Quantitative Determination of High Molecular Weight Glutenin Subunits of Hard Red Spring Wheat by SDS-PAGE. I. Quantitative Effects of Total Amounts on Breadmaking Quality Characteristics

Thirteen hard red spring wheat genotypes in which seven genotypes had the same high molecular weight (HMW) glutenin subunits (2*, 7+9, 5+10) were compared for their physical-chemical and breadmaking properties. These samples were categorized into three groups based on their dough mixing and baking performances as follows: the strong dough (SD) group (six genotypes), characterized by the strongest dough mixing (average stability, 35 min); the good loaf (GL) group (four genotypes), characterized by the largest loaf volume; and the poor loaf (PL) group (three genotypes), characterized by the smallest loaf volume. Total flour proteins were fractionated into 0.5M salt-soluble proteins, 2% SDS-soluble proteins, and residue proteins (insoluble in SDS buffer). SDS-soluble proteins, residue proteins, and total flour proteins were analyzed by SDS-PAGE and densitometry procedures to determine the proportions of HMW glutenin subunits, medium molecular weight proteins, and low molecular weight proteins in relation to the total amount of proteins. No differences in the amount of salt-soluble proteins were found among the different groups of samples. Solubilities of gluten proteins (total proteins minus salt-soluble proteins) in SDS buffer were related to the differences in dough strength and baking quality among the three groups. The SD group had the lowest solubility and the PL group had the highest. SDS-PAGE analysis showed that SDS-soluble proteins of the SD group contained a smaller amount of HMW glutenin subunits than those of the GL and PL groups. The highest proportions of HMW glutenin subunits in total flour proteins were found in the SD group, while the PL group had the lowest percentage of HMW glutenin subunits in their total flour proteins. These results showed that the total quantities of HMW glutenin subunits played an important role in determining the dough mixing strength and breadmaking performance of hard red spring wheats.     

Characterization of Monomeric and Glutenin Polymeric Proteins of Hard Red Spring Wheats During Grain Development by Multistacking SDS-PAGE and Capillary Zone Electrophoresis

Three cultivars of hard red spring (HRS) wheats with identical high molecular weight (HMW) glutenin subunit composition (5+10 type, Glu-D1d) but different dough properties and breadmaking quality were used in this study. The synthesis and accumulation characteristics of different protein fractions during grain development were examined. Samples were collected at three-day intervals from anthesis to maturity between day 10 to day 37. The nonreduced SDS-extractable glutenin aggregates of developing grains were characterized by a multistacking SDS-PAGE procedure to obtain information on the size distribution and polymerization of glutenin aggregates. The HMW to low molecular weight (LMW) glutenin subunit ratio was determined for its relationship to polymerization of the various glutenin aggregates of different molecular sizes. Glutenin proteins were quantified using an imaging densitometer. In addition, albumins and globulins, α- and β-gliadins, γ-gliadins, and ω-gliadins were separated by capillary zone electrophoresis. The results indicated that albumins-globulins, gliadins, and glutenins in developing grains were present at 10 days after anthesis or earlier. Albumin-globulins decreased in proportion, while gliadins increased in proportion during grain development. Polymerization of glutenin aggregates occurred 10 days after anthesis or earlier and increased significantly throughout the grain-filling period until maturity. Larger aggregates of glutenin increased in proportion, while smaller ones decreased in proportion during grain development. Ratio of polymers to monomers increased significantly from day 10 to day 22 of grain development and then remained constant until grain maturity. Glutenin polymers arrived at their maximum in proportion to total SDS-extractable proteins or monomers at day 22 after anthesis while the molecular size of these polymers continued to increase, as indicated by a rapid increase in proportion of HMW to LMW glutenin subunits. Significant differences were found in accumulation rates of glutenin polymers among the three cultivars. Cultivars Kulm and Grandin, with better breadmaking quality, appeared to have greater rates of accumulation and HMW subunit synthesis or formation of larger polymers than did Sharp, a cultivar with poorer quality. Significant differences were found among the three cultivars in the proportion of albumins-globulins and gliadins during grain development. However, no significant differences were found among the cultivars in the proportion of albumins-globulins, α-, β-, γ-, and ω-gliadins at grain maturity. Varietal differences in breadmaking quality were due mainly to the differences in glutenin polymers such as ratio of polymeric to monomeric proteins, molecular size distribution, and ratio of HMW to LMW glutenin subunits among wheat cultivars of 2*, 7+9, and 5+10 subunit types. The better breadmaking cultivars might be characterized with higher proportions of glutenins and greater proportion of HMW subunits in total SDS-extractable proteins than the poorer quality cultivar. However, more genotypes need to be examined.     

