Separation and Quantification of HMW Glutenin Subunits by Capillary Electrophoresis

The use of capillary electrophoresis in SDS (SDS‐CE) for separation and quantification of HMW glutenin subunits (HMW‐GS) was investigated. HMW‐GS were precipitated with 40% acetone from 50% 1‐propanol extract of flour under reducing conditions after removal of monomeric proteins with 50% 1‐propanol. Poly (ethylene oxide) was used in the running buffer (3% w/v) for SDS‐CE. The results indicated that HMW‐GS could be well separated by SDS‐CE, including subunits 7+8, 7+9, 2+12, 5+10, and 17+18. However, HMW‐GS showed delayed migration times compared with molecular weight protein standards. Some HMW‐GS were reversed in their mobilities in SDS‐CE compared with their mobility and molecular weights by SDS‐PAGE. Therefore, the SDS‐CE was unsuitable for MW determination of HMW‐GS. A linear response was obtained from SDS‐CE of a plot of the concentration of HMW‐GS of the 40% acetone precipitate versus corrected areas for absorbance at 214 nm. Quantification of HMW‐GS for the two biotypes (subunits 5+10 vs. 2+12) of an Australian wheat cultivar Warigal confirmed the differences between the two biotypes in their quantity of HMW‐GS. Therefore, the technique could be used to quantify HMW‐GS in conjunction with SDS‐PAGE.     

A Novel Pair of HMW Glutenin Subunits from Aegilops searsii Improves Quality of Hexaploid Wheat

This study involved screening of wild species of wheat in search of functionally useful seed storage proteins for improvement of breadmaking quality of wheat (Triticum aestivum). After screening of 177 disomic addition lines (DALs) of wheat belonging to different wild species, Aegilops searsii DALs were selected and studied in detail. These DALs of Ae. searsii were from chromosome 1S^s to 7S^s in the background of cultivated wheat cv. Chinese Spring (CS). By analyzing these addition lines, genetic loci of actively expressed genes for the high molecular weight glutenin subunits (HMW‐GS) and gliadin were found on the chromosome 1S^s for the first time and have been designated as Glu‐S^s1 and Gli‐S^s1, respectively. Disomic addition line of chromosome 1S^s (DAL1S^s) showed improved dough strength in different generations compared with CS. SDS sedimentation value and specific sedimentation of DAL1S^s were higher than CS. Mixograph peak height and band width were higher, with no difference in mixing peak time from CS. All these factors indicate a positive effect of quantity as well as quality of gluten proteins of Ae. searsii. This was further supported by increased polymer formation in DAL1S^s because the ratio of unextractable polymeric protein to total polymeric protein (UPP/TPP%) of DAL1S^s was significantly higher than CS. Genes for HMW‐GS (major determinant of end‐product quality in wheat) of Ae. searsii were cloned and sequenced from the DAL1S^s. Phylogenetic analysis of deduced amino acid sequences showed that both x and y HMW‐GS were more similar to that of D genome rather than B genome of wheat. Although S genome is structurally more similar to B genome of wheat, functionally it is more similar to the D genome of wheat and possesses good quality HMW‐GS required for improvement of breadmaking quality of wheat.     

Quantitative Variation of HMW Glutenin Subunits from Hard Red Spring Wheats Grown in Different Environments

The objective of this study was to investigate the quantitative variation of HMW glutenin subunits in relation to glutenin polymers and hence breadmaking quality across different environments. Six genotypes of hard red spring (HRS) wheat were grown at seven locations in North Dakota in 1998 in a randomized complete‐block experimental design with three replicates at each location. Unreduced SDS‐soluble glutenins of flour were fractionated by multistacking SDS‐PAGE into different sized glutenin polymers, followed by SDS‐PAGE and imaging densitometry to determine the quantitative variation of HMW glutenin subunits. SDS‐insoluble glutenin polymers also were examined for their quantitative composition of HMW glutenin subunits. The results showed that the percentage of HMW glutenin subunits was significantly affected by growing locations. The quantity of HMW glutenin subunits in SDS‐insoluble glutenins was significantly and positively correlated with loaf volume. SDS‐insoluble glutenin polymers had a higher percentage of HMW glutenin subunits than did SDS‐soluble glutenins. SDS‐insoluble glutenin polymers in flour were positively and significantly correlated in proportions of both total and individual HMW glutenin subunits in total SDS glutenins. SDS‐insoluble glutenin polymers also were positively and significantly correlated with the combined proportion of HMW glutenin subunits 2* + 5. The results of this study indicated that either subunit 2* or 5 might be more important in forming a greater quantity of larger SDS‐insoluble glutenin polymers than other subunits. SDS‐insoluble glutenin polymers from different cultivars or locations could have different quantities of HMW glutenin subunits in their composition. SDS‐insoluble glutenin polymers with more HMW glutenin subunits might be larger sized than those with less HMW glutenin subunits. Environment significantly influenced the quantitative variation of HMW glutenin subunits, which in turn affected the size distribution of glutenin polymers, and hence breadmaking quality.     

