored at −70°C until use. Proteins were separated in the second dimension by SDS gel electrophoresis using an XCell SureLock Mini-Cell electrophoresis system with Novex NuPage 4–12% acrylamide Bis-Tris gels and NuPAGE MES SDS running buffer (Invitrogen Corp., Carlsbad, CA). Gels were stained with Coomassie G-250 (Sigma, St. Louis, MO), destained in water for 2 h at room temperature and stored at 4°C in 20% ammonium sulfate.
Statistical analysis of 2-D gel images
2-DE gels were digitized with a calibrated scanner (UMAX Powerlook III; Dallas, TX) at 300 dpi using the same settings for all gels. Flour proteins were identified previously by MS/MS  and additional proteins in the endosperm were identified by the same methods. Progenesis SameSpots Ver. 4.0 (Nonlinear Dynamics Limited, Newcastle upon Tyne, UK) was used to detect spots, match spot patterns, normalize and quantify spot volumes, and create protein accumulation profiles. The means and standard deviations for individual spot volumes were calculated and found to be quite similar among the replicate gels for each time point. All of the gluten proteins had Anova p-values less than 0.05 and Power greater than 0.8. Progenesis SameSpots was also used to analyze 2-D gel patterns by principal component analysis and match protein accumulation patterns by correlation analysis. The spot volume data were analyzed by hierarchical cluster analysis using the TIGR MultiExperimentViewer (MeV) v. 4.0 (, http://www.tm4.org/mev/]).
Two-dimensional gel electrophoresis
High molecular weight-glutenin subunits
Low molecular weight-glutenin subunits
Large group of LMW-GS
Small group of LMW-GS
Tandem mass spectrometry
This research was funded by USDA Agricultural Service CRIS Project 5325-43000-027-00D. Mention of trade names or commercial products in this report is solely for the purpose of providing specific information and does not imply recommendation over or endorsement by the U.S. Department of Agriculture. The U.S. Department of Agriculture is an equal opportunity provider and employer.
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