Synthesis of gluten-forming polypeptides. 1. Biosynthesis of gliadins and glutenin subunits

Five winter wheat cultivars--GK Othalom (HMW-GS composition 2*, 7+8, 5+10), Ukrainka (1, 7+8, 5+10), Palotás (2*, 7+9, 5+10), K?dm?n (2*, 7+8, 5+10), and Csongrád (2*, 7+9, 2+12)--grown in Hungary and harvested in the year 2005 were studied. The biosynthesis of gluten-forming polypeptides was followed starting at the 12th day after anthesis to the 53rd. Fresh kernel weight, moisture, and dry matter content of fresh kernels and gliadin and glutenin contents were determined. Gliadin components, total amounts of HMW and LMW polypeptides, and individual HMW polypeptides were determined using a RP-HPLC technique. Although considerable quantitative differences were observed concerning the content of total protein, gliadin, glutenin, and individual gluten-forming polypeptides, the character of accumulation of protein components--determined on the basis protein mass/kernel--was the same for the all of the cultivars studied and could be presented by a sigmoid curve. Small quantities of the gliadin and glutenin monomers may be detected in early stages of kernel development, but the bulk of these proteins is synthesized in later stages of development. It is generally suggested by specialists that the formation and accumulation of glutenin polymers starts later than the synthesis of monomers. Experimental data presented in this paper confirm this suggestion and show that in the first phase of protein synthesis the monomers are in "free" form; polymeric glutenin is detected only later. HMW glutenin subunits are synthesized synchronously, and quantitatively the polypeptides coded by chromosomes D and B dominate.     

Depolymerization of the Glutenin Macropolymer During Dough Mixing: I. Changes in Levels,Molecular Weight Distribution,and Overall Composition

Changes in the amounts, molecular weight distributions, and levels of major groups of subunits in the glutenin macropolymer (GMP) of doughs during mixing were investigated. The GMP (gel protein) is the unreduced fraction of gluten protein that remains as a layer on top of the starch after extraction of SDS-soluble proteins and centrifugation. Experiments involved doughs prepared from flours derived from one weak and one strong cultivar and lines derived from cv. Olympic that were null for specific high molecular weight glutenin subunits (HMW-GS). During mixing, the amount of GMP decreased; the major changes occurred before peak mixing time (MT, achievement of peak resistance). In addition, the average apparent molecular weight of GMP (determined by both size-exclusion HPLC and multilayer gel electrophoresis) decreased during mixing, but in this case, the major changes were seen later in the mixing process, during dough breakdown. Even after extensive mixing, polymers and oligomers were released, not free glutenin subunits. During dough breakdown, the composition of GMP also changed, such that the proportion of HMW-GS decreased but β-amylases/D low molecular weight glutenin subunits (LMW-GS) increased. Changes in the total amounts of other LMW-GS typically were smaller with a decrease in the proportion of B subunits and an increase in the proportion of C subunits. The major changes in GMP composition were observed after peak MT (peak resistance) occurring earlier and to a greater extent in the weaker dough. Our results suggest that dough breakdown during mixing may be triggered by loss of HMW-GS, leading to changes in the molecular weight distribution and composition of the disulfide-bonded GMP.     

