High Molecular Weight Glutenin Research Articles

Evaluation of Baking Quality in Iranian Bread Wheat Cultivars using High Molecular Weight Glutenin Subunits

Evaluation of Baking Quality in Iranian Bread Wheat Cultivars using High Molecular Weight Glutenin Subunits

Molecular breeding of wheat lines for multiple rust and Fusarium head blight resistance

Bread wheat is a widely cultivated crop with production that is often negatively influenced by disease outbreaks. Biological constraints in production include the wheat rusts (leaf rust, stem rust and stripe rust) and Fusarium head blight (FHB). Different strategies are available in the control of these important fungal diseases. However, resistance breeding remains the preferred option of control as it lowers the risk of disease outbreaks and is a more cost effective and environmentally friendly approach. Many effective rust and FHB resistance genes have been discovered and successfully deployed through resistance breeding worldwide. However, some of these genes, when deployed singly, are vulnerable to evolving pathogens. In this study we aimed to develop wheat lines with the prospect of durable resistance against the rusts and FHB through combining eight resistance genes/quantitative trait loci; Lr19, Lr34/Yr18/Sr57/Pm38/Ltn1, Sr2/Yr30, Sr26, Sr39, Fhb1, Qfhs.ifa-5A-1 and Qfhs.ifa-5A-2 into a single wheat plant using marker-assisted selection. Cross and self-pollinated populations were developed to increase the frequency and homozygosity levels of resistance genes in progeny. Molecular markers were furthermore applied to determine the identity of the high molecular weight-glutenin subunits (HMW-GS) and to screen for the presence of the 1BL.1RS translocation in the final populations. We were successful in developing wheat plants containing complex sources of rust and FHB resistance and confirmed the presence of HMW-GS markers linked to strong dough strength and good bread making qualities in these genotypes.

The wPDI Redox Cycle Coupled Conformational Change of the Repetitive Domain of the HMW-GS 1Dx5-A Computational Study.

The repetitive sequence of glutenin plays an important role in dough rheology; however, its interaction with wheat protein disulfide isomerase (wPDI) remains unclear. In this study, the conformations of wild type glutenin repetitive sequence (WRS) from the high molecular weight glutenin subunit (HMW-GS) 1Dx5, an artificially designed glutenin repetitive sequence (DRS) of which the amino acid composition is the same but the primary structure is different, and wPDI under different redox states were simulated. The molecular interactions between the aforementioned repetitive sequences with wPDI under different redox states were further investigated. The results indicated that the repetitive sequences bind to the b and b′ domains of an “open”, oxidized wPDI (wPDIO) which serves as the acceptor state of substrate. The repetitive sequence is partially folded (compressed) in wPDIO, and is further folded in the thermodynamically favored, subsequent conformational transition of wPDIO to reduced wPDI (wPDIR). Compared with the artificially designed one, the naturally designed repetitive sequence is better recognized and more intensively folded by wPDI for its later unfold as the molecular basis of dough extension.

RNA interference targeting ω-secalin genes differentially affects the processing quality in a wheat T1BL·1RS translocation line

Wheat-rye T1BL·1RS translocation lines are widely used, especially in China, but their processing quality is generally poor. An interfering expression vector targeting the ω-secalin genes was constructed with the 1Bx7 seed-specific promoter. Biolistic-mediated genetic transformation of the wheat cultivar KN199 carrying the T1BL·1RS translocation generated 10 transgenic lines. Two representative transgenic lines, 8-2 and 13-7, were selected for analysis. Compared with the control, the two transformants showed an up to 4.5-fold decrease in total ω-secalins and various levels of decrease in ω-gliadins, γ-gliadins, and low-molecular-weight glutenins. A decrease in high molecular weight (HMW) glutenin 1Bx7 was detected only in 8-2, owing possibly to promoter methylation. Increased levels of α-gliadins were observed in both transformants, but increased levels of HMW glutenins were observed only in 13-7. Line 13-7 showed increases in gluten index, Zeleny sedimentation value, stabilization time, and maximum resistance. Its bread volume was 849.6 mL, an 11.9% increase over that of the control. Line 8-2 showed decreases in these parameters, but its total cake-making quality score was 88, an 17.3% increase over that of the control. The study demonstrates that the same RNAi construct may produce different effects on wheat processing quality and highlights the influence of the vector promoter in RNA interference.

