Molecular characterization of two novel Glu-D1-encoded subunits from Chinese wheat (Triticum aestivum L.) landrace and functional properties of flours possessing the two novel subunits

The high molecular weight glutenin subunits (HMW-GS) can be used for wheat quality improvement. Two novel alleles (designated 1Dx1.5* and 1Dy12.2*, respectively) at the Glu-D1 locus were identified in the Chinese wheat landrace variety ‘Jiuquanjinbaoyin’ by comparison of subunit mobility with that previously identified in several standard hexaploid wheats. The 1Dx1.5* and 1Dy12.2* genes were isolated using the allele-specific PCR primers and the complete open reading frames (ORFs) were obtained. Allele 1Dx1.5* consists of 2487 bp encoding a mature protein of 827 amino acid residues, whereas 1Dy12.2* consists 1980 bp encoding 658 residues. Comparisons of amino acid sequences analysis showed that 1Dx1.5* had higher similarity with the HMW-GS isolated from the wheat related species (Aegilops tauchi Coss.) than from the bread wheat varieties (Triticum aestivum L.). The 1Dy12.2* amino acid sequence showed a generally similar to the 1Dy12* isolated from Chinese endemic wheats. Meanwhile, the dough properties of the lines expressing (null, 7+8, 1.5*+10), (null, 7+8, 2+12.2*) and (null, 7+8, 2+12), respectively, were measured by a Mixograph, which demonstrates that the alleles 1.5*+10 can be considered as having positive impact on dough strength when compared with the alleles 2+12. In addition, the subunits 2+12.2* also showed a greater impact on dough strength than 2+12.     

Characterization of a monoclonal antibody specific for HMW subunits of glutenin and its use to investigate glutenin polymers

A monoclonal antibody, IFRN 1602, has been developed to a synthetic peptide based on the sequence (94)GSVTCPQQV(101) of HMW subunit 1Dx5. The antibody bound strongly to the synthetic peptide based on the cognate sequence of HMW subunit 1Dx2 which contains a serine instead of a cysteine residue. However, it recognized the immunizing peptide by enzyme-linked immunosorbent assay (ELISA) only poorly, probably because the peptide exists as a disulfide-bonded dimer under the assay conditions. From immunoblotting studies against a wide range of wheat varieties, IFRN 1602 was shown to primarily recognize x-type HMW subunits of glutenin encoded on chromosomes 1A and 1D, cross-reacting weakly with the 1A and 1D y-type subunits. It did not bind to any of the 1B-encoded subunits. The Mab also recognized a small number of polypeptides of greater mobility than HMW subunits which were not visible on the stained gels and occurred only in the presence of specific 1A and 1D x-type HMW subunits. Such polypeptides were not present in a preparation of recombinant subunit 2, suggesting that they are modified forms of the subunits which arise in the seed perhaps by processing of the associated subunits. When used to probe partially reduced glutenin, IFRN 1602 bound to 1Dx5-1Dy10 dimers. As the Mab reacted primarily with Cys(97) of 1Dx5 in a reduced form, these data suggest that this residue is not involved in either intra- or intermolecular disulfide bond in the HMW subunit dimers. Thus, Cys(97) of 1Dx5 may be present in gluten in a reduced form, involved in intramolecular disulfide bonds, or linking of the HMW subunit dimers into larger polymers.     

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.     

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.     

Molecular characterization of a novel HMW-GS 1Dx5′ associated with good bread making quality (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) and the study of its unique inheritance

A novel 1Dx type high molecular weight glutenin subunit (HMW-GS) associated with good bread-making quality was identified in the bread wheat line W958. This glutenin subunit was designated as 1Dx5′ here for the same electrophoretic mobility as the traditional one 1Dx5 in the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. In this work, the 5′ flanking region, N-terminal as well as partial central repetitive domain of this allele was cloned and sequenced. Comparison of the amino acid sequence of 1Dx5′ with that of 1Dx5 and 1Dx2 showed the main difference being the substitution of one cysteine residue located in the central repetitive domain of 1Dx5 with serine residue in 1Dx5′ or an additional proline being inserted at the N-terminal of 1Dx5′ compared with 1Dx2. Allelic specific-polymerase chain reaction (AS-PCR) molecular markers discriminating the alleles of Glu-D1 locus were designed and applied to two F₂ segregation populations. Meanwhile, the dough properties of the F₂ population were measured and analyzed, showing that the 1Dx5′ subunit is as good as 1Dx5 and superior than 1Dx2 in terms of dough property. The result illustrated that the inter-chain hydrogen bonds formed between the subunit repetitive domains rather than the cysteine may play the main role in bread making quality. Notably, the novel 1Dx5′ have a great portion in the F₂ segregation population, suggesting the phenomenon of segregation distortion which could facilitate the introduction of 1Dx5’ into the wheat breeding program.     