Effect of the Molecular Weight Distribution of Glutenin Protein from an Extra‐Strong Wheat Flour on Rheological and Breadmaking Properties Through Reconstitution Studies

Ten glutenin fractions were separated by sequential extraction of wheat gluten protein with dilute hydrochloric acid from defatted glutenin‐rich wheat gluten of the Canadian hard red spring wheat (HRSW) cultivar Glenlea. The molecular weight distribution (MWD) of 10 different soluble glutenin fractions was examined by multistacking SDS‐PAGE under nonreduced conditions. Also, the subunit composition of the different glutenin fractions was determined by SDS‐PAGE under reduced conditions. The MWD of the fractions (especially HMW glutenins) varied from fraction to fraction. From early to later fractions, the MWD shifted from low to high. The early extracted fractions contained more LMW glutenin subunits (LMW‐GS) and less HMW glutenin subunits (HMW‐GS). The later extracted fractions and the residue fraction contained much more HMW‐GS (2*, 5, and 7 subunits) than the early extracted fractions. The trend in the amounts of 2*, 5, and 7 subunits in each fraction from low to high matched the extraction solvent sequence containing from lower to higher levels of HCl. The influence of glutenin protein fractions from the extra‐strong mixing cultivar, Glenlea, on the breadmaking quality of the weak HRSW, McVey, was assessed by enriching (by 1%) the McVey base flour with isolated glutenin protein fractions from Glenlea. The mixograph peak development times and loaf volumes of enriched flour were measured in an optimized baking test. The results indicated that the higher content in Glenlea glutenin of HMW‐GS with higher molecular weight, such as 2*, 5, and 7, seem to be the critical factor responsible for the strong mixing properties of Glenlea. Our results confirmed that subunit 7 occurred in the highest quantity of all the HMW‐GS. Therefore, it seems that the greater the content of larger molecular weight glutenin subunits, the larger the glutenin polymers and the stronger the flour.     

新疆小麦地方品种资源HMW-GS的遗传多样性组成分析

为了明确新疆小麦地方品种高分子量麦谷蛋白亚基的遗传多样性,并为小麦品质改良提供基础材料,利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE) 技术,分析了源自新疆地区的282份小麦地方品种的高分子量麦谷蛋白亚基组成。结果表明,在Glu-A1、Glu-B1和Glu-D1位点上的等位变异分别为3、6和 5种,三个位点上的优势亚基依次为null、7+8和2+12,其频率分别是75.5%、90.8%和72.0%。在Glu-1位点共检测到20种亚基组合,其中(null, 7+8, 2+12)组合的频率最高,为52.8%, 其次是(null, 7+8, 2.6+12)和(2*, 7+8, 2+12)组合,其频率分别为14.1%和11.0%,其它亚基组合的频率均低于10%。另外,在Glu-D1位点上还检测到一个新的亚基2.6+12。在供试的282份新疆地方品种中发现了两份具有优质亚基组合的材料,它们的亚基组成为(2*, 7+9, 5+10)和(1, 7+9, 5+10),这些地方品种可作为改良小麦品质性状的重要遗传资源。     

Genetic variability at the Glu-1 loci in old and modern wheats (triticum aestivum L.) cultivated in Slovakia

The high molecular weight glutenin subunits (HMW-GS) composition at the Glu-1 complex loci, in 23 old original wheat genotypes cultivated in Slovakia several decades ago and 32 modern Slovak and Czech wheat cultivars growed in Slovakia at present were studied by SDS-PAGE. Some of the HMW-GS – subunit pairs 3+12, 17+18, and subunit 20, present in old historical wheats were missing in modern cultivars utilized in Slovakia nowadays. There were observed 15 different HMW-GS encoded by 11 alleles or allelic pairs in old genotypes. Lower number of different HMW-GS and competent alleles were observed in a set of modern wheat cultivars – 11 different HMW glutenin subunits encoded by 8 alleles or allelic pairs. The same number of different HMW-GS patterns was revealed in both sets of wheats. From the point of view of genetic variability, it could be concluded that long-term effort of breeders and decreasing of cultivation of landraces and old cultivars are associated with the loss of several HMW-GS alleles and decreasing of genetic variability of wheats. Molecular characterization can reveal broad allelic variability of old wheat genotypes and landraces. Their maintenance in genetic resource collections can prevent losses of these interesting genes.     

