High Molecular Weight Glutenin Research Articles

Effects of heat exposure from late sowing on agronomic traits and the technological quality of hexaploid wheat

Heat stress reduces wheat grain yield and affects quality traits relevant to trading and end use. In this study, 98 landraces and 152 elite hexaploid wheat varieties and breeding lines were trialled at two irrigated field sites in southern NSW to better understand heat stress responses and identify promising genotypes for heat tolerance breeding. Potential heat tolerance was defined using performance in heat-exposed late-sown (LS) plots relative to normal-sown (NS) plots, using a regression approach to calculate response indices uncorrelated with awn emergence time and performance per se in NS. The best individual responders for grain yield included genotypes from both Australia and Mexico. On average, lines selected in CIMMYT’s High Temperature Wheat Yield Trials (HTWYT) performed well for yield per se in NS as well as for yield response to LS. CIMMYT lines previously selected for large grains under heat stress conditions performed well for grain size in NS and for LS responses of grain size and test weight. Dough peak resistance and time to peak resistance in a mixograph were increased by LS and these traits were positively correlated with grain protein and the occurrence of the Glu-D1d high molecular weight glutenin locus allele, respectively.

The benefits of experimental breeding research genotypes for the genetic resources of spring durum wheat

AbstractOur study aimed to incorporate 60 newly acquired spring durum wheat genotypes into the Prague Gene Bank collection, focusing on 54 experimental breeding research genotypes and their technological quality contributions for conservation and future use. We analyzed a total of 11 field and 6 technological parameters of grain quality. Additionally, we identified the composition of high molecular weight glutenin subunits (HMW-GSs) in 13 accessions with contrasting technological characteristics. Two different extraction procedures were employed to detect HMW-GSs: one based on total protein extraction and the other on gliadin pre-extraction. The tested parameters exhibited a significant range of variability, with the relative standard deviation ranging from 2.1% for starch content to 96.7% for the gluten index. Additionally, six breeding research genotypes exhibited high technological grain quality comparable to the two modern durum wheat cultivars. Two accessions: M90-99–2 and IG 142076 showed above-average Zeleny sedimentation values, probably positively influenced by the presence of HMW-GSs A1: (2*). Genotypes PI 675012 and IG 142039 displayed the highest grain stability in technological parameters. Both extraction methods also detected the specific subunit B1: (6 + 8*) in the genotype IG 142039. Preserving these genotypes thus represents a substantial expansion of genetic resources in the genebank.

Manipulation of the post-flowering source/sink ratio differentially affects protein composition and gluten quality in contrasting bread wheat genotypes

Studies of the impact of assimilate availability during grain filling on protein composition and gluten quality of bread wheat are scarce. Therefore, this work aimed to analyze the responses of wet gluten percentage, SDS sedimentation index, and protein composition to changes in the source/sink (S/S) ratio (post-flowering growth per grain) in six Argentinean wheat genotypes contrasting in baking aptitude (three quality groups), as well as to evaluate its interaction with early nitrogen-sulfur fertilization. For two years, rainfed field trials were carried out in the Humid Pampas in Southern South America, applying manipulative S/S treatments (shading, defoliation, thinning, and trimming). Gluten percentage showed a negative linear trend against the increase in S/S ratio with differences among genotypes even within the same quality group (slope range: −0.0248 to −0.6566). The associations between the main protein fractions (i.e., GLI/GLU, HMW/LMW) with the S/S ratio were not significant, while the proportions of specific high molecular weight glutenin subunits were relevant, such as Glu-A1x/HMW (slope range: −0.00027 to −0.00147) and Glu-D1y/HMW (0.00029–0.00139). Early nitrogen-sulfur fertilization increased the influence of the S/S ratio on gluten percentage while stabilizing the sedimentation index. These results apport another approach, based on S/S ratio, to understanding the determination of grain quality under changing contexts.