Reduction of Allergenic Potential in Bread Wheat RNAi Transgenic Lines Silenced for CM3, CM16 and 0.28 ATI Genes

Although wheat is used worldwide as a staple food, it can give rise to adverse reactions, for which the triggering factors have not been identified yet. These reactions can be caused mainly by kernel proteins, both gluten and non-gluten proteins. Among these latter proteins, α-amylase/trypsin inhibitors (ATI) are involved in baker’s asthma and realistically in Non Celiac Wheat Sensitivity (NCWS). In this paper, we report characterization of three transgenic lines obtained from the bread wheat cultivar Bobwhite silenced by RNAi in the three ATI genes CM3, CM16 and 0.28. We have obtained transgenic lines showing an effective decrease in the activity of target genes that, although showing a higher trypsin inhibition as a pleiotropic effect, generate a lower reaction when tested with sera of patients allergic to wheat, accounting for the important role of the three target proteins in wheat allergies. Finally, these lines show unintended differences in high molecular weight glutenin subunits (HMW-GS) accumulation, involved in technological performances, but do not show differences in terms of yield. The development of new genotypes accumulating a lower amount of proteins potentially or effectively involved in allergies to wheat and NCWS, not only offers the possibility to use them as a basis for the production of varieties with a lower impact on adverse reaction, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has not been established yet.

Effects of 1Dy12 subunit silencing on seed storage protein accumulation and flour-processing quality in a common wheat somatic variation line

Dissecting the functions of high molecular weight glutenin subunits (HMW-GSs) is helpful for improving wheat quality via breeding. In this study, we used a wheat mutant AS273 in which HMW-GS 1Dy12 was silenced to investigate the silencing mechanism of 1Dy12 and its effects on gluten accumulation and flour-processing quality. Results suggested that the expression of 1Dy12 in AS273 was decreased by one fifth during grain development; a stop codon produced by a base mutation (C/T) led to truncated translation; the absence of 1Dy12 stimulated the accumulation of low molecular weight glutenin subunits (LMW-GSs), gliadins, and glutenin macropolymers, and was resulted in larger protein bodies; AS273 had an inferior flour-processing performance. Based on the outputs achieved in this study it is concluded that 1Dy12 makes important contributions to bread, sponge cake and biscuit-processing quality.

Changes in yield determinants and quality traits in wheat after 12 cycles of recurrent selection

The objective of this study was to test the genetic response to 12 cycles of a recurrent selection program for grain yield and related traits. During two seasons the 39 S-derived families (three/C0–C12 populations) were evaluated under conventional (CT) and non-tillage (NT) systems. Grain and biomass yield were determined. The grain number per square meter and harvest index were also estimated. Flour protein content, sodium dodecyl sulphate sedimentation, and lactic acid solvent retention capacity were considered as end-use quality predictive tests. High molecular weight glutenin subunits (HMW-GS) variation was also analyzed. A significant increase in grain yield per selection cycle, estimated in 3.25 g m−2 (1.3%) per year, was found under NT, but there was a not significant genetic progress under CT. The decrease in the percentage of protein in the most advanced selection cycles (11.0% vs. 9.7%) did not negatively affect its quality. HMW-GS variation analysis showed the erosion of extreme subunits related to gluten strength. After 24 years of recurrent selection, the grain yield was improved without affecting its industrial quality.

