Quality and Agronomic Effects of Three High‐Molecular‐Weight Glutenin Subunit Transgenic Events in Winter Wheat

Quality and agronomic effects of three transgenic high molecular weight glutenin subunit (HMW‐GS) events were characterized in advanced‐generation breeding lines of hard winter wheat (Triticum aestivum L.) in three Nebraska crop years. Two of the transgenic events studied, Dy10‐E and B52a‐6, overexpress HMW‐GS 1Dy10, while the third event, Dx5 +Dy10‐H, overexpresses HMW‐GS 1Dx5 and, to a much lesser extent, 1Dy10. In addition, novel proteins possessing solubility characteristics defined as HMW‐GS were present in Dx5+Dy10‐H and B52a‐6. Average grain yield of lines derived from the three transgenic events was statistically lower than that of a group of control cultivars and advanced breeding lines, but not lower than the mean values of respective nontransgenic siblings. Grain hardness was influenced by one of the events. Dx5+Dy10‐H produced harder kernels than controls, its nontransgenic siblings, and the two additional transgenic events. All three events produced doughs with unusual mixing properties, although not likely to be directly useful in commercial applications. As a consequence, loaf volumes were depressed to variable degrees by the three events. The results indicated that over‐expression of HMW‐GS could eventually lead to improved breadmaking quality by optimizing the level of overexpression or by development and characterization of additional events.     

Flour Protein Composition and Functional Properties of Transgenic Rye Lines Expressing HMW Subunit Genes of Wheat

Flours from nonsprouted (ns) kernels and dried sprouted (s) kernels of transgenic rye expressing HMW glutenin subunits (HMW‐GS) 1Dy10 (L10) or 1Dx5+1Dy10 (L5+10) from wheat were compared with flours from the corresponding wildtype rye (Lwt). The crude protein content of nonsprouted flours ranged from 9.2% (Lwt) to 10.4% (L5+10) and was lowered by ≈1% due to sprouting. Flour proteins were separated into albumins/globulins, prolamins, and glutelin subunits by a modified Osborne fractionation and into SDS‐soluble and insoluble fractions. Portions of the prolamin fractions were reduced in the same manner as glutelins. The different fractions were then characterized and quantified by RP‐HPLC on C₈ silica gel. The proportion of albumins/globulins did not significantly differ between transgenic lines and wildtype. The proportions of alcohol‐insoluble glutelins and SDS‐insoluble proteins drastically increased in transgenic rye due to a shift of HMW and γ‐75k secalins into the polymeric fractions. Significant differences in the proportion of highly polymeric proteins between nonsprouted and sprouted flours could not be detected. The quantitative data demonstrated that the expression of HMW‐GS led to a higher degree of polymerization of storage proteins in rye flour. The HMW‐GS combination 1Dx5+1Dy10 showed stronger effects than 1Dy10 alone. The analyzed flours contained two HMW secalins (R1, R2), whose amino acid compositions were closely related to those of 1Dy10 and 1Dx5, respectively. The amounts of R1 in Lwt flours determined by RP‐HPLC were 221 mg (ns) and 186 mg (s) per 100 g and those of R2 were 344 mg (ns) and 298 mg (s), respectively. These amounts increased to 240 mg (ns)/201 mg (s) (R1) and 479 mg (ns)/432 mg (s) (R2) in L10 flours. In L5+10 flours, the amount of R1 decreased to 150 mg (ns)/132 mg (s) while R2 increased to 432 mg (ns)/338 mg (s). The amount of HMW‐GS 1Dy10 was almost the same as that of R2 in L10 flours but was strongly increased in L5+10 flour (633 mg [ns]/538 mg [s]). HMW‐GS 1Dx5 was, by far, the major subunit in L5+10 flours (987 mg 7[ns]/896 mg [s]). The summarized amounts of all HMW subunits increased from ≈0.5 g (Lwt) to ≈1.1 g (L10) and ≈2.0 g (L5+10). Thus only L10 flours were similar to wheat flours in HMW subunit content. The baking performance of L10 flour determined by a microbaking test was improved compared with Lwt flour, whereas L5x10 flour showed very poor properties obviously due to the strongly increased proportion of highly cross‐linked glutelins. The breadmaking quality of flours from 1Dy10 seeds and wildtype seeds was reduced by the same degree when flours from sprouted seeds were analyzed.     