Identification of RNAi hypoallergic bread wheat lines for wheat-dependent exercise-induced anaphylaxis patients.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is one of the most severe forms of wheat allergy. It occurs in patients when they exercise after ingesting wheat-containing foods. Nowadays, the only possible alternative for WDEIA patients is to avoid such foods. This study investigated the potential of six RNA of interference (RNAi) wheat lines with low-prolamin content as alternatives for WDEIA patients. For that purpose, a high performance-liquid chromatography (HPLC) analysis was performed to evaluate differences in gluten protein fractions among these lines. Next, western blots were conducted to measure the immunoglobulin E (IgE) reactivity to wheat proteins in sera from five WDEIA patients. Additionally, monoclonal antibodies (moAb) recognition sites and the IgE binding sites were searched in all peptides identified by LC-MS/MS after protein digestion. The results showed a 61.4%-81.2% reduction in the gliadin content of the RNAi lines, accompanied by an increase in their high-molecular weight (HMW) glutenin content compared to the wild type bread wheat line (WT). In all cases, the reduction in gliadin content correlated with a decrease in IgE reactivity observed in the sera of WDEIA patients, highlighting the E82 and H320 lines. These two RNAi lines exhibited a ≤90% reduction in IgE reactivity. This reduction could be attributed to an absence of IgE binding sites associated with α- and ω5-gliadins, which were present in the WT. Overall, these lines offer a potential alternative for foodstuff for individuals with WDEIA.

Unveiling the binding mechanism between starch granule-surface proteins and glutenins during dough mixing process

Starch granule-surface proteins (SGSPs) and gluten proteins can interact to influence dough quality. Despite their pronounced effects, the interaction mechanism remains insufficiently understood. Herein, the binding mechanism between SGSPs and different molecular weight glutenin was explored by experimental methods and single-molecule techniques. Our findings revealed that SGSPs promoted strong binding with both high and low molecular weight glutenin (HMW-GS and LMW-GS). When the ratio of SGSPs and LMW-GS increased to 2:1, the content of disulfide bond increased from 0.03 μmol/L to 0.21 μmol/L while the free sulfhydryl showed the opposite pattern, indicating a tightly packed composite protein structure. A combination of unbiased and steered molecular dynamics simulations further revealed that SGSPs exhibited a higher binding affinity towards LMW-GS in comparison to HMW-GS due to the contributions of solvation and specific interaction energies. This endows the SGSPs-LMW-GS complex with significantly enhanced stability at free state and much higher unbinding force upon stretching, substantially improving the mechanical quality of dough. These findings indicate that SGSPs predominantly affects the interactions between starch and gluten, which will shed light on the dough formation and its properties.

Nanopore Amplicon Sequencing Allows Rapid Identification of Glutenin Allelic Variants in a Wheat Collection

Genetic variation in high molecular weight glutenin (HMW-GS) genes is tightly linked with the breadmaking quality of wheat. Hundreds of different alleles have been identified in HMW-GS genes worldwide. Such huge variability makes it difficult to distinguish them using conventional genotyping methods (for example, SDS-PAGE, SNP detection, etc.). Here, we exploited the nanopore amplicon sequencing technique (Amplicon-Seq) to uncover genetic variants distributed along the full-length sequence of six HMW-GSs, including the promoter and protein-coding regions. We analyzed 23 wheat accessions for allelic variants of HMW-GSs using the Amplicon-Seq and SDS-PAGE methods. We obtained sufficient (>50×) target gene coverage by ONT reads in just one hour. Using the obtained data, we identified numerous single nucleotide polymorphisms and InDels in the protein coding and promoter regions. Moreover, Amplicon-Seq allowed for the identification of new alleles (Glu-A1x1-T) of the Glu-1Ax gene that could not be recognized by SDS-PAGE. Collectively, our results showed that Amplicon-Seq is a rapid, multiplexed, and efficient method for high-throughput genotyping of full-length genes in large and complex genomes. This opens new avenues for the assessment of target gene variation to select novel alleles and create unique combinations of desirable traits in plant breeding programs.

