Prolamin aggregation, gluten viscoelasticity, and mixing properties of transgenic wheat lines expressing 1Ax and 1Dx high molecular weight glutenin subunit transgenes

The composition of high molecular weight (HMW) subunits of glutenin determines the gluten strength and influences the baking quality of bread wheat. Here, the effect of transgenes coding for subunits 1Ax1 and 1Dx5 was studied in two near-isogenic wheat lines differing in their HMW subunit compositions and mixing properties. The subunits encoded by the transgenes were overexpressed in the transformed lines and accounted for 50-70% of HMW subunits. Overexpression of 1Ax1 and 1Dx5 subunits modified glutenin aggregation, but glutenin properties were much more affected by expression of the 1Dx5 transgene. This resulted in increased cross-linking of glutenin polymers. In dynamic assay, the storage and loss moduli of hydrated glutens containing 1Dx5 transgene subunits were considerably enhanced, whereas expression of the 1Ax1 transgene had a limited effect. The very high strength of 1Dx5 transformed glutens resulted in abnormal mixing properties of dough. These results are discussed with regard to glutenin subunit and glutenin polymer structures.     

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.     

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.     

The relationship between high-molecular-weight glutenin subunit composition and the quality of Japanese hexaploid wheat lines

To reveal the high-molecular-weight (1-1MW) glutenin subunit composition, the seed storage proteins of 40 Japanese wheat (Triticum aestivum) lines were fractionated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis to determine their HMW glutenin subunit composition. These were identified by comparison of subunit mobility with that previously found in hexaploid wheat. Twelve different, major glutenin HMW subunits were identified. Each line contained three to five subunits, and 11 different glutenin subunit patterns were observed for 11 alleles in Japanese lines. The Glu-1 quality scores were not particularly high for most of the Japanese wheats in the southern part of Japan (Kyushu district). However, the Glu-1 quality scores of several wheat lines in the Hokkaido area (north Japan) were high. South Japanese wheat lines showed specialty allelic variation in the glutenin HMW 145 kfla subunit, different from those in non-Japanese hexaploid wheats.     

Identification of alleles for complex gene loci Glu-A1, Glu-B1, and Glu-D1, which code for high molecular weight subunits of glutenin in Japanese hexaploid wheat varieties

Seed storage proteins of Japanese wheat (Triticum aestivum) varieties were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to identify the alleles for complex gene loci, Glu-A1, Glu-B1, and Glu-D1, which code for high molecular weight (HMW) subunits of glutenin in Japanese hexaploid wheat varieties. These were identified by comparison of subunit mobility with those previously found in hexaploid wheat. Twenty-four different, major glutenin HMW subunits were identified, and each variety contained three to five subunits. Seventeen different glutenin subunit patterns were observed for 14 alleles in Japanese varieties. A catalog of alleles for the complex gene loci, Glu-A1, Glu-B1, and Glu-D1, that code for HMW subunits of glutenin in hexaploid wheat was compiled. Japanese varieties showed some special allelic variation in glutenin HMW subunits that was different from those in hexaploid wheats of other countries.     

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.     

Effect of Multiple Substitution of Glutenin and Gliadin Proteins on Flour Quality of Canada Prairie Spring Wheat

The effect of genetic substitution of two to four glutenin and gliadin subunits from a Canada Prairie Spring (CPS) cv. Biggar BSR into Alpha 16, another CPS wheat line, was studied for rheological and baking quality. Results from double substitution showed that the presence of a gliadin component from Biggar BSR (BGGL) and low molecular weight glutenin subunit 45 (LMW 45) contributed to improved dough strength characteristics. Presence of BGGL in combination with high molecular weight glutenin subunit 1 (HMW 1) or 17+18 (HMW 17+18) also showed improved dough strength over control Alpha lines. When three or four protein subunits were substituted, even though improved quality performance was observed, it was associated with the negative effect of lowered flour water absorptions in spite of similar protein contents. The study confirms that LMW glutenins, as well as gliadins, play an important role along with HMW glutenins in wheat flour quality. CPS wheat lines with improved dough strength properties can be selected from the double substitution lines with the combination of BGGL/LMW 45 and BGGL/HMW 1.     