Atomic Force Microscopy (AFM) Study of Interactions of HMW Subunits of Wheat Glutenin

Atomic force microscopy (AFM) has been used to study the noncovalent interactions of alkylated HMW subunit 1Dx5 and a M _r 58,000 peptide derived from the central repetitive domain. Both protein and peptide align side‐by‐side to form fibrils, the HMW subunit forming a branched network, and the peptide forming linear rods. The N‐ and C‐terminal domains of the subunit would, therefore, appear to contain regions that interact through noncovalent interactions in the absence of disulfide bond formation. These regions may be of importance in facilitating disulfide bond formation during protein body development.     

Purification and Characterization of the Glutenin Subunits of Triticum tauschii,Progenitor of the D Genome in Hexaploid Bread Wheat

N-terminal amino acid sequences and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) molecular weights have been determined for high-performance liquid chromatography (HPLC)-purified high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits (GS) of Triticum tauschii ssp. strangulata, contributor of the D genome to hexaploid bread wheat. The use of three different extraction procedures resulted in similar glutenin preparations. On the basis of N-terminal sequences, the same types of glutenin subunits that have been reported in bread and durum wheats (HMW-GS of both the x and y types and LMW-GS of the LMW-s, LMW-m, α-, and γ-types) were found in T. tauschii. However, the HMW-GS in T. tauschii were in greater proportion relative to LMW-GS when compared to reported values for a bread and durum wheat. Our results support the likelihood that differences in the proportions of the various subunits contributed by the A, B, and D genomes, rather than qualitative differences in the types of subunits, are responsible for the major differences in quality characteristics between bread wheat and durum wheat.     

(英文)

对来源于美、中、俄及埃塞阿比亚等22个国家的142份硬粒小麦材料的种子贮藏蛋白位点及遗传变异进行了研究。供试的硬粒小麦(Triticum durum Desf )材料共检测出37条醇溶蛋白条带，无1条带纹为所有材料共有，多态性达到100％，说明硬粒小麦具有丰富的醇溶蛋白等位变异。聚类分析将142份供试材料分为3个大类，材料间遗传差异大小在不同的国家有所不同，表明醇溶蛋白带型与地理来源有一定关系。高分子量谷蛋白电泳共分离出14种亚基和15种亚基组合，但是优质亚基所占比例不高，这可能是因为硬粒小麦加工用途的特殊性，使得多年的育种并未太多改变硬粒小麦高分子量谷蛋白亚基等位变异的频率，促成优质亚基的累计。     

Preparative Method for In Vitro Production of Functional Polymers from Glutenin Subunits of Wheat

An in vitro method for preparative‐scale production of artificial glutenin polymers utilizes a controlled environment for the oxidation of glutenin subunits (GS) isolated from wheat flour to achieve high polymerization efficiency. The functionality of in vitro polymers was tested in a 2‐g model dough system and was related to the treatment of the proteins before, during, and after in vitro polymerization. When added as the only polymeric component in a reconstituted model dough (built up from gliadin, water solubles, and starch fractions), in vitro polymers could mimic the behavior of native glutenin, demonstrating properties of dough development and breakdown. Manipulating the high molecular weight (HMW)‐GS to a low molecular weight (LMW)‐GS ratio altered the molecular weight distribution of in vitro polymers. In functional studies using the 2‐g mixograph, simple doughs built up from homopolymers of HMW‐GS were stronger than those using homopolymers of LMW‐GS. These differences may be accounted for, at least in part, by different polymer size distributions. The ability to control the size and composition of glutenin polymers shows the potential of this approach for investigating the effects of glutenin polymer size on dough function and flour end‐use quality.     