Physical Characteristics of Genetically Altered Wheat Related to Technological Protein Separation

Wheat protein is a technologically challenging substrate for food and nonfood applications because of its compositional diversity and susceptibility to denaturation. Genetic modification could be used to create cultivars capable of producing more uniform or focused and novel protein compositions targeted to nonfood uses. These lines could serve as expression systems for specific high‐molecular‐weight (HMW) protein polymers and would be new crops leading to more diverse agricultural opportunities. However, fundamental changes to the molecular architecture in such wheat seeds could also result in separation and processing issues, such that conventional methods of protein enrichment may need modification or even reinvention. Enriched gluten protein fractions were prepared from Bobwhite lines modified to overproduce HMW glutenin subunits Dx5 and/or Dy10. These lines serve as experimental models to test various approaches that may be taken for protein polymer enrichment. However, conventional wheat gluten enrichment based on the glutomatic as a small model of industrial methods was incapable of producing enrichment for any of the tested meal or flour, including that from the non‐transformed parent Bobwhite. Mixing in the mixograph or farinograph failed to produce standard patterns for whole kernel meal and straight‐run flour, and the normal cohesiveness of dough expected from these devices was not observed. Microscopy of stained dough samples revealed severely limited formation of normal protein networks, a capability crucial to conventional separation technology. Particle size analysis of whole kernel meal revealed a higher resistance to milling for the altered lines. Higher drying rates, lower farinograph moisture absorption, and increased thermal transition temperatures were observed. These data suggested that the native architecture of these new forms was more tightly constructed with reduced capacity for alteration by hydration and input of mechanical energy. An alternative enrichment method featuring solvation in SDS and precipitation in acetone produced coagulated (Bobwhite) or partially coagulated protein (transgenic lines producing Dx5 or Dy10) enriched to 78–85% protein with high yield.     

Quantitation of LMW‐GS to HMW‐GS Ratio in Wheat Flours

A comparison was made of methods for measuring the LMW/HMW glutenin subunit (GS) ratio for glutenin. A set of near‐isogenic wheat lines with the number of HMW‐GS varying from 0 to 5 was utilized to provide a wide range of LMW/HMW‐GS. Glutenin preparations were obtained from ground whole meal after solubilization of monomeric proteins by dimethyl sulfoxide (DMSO) or 50% propanol or by fraction collection from a preparative SE‐HPLC column. Analyses were made on the reduced glutenin from each of the three preparations by RP‐HPLC, SE‐HPLC, and SDS‐PAGE. Both solvents, DMSO and 50% propanol, extracted appreciable amounts of polymeric protein, thus casting some doubts on the accuracy of the determinations. This problem was largely avoided when the polymeric fraction was collected from the eluate of a total glutenin extract run on a preparative SE‐HPLC column. Less glutenin was removed by the two solvents for lines with a greater number of HMW‐GS or with strength‐associated HMW‐GS 5+10 coded by the 1D chromosome. Collection of the polymeric protein in SE‐HPLC, followed by separation of the glutenin subunits in RP‐HPLC, was the best method for quantitating the LMW/HMW‐GS ratio. SE‐HPLC gave a clear separation of the two groups of subunits as well as HMW albumins. RP‐HPLC has the potential advantage of being able to quantitate individual subunits.     