Genetic Variance for Gluten Strength Contributed by High Molecular Weight Glutenin Proteins

A total of 162 doubled haploid (DH) lines were produced from a cross between Triticum aestivum L. ‘AC Karma’ and line 87E03‐S2B1 to study the genetic contribution of high molecular weight (HMW) glutenin subunits to gluten strength. HMW glutenin subunit composition of each DH line was determined by SDS‐PAGE. The population was grown in the field at one location in 1999 and at three locations in 2000. Gluten strength and dough mixing properties were measured by mixograph test and SDS‐sedimentation test. Variance components were estimated for each measurement to determine the variability contributed by HMW glutenin subunits. Results indicated significant environmental impact on tested mixograph parameters, SDS‐sedimentation volumes and grain and flour protein concentration. Significant main effects of Glu‐1D loci encoded subunits were obtained for mixograph development time, energy to peak, slope after peak, and first minute slope. Lines containing 5+10 combination of subunits had higher values for mixograph development time and energy to peak, while slope after peak and first minute slope were lower as compared with 2+12 containing lines. Low intergenomic interactions were observed for bandwidth energy (BWE), total energy (TEG), and SDS‐sedimentation test, involving B and D genomes only. A portion of the genetic variability for gluten strength was accounted for overexpression of Bx7 subunit originating from the cultivar Glenlea derived line 87E03‐S2B1. There was no significant effect of Glu‐A1 encoded subunits on any of the tested parameters. Estimated genetic variability for gluten strength contributed by Glu‐B1 and Glu‐D1 encoded HMW glutenins was 55% for mixing development time and 51% for energy to peak.     

Synergistic and Additive Effects of Three High Molecular Weight Glutenin Subunit Loci. I. Effects on Wheat Dough Rheology

The high molecular weight glutenin subunits (HMW‐GS) play an important role in governing the functional properties of wheat dough. To understand the role of HMW‐GS in defining the basic and applied rheological parameters and end‐use quality of wheat dough, it is essential to conduct a systematic study where the effect of different HMW‐GS are determined. This study focuses on the effect of HMW‐GS on basic rheological properties. Eight wheat lines derived from cvs. Olympic and Gabo were used in this study. One line contained HMW‐GS coded by all three loci, three lines were each null at one of the loci, three lines were null at two of the loci and one line null at all three loci. The flour protein level of all samples was adjusted to a constant 9% by adding starch. In another set of experiments, in addition to the flour protein content being held at 9%, the glutenin‐to‐gliadin ratio was maintained at 0.62 by adding gliadin. Rheological properties such as elongational, dynamic, and shear viscometric properties were determined. The presence of Glu‐D1 subunits (5+10) made a significantly larger contribution to dough properties than those encoded by Glu‐B1 (17+18), while subunit 1, encoded by Glu‐A1, made the least contribution to functionality. Results also confirmed that HMW‐GS contributed to strength and stability of dough.     

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.     

Rheological Properties of Full-Formula Doughs Derived from Near-Isogenic 1BL/1RS Translocation Lines

Four pairs of near-isogenic wheat lines, with and without the 1BL/1RS translocation, and differing at the Glu-1 loci (coding for high molecular weight [HMW] glutenin subunits) were evaluated for their dough mixing properties, dough stickiness, and baking performance. In all 1BL/1RS translocation lines, weakening of the dough consistency occurred within 2 min past peak time. The full-formula dough from every 1BL/1RS translocation line exhibited poor dough mixing characteristics and increased stickiness compared to the corresponding wheat control. The HMW glutenin subunits coded by the Glu-A1 locus had no apparent effect on mixing properties, but did have a slight effect on the dough stickiness at two of the four stages of dough mixing. Glu-B1 and Glu-D1 loci encoded glutenin subunits produced significant changes in dough mixing properties and dough stickiness, respectively. With respect to baking performance, there was no significant difference between loaf volumes of 1BL/1RS versus control wheats for three of four near-isogenic pairs. Within the 1RS-group, the translocation lines containing HMW glutenin subunits 5+10 produced bread with greater loaf volumes than the pairs containing its allelic counterpart 2+12. Loaf volume was not influenced by the subunits associated with the Glu-B1 loci. In general, the breads baked from 1BL/1RS translocation lines had a relatively poor crumb and crust quality and contained larger gas cells than the wheat controls. In comparing isogenic pairs, the magnitude of the difference in loaf volume between the control wheat and the corresponding 1BL/1RS translocation line was greater in the pair unique for HMW subunits 5+10; the difference was primarily due to the stronger mixing properties of the wheat control.     