卫星搭载小麦SP3代高分子麦谷蛋白亚基的SDS-PAGE分析

利用十二烷基磺酸钠-聚丙烯胺凝胶电泳（SDS-PAGE）技术对返回式卫星搭载小麦两个品种SP3代籽粒的高分子麦谷蛋白亚基（HMW-GS）进行了分析,并按照相关评分方法计算了高分子量麦谷蛋白Glu-1位点的品质得分。结果表明,经卫星搭载可产生较高频率的HMW-GS基因变异,陕253和西农1043 两个品种SP3代HMW-GS组成的变异频率分别为27.08%和27.45%。陕253和西农1043 SP3代的小麦品质得分分别为7分和6分,陕253 SP3代变异株为优质小麦。     

Allelic Variation at Glu-D1 Locus for High Molecular Weight (HMW) Glutenin Subunits: Quantification by Multistacking SDS-PAGE of Wheat Grown Under Nitrogen Fertilization

Two biotypes of an Australian wheat cultivar, Warigal, differing only in the Glu-D1 high molecular weight (HMW) glutenin subunits 5+10 and 2+12 were used in this study. The objective was to examine the effects of nitrogen fertilization and allelic variation at the Glu-D1 locus on the characteristics of glutenin polymers. Unreduced proteins containing the SDS-soluble glutenins and the other protein classes were analyzed by multistacking SDS-PAGE which separates the glutenin into six distinctly different-sized aggregates. The results showed that nitrogen fertilization significantly increased protein quantity, ratio of polymers to monomeric proteins, and sizes of SDS-soluble glutenins. Nitrogen fertilization affected the proportions of HMW subunits in both SDS-soluble and SDS-insoluble glutenin polymers and the ratio of x to y subunits in SDS-insoluble glutenin polymers. Nitrogen fertilization, however, did not cause a significant change in ratio of SDS-soluble to SDS-insoluble glutenins. SDS-insoluble glutenins had a greater ratio of HMW to LMW and x to y subunits, especially with a higher increase of 1Dx subunits, than SDS-soluble glutenins. The HMW/LMW subunit ratio and the x/y subunit ratio may be used to predict sizes of glutenin polymers. The biotype with 5+10 subunits had a greater x/y subunit ratio in the SDS-insoluble glutenins than the 2+12 type. A greater proportion of subunit 5 was formed than subunit 2 in the SDS-insoluble glutenin polymers. Both nitrogen fertilization and allelic variation at Glu-D1 loci could affect the characteristics of glutenin polymers.     

新疆小麦高分子量麦谷蛋白亚基组成及主效亚基分析

为探索新疆强筋小麦的主效亚基,本文采用SDS-PAGE方法对新疆主栽的21种中筋小麦和17种强筋小麦的高分子量麦谷蛋白亚基进行了分析。结果表明:供试材料中共出现12种类型的亚基和12种亚基组合。中筋小麦Glu-A1位点亚基主要为N,Glu-B1位点亚基主要为7+8,Glu-D1位点亚基主要为2+12。强筋小麦Glu-A1位点亚基主要为1和N,Glu-B1位点亚基主要为7+8和7+9,Glu-D1位点亚基主要为5+10。聚类分析结果显示:在相似系数为0.43时可将供试小麦分为2类:第1类包括28个品种,均含有2+12亚基,其中既有强筋小麦也有中筋小麦;第2类包括12个品种,均含有5+10亚基,全部为强筋小麦。5+10亚基对小麦品质贡献明显大于其他亚基,是新疆强筋小麦的主效亚基。本研究可为提高新疆小麦面筋强度的分子育种提供理论依据。     

Storage of Wheat Grains at Elevated Temperatures Increases Solubilization of Glutenin Subunits

Grains of two wheat (Triticum aestivum L.) cultivars, Sunco and Sunsoft, were stored at 4°C and 30°C for 270 days to examine changes in proteins during storage. When whole meal flour extracted from the grains was analyzed using an unfractionated protein extraction procedure, no significant changes were found in protein content or SDS‐PAGE profile for either cultivar in samples stored at 30°C compared with those stored at 4°C. Fractionation of the flour samples from stored grain into soluble and insoluble proteins revealed increases in soluble protein content for both cultivars stored at 30°C compared with 4°C. The soluble protein content, expressed as a percentage of the total protein, increased by 1.5% (P = 0.032) for Sunco and by 8.0 % (P = 0.158) for Sunsoft during storage at 30°C compared with those samples stored at 4°C. Analysis by SDS‐PAGE and subsequent protein identification revealed that the most evident change that occurred during storage at 30°C was an increase in the content of high molecular weight glutenin subunits (HMW‐GS) in the soluble fraction. The potential effect of changes in solubility of HMW‐GS on functional properties is discussed.     

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.     