Quantitative Glutenin Composition from Gel Electrophoresis of Flour Mill Streams and Relationship to Breadmaking Quality

Three samples of Nekota (hard red winter wheat) were milled, and six mill streams were collected from each sample. The 18 mill streams were analyzed separately as well as recombined to form three patent flours. The methods of multistacking (MS)‐SDS‐PAGE and SDS‐PAGE were used to separate the unreduced SDS‐soluble glutenins and the total reduced proteins, respectively. The separated proteins were quantified by densitometry. The quantity of unreduced SDS‐soluble proteins was significantly different among the mill streams at the 4% (largest molecular weight polymeric glutenins) and at the 10 and 12% (smaller molecular weight polymeric glutenins) origins of the MS‐SDS‐PAGE gels. The quantities of total HMW‐GS, LMW‐GS, 2*, 7+9, and 5+10 subunits and the ratio of HMW‐GS to LMW‐GS in polymeric protein samples isolated using preparative MS‐SDS‐PAGE and in total reduced protein extracts were significantly different among mill streams. The quantities of HMW‐GS, LMW‐GS, 2*, 7+9, and 5+10 subunits from total reduced proteins were positively and significantly correlated with loaf volume. The quantities of glutenin subunits (both HMW‐GS and LMW‐GS) from unreduced SDS‐soluble proteins were positively or negatively correlated with loaf volume at the various MS‐SDS‐PAGE gel origins but the levels of correlation were not significant. These results showed that the glutenin protein composition was different among the various mill streams and demonstrated that electrophoretic analysis of the proteins in these fractions is a useful tool for studying the variation in functional properties of flour mill streams.     

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.     

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.     

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.     

Temperature‐Induced Changes in the Dynamic Rheological Behavior and Size Distribution of Polymeric Proteins for Glutens from Wheat Near‐Isogenic Lines Differing in HMW Glutenin Subunit Composition

Viscoelasticity of hydrated gluten depends on composition of HMW gluten subunits (GS), size distribution of glutenin polymers, and proteinprotein interactions. Glutens extracted from four near‐isogenic lines with differing HMW‐GS were analyzed. Rheological properties were studied by dynamic assay in shear. Size distribution of prolamins was determined by sequential extraction and size‐exclusion HPLC. Assays performed at 20°C confirmed that viscoelasticity was determined by large glutenin polymers. The abundance of large glutenin polymers depended on the HMW‐GS composition of the lines. Difference of functionality linked to subunit structure was highlighted by comparing the behaviors of the 1A/1B null and 1A/1D null lines. Glutens were submitted to heating and cooling cycles, with or without an SH‐blocking agent (N‐ethylmaleimide [NEMI]). At 20–40°C, no irreversible changes of the mechanical properties occurred. Thermal treatment affected chain mobility, and possibly H bonds, but not the chemical structure of the network. At >40°C, irreversible rheological changes were observed without NEMI. Irreversibility was mainly due to chemical modifications affecting the polymer size distribution through SH‐SS exchange reactions. The sensitivity of gluten to temperature depended on subunit composition.     

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.     

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.     

Protein and Quality Characterization of Complete and Partial Near‐Isogenic Lines of Waxy Wheat

The objective of this study was to evaluate protein composition and its effects on flour quality and physical dough test parameters using waxy wheat near‐isogenic lines. Partial waxy (single and double nulls) and waxy (null at all three waxy loci, Wx‐A1, Wx‐B1, and Wx‐D1) lines of N11 set (bread wheat) and Svevo (durum) were investigated. For protein composition, waxy wheats in this study had relatively lower albumins‐globulins than the hard winter wheat control. In the bread wheats (N11), dough strength as measured by mixograph peak dough development time (MDDT) (r = 0.75) and maximum resistance (R_max) (r = 0.70) was significantly correlated with unextractable polymeric protein (UPP), whereas in durum wheats, moderate correlation was observed (r = 0.73 and 0.59, respectively). This may be due to the presence of high molecular weight glutenin subunits (HMW‐GS) Dx2+Dy12 at the Glu‐D1 locus instead of Dx5+Dy10, which are associated with dough strength. Significant correlation of initial loaf volume (ILV) to flour polymeric protein (FPP) (r = 0.75) and flour protein (FP) (r = 0.63) was found in bread wheats, whereas in durum wheats, a weak correlation of ILV was observed with FP (r = 0.09) and FPP (r =0.51). Significant correlation of ILV with FPP in bread wheats and with % polymeric protein (PPP) (r = 0.75) in durum lines indicates that this aspect of end‐use functionality is influenced by FPP and PPP, respectively, in these waxy wheat lines. High ILV was observed with 100% waxy wheat flour alone and was not affected by 50% blending with bread wheat flour. However, dark color and poor crumb structure was observed with 100% waxy flour, which was unacceptable to consumers. As the amylopectin content of the starch increases, loaf expansion increases but the crumb structure becomes increasingly unstable and collapses.     