Protein and Quality Characterization of Triticale Translocation Lines in Breadmaking

Introduction of high molecular weight glutenin subunits (HMW‐GS) from the Glu‐D1d locus of wheat into triticale restores the genetic constitution of high molecular weight glutenin loci to that of wheat and subsequently improves the breadmaking quality of triticale. One means of achieving such restoration of the genetic constitution is through the use of translocation lines. The aim of this study was to evaluate and compare the performance of translocations 1A.1D and 1R.1D with HMW‐GS 5+10 and 2+12 in terms of physical dough tests and baking quality using four different sets of triticale lines, GDS7, Trim, Rhino, and Rigel. In general, significantly lower milling quality (flour yield), very low mixing times with lower loaf volume were typical of all the triticales studied except 1A.1D 5+10 lines, when compared to hard wheat flour (Pegaso). Among the lines studied, significantly higher loaf volume, mixograph dough development time (MDDT), and maximum resistance to extension (R_max) were observed with 1A.1D 5+10 lines indicating that translocation of the Glu‐D1d allele with HMW‐GS 5+10 was beneficial in terms of improving the quality attributes. Although pure triticale flour from these lines did not possess the functional characteristics for good quality bread, the translocation 1A.1D that contains HMW glutenin subunits 5+10 showed significant improvement in quality characteristics, and could reasonably be expected to yield commercially satisfactory bread loaves when combined with bread wheat flour. Significantly higher UPP, R_max, and MDDT values along with a lower gliadin‐to‐glutenin ratio in 1A.1D 5+10 of GDS7 and Rigel sets indicate that the molecular weight distribution was shifted to higher molecular weights, resulting in greater dough strength associated with 5+10 subunits.     

Isolation and Characterization of High‐Molecular‐Weight (HMW) Gliadins from Wheat Flour

The aim of this study was to isolate high‐molecular‐weight (HMW) gliadins from wheat flour and to characterize the protein components that contribute to HMW gliadins. Wheat flour Akteur was extracted with a modified Osborne procedure, and the fraction soluble in 60% ethanol (total gliadins) was separated by gel‐permeation HPLC, yielding three fractions, GP1–GP3. GP1 (21.5%) consisted of oligomeric HMW gliadins, GP2 (15.2%) of ω5‐gliadins, and GP3 (63.3%) of ω1,2‐, α‐, and γ‐gliadins. Two‐dimensional SDS‐PAGE of HMW gliadins showed that interchain disulfide bonds were present in HMW gliadins. The molecular mass distribution of HMW gliadins determined by gel‐permeation HPLC was in a range from 66,000 to 680,000 with an average degree of polymerization of 13. Reduced HMW gliadins were further separated by preparative reversed‐phase HPLC into four subfractions (RP1, RP2, RP3, and RP4), which were characterized by SDS‐PAGE and semiquantitative N‐terminal sequencing. HMW gliadins of the wheat flour Akteur contained all types of gluten proteins: 48% low‐molecular‐weight glutenin subunits, 18% γ‐gliadins, 13% α‐gliadins, 9% ω1,2‐gliadins, 8% HMW glutenin subunits, and 4% ω5‐gliadins. We postulate that the existence of HMW gliadins can be explained by the presence of terminators, which interrupt the polymerization of glutenin subunits during biosynthesis and lead to polymers of limited size (oligomers) that are still soluble in aqueous ethanol.     