Use of near-isogenic wheat lines to determine the glutenin composition and functionality requirements for flour tortillas

In wheat ( Triticum aestivum L), the synthesis of high molecular weight (HMW) glutenins (GS) is controlled by three heterologous genetic loci present on the long arms of group 1 wheat chromosomes. The loci Glu-A1, Glu-B1, and Glu-D1 and their allelic variants play important roles in the functional properties of wheat flour. This study focused on understanding the functionality of these protein subunits on tortilla quality. Near-isogenic wheat lines in which one or more of these loci were absent or deleted were used. Tortillas were prepared from each deletion line and the parent lines. The elimination of certain HMW-GS alleles alter distinct but critical aspects of tortilla quality such as diameter, shelf stability, and overall quality. Two deletion lines possessing HMW-GS 17 + 18 at Glu-B1 and deletions in Glu-A1 and Glu-D1 had significantly larger tortilla diameters, yet tortilla shelf life was compromised or unchanged from the parent lines used to develop the deletion lines or the commercial tortilla flour used as a control. Alternatively, a deletion line possessing Glu-A1 and Glu-D1 (HMW-GS 1, 5 + 10) and a deletion in Glu-B1 also significantly improved tortilla diameters. Whereas the increase in diameter was less than the line possessing only HMW-GS 17 + 18 at Glu-B1, the stability of the tortillas were, however, maintained and improved as compared to the parent lines containing a full compliment of HMW-GS. Thus, the presence of subunits 5 + 10 at Glu-D1 alone or in combination with subunit 1 at Glu-A1 appears to provide a compromise of improvement in dough extensibility for improved tortilla diameters while also providing sufficient gluten strength to maintain ideal shelf stability.     

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

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

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.     

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.     

小麦1Ax1和1Ax2*近等基因系性状差异的研究

以小麦转HMW-GS1Dx5+1Dy10基因获得的1Ax1和1Ax2*近等基因系08K860(HWM-GS组成为1,7+9,5+10)和08K871(HWM-GS亚基组成为2*,7+9,5+10)为材料,研究了近等基因系籽粒品质、粉质仪参数、拉伸仪参数和色度仪参数等方面的差异。研究结果表明,近等基因系08K860和08K871的上述性状差异很小,经统计分析差异未达显著水平。近等基因系08K860和08K871遗传背景相近,1Ax1和1Ax2*亚基对品质的贡献率相同。在小麦品质育种上应同样重视具有1Ax1亚基后代的选择。     

Novel and ancient HMW glutenin genes from Aegilops tauschii and their phylogenetic positions

A pair of novel high-molecular-weight glutenin subunits (HMW-GS) 1Dx3.1^t and 1Dy11*^t were revealed and characterized from Aegilops tauschii Coss. subspecies tauschii accession AS60. SDS-PAGE band of 1Dx3.1^t was between those of 1Dx2 and 1Dx3, while 1Dy11*^t was between 1Dy11 and 1Dy12. The lengths of 1Dx3.1 ^t and 1Dy11* ^t were 2,514?bp and 1,968?bp, encoding 836 and 654 amino acid residues, respectively. Their authenticity was confirmed by successful expression of the coding regions in Escherichia coli. Network analysis indicated that 1Dx3.1 ^t together with other five rare alleles only detected in Asia common wheat populations represented the ancestral sequences in Glu-D1 locus. Neighbor-joining tree analysis of previously cloned x-type and y-type alleles in the Glu-D1 locus supported the hypothesis that more than one Ae. tauschii genotypes were involved in the origin of hexaploid wheat and that different Ae. tauschii accessions contributed the D genome to common wheat and Ae. cylindrical Host, respectively. An Ae. tauschii accession with 1Dx3.1 ^t or a closely related allele probably have involved in the origin of common wheat. Since accession AS60 used in this study belonged to typical ssp. tauschii, present results suggested the possibility that ssp. tauschii was involved in the evolution of common wheat.     