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.     

Immunocytochemical Localization of Gluten Proteins Uncovers Structural Organization of Glutenin Macropolymer

The content and composition of the disulfide‐bonded glutenin macropolymer has been shown to influence dough properties, although its structural organization is poorly characterized. The structure of the glutenin macropolymer in dough was studied using an immunolocalization transmission electron microscopy (TEM) technique by localizing gliadins, low molecular weight glutenin subunits (LMW‐GS), and high molecular weight glutenin subunits (HMW‐GS) in sections of dough using antibody probes selective for each of the three classes of gluten polypeptides. Distinct differences in the distribution patterns of gliadins, LMW‐GS, and HMW‐GS were observed, which suggests that proteins have different roles in the structural organization of the gluten matrix. On the basis of the observed distribution of the proteins in dough, it is speculated that gliadins, which are randomly distributed as individual particles, fill space within the glutenin macropolymer; LMW‐GS, which are present as clusters, are speculated to form aggregated branch structures; and HMW‐GS, which are present as chains, are speculated to form a network from which the LMW‐GS branches are formed. Changes in the distribution of gliadins, LMW‐GS, and HMW‐GS in dough during mixing were also noted. Such an arrangement supports previous biochemical evidence which has established that gliadins, LMW‐GS, and HMW‐GS have specific roles in the structural organization of the glutenin macropolymer in doughs.     

Accumulation of High‐Molecular‐Weight Glutenin Subunits in Superior and Inferior Grains of a Winter Wheat,Yangmai 158

The difference in accumulation of high‐molecular‐weight glutenin subunits (HMW‐GS) in superior (basal) and inferior (distal) grains results in the nonuniformity of grain quality in a winter wheat (Triticum aestivum L. ‘Yangmai 158’). The HMW‐GS accumulation and glutenin macropolymer (GMP) content were studied in superior and inferior grains during the grain‐filling period. Compared with inferior grains, HMW‐GS was formed earlier and total accumulation amount was higher in superior grains. The total HMW‐GS content was higher in superior grain than inferior grain, except at maturity. For individual HMW‐GS types, the accumulation and content of subunit 7 were the highest, followed by subunit 12, and those of subunit 8 were the lowest, followed by subunit 2 in superior grain. In contrast, the accumulation and content of subunit 7 at maturity were significantly higher than subunit 8 but similar between subunit 2 and subunit 12 in inferior grain. Moreover, the accumulation of subunit 7 and 12 in superior grain was significantly higher than in inferior grain. However, compared with the inferior grain, the GMP accumulation was higher but content was lower in superior grain at maturity.     