The Effect of High Molecular Weight Glutenin Subunit Encoded by Glu-B1k Allele on Bread-Making Quality of Near-Isogenic Lines of Bread Wheat

: In this study, the effect of the 22 glutenin subunit encoded by the Glu-B1k allele on the chromosome B on the quality of wheat was investigated. Nevzatbey and a genotype of Triticum aestivum L. subsp. sphaerococcum were used as parents and produced the near-isogenic lines (NILs) in the generation of BC4F3. Plant morphological traits and protein content, sedimentation volume, lactic acid solvent retention capacity (SRC), and glutenin swelling index (GSI) of the NILs were determined. The mean protein content of the NILs carrying 22 glutenin subunit was higher than that of the NILs carrying the 7+9 glutenin subunit (20.4% and 16.2%, respectively). In contrast, the NILs with 22 glutenin subunit had a lower sedimentation volume than those of the NILs with 7+9 glutenin subunits. The 22 glutenin subunit decreased the sedimentation volume from 19.47 to 13.49 mL. The average GSI value of the NILs carrying 7+9 glutenin subunits was higher than that of the NILs carrying 22 glutenin subunit (3.05 and 2.92). In conclusion, in this study we were able to detect a quality difference between NILs with 22 and 7+9 glutenin subunits in a small amount of samples. These findings suggest that glutenin subunit 22 may be associated with low gluten strength.

Study of Baking Quality and Determination of High Molecular Weight Glutenin Subunits (HMW-GS) of Local and Introduced Wheat Varieties

The baking qualities of 36 varieties of wheat are considered. Storage proteins — high molecular weight glutenin subunits (HMW-GS) — were studied. Electropherograms were processed in the GelPro Analyzer 4.0 computer program. The molecular weight of glutenin subunits was determined according to the method of G. Galili, M. G. Feldman using a marker variety. The quality of glutenin was assessed in points according to the method of O. Lukov. The bread was baked using the straight method, in accordance with the generally accepted method of laboratory baking; the fermentation of the dough and further baking were carried out on Domino and Arianna devices. As a result of the research, it was revealed that only 7 test samples (Askeran, Azemetli 95, Gonen, Gyrmyzy Gul, Nurlu99, Tale38, Ugur) with a null allele, having subunit 1 (Glu-A1a allele), have a fragment of 344 bp in size, in the remaining 29 samples encoded by the Glu-A1 locus, having subunit 2* (Glu-A1b allele), a fragment of 362 bp in size is amplified. The presence of a null allele does not always have a negative effect on baking quality. The reason for this remains to be determined.

Partial C-terminal truncation of Bx and Dy high molecular weight glutenin subunits after conserved aspartate

The composition of polymeric gluten proteins and interaction with non-covalently associated gliadins define the functional utility of a flour. This composition is dictated by the timing of cellular and genetic events in the plant and the developing seed. This interplay and its effect on gluten functionality is poorly understood and could provide a new opportunity for improving wheat quality in the future. Truncated proteoforms of high-molecular weight glutenin subunits (HMWGS) were observed for Bx and Dy subtypes across 49 Canadian bread wheat and 16 durum varieties. The relative abundances of these variants ranged from 7.5% to 37.7% and abundances of Bx and Dy variants were highly correlated (r2 = 0.85). HMWGS Bx7* and Dy10 were purified and the mass difference was shown to be from the loss of a C-terminal hexapeptide after a conserved aspartate residue. This aspartate was present at the ω-7 position in >90% of HMWGS accessions and the C-terminal sequence was highly conserved. Importantly, all HMWGS subtypes contain the same conserved sequences, but truncated variants were never observed for By, Dx and Ax HMWGS. The potential impact of these truncated HMWGS on gluten form and function is discussed.

Identification of new nucleotide sequences of the Glu-B1-1 gene encoding x-type glutenins in bread wheat.

Studies of the genetic base and polymorphism of bread wheat cultivars aimed at identifying alleles of genes associated with high baking and other economically valuable traits seem to be relevant, since bread wheat, along with all representatives of the Triticeae tribe, has a huge genetic potential for creating cultivars with high technological and rheological properties of grain flour. The aim of this study was sequencing and analysis of the nucleotide sequences of the Glu-B1-1 gene, and analysis of the predicted amino acid sequences of its protein product in three cultivars of bread wheat. Thus, in the course of genotyping cultivars and lines of bread wheat for the Glu-B1-1 gene, in the cultivars 'Avesta', 'Leningradka krupnozernaya' and line C-75094, previously undescribed changes in the size of amplifiable regions of the Glu-B1-1 gene for high-molecular weight glutenins were found. Comparative analysis of the nucleotide sequences of these genes with known sequences showed the presence of two deletions in 'Avesta' and C-75094 and the presence of seven single-nucleotide substitutions in 'Leningradka krupnozernaya'. Alignment of the predicted Glu-B1 amino acid sequences of the studied accessions and the standard cultivar carrying the Glu-B1-a allele showed that deletions in the amino acid sequences of 'Avesta' and C-75094 accessions are localized in the central domain of the protein and affect the amount of tri-, hexa-, and nonapeptides, and in 'Leningradka krupnozernaya', a decrease in GQQ and PGQGQQ by one unit was revealed. In addition, substitutions of five amino acids were found in 'Leningradka krupnozernaya'. Thus, we have found previously undescribed deletions and substitutions in the nucleotide sequences of the Glu-B1-1 gene for high-molecular-weight glutenins, which lead to changes in amino acid sequences in functionally important regions, namely, in the central domains of protein molecules. The identified mutations can be used for genotyping bread wheat cultivars.