Studies on Effects of Microbial Transglutaminase on Gluten Proteins of Wheat. I. Biochemical Analysis

The enzyme transglutaminase (TG) is known to have beneficial effects on breadmaking. However, only limited information is available on the structural changes of gluten proteins caused by TG treatment. The effect of TG has, therefore, been systematically studied by means of model peptides, suspensions of wheat flours and doughs. The treatment of synthetic peptides mimicking amino acid sequences of HMW subunits of glutenin with TG results in isopeptide bonds between glutamine and lysine residues. To study the effect on gluten proteins, different amounts of TG (0 to 900 mg enzyme protein per kg) were dissolved in a buffer and added to wheat flour. The flour suspensions were incubated and centrifuged and the residues were successively extracted with water, a salt solution, 60% aqueous ethanol (gliadin fraction) and SDS solution including a reducing agent (glutenin fraction). The characterization of the fractions by amino acid analysis, SDS‐PAGE, gel permeation HPLC and reversed‐phase HPLC has indicated that the quantity of extractable gliadins decreases by increasing TG amounts. Among gliadins, the ω5‐type was affected to the greatest extent by the reduction of extractability, followed by the ω1,2‐, α‐ and γ‐types. The oligomeric portion of the gliadin fractions (HMW gliadin) was strongly reduced when flour was treated with 450 and 900 mg TG per kg of flour, respectively. In the first instance, the quantity of the glutenin fractions increased by the treatment of flour with 90 and 450 mg TG per kg of flour, and significantly decreased by the treatment of flour with 900 mg TG per kg of flour. Parallel to an increase in TG concentration, the amounts of glutenin‐bound ω‐gliadins and HMW subunits were strongly reduced, whereas the LMW subunits reached a maximal amount after treatment with 450 mg TG per kg of flour. The insoluble residue was almost free of protein when flour was treated with lower amounts of TG. Higher amounts led to a great increase of protein in the residues. The effects of TG on doughs were similar to those of flour suspensions, but less strongly pronounced probably due to the lower water content of the dough system. Sequence analysis of peptides from a thermolytic digest of the insoluble residue revealed that HMW subunits of glutenin and α‐gliadins were predominantly involved in cross‐links formed by TG treatment.     

Application of a Micro Z‐Arm Mixer to Characterize Mixing Properties and Water Absorption of Wheat Flour

This study applied the use of a new small‐scale apparatus, the micro Z‐arm mixer, which has analogous mixing action to that of the traditional valorigraf and farinograph. A novel methodology has been developed for prediction of water absorption replacing the traditional titration method. The basis of this technique is a common characteristic of wheat flour samples: a reasonably constant slope (20–25.7 BU%) of the relationship between dough resistance and the amount of water present during mixing. Using an average slope value, prediction of water absorption was possible from a single measurement using a simple equation and with a standard error of 1.65%. Applications of the new mixer to cereal research are highlighted, including investigation of the effects of flour protein content and protein composition on mixing properties and water absorption. When protein content and protein composition have been systematically altered by the addition of isolated proteins into the flour, both dough development time (DDT) and water absorption increased when protein content was increased by glutenin addition and decreased when protein content was decreased by starch addition. Gliadin addition decreased DDT; gluten addition slightly increased DDT; glutenin addition significantly increased DDT. Water absorption was not affected by altering the glutenin‐to‐gliadin ratio, but it changed in proportion to the amount of protein added. The effect of HMW‐GS composition on the mixing requirement obtained with the micro Z‐arm mixer and with the 2‐g mixograph was also investigated using a set of single‐, double‐, and triple‐null lines for HMW‐GS coding genes. While subunits coded on the GluD1 locus were most important for determining the mixing requirement in both cases, the sample ranking was different in the two mixing actions. A better differentiation ability of the micro Z‐arm mixer was established for triple‐ and double‐null lines.     