In Vitro Polymerization of Wheat Glutenin Subunits with Inorganic Oxidizing Agents. I. Comparison of Single‐Step and Stepwise Oxidations of High Molecular Weight Glutenin Subunits

High molecular weight glutenin subunits (HMW‐GS) were isolated from wheat flour and polymerized in vitro at pH 3.0 with different oxidizing agents (KBrO₃, KIO₃, H₂O₂). An oxidation protocol with single addition of oxidant (single‐step oxidation) was compared with a set‐up in which the oxidant was added in multiple steps (stepwise oxidation). Changes in size distribution were evaluated with size‐exclusion HPLC, multilayer SDS‐PAGE, and flow‐field flow fractionation (flow‐FFF). Flow‐FFF is particularly suitable for measuring changes in glutenin size in the very high size ranges. In order of increasing sizes of the resulting polymers, the different oxidizing agents could be ranked as KBrO₃ < KIO₃ < H₂O₂. However, none of the oxidation conditions allowed for a complete polymerization of HMW‐GS. Interestingly, it was found that high concentrations of KIO₃ negatively affect the degree of polymerization. A similar observation was not made with KBrO₃ or H₂O₂. SDS‐PAGE showed that y‐type HMW‐GS particularly failed to incorporate in glutenin polymers. Simultaneously, these HMW‐GS displayed higher mobilities on SDS‐PAGE that can be ascribed to the formation of intrachain SS bonds. Possible explanations for the incomplete polymerization of HMW‐GS are given.     

Influence of High Molecular Weight (HMW) and Low Molecular Weight (LMW) Glutenin Subunits Controlled by Glu‐1 and Glu‐3 Loci on Durum Wheat Quality

The progenies of four intervarietal durum wheat crosses were used to determine the effects of glutenin variants coded at Glu‐1 and Glu‐3 loci on durum wheat quality properties. The F₂ lines were analyzed for high molecular weight (HMW) and low molecular weight (LMW) glutenin composition by electrophoresis. Whole grain derived F₃ and F₄ samples were analyzed for vitreousness, protein, and dry gluten contents, gluten index, SDS sedimentation volume, mixograph, and alveograph properties. Allelic variation at the Glu‐B1 and Glu‐B3 loci affected gluten quality significantly. Comparisons among the Glu‐B3 and Glu‐B1 loci indicated that the LMW glutenin subunits controlled by Glu‐B3 c and j made the largest positive contribution, followed by the alleles a, k, and b. HMW glutenin subunits 14+15 gave larger SDS values and higher mixing development times than subunits 7+8 and 20. The positive effects of the glutenin subunits LMW c and HMW 14+15 were additive. Flour protein content, vitreousness, and mixograph peak height values were positively correlated with each other as well as with Dglut values, whereas the SDS sedimentation highly correlated with mixing development time, alveograph strength, and extensibility but was not correlated with the other parameters. The results of quality analysis, together with the results of the genetic analysis, led to the conclusion that SDS sedimentation, mixograph mixing development time, and peak breakdown are the tests more influenced by allelic variation of prolamin. The uses of the results in durum wheat quality breeding programs are discussed.     

Separation of Wheat Proteins by Two-Dimensional Reversed-Phase High-Performance Liquid Chromatography Plus Free Zone Capillary Electrophoresis

Gliadins and glutenins from four hard red winter wheat cultivars were separated by a novel two-dimensional (2D) technique. Protein extracts were separated by reversed-phase high performance liquid chromatography as the first dimension with each 30-sec interval collected separately. Those fractions were then separated by free-zone capillary electrophoresis (FZCE) for the second dimension. Data was combined into 2D surface contour plots similar to traditional gel electrophoresis 2D maps. For HPLC, C₈ and C₁₈ columns were used in the first dimension to separate gliadins and glutenins, respectively. Uncoated fused silica capillaries (27 cm × 25 μm, i.d.) were used for the 2D FZCE separations. Differences in the 2D maps of both gliadin and glutenin fractions were found between pairs of both closely related and sister lines that varied in quality.     

Composition of HMW and LMW Glutenin Subunits and Their Effects on Dough Properties,Pan Bread,and Noodle Quality of Chinese Bread Wheats