Assessment of the Glutenin Subunits Diversity in a Durum Wheat (T. turgidum ssp. durum) Collection from Morocco

Landraces and old wheat cultivars display great genetic variation and constitute a valuable resource for the improvement of modern varieties, especially in terms of quality. Gluten quality is one of the major determinants of wheat quality, and it is greatly influenced by variation in the high molecular weight and low molecular weight glutenin subunits (HMW-GS and LMW-GS). Identification of novel allelic variants for either of the two groups of the gluten-forming proteins could greatly assist in the improvement of wheat gluten quality. In the present study, the allelic composition of the HMW- and LMW-GS of ninety-five durum wheat accessions was evaluated. These accessions included Moroccan cultivars and landraces and North American cultivars and were all conserved in the National Gene Bank from Morocco. In total, 20 cataloged alleles and 12 novel alleles were detected. For the HMW-GS, two alleles were found at the Glu-A1 locus, and seven different allelic variants were identified at the Glu-B1 locus. Among them, two alleles were new (alleles Glu-B1cp and co). Additionally, two of the analyzed accessions exhibited the Glu-D1d allele, suggesting the presence of the Glu-D1 locus introgression. For the LWM-GS, eight, ten and two alleles were identified at the Glu-A3, Glu-B3 and Glu-B2 loci, respectively. Among them, two new allelic variants were identified at the Glu-A3 locus, and seven new allelic variants were identified at the Glu-B3 locus. Overall, the Moroccan landraces exhibited a greater genetic diversity and a greater number of glutenin alleles compared to the Moroccan and North American durum wheat cultivars. The novel germplasm and glutenin alleles detected in this study could contribute to the improvement of durum wheat quality and the expansion of modern durum wheat genetic diversity.

Quality differences between NILs of wheat variety Longmai 20 possessing HMW-GS 7OE + 8* and 17 + 18

In order to determine the genetic differences between high-molecular weight glutenin subunits (HMW-GS) 7OE + 8* and 17 + 18, HMW-GS 7OE + 8* from a Canadian extra-strong wheat cultivar Glenlea was introduced to Longmai 20 (1, 17 + 18, 5 + 10) by 6 consecutive backcrosses with biochemical marker-assisted selection. The near-isogenic lines (NILs) of Longmai 20 possessing HMW-GS 7OE + 8* and 17 + 18 were grown in the experimental field of the Crop Breeding Institute of Heilongjiang Academy of Agricultural Sciences from 2006 to 2009. The field experiments were conducted in a two-column contrast arrangement with four replicates. Compared with the NILs possessing subunits 17 + 18, the quality parameters of NILs with subunits 7OE + 8*, which include protein content, dry gluten content, ratio of wet gluten/dry gluten, ratio of Zeleny sedimentation/dry gluten, stability, maximum resistance and area of extensogram, increased by 1.5% (P = 0.06), − 0.7% (P = 0.47), − 1.9% (P < 0.01), 6.6% (P = 0.02), 11.2% (P = 0.6), 20.3% (P < 0.01) and 19.9% (P < 0.01), respectively. HMW-GS 7OE + 8* have a greater positive effect on the rheological properties of flour, such as the maximum resistance and area of extensogram, which are less influenced by environmental conditions compared to stability of Farinogram.

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Transfer of the ph1b gene of ‘Chinese Spring’ into a common wheat cultivar with excellent traits

In wheat/alien species hybrids, pairing homoeologous (Ph1) suppresses the pairing and recombination of wheat and alien chromosomes. Ph1b has been found to be the most effective allele in inducing meiotic homoeologous pairing/recombination and is widely used in gene introgression from wild Triticeae species into wheat. However, due to the poor agronomic traits of the ‘Chinese Spring’ ph1b (CS ph1b) mutant, which is more susceptible to disease, the application of CS ph1b is limited. In this study, we transferred the ph1b gene from the CS mutant to a commercial cultivar Shumai 126 (SM126), which has excellent agronomic traits, by crossing and continuous backcrossing. The results showed that the ph1b was present in 11 lines in the BC2F2 generation and was transferred to SM126. Further study indicated that seven homozygous ph1b mutant allele lines were identified from the BC2F3 generation. Two of these lines, SM126ph1b line 1 and 24, are homozygous at loci encoding high molecular weight glutenin subunits (HMW-GS) and the Wx protein, and the line 24 possesses the excellent agronomic traits of SM126. The results of meiotic metaphase I pairing showed that the SM126ph1b pollen mother cells (PMCs) are slightly higher pairing than the level of the original CSph1b stock. A progeny line of wild cross between wheat and Aegilops sharonensis was crossed with CSph1b and line 24 of SM126ph1b, respectively; the results of ND-FISH further proved that SM126ph1b and CSph1b have same effect on inducing homoeologous pairing/recombination, which will facilitate the use of wheat alien chromosome recombinations in wheat improvement.