Gluten subfractions of wheat storage proteins are affected by high night temperature during grain formation

AbstractGluten (gliadin + glutenin) protein in wheat flour is affected by high temperature (day and/or night) resulting in undesirable consequences on dough quality. A study was conducted with early and late‐maturing wheat genotypes, to assess the spatial (superior‐ central and inferior‐ apical and basal spikelets) variation in the composition of gluten subfractions in the developing ear under high night temperature (HNT). We hypothesised that protein content in the superior and inferior grains may show a differential quantitative and qualitative response to HNT. HNT resulted in a significant increase in protein content which exhibited a strong (r = −0.44*) negative correlation with sedimentation volume (SV) that determines baking quality. The late‐maturity genotypes were more responsive to HNT with changes in ω‐5 and γ gliadin subfractions of both superior and inferior spikelets, though a consistent trend was not established. The proportion of high molecular weight (HMW) glutenins increased, whereas low molecular weight (LMW) glutenins reduced in most of the genotypes under HNT. Both HMW and LMW glutenins revealed significant positive (r = 0.43* and r = 0.81***, respectively) correlation with SV. The expression analysis of genes for gluten subfractions showed a significant decrease in transcript abundance of α, ω‐5, γ, HMW, and LMW fractions under HNT.

Wheat DOF transcription factors TaSAD and WPBF regulate glutenin gene expression in cooperation with SPA.

Grain storage proteins (GSPs) quantity and composition determine the end-use value of wheat flour. GSPs consists of low-molecular-weight glutenins (LMW-GS), high-molecular-weight glutenins (HMW-GS) and gliadins. GSP gene expression is controlled by a complex network of DNA-protein and protein-protein interactions, which coordinate the tissue-specific protein expression during grain development. The regulatory network has been most extensively studied in barley, particularly the two transcription factors (TFs) of the DNA binding with One Finger (DOF) family, barley Prolamin-box Binding Factor (BPBF) and Scutellum and Aleurone-expressed DOF (SAD). They activate hordein synthesis by binding to the Prolamin box, a motif in the hordein promoter. The BPBF ortholog previously identified in wheat, WPBF, has a transcriptional activity in expression of some GSP genes. Here, the wheat ortholog of SAD, named TaSAD, was identified. The binding of TaSAD to GSP gene promoter sequences in vitro and its transcriptional activity in vivo were investigated. In electrophoretic mobility shift assays, recombinant TaSAD and WPBF proteins bound to cis-motifs like those located on HMW-GS and LMW-GS gene promoters known to bind DOF TFs. We showed by transient expression assays in wheat endosperms that TaSAD and WPBF activate GSP gene expression. Moreover, co-bombardment of Storage Protein Activator (SPA) with WPBF or TaSAD had an additive effect on the expression of GSP genes, possibly through conserved cooperative protein-protein interactions.

Evaluating Total Phenolic Content, Antioxidant Activity, High Molecular Weight Glutenin Subunits (HMW-GS), and Grain Yield Parameters of Cultivated Wheat and Hybrids

Evaluating Total Phenolic Content, Antioxidant Activity, High Molecular Weight Glutenin Subunits (HMW-GS), and Grain Yield Parameters of Cultivated Wheat and Hybrids

Effect of high molecular weight glutenin subunits on wheat quality properties, across a wide range of climates and environments in Iran.

The online version contains supplementary material available at 10.1007/s12298-023-01324-6.