Characterization of Glutenin Protein Fractions from Sequential Extraction of Hard Red Spring Wheats of Different Breadmaking Quality

Gluten proteins from two cultivars of hard red spring (HRS) wheat with good and poor breadmaking quality were fractionated into 13 fractions by sequential extraction with dilute hydrochloric acid. Each subfraction was characterized by multistacking (MS) SDS‐PAGE under nonreducing conditions, followed by imaging densitometry. The glutenin polymers from the origins of MS‐SDS‐PAGE were analyzed by SDSP‐PAGE under reducing conditions to determine the composition of high and low molecular weight subunits. The results showed that fractions differed significantly in glutenin‐to‐gliadin ratios and in the size distribution of glutenin polymers. The earlier precipitated fractions were composed of more gliadins but fewer glutenin polymers. However, the glutenin polymers gradually increased in their relative quantities with the residue having the largest glutenin‐to‐gliadin ratio. The size distribution of glutenin polymers differed significantly from early precipitated to later fractions. The relative quantities of glutenin aggregates at the 4% origins increased significantly. The ratio of high molecular weight (HMW) to low molecular weight (LMW) glutenin subunits increased significantly from early to intermediate fractions. Between the two cultivars, significant differences were found in the ratio of HMW to LMW glutenin subunits and quantity of SDS insoluble glutenin polymers in the residue fraction with the better breadmaking quality cultivar ND706 having a greater ratio than the cultivar Sharp. It was concluded that the size distribution of glutenin polymers played an important role in determining the differences in breadmaking quality between the good and poor HRS wheat cultivars.     

Effect of Climate Change on Wheat Quality and HMW Glutenin Subunit Composition in the Pannonian Plain

The primary goal of this study is to improve our understanding of the extent of influence of climatic factors in Serbia and high‐molecular‐weight glutenin subunit (HMW‐GS) composition upon wheat end‐use quality. In‐depth analyses were performed on four bread wheat cultivars that are the most common in agricultural practice in Serbia. Total glutenin content showed significant difference between the production years, in opposition to gliadins. Cluster analysis of different percentages of glutenin and gliadin subunit molecular weight ranges (<40,000, 40,000–80,000, 81,000–120,000, and >120,000) indicated that the year of production and the cultivar did not have a significant effect on the percentage ranges for glutenins. However, they had a considerable impact on the percentage ranges for gliadins. Production year and the interaction of year and cultivar had the strongest influences on the percentage of SDS‐unextractable polymeric proteins. A synergistic effect of the HMW‐GS composition and climatic conditions revealed that all eight samples with HMW‐GS composition 2*, 5 + 10, 7 + 9 along with the highest Glu 1 score of 9 (out of a maximum of 10) produced in the year 2011 belonged to two clusters with the best wheat end‐use quality. Furthermore, the climate conditions in 2011 made it possible for the wheat cultivars with HMW‐GS composition –, 2 + 12, 7 + 9 to possess similar qualities as cultivars with HMW‐GS composition 2*, 5 + 10, 7 + 9 produced in 2012.     

Heat-Shock Protein 70 and Dough-Quality Changes Resulting from Heat Stress During Grain Filling in Wheat