SNP markers for low molecular glutenin subunits (LMW-GSs) at the Glu-A3 and Glu-B3 loci in bread wheat.

The content and composition of seed storage proteins is largely responsible for wheat end-use quality. They mainly consist of polymeric glutenins and monomeric gliadins. According to their electrophoretic mobility, gliadins and glutenins are subdivided into several fractions. Glutenins are classified as high molecular weight or low molecular weight glutenin subunits (HMW-GSs and LMW-GSs, respectively). LMW-GSs are encoded by multigene families located at the orthologous Glu-3 loci. We designed a set of 16 single-nucleotide polymorphism (SNP) markers that are able to detect SDS-PAGE alleles at the Glu-A3 and Glu-B3 loci. The SNP markers captured the diversity of alleles in 88 international reference lines and 27 Mexican cultivars, when compared to SDS-PAGE and STS markers, however, showed a slightly larger percent of multiple alleles, mainly for Glu-B3. SNP markers were then used to determine the Glu-1 and Glu-3 allele composition in 54 CIMMYT historical lines and demonstrated to be useful tool for breeding programs to improve wheat end product properties.

High‐molecular‐weight glutenin subunit compositions in current Chinese commercial wheat cultivars and the implication on Chinese wheat breeding for quality

AbstractBackground and objectivesGenetic diversity of high‐molecular‐weight (HMW) glutenin subunits is often used for wheat improvement because of its correlation with end‐use quality of wheat. The objectives were (a) to characterize and analyze the HMW‐GS composition of common wheat, (b) determine the diversity of high‐molecular‐weight glutenin subunits, and (c) elucidate the distribution characteristics of wheat good‐quality subunit and quality classification.FindingsThe results revealed a total of 15 different alleles (3 at Glu‐A1, 8 at Glu‐B1, and 4 at Glu‐D1) and 35 allele combinations. Glu‐A1c (57.92%), Glu‐A1a (62.94%), Glu‐A1c (55.86%) appeared to be the most frequent alleles at Glu‐A1 in Hebei, Henan, and Sichuan provinces, respectively. The Glu‐B1c was the most common allele at the Glu‐B1 locus in all three provinces, with frequencies of 48.75%, 55.24%, and 29.66%, respectively. Glu‐D1a (66.25%), Glu‐D1d (53.85%), Glu‐D1a (55.17%) at the Glu‐D1 locus appeared to be the common alleles in Hebei, Henan, and Sichuan provinces, respectively. Cluster analysis based on allelic similarity of Glu‐1 loci classified the 528 wheat cultivars into two major categories.ConclusionsThese results indicated that the HMW glutenin allele compositions are not optimal for the current varieties in these provinces. A high level of quality improvement may be implemented by increasing the frequencies of Glu‐A1a (1) or Glu‐A1b (2*), Glu‐B1i (17 + 18), Glu‐D1d (5 + 10).Significance and noveltyOur results revealed clear directions for wheat quality improvement in these provinces.