Knowledge of composition of high molecular weight glutenin subunits (HMW‐GS) and low molecular weight glutenin subunits (LMW‐GS) and their associations with pan bread and noodle quality will contribute to genetically improving processing quality of Chinese bread wheats. Two trials including a total of 158 winter and facultative cultivars and advanced lines were conducted to detect the allelic variation at Glu‐1 and Glu‐3 loci by SDS‐PAGE electrophoresis and to understand their effects on dough properties, pan bread, and dry white Chinese noodle (DWCN) quality. Results indicate that subunits/alleles 1 and null at Glu‐A1, 7+8 and 7+9 at Glu‐B1, 2+12 and 5+10 at Glu‐D1, alleles a and d at Glu‐A3, and alleles j and d at Glu‐B3 predominate in Chinese germplasm, and that 34.9% of the tested genotypes carry the 1B/1R translocation (allelic variation at Glu‐D3 was not determined because no significant effects were reported previously). Both variations at HMW‐GS and LMW‐GS/alleles and loci interactions contribute to dough properties and processing quality. For dough strength related traits such as farinograph stability and extensigraph maximum resistance and loaf volume, subunits/alleles 1, 7+8, 5+10, and Glu‐A3d are significantly better than those of their counterpart allelic variation, however, no significant difference was observed for the effects of d, b, and f at Glu‐B3 on these traits. For extensigraph extensibility, only subunits 1 and 7+8 are significantly better than their counterpart alleles, and alleles d and b at Glu‐B3 are slightly better than others. For DWCN quality, no significant difference is observed for HMW‐GS at Glu‐1, and Glu‐A3d and Glu‐B3d are slightly better than other alleles. Glu‐B3j, associated the 1B/1R translocation, has a strong negative effect on all quality traits except protein content. It is recommended that selection for subunits/alleles 1, 7+8, 5+10, and Glu‐A3d could contribute to improving gluten quality and pan bread quality. Reducing the frequency of the 1B/1R translocation will be crucial to wheat quality improvement in China.     

Effects of High and Low Molecular Weight Glutenin Subunits on Rheological Dough Properties and Breadmaking Quality of Wheat

High molecular weight (HMW) or low molecular weight (LMW) subunits of different chemical state (reduced, reoxidized with KBrO₃, or KIO₃) or gliadins were added in 1% amounts to a base flour of the wheat cultivar Rektor and mixed with water. The corresponding doughs were then characterized by microscale extension tests and by microbaking tests and were compared to doughs from the base flour without additives. The maximum resistance of dough was strongly increased by HMW subunits in a reduced state and by HMW subunits reoxidized with KBrO₃. A moderate increase of resistance was caused by HMW subunits reoxidized with KIO₃ and by LMW subunits reoxidized with KBrO₃ or KIO₃. This resistance was strongly lowered by LMW subunits in a reduced state and by gliadins. The extensibility of dough was significantly increased only by gliadins and reduced HMW subunits; HMW subunits reoxidized with KBrO₃ had no effect, and all other fractions had a decreasing effect. In particular, glutenin subunits reoxidized with KIO₃ induced marked decrease of extensibility, resulting in bell‐shaped curve extensigrams, which are typical for plastic properties. The effect of reoxidized mixtures (2:1) of HMW and LMW subunits on maximum resistance depended on the oxidizing agent and on the conditions (reoxidation separated or together); extensibility was generally decreased. Bread volume was increased by addition of HMW subunits (reduced or reoxidized with KBrO₃) and decreased by LMW subunits (reoxidized with KBrO₃ or KIO₃) and by a HMW‐LMW subunit mixture (reoxidized with KBrO₃). The volume was strongly decreased by addition of reduced LMW subunits. A high bread volume was related to higher values for both resistance and extensibility.     

Expression,Purification, and Functional Analysis of Three Low‐Molecular‐Weight Glutenin Subunits from Wheat Cultivar Cheyenne

Glutenins, which form the network of gluten protein, are of great importance for the quality of flour products. Glutenins can be divided into HMW and LMW subunits according to molecular weight. Three genes for LMW glutenin subunits (LMW‐GS), named lmw‐cnd1, lmw‐cnd2, and lmw‐cnd3 with open reading frames of 1,053, 903, and 969 bp, respectively, were cloned from wheat cultivar Cheyenne. Heterologous expression vectors of the three LMW‐GS were constructed, and the recombinant proteins LMW‐CND1, LMW‐CND2, and LMW‐CND3 were overexpressed in Escherichia coli. After cell disruption with ultrasound, target proteins of high purity were obtained by using Ni²⁺ affinity chromatography. Farinograph and TAPlus measurements were used to investigate the effects of the three LMW‐GS on the characteristics of flour and dough. The results showed that the addition of each LMW‐GS can lead to an increase in the elasticity of the dough. Moreover, LMW‐CND2 and LMW‐CND3 promoted the strength of the dough. All three LMW‐GS caused a decrease of hardness and increase of springiness and cohesiveness of dough according to texture profiling results. Consequently, all three LMW‐GS have positive effects on the processing characteristics of dough and can improve bread quality to different extents.