Interactions of Genotype and Glutenin Subunit Composition on Breadmaking Quality of Durum 1AS•1AL‐1DL Translocation Lines

Dual‐purpose durum (Triticum turgidum L. subsp. durum) wheat, having both good pasta and breadmaking quality, would be an advantage in the market. In this study, we evaluated the effects of genotype and varying HMW and LMW glutenin subunit composition on durum breadmaking quality. Genotypes included five near‐isogenic backgrounds that also differed by variability at the Glu‐D1d (HMW subunits 1Dx5+1Dy10), Glu‐B1 (presence or absence of subunit 1By8), and Glu‐B3 (LMWI or LMWII pattern) loci. Quality tests were conducted on genotypes grown at five North Dakota locations. Genotype had a stronger influence on free asparagine content than glutenin subunit composition. Genotypes carrying Glu‐D1d had higher glutenin content than lines that did not carry Glu‐D1d. Among Rugby translocation genotypes, lines carrying LMWI had higher gliadin content and better loaf volume than genotypes carrying LMWII. Absence of 1By8 produced major reductions in loaf volume in nontranslocation lines regardless of whether LMWI or LMWII was present. In contrast, the presence of Glu‐D1d compensated well for the absence of 1By8 regardless of which LMW pattern was present. The durum genotypes did not have loaf volumes equal to bread wheat cultivars, and results suggest that improved extensibility is needed to improve durum breadmaking quality.     

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.     

Role of Gluten and Its Components in Determining Durum Semolina Dough Viscoelastic Properties

Gluten was isolated from three durum wheat cultivars with a range in strength. Gluten was further fractionated to yield gliadin, glutenin and high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits (GS). The gluten and various fractions were used to enrich a base semolina. Enriched dough samples were prepared at a fixed protein content using a 2‐g micromixograph. Mixing strength increased with addition of gluten. Dynamic and creep compliance responses of doughs enriched with added gluten ranked in order according to the strength of the gluten source. Gliadin addition to dough resulted in weaker mixing curves. Gliadin was unable to form a network structure, having essentially no effect on dough compliance, but it did demonstrate its contribution to the viscous nature of dough (increased tan δ). Source of the gliadin made no difference in response of moduli or compliance. Addition of glutenin to the base semolina increased the overall dough strength properties. Glutenin source did influence both dynamic and compliance results, indicating there were qualitative differences in glutenin among the three cultivars. Enrichment with both HMW‐GS and LMW‐GS increased overall dough strength. Source of HMW‐GS did not affect compliance results; source of LMW‐GS, however, did have an effect. The LMW‐2 proteins strengthened dough to a greater extent than did LMW‐1. Mechanisms responsible for dough viscoelastic properties are described in terms of reversible physical cross‐links.     

Transgenic Enhancement of High‐Molecular‐Weight Glutenin Subunit 1Dy10 Concentration: Effects in Wheat Flour Blends and Sponge and Dough Baking

Dough strength is needed for efficient breadmaking quality. This property is strongly influenced in wheat (Triticum aestivum L.) by gluten seed storage proteins and, in particular, by high‐molecular‐weight (HMW) glutenin subunit composition. Experiments were designed to elevate expression of a key native HMW glutenin subunit (1Dy10) via genetic engineering and to determine whether resultant flours can be used in sponge and dough applications, the most common commercial bread‐baking procedure. Both unblended and blended samples from transgenic and nontransgenic sister lines were tested, with blended samples being formed by addition to a control sample. Dough properties, as determined by farinograph evaluation, were improved by the transgene‐encoded increases in 1Dy10 in both undiluted and blended flours. Mean farinograph stability of transgenic samples was twice that of the control, and blends with transgenic samples demonstrated increases in stabilities proportional to the amount of transgenic flour included. Mean farinograph quality numbers of transgenic samples, and of all blends containing transgenic flour, were significantly higher than both the control and all nontransgenic treatments. In the sponge and dough bake procedure, undiluted transgenic samples induced lower scores, relative to both control and undiluted nontransgenic samples, for water absorption, crumb body firmness, and loaf volume. In blends, however, the transgenic samples resulted in improvements in some sponge and dough loaf attributes, including loaf symmetry and crumb color score, without any concomitant loss of loaf volume in transgenic blends. These improved variables relate to finished product appearance and to consumer selection in markets. The use of transgenic flours with increased 1Dy10 glutenin content in commercial blends could provide advantages in sponge and dough bake applications.     

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.