In an attempt to further elucidate the molecular mechanisms that determine the loss of dough strength associated with heat stress of growing wheat, the roles of heat-shock proteins (HSP) and heat-shock elements upstream of glutenin genes were investigated. A range of genotypes differed in the extent of synthesis of high molecular weight glutenin subunits (HMW-GS) and HSP during heat stress. The concentration of HSP 70 remaining in mature grain increased as a result of a few days' heat stress of wheat plants. The amount of HSP 70 in mature grain samples from heat-stressed plants of 45 genotypes was not strongly correlated with loss of dough strength. There was much less evidence for this mechanism than for other molecular hypotheses from the literature, particularly, changes in glutenin-to-gliadin ratio, size distribution of the glutenin polymer, and the involvement of HSP and chaperones during grain-protein synthesis. HSP 70 was purified from heat-stressed grain, and was added to (or incorporated into) dough in the direct-drive mixograph. The HSP behaved similarly to several other hydrophilic proteins when added at a level of 2 mg/2 g of flour. It showed no dramatic effects on dough properties that could constitute a major explanation for the dough-weakening effects of heat stress, even though the level of addition was well above the maximum levels that might be encountered in field-grown, mature grain. Furthermore, sequencing of the genes (upstream of the coding region) for HMW-GS failed to show the presence of heat-shock promoters, even for genotypes that differed considerably in their reactions to heat stress. The findings simplify the range of possibilities that cause heat-related loss of dough strength, focusing attention on the degree of polymerization of the glutenin chains, and on the roles of HSP and chaperones in the developing grain.     

Detection of high molecular weight glutenin subunits in triticale (x Triticosecale Wittm.) Cultivars by capillary zone electrophoresis

An improved method for separating and characterizing high molecular weight glutenin subunits (HMW-GS) in hexaploid triticale by capillary zone electrophoresis (CZE) was developed. A low-concentrate mixture of hydrophilic polymers, poly(vinylpyrrolidone) (PVP) and hydroxypropylmethylcellulose (HPMC), in an isoelectric buffer was employed for dynamic coating of the capillary inner wall. In separation buffer PVP with lower concentrated poly(ethylene oxide) (PEO) was replaced. The CZE electropherograms of HMW-GS showed two group peaks in accordance with x- and y-type subunits with migration times of 6.8-7.8 and 8.4-11.5 min, respectively. In total, 14 HMW subunits (2 subunits encoded by Glu-A1 locus and 12 by Glu-B1) were identified. The CZE analyses revealed that each of the subunits Bx7 and By8 determined by SDS-PAGE makes up three subunits (Bx6.8, Bx7, and Bx7* and By8, By8*, and new By8**, respectively), with different migration times. It was also shown that the subunits By18 and By20 in triticale determined by SDS-PAGE have different migration times in comparison with the same subunits in bread wheat. For these new HMW-GS, the following names were assigned: By18* instead of By18 and By20* instead of By20. The presented CZE method is an efficient alternative to the SDS-PAGE procedure for early selection of useful triticale genotypes with good breadmaking quality.     

Effect of Foliar Sulfur and Nitrogen Fertilization on Wheat Storage Protein Composition and Dough Mixing Properties

Nitrogen (N) and sulfur (S) supplies have a strong influence on the quality and quantity of wheat storage proteins, which play an important role in the breadmaking process. Nitrogen derived from urea, S from micronized elemental sulfur, and a mixture of both (N+S) were applied at anthesis stage on wheat by foliar spray. To relate N and S incorporation in storage proteins to the quality of dough, their incorporation into each storage protein fraction was measured: monomers, low molecular weight glutenin subunits (LMW‐GS), and high molecular weight glutenin subunits (HMW‐GS). Then protein fraction quantities, molecular weight distribution (MWD), polymerization index (PI), and molecular dimensions of unextractable polymeric protein (UPP), as well as dough mixing properties were determined. Fertilizers N and S were differentially incorporated into each storage protein fraction, influencing protein synthesis. Moreover, after the N+S fertilization, the increase of the polymeric proteins induced an increase in molecular weight and compactness, as well as in dough strength and consistency. These results provide evidence that N and S fertilizers applied by foliar spray route at anthesis, simultaneously, play an important role in controlling the storage protein synthesis and the degree of polymerization, which in turn influence dough mixing properties.     