Characterization of High Molecular Weight Glutenin Subunits in Wild Emmer Wheat (Triticum dicoccoides)

Identification and molecular analysis of high molecular weight glutenin subunits (HMW-GS) of wild emmer wheat T. dicoccoides K5199 were carried out. Novel HMW glutenin subunits 1AxTd and 1ByTd were identified. Nucleotide sequences of 1AxTd and 1ByTd genes were detected (GenBank accession numbers MH475136 and MG897125, respectively). The coding sequence of 1AxTd was similar to 1Ax1 of T. aestivum (99.7%), while the 1ByTd gene sequence was similar to the genes of other T. dicoccoides samples (98.5 and 97.6%). Primary and secondary protein structures of the studied HMW-GS allowed for predicting a high contribution of the 1AxTd subunit to the breadmaking qualities and the average contribution of 1ByTd thereto. Evaluation of the most important grain quality traits identified increased protein and gluten content as well as reduced rheological properties of T. dicoccoides K5199 gluten.

Effect of extrusion on the polymerization of wheat glutenin and changes in the gluten network

The changes in the gluten network during extrusion treatment were studied by assessing the polymerization behavior of glutenin. Gluten samples were extruded at different barrel temperatures, screw speeds, and flow rates. The results indicated that high molecular weight glutenin subunits increased while free sulfhydryl groups and low molecular weight glutenin subunits decreased as the screw speeds and flow rates increased during extrusion treatment. Specific β-sheet structures of gluten clearly increased, while α-helices and β-turns fluctuated during extrusion processing, thus forming a tight gluten network. The characteristics of the protein network were evaluated by confocal laser scanning microscopy. The results showed that a homogeneous and denser gluten network was formed at higher extrusion temperatures during the extrusion process, which may be related to the polymerization of low-molecular-weight glutenin subunits. This study provides a theoretical basis for the improvement and regulation of extrusion quality during the gluten extrusion process.

Relationships of protein composition, gluten structure, and dough rheological properties with short biscuits quality of soft wheat varieties

AbstractUnderstanding the contribution of protein composition and gluten structure to dough property and biscuit baking is crucial to improve soft wheat (Triticum aestivum L.) grain quality. Thus, the relationships of protein composition, gluten structure, and dough rheological properties with short biscuits quality was investigated using 10 Chinese soft wheat varieties. Content of grain protein, flour protein, gliadin, glutenin, and high and low molecular weight glutenin subunits were significantly positively correlated with hardness of biscuit, but negatively correlated with spread factor and sensory score. Gluten of wheat varieties with lower dough strength and better biscuit quality showed thinner walls of apertures and more regular apertures in its microstructure. The gluten with higher strength had more apertures and antiparallel β‐sheets and fewer α‐helices. Moreover, dough with a higher softening degree was easier to stretch and tended to produce biscuits with larger diameter and crispier taste during baking. In conclusion, main wheat quality characteristics that can be used to select grain samples suitable for making tasty biscuits were recommended as follows: flour protein content, wet gluten content and microstructure, Zeleny sedimentation value, and dough softening degree.

Effects of species and breeding on wheat protein composition

Wheat proteins are characterized by their excellent contribution to technological and baking properties. However, wheat proteins, especially gluten and amylase-trypsin inhibitors (ATIs), are also known to be responsible for a broad range of intolerances and allergies. In order to evaluate the impact of genetic variability on the composition of these functional but immunogenic protein types, a set of different Triticum species, including common wheat, durum, spelt, emmer and einkorn, was examined regarding ATI and gluten concentrations by RP-HPLC. Additionally, inhibition towards trypsin was determined by an enzymatic assay. Based on the results, none of the investigated wheat species can be considered to be less ‘immunogenic’. Nevertheless, due to the large variability of ATI and gluten amounts among different genotypes, the selection of less-immunoreactive wheat varieties for individuals that suffer from wheat related diseases (WRDs) might be possible. The impact of breeding was assessed for different varieties of common wheat from different breeding periods. Results revealed significant improvement of technologically valuable parameters such as the amount of high molecular weight (HMW) glutenins and the gliadin to glutenin ratio, but no increase in immunogenic proteins.