DHPLC scoring of a SNP between promoter sequences of HMW glutenin x-type alleles at the Glu-D1 locus in wheat

The promoter regions of HMW glutenin x-type genes at the Glu-D1 locus were surveyed for SNPs within a subpopulation of German bread wheat cultivars. On the basis of the promoter sequences of HMW glutenin subunit genes Glu-A1-x1, Glu-A1-x2, Glu-B1-x1, Glu-B1-x7, Glu-D1-x2, and Glu-D1-x5, an amplification refractory mutation system assay was designed to selectively amplify Dx-specific PCR fragments. Comparative sequence analysis among seven Glu-D1-x2 and seven Glu-D1-x5 wheat cultivars only confirmed a G-A transition in the promoter sequence to be a true polymorphism. SNP scoring by DHPLC of 95 German bread wheat cultivars, with the exception of cv. Anemos, showed that the transition completely agreed with the presence of HMW glutenin subunits 1Dx5 + 1Dy10 in SDS-PAGE. Therefore, the developed DHPLC assay is suitable for high-throughput genotyping to assist the selection of HMW glutenin genes in wheat quality breeding programs.     

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.     

High molecular weight glutenin subunits in some durum wheat cultivars investigated by means of mass spectrometric techniques

The primary structures of high molecular weight glutenin subunits (HMW-GS) of 5 Triticum durum Desf. cultivars (Simeto, Svevo, Duilio, Bronte, and Sant'Agata), largely cultivated in the south of Italy, and of 13 populations of the old spring Sicilian durum wheat landrace Timilia (Triticum durum Desf.) (accession nos. 1, 2, 3, 4, 7, 8, 9, 13, 14, 15, SG1, SG2, and SG3) were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high performance liquid chromatography/nanoelectrospray ionization mass spectrometry (RP-HPLC/nESI-MS/MS). M(r) of the intact proteins determined by MALDI mass spectrometry showed that all the 13 populations of Timilia contained the same two HMW-GS with 75.2 kDa and 86.4 kDa, whereas the other durum wheat cultivars showed the presence of the expected HMW-GS 1By8 and 1Bx7 at 75.1 kDa and 83.1 kDa, respectively. By MALDI mass spectrometry of the tryptic digestion peptides of the isolated HMW-GS of Timilia, the 1Bx and 1By subunits were identified as the NCBInr Acc. No AAQ93629, and AAQ93633, respectively. Sequence verification for HMW-GS 1Bx and 1By both in Simeto and Timilia was obtained by MALDI mass mapping and HPLC/nESI-MSMS of the tryptic peptides. The Bx subunit of Timila presents a sequence similarity of 96% with respect to Simeto, with differences in the insertion of 3 peptides of 5, 9, and 15 amino acids, for a total insertion of 29 amino acids and 25 amino acid substitutions. These differences in the amino acidic sequence account for the determined Δm of 3294 Da between the M(r) of the 1Bx subunits in Timilia and Simeto. Sequence alignment between the two By subunits shows 10 amino acid substitutions and is consistent with the Δm of 148 Da found in the MALDI mass spectra of the intact subunits.     

Synergistic and Additive Effects of Three High Molecular Weight Glutenin Subunit Loci. II. Effects on Wheat Dough Functionality and End‐Use Quality

Understanding the relationship between basic and applied rheological parameters and the contribution of wheat flour protein content and composition in defining these parameters requires information on the roles of individual flour protein components. The high molecular weight glutenin subunit (HMW‐GS) proteins are major contributors to dough strength and stability. This study focused on eight homozygous wheat lines derived from the bread wheat cvs. Olympic and Gabo with systematic deletions at each of three HMW‐GS encoding gene loci, Glu‐A1, Glu‐B1, and Glu‐D1. Flour protein levels were adjusted to a constant 9% by adding starch. Functionality of the flours was characterized by small‐scale methods (2‐g mixograph, microextension tester). End‐use quality was evaluated by 2‐g microbaking and 10‐g noodle‐making procedures. In this sample set, the Glu‐D1 HMW‐GS (5+10) made a significantly larger contribution to dough properties than HMW‐GS coded by Glu‐B1 (17+18), while subunit 1 coded by Glu‐A1 made the smallest contribution to functionality. These differences remained after removing variations in glutenin‐to‐gliadin ratio. Correlations showed that both basic rheological characteristics and protein size distributions of these flours were good predictors of several applied rheological and end‐use quality tests.     

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.