Distribution of Protein Composition in Bread Wheat Flour Mill Streams and Relationship to Breadmaking Quality

Wheat protein quantity and composition are important parameters for wheat baking quality. The objective of this study was to use fractionation techniques to separate the proteins of flour mill streams into various protein fractions, to examine the distribution of these protein fractions, and to establish a relationship between protein composition and breadmaking quality. Nine break streams, nine reduction streams, and three patent flours obtained from three samples of Nekota (a hard red winter wheat) were used in this study. A solution of 0.3M NaI + 7.5% 1-propanol was used to separate flour protein into monomeric and polymeric proteins. The protein fractions, including gliadin, albumin+globulin, HMW-GS, and LMW-GS, were precipitated with 0.1M NH₄Ac-MeOH or acetone. The fractions were statistically analyzed for their distribution in the mill streams. The quantities of total flour protein and protein fractions in flour were significantly different among mill streams. The ratio of polymeric to monomeric proteins in break streams was significantly greater than in the reduction streams. The relationship between protein composition and breadmaking quality showed that the quantities of total flour protein, albumin+ globulin, HMW-GS, and LMW-GS in flour were significantly and positively correlated with loaf volume. The ratio of HMW-GS to LMW-GS had little association with loaf volume. The gliadin content in total flour protein was negatively and significantly correlated with loaf volume. These results indicated that the quantity and composition of protein among the mill streams was different, and this resulted in differences in breadmaking quality.     

Protein heterogeneity in European wheat landraces and obsolete cultivars

Identity and present degree of genetic homogeneity and heterogeneity, respectively of 52 European wheat accessions, maintained in the collection of wheat genetic resources, have been characterized using analyses of glutenins by sodiumdodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Six of the analyzed wheat accessions were observed to be homogeneous, while 46 (88.5%) of them were heterogeneous in protein profiles. Heterogeneous accessions possessed 2 to 13 different protein lanes. Together, 17 high molecular weight glutenin subunit (HMW-GS) alleles have been found. The most frequent HMW-GS alleles at the Glu-A1, Glu-B1, and Glu-D1 complex loci were 1, 7+9, and 2+12, respectively. However, also low frequented HMW-GS alleles or allelic combinations, such as 7+15, 13+16, 20, 6, 7, and 9 were observed. Furthermore, another new allele encoding HMW glutenin subunit with relative molecular weight 98.6 kDa has been found in one of the lines of the cultivar Eritrospermum 917. The Glu-score in the examined accessions varied in broad range, some of the lines reached the maximum value 10.     

Effects of Prolamins Encoded by Chromosomes 1B and 1D on the Rheological Properties of Dough in Near-Isogenic Lines of Bread Wheat

Fourteen bread wheat near-isogenic lines (NILs) with different alleles at 1B- and 1D-chromosome loci Glu-1, Glu-3 and Gli-1 coding for high molecular weight glutenin subunits (HMW-GS), low molecular weight-GS, and gliadins, respectively, were grown in replicated plots to investigate the individual and combined effects of glutenin and gliadin components on the rheological properties of dough as determined by the Chopin alveograph. NILs did not reveal significant differences in seed yield, protein content, kernel weight, test weight, flour yield, and starch damage. On the contrary, they had a large variation in alveograph dough tenacity P (55–93 mm), swelling G (17–26 mL) and strength W (140–252 J × 10^-4). The null alleles at the Gli-D1/Glu-D3 loci, and allele Glu-D1d (HMW-GS 5+10) were found to have a strong positive influence on dough tenacity and a remarkable negative influence on dough swelling (extensibility) when compared to alleles Gli-D1/Glu-D3b and Glu-D1a (HMW-GS 2+12), respectively. On the other hand, alleles Glu-B1c (HMW-GS 7+9) and Gli-B1/Glu-B3k gave greater G values than alleles Glu-B1u (HMW-GS 7*+8) and Gli-B1/Glu-B3b. The effects of individual Glu-1, Gli-1, or Glu-3 alleles on P and G values were largely additive. The impact of the null allele at Gli-D1/Glu-D3 on gluten strength was highly positive in NILs possessing HMW-GS 2+12, and negligible or negative in NILs containing HMW-GS 5+10, suggesting that there is scope for improving dough quality by utilizing this allele in combination with HMW-GS 2+12. Gli-D1/Glu-D3-encoded prolamins were shown to play a major role in conferring extensibility to dough, and could account for the superior breadmaking characteristics of bread wheat as compared to durum wheat.     