Insights into the particle size, protein form, pasting and dough rheological properties of flour obtained from wheat grown at high and low altitude

SummaryFlours from four wheat varieties grown at high (~3180 m) and low altitude (~247 m) were compared for particle size distribution, protein composition (monomeric and polymeric), amino acids, pasting and dough rheological properties. High altitude wheat (HAW) flours showed greater proportion of large size particles and higher protein content, gluten, ash content and each category of gliadins and high molecular weight glutenins but revealed lower gluten index than low altitude wheat (LAW) flours. HAW showed higher extractable polymeric, unextractable monomeric proteins, pasting temperature, damaged starch and arabinoxylans while lower unextractable polymeric, extractable monomeric protein, paste viscosity, dough stability and development time than LAW flours. HAW flours from PBW725 and HD3086 showed higher concentration of total aromatic and lower of total sulphur amino acids than their counterpart LAW flour. Differences in flours characteristics of LAW and HAW were clearly due to differences in accumulation of protein, starch and arabinoxylans.

Effect of High-Molecular Weight Glutenin Subunits (HMW-GSs) on Gluten Polymerization during Biscuit Making: Insights from Experimental and Molecular Dynamics Simulation Study.

The effect of high-molecular weight glutenin subunits (HMW-GSs) on gluten polymerization during biscuit making was investigated using a set of HMW-GS deletion lines. Results showed that the deletion of HMW-GSs improved the biscuit quality compared with the wild type (WT), especially in x-type HMW-GS deletion lines. Slight gluten depolymerization was observed during dough mixing, while progressive gluten polymerization occurred during biscuit baking. The deletion of HMW-GSs suppressed the polymerization of glutenin and gliadin compared with the WT during biscuit baking, especially in x-type HMW-GS deletion lines. These actions resulted in less elevation of the intermolecular β-sheet and ordered α-helix and altering the disulfide (SS) conformation to a less stable conformation in HMW-GS deletion lines compared with the WT during baking. Molecular dynamics simulation analysis further demonstrated that x-type HMW-GSs had higher thermal stability compared with y-type HMW-GSs during heating.

Association of High and Low Molecular Weight Glutenin Subunits with Gluten Strength in Tetraploid Durum Wheat (Triticum turgidum spp. Durum L.)

The gluten strength and the composition of high- and low-molecular-weight glutenin subunits (HMWGSs and LMWGSs) of fifty-one durum wheat genotypes were evaluated using sodium dodecyl sulfate (SDS) sedimentation testing and SDS polyacrylamide gel electrophoresis (SDS-PAGE). This study examined the allelic variability and the composition of HMWGSs and LMWGSs in T. durum wheat genotypes. SDS-PAGE was proven to be a successful method for identifying HMWGS and LMWGS alleles and their importance in determining the dough quality. The evaluated durum wheat genotypes with HMWGS alleles 7+8, 7+9, 13+16, and 17+18 were highly correlated with improved dough strength. The genotypes containing the LMW-2 allele displayed stronger gluten than those with the LMW-1 allele. The comparative in silico analysis indicated that Glu-A1, Glu-B1, and Glu-B3 possessed a typical primary structure. The study also revealed that the lower content of glutamine, proline, glycine, and tyrosineand the higher content of serine and valine in the Glu-A1 and Glu-B1 glutenin subunits, and the higher cysteine residues in Glu-B1 and lower arginine, isoleucine, and leucine in the Glu-B3 glutenin, are associated with the suitability of durum wheat for pasta making and the suitability of bread wheat with good bread-making quality. The phylogeny analysis reported that both Glu-B1 and Glu-B3 had a closer evolutionary relationship in bread and durum wheat, while the Glu-A1 was highly distinct. The results of the current research may help breeders to manage the quality of durum wheat genotypes by exploiting the allelic variation in glutenin. Computational analysis showed the presence of higher proportions of glutamine, glycine, proline, serine, and tyrosine than the other residues in both HMWGSs and LMWGSs. Thus, durum wheat genotype selection according to the presence of a few protein components effectively distinguishes the strongest from the weakest types of gluten.