Proteomic Analysis of Proteins Responsive to Drought and Low Temperature Stress in a Hard Red Spring Wheat Cultivar.

Drought stress is becoming more prevalent with global warming, and has been shown to have large effects on gluten proteins linked to wheat bread making quality. Likewise, low temperature stress can detrimentally affect proteins in wheat. This study was done to determine the differential abundance of high molecular weight (HMW) glutenin proteins in a drought and low temperature stressed high quality hard red spring wheat cultivar (PAN3478), against a control. The treatments were applied in the greenhouse at the soft dough stage. HMW glutenin proteins were extracted from the flour, and were separated by using two-dimensional gel electrophoresis. Protein spots that had p values lower than 0.05 and fold values equal to or greater than 1.2 were considered to be significantly differentially abundant. These proteins were further analyzed by using tandem mass spectrometry. There was a 1.3 to 1.8 fold change in 17 protein spots due to the cold treatment. The drought treatment caused a 1.3 to 3.8 fold change in 19 protein spots. These spots matched either HMW or low molecular weight (LMW) glutenin subunits. In the latter case, the C subunits of LMW glutenins were notably found to be up-regulated under both stress conditions. All the proteins that have been identified can directly influence dough characteristics. Data are available via ProteomeXchange with the identifier PXD017578.

Natural variants of α-gliadin peptides within wheat proteins with reduced toxicity in coeliac disease.

The only generally accepted treatment of coeliac disease (CD) is a lifelong gluten-free diet. Wheat gluten proteins include gliadins, low and high molecular weight glutenins. However, we have found significant structural variations within these protein families among different cultivars. To determine which structural motifs might be less toxic than others, we assessed five variants of α-gliadin immunodominant CD-toxic peptides synthesised as 16mers in CD T cell stimulation assays with gluten-sensitive T cell lines generated from duodenal biopsies from CD-affected individuals. The peptides harboured the overlapping T cell epitopes DQ 2.5-glia-α-2 and naturally occurring variants that differed in certain amino acids (AA). The results revealed that introduction of two selected AA substitutions in α-gliadin peptides reduced immunogenicity. A peptide with three AA substitutions involving two glutamic acids (E) and one glutamine residue (G) revealed the peptide was negative in 5:5 samples. We used CD small-intestinal organ culture to assess CD toxicity that revealed two peptides with selected substitution of both glutamic acid (E) and proline (P) residues abrogated evidence of CD toxicity.

Reducing the Immunogenic Potential of Wheat Flour: Silencing of Alpha Gliadin Genes in a U.S. Wheat Cultivar.

The alpha gliadins are a group of more than 20 proteins with very similar sequences that comprise about 15%–20% of the total flour protein and contribute to the functional properties of wheat flour dough. Some alpha gliadins also contain immunodominant epitopes that trigger celiac disease, a chronic autoimmune disease that affects approximately 1% of the worldwide population. In an attempt to reduce the immunogenic potential of wheat flour from the U.S. spring wheat cultivar Butte 86, RNA interference was used to silence a subset of alpha gliadin genes encoding proteins containing celiac disease epitopes. Two of the resulting transgenic lines were analyzed in detail by quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry. Although the RNA interference construct was designed to target only some alpha gliadin genes, all alpha gliadins were effectively silenced in the transgenic plants. In addition, some off-target silencing of high molecular weight glutenin subunits was detected in both transgenic lines. Compensatory effects were not observed within other gluten protein classes. Reactivities of IgG and IgA antibodies from a cohort of patients with celiac disease toward proteins from the transgenic lines were reduced significantly relative to the nontransgenic line. Both mixing properties and SDS sedimentation volumes suggested a decrease in dough strength in the transgenic lines when compared to the control. The data suggest that it will be difficult to selectively silence specific genes within families as complex as the wheat alpha gliadins. Nonetheless, it may be possible to reduce the immunogenic potential of the flour and still retain many of the functional properties essential for the utilization of wheat.