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.     

Comparative study of the effect of incorporated individual wheat storage proteins on mixing properties of rice and wheat doughs

The aim of this work was to compare the effects of incorporated wheat storage proteins on the functional properties of rice and wheat flours. The advantage of rice as a base flour compared to wheat is that it does not contain any wheat flour components and, therefore, has no interactive effect between wheat glutenin proteins. The incorporation of individual HMW glutenin subunit proteins (Bx6, Bx7, and By8) in different ratios had significant positive effects on the mixing requirements of both rice and wheat doughs. Reconstitution experiments using two x+y type HMW-GS pairs together with a bacterially expressed LMW-GS have been also carried out in this study. The largest effects of polymer formation and mixing properties of rice flour dough were observed when Bx and By subunits were used in a 1:1 ratio and HMW and LMW glutenin subunits in a 1:3 ratio. However, using the same subunit ratios in wheat as the base flour, these synergistic effects were not observed.     

Depolymerization of the Glutenin Macropolymer During Mixing: II. Differences in Retention of Specific Glutenin Subunits

The depolymerization of individual high and low molecular weight (HMW and LMW, respectively) glutenin subunits (GS) from the glutenin macropolymer (GMP) in doughs during mixing was investigated by reversed-phase (RP) HPLC and SDS-PAGE. Cultivars with different dough strengths, as well as lines null for specific HMW-GS and biotypes differing at individual HMW-GS and LMW-GS encoding loci, were studied. During mixing, the proportion of total HMW-GS in GMP decreased, and the ratios of different subunits in the GMP in doughs changed. There was a loss of chromosome 1B- and 1D-encoded x-HMW-GS, while the relative proportions of y-HMW-GS (among HMW-GS) increased. Changes in 1B subunits occurred first, while most of the changes in 1D HMW-GS content occurred during dough breakdown. Changes were more pronounced for doughs of weak to average strengths than for stronger doughs. RP-HPLC analysis demonstrated a consistent increase in the retention times (surface hydrophobicity) of chromosome 1D-encoded HMW-GS but not of other HMW-GS or LMW-GS during mixing. SDS-PAGE and RP-HPLC demonstrated that specific B subunits, typically those with lower hydrophobicity, were selectively depolymerized from the GMP during dough breakdown, while the proportions of specific C subunits, typically those with greater hydrophobicity, increased. Similar trends were seen in analyses of several pairs of biotypes differing at single LMW-GS encoding loci, although there were slight differences in the depolymerization behavior of wheats with different allelic compositions. The results suggest that dough breakdown may be triggered by the loss of specific HMW-GS from the GMP, and a structural hierarchy may exist for different LMW-GS within glutenin in doughs.     

普通小麦背景中长穗偃麦草高分子量麦谷蛋白基因的表达、染色体定位与分子标记

摘要：以普通小麦（Triticum aestivum）中国春、长穗偃麦草?穴Thinopyrum elongatum ?雪及其双二倍体、二体异附加系、二体异代换系为材料，采用SDS-PAGE分析了种子高分子量麦谷蛋白亚基。长穗偃麦草高分子量麦谷蛋白基因在中国春背景中编码一条高分子量麦谷蛋白亚基，其迁移率与中国春1By8亚基相同，命名为1E8亚基，控制该亚基的基因位点Glu-E1位于长穗偃麦草E组染色体第一同源群的长臂上。用高分子量麦谷蛋白y亚基基因重复区域的特异引物进行扩增，长穗偃麦草1E8亚基编码基因（Glu-E1）扩增出1 300 bp的片段，而中国春1By8亚基编码基因（Glu-B1y）扩增出1 950 bp的片段。     

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.     

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.     

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.     

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.     

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.