A Narrative Mini Review on Current Status of Hypoallergenic Wheat Development for IgE-Mediated Wheat Allergy, Wheat-Dependent Exercise-Induced Anaphylaxis

Immunoglobulin E (IgE)-mediated food allergies to wheat that develop after school age typically shows a type of wheat-dependent exercise-induced anaphylaxis (WDEIA). At present, avoidance of wheat products or postprandial rest after ingesting wheat is recommended for patients with WDEIA, depending on the severity of the allergy symptoms. ω5-Gliadin has been identified as the major allergen in WDEIA. In addition, α/β-, γ-, and ω1,2-gliadins, high and low molecular weight-glutenins, and a few water-soluble wheat proteins have been identified as IgE-binding allergens in a small proportion of patients with IgE-mediated wheat allergies. A variety of approaches have been manufactured to develop hypoallergenic wheat products that can be consumed by patients with IgE-mediated wheat allergies. In order to analyze such approaches, and to contribute to the further improvement, this study outlined the current status of these hypoallergenic wheat productions, including wheat lines with a reduced allergenicity that are mostly constructed for the patients sensitized to ω5-gliadin, hypoallergenic wheat by enzymic degradation/ion exchanger deamidation, and hypoallergenic wheat by thioredoxin treatment. The wheat products obtained by these approaches significantly reduced the reactivity of Serum IgE in wheat-allergic patients. However, either these were not effective on some populations of the patients, or low-level IgE-reactivity to some allergens of the products was observed in the patients. These results highlight some of the difficulties faced in creating hypoallergenic wheat products or hypoallergenic wheat lines through either traditional breeding or biotechnology approaches in developing hypoallergenic wheat completely safe for all the patients allergic to wheat.

Multiomics Based Association Mapping in Wheat Reveals Genetic Architecture of Quality and Allergenic Related Proteins.

Wheat is an important staple crop since its proteins contribute to human and animal nutrition and are important for its end-use quality. However, wheat proteins can also cause adverse human reactions for a large number of people. We performed a genome wide association study (GWAS) on 114 proteins quantified by LC-MS-based proteomics and expressed in an environmentally stable manner in 148 wheat cultivars with a heritability > 0.6. For 54 proteins, we detected quantitative trait loci (QTL) that exceeded the Bonferroni-corrected significance threshold and explained 17.3−84.5% of the genotypic variance. Proteins in the same family often clustered at a very close chromosomal position or the potential homeolog. Major QTLs were found for four well-known glutenin and gliadin subunits, and the QTL segregation pattern in the protein encoding the high molecular weight glutenin subunit Dx5 could be confirmed by SDS gel-electrophoresis. For nine potential allergenic proteins, large QTLs could be identified, and their measured allele frequencies open the possibility to select for low protein abundance by markers as long as their relevance for human health has been conclusively demonstrated. A potential allergen was introduced in the beginning of 1980s that may be linked to the cluster of resistance genes introgressed on chromosome 2AS from Triticum ventricosum. The reported sequence information for the 54 major QTLs can be used to design efficient markers for future wheat breeding.

BZIP transcription factors repress the expression of wheat (Triticum aestivum L.) high molecular weight glutenin subunit genes in vegetative tissues

High molecular weight glutenin subunits (HMW GS) represent an important fraction of endosperm-specific seed-storage proteins that provide elasticity to bread dough. Previously, the second cis-regulatory module (CRM2) was found to be one of the most conserved part of HMW GS promoters, which indicated its pre-eminent role in their gene regulation. Here, we observed that deletion of CRM2 from the promoters of the Bx7 and By8 HMW GS genes increased the leakage of their transient expression in wheat leaf tissue. The effect of a VP1, an Myb and an antisense bZIP transcription factor (TF)-binding site, potentially involved in endosperm-specific regulation within CRM2, was then studied. The activity of a Bx7 gene promoter containing a mutant CRM2 with altered VP1 and Myb TF-binding sites, but an intact bZIP TF-binding site, was similarly low to that of the wild type in leaves. Transactivation analysis and EMSA indicated the binding of TFs TabZIP34 and TabZIP115 to the Skn-1 motif GTCAT in CRM2 and the repression of Bx7 and By8 HMW GS gene promoter activity in leaves. TabZIP34 and TabZIP115 may be involved in the downregulation of HMW GS gene expression in vegetative tissues and early-stage endosperm as well its modulation during seed maturation.