Genetic characteristic of high molecular weight glutenin subunits in somatic hybrid wheat lines -- potential application to wheat breeding

Analysis of 17 derivatives from a somatic fusion between common wheat (Triticum aestivum) and tall wheat grass (Thinopyrum ponticum) showed a diversity of high molecular weight glutenin subunit (HMW-GS) compositions. On the basis of the inheritance of HMW-GS patterns, the derivatives were either (i) bred true over four successive generations, (ii) generated a few novel HMW-GS combinations at each generation, or (iii) showed highly unstable HMW-GS compositions. HMW-GS analysis of F(5) seed and each single seed-generated F(6) progenies further revealed that most of the HMW-GS had genetic stability. The variations of HMW-GS were inferred to occur in early generations and were maintained thereafter. Low molecular weight glutenin subunits (LMW-GS) in hybrid lines with high or low bread-making quality, classified into the first pattern, were compared. The result showed that hybrid lines with the uniform HMW-GS patterns also have identical LMW-GS patterns. The Glu-1 quality score was inferred to be relatively significant to the sodium dodecyl dulfate sedimentation value, as well as to correlate with the gluten exponent and contents of dry gluten and proteins. Sexual hybridization between high-quality somatic hybrid progeny II-12 and Chinese Spring (CS) showed that these high-quality HMW-GS genes could entail progenies. There was not subunit variation in the progenies of II-12 x CS. Therefore, sexual hybridization between somatic hybrid line and cultivars can transfer novel high-quality HMW-GS of somatic hybrids and benefit wheat breeding.     

Molecular Weight Distribution of Wheat Proteins

The molecular weight distribution (MWD) of wheat proteins is becoming recognized as the main determinant of physical dough properties. Studies of high polymers have shown that properties such as tensile strength are related to a fraction of polymer with molecular weight above a critical value and the MWD of this fraction. Elongation to break is treated as a kinetic process with energies of activation for breaking noncovalent bonds and for chain slippage through entanglements. These considerations are related to tensile properties of wheat flour doughs such as those measured by the extensigraph. The MWD of wheat proteins is determined by the relative amounts of monomeric and polymeric proteins and the MWD of the polymeric proteins. The latter, in turn, depends on the ratio of high molecular weight glutenin subunits (HMW-GS) to low molecular weight glutenin subunits (LMW-GS), the specific HMW-GS that result from allelic variation, and the presence of modified gliadins that act as chain terminators. The role of these compositional variables in determining dough extensional properties is discussed in terms of present knowledge. Determination of MWD of wheat proteins is hindered by the difficulty of their solubilization and the lack of methods for reliably measuring very high molecular weights. Among the promising techniques for achieving these measurements are multiangle laser light scattering (MALLS) and field flow fractionation (FFF).     

Development of a Panel of Specific Monoclonal Antibodies to High Molecular Weight Glutenin Subunits and Their Application in Genetic Screening

High molecular weight glutenin subunits (HMW-GS) encoded by different chromosomal loci and alleles (1, 2, 5, 7, 10, and 12) were purified using reversed-phase HPLC from reduced, aqueous propanol extracts of flour from aneuploid or null wheat lines. Unlike previous libraries of monoclonal antibodies developed in our laboratory to SDS-extracted or alkylated HMW-GS, several of the monoclonal antibodies (mAb) developed in this study had a range of specificity patterns for HMW-GS in enzyme-linked immunosorbent assay (ELISA) and on immunoblots. A subset of the mAb bound either x- or y-type HMW-GS but not other gluten proteins, while a few antibodies bound one (mAb 110622, 110421, 140820), or two (mAb 101319, 110804, 140705, 1410460) HMW-GS expressed in each cultivar tested. In most cases, antibodies bound equally to the subunits encoded by different HMW-GS alleles. The more specific antibodies should be useful in research on the quantitative variation of HMW-GS expression and in studies of the role of particular HMW-GS in dough structure. The mAb 101319, which was prepared to subunit 1, bound to HMW-GS 1Bx subunits in ELISA and on immunoblots. This antibody also provided a higher absorbance value in ELISA with extracts of wheat lines expressing the Glu-Ble allele (HMW-GS 20) compared with the Glu-Bli allele (HMW-GS 17+18). Another mAb (110622) detected subunit 2 more strongly than subunit 5 in ELISA and produced a higher signal in immunoblots with subunit 2 even though these subunits are >98.7% homologous in amino acid sequence. An ELISA assay using this antibody was optimized for discrimination of wheat lines with the allelic pairs of subunits 1Dx5-1Dy10 from those with 1Dx2-1Dy12, with the former lines providing stronger dough properties and superior breadmaking quality. The performance of this assay was unaffected by other variations at HMW-GS loci and was demonstrated in sets of biotypes, doubled haploid, and cross-bred breeder's lines.     

Effects of Wheat Bug (Eurygaster maura) Protease on Glutenin Proteins

Proteolytic degradation of 50% 1-propanol insoluble (50PI) glutenin of six common wheat cultivars by wheat bug (Eurygaster maura) protease was investigated using reversed-phase HPLC. Wheat at the milk-ripe stage was manually infested with adult bugs. After harvest, bug-damaged kernels were blended (2:1, kernel basis) with undamaged grain of the same cultivar. Samples of ground wheat were incubated in distilled water for different times (0, 30, 60, and 120 min). The incubated whole meal samples were subsequently freeze-dried and stored until analysis. The degree of proteolytic degradation of 50PI glutenin was determined based on the quantity of total glutenin subunits (GS), high molecular weight GS (HMW-GS), and low molecular weight GS (LMW-GS). For ground wheat samples incubated for ≥30 min, 50PI glutenin was substantially degraded as evidenced by a >80% decrease on average in total GS, HMW-GS, and LMW-GS. Some cultivars showed different patterns of glutenin proteolysis as revealed by differences in the ratios of HMW-GS to LMW-GS between sound and bug-damaged samples; a significant decrease in this ratio was found for four cultivars. This evidence, combined with other observations, indicated that there were intercultivar differences in polymeric glutenin resistance to the protease of the wheat bug Eurygaster maura. While the nature of this resistance is unknown, it should be possible to select and develop wheat cultivars with improved tolerance for wheat bug damage. Propanol insoluble glutenin, which corresponds to relatively large glutenin polymers, appears to be an excellent quantitative marker for this purpose.     

Protein heterogeneity in European wheat landraces and obsolete cultivars: Additional information

The objective of this study was to determine the composition of high molecular weight glutenin subunits of landraces and obsolete cultivars. Altogether glutenin profiles of 67 European wheats were analyzed by sodiumdodecylsulphate polyacrylamide gel electrophoresis. Nineteen of them were observed to be homogeneous, whereas 48 (71%) were heterogeneous in glutenin profiles. Heterogeneous accessions possessed from 2 to 9 different glutenin phenotypes. Seventeen high molecular weight (HMW)-glutenin subunits have been found, three belonged to Glu-1A, 11 to Glu-1B, and three to Glu-1D locus. The most frequented HMW-GS at the Glu-A1, Glu-B1, and Glu-D1 complex loci were 0, 7+9, and 2+12, respectively. However, allele low frequented in wheat such as 13+16, 20, 6, 7, 8, and 9 were observed also. Furthermore, other new alleles encoding HMW-GS at the locus Glu-1B with relative molecular weight 120 and 104 kDa have been found in one of the line of the Swedish cultivar Kotte. TheGlu-1 quality score in the examined accessions varied broadly with some lines reaching the maximum value of 10.