High Molecular Weight Glutenin Research Articles

The transcription factor CAMTA2 interacts with the histone acetyltransferase GCN5 and regulates grain weight in wheat.

Grain weight and size are major traits targeted in breeding to improve wheat (Triticum aestivum L.) yield. Here, we find that the histone acetyltransferase GENERAL CONTROL NONDEREPRESSIBLE 5 (GCN5) physically interacts with the calmodulin-binding transcription factor CAMTA2 and regulates wheat grain size and weight. gcn5 mutant grains were smaller and contained less starch. GCN5 promoted the expression of the starch biosynthesis genes SUCROSE SYNTHASE 2 (Sus2) and STARCH-BRANCHING ENZYME Ic (SBEIc) by regulating H3K9ac and H3K14ac levels in their promoters. Moreover, immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CAMTA2 physically interacts with GCN5. The CAMTA2-GCN5 complex activated Sus2 and SBEIc by directly binding to their promoters and depositing H3K9ac and H3K14ac marks during wheat endosperm development. camta2 knockout mutants exhibited similar phenotypes to gcn5 mutants, including smaller grains that contained less starch. In gcn5 mutants, transcripts of high molecular weight (HMW) Glutenin (Glu) genes were downregulated, leading to reduced HMW glutenin protein levels, gluten content, and sodium dodecyl sulfate (SDS) sedimentation volume. However, the association of GCN5 with Glu genes was independent of CAMTA2, since GCN5 enrichment on Glu promoters was unchanged in camta2 knockouts. Finally, we identified a CAMTA2-AH3 elite allele that corresponded with enhanced grain size and weight, serving as a candidate gene for breeding wheat varieties with improved grain weight.

Selection of marker systems for determining the allelic composition of high molecular glutenin loci in winter wheat

Aim. Selection of marker systems and conducting multiplex polymerase chain reactions to determine the allelic composition of the Glu-A1, Glu-B1 and Glu-D1 loci, which determine the high quality of flour in common wheat samples. Methods. Polymerase chain reaction (PCR) using specific primers for the Glu-A1, Glu-B1 and Glu-D1 loci of wheat. Results. PCR analysis was performed to detect the insertion at 43 b. p. in the MAR region, in varieties with the Glu-B1al allele, the presence of an amplicon 563 b. p. long was observed, for varieties that carry other alleles of the locus – an amplicon 520 b. p. long. The results on the Glu-D1 locus showed that for № 2, № 3, № 4, № 7, № 8, № 10, № 11, № 19, № 20, № 21 subunits (5 + 10) were identified. Conclusions. With the help of allele-specific primers, the allelic composition of loci of high molecular weight glutenins was determined in 22 lines wheat. Three alleles Glu-A1a, Glu-A1b, Glu-A1c were found in the Glu-A1 locus; in the Glu-B1 locus, only the Glu-B1al allele was detected, there are two alleles at the Glu-D1 locus: Glu-D1a and Glu-D1d (the Glu-D1a allele predominated). As a result of our research, samples of soft wheat (lines № 2, № 7, № 21) were selected, which can be attributed to genotypes with high baking quality of the flour.

Molecular Mechanism of Egg White Protein for Strengthening the Cross‐Linking Properties of Heat‐Induced Wheat Gluten Gel

AbstractThe chemical interaction between egg white protein (EWP) and wheat gluten (WG) is significantly influenced by the amount of EWP added. Therefore, the effects of EWP addition on the gelling properties and chemical interactions of heat‐induced WG–EWP gels are thoroughly examined. The results demonstrate that the enhancement in gel strength of WG–EWP gels is positively correlated with the amount of EWP added. EWP addition improves protein–protein interactions by reducing the freedom of water. The enhanced aggregation between WG and EWP is likely due to a decrease in surface hydrophobicity and an increase in β‐sheet content. EWP enhances cross‐linking with low molecular weight glutenin subunit (LMW‐GS), high molecular weight glutenin subunit (HMW‐GS), and gliadin (Gli). Specifically, EWP primarily cross‐links with ω‐, α‐, and γ‐Glis through S–S bonds and interacts with GS through hydrophobic interactions and S–S bonds. This study provides a theoretical foundation for improving the WG network structure in wheat‐based food production.

Selection and characterisation of DNA aptamer targeting immunogenic peptide sequence of high molecular weight glutenin (HMW-GS) of gluten

Selection and characterisation of DNA aptamer targeting immunogenic peptide sequence of high molecular weight glutenin (HMW-GS) of gluten

Image-assisted quantification of high and low molecular weight glutenin fractions in wheat by SDS-PAGE

Gluten quality in bread wheat is mainly determined by gluten strength and protein content. Traditionally gluten quality has been predicted based only on allelic variation for HMW-GS characterized by SDS-PAGE- The ratio of high to low molecular weight glutenin subunits has received less attention despite its influence on gluten properties is acknowledge. That is mainly because accurate quantification of glutenin fractions is made by RP-HPLC, a more complex, expensive and time-consuming technology. In the present study, a methodology to estimate HMW/LMW glutenin subunit ratio by SDS-PAGE results was assessed. We used a segregating population derived from a cross between two Spanish bread wheat landraces (Richela Blanca and Jeja Pardilla) showing striking differences according to SDSS tests. Quantification of prolamins by RP-HPLC confirmed a wide range of variation in the HMW/LMW glutenin subunit ratio within the population. Then, we selected 22 lines that represented the available variation. Subsequently, we obtained high-quality SDS-PAGE images following a standard protocol to characterize glutenin allelic variants. Estimation of HMW/LMW-GS ratio was obtained by analyzing the intensity of HMW-GS and LMW-GS gel bands. Correlation between RP-HPLC and SDS-PAGE values was greater than 0.85 proving the suitability of the much simpler and cheaper electrophoretic-based alternative.

Development of a germplasm master set covering variability of high molecular weight glutenin subunits for the GLU-A1 locus in Triticum sp.

Gluten quality is an important characteristic of wheat. Gluten is a viscoelastic protein network that is formed after hydrating and mixing wheat flour. This protein network is mainly composed of glutenins (High and Low Molecular Weight Glutenins (HMW-GS and LMW-GS, respectively)) and gliadins. HMW-GS are codified by the loci GLU-A1, GLU-B1 and GLU-D1 in common wheat. For the GLU-A1 locus, many alleles have been described and collected in the Wheat Gene Catalogue (WGC). Many of those alleles have not been compared in conjunction and it is not possible to know if the current GLU-A1 variability described is real or it is overestimated. This study has focused on collecting all the germplasm associated with GLU-A1 variability, to verify the GLU-A1 allele of each accession and if the current variability described in the WGC is real. It was possible to collect and compare by SDS-PAGE most of the germplasm described in the WGC. The identity of many alleles was confirmed while there are other cases that should be reviewed in more detail. This study contributes to the correct classification of the GLU-A1 diversity and will promote the use of different alleles in future breeding programs aiming to improve gluten quality.

Genome wide association mapping of end-use gluten properties in bread wheat landraces (Triticum aestivum L.)

Numerous studies have aimed to dissect the genetic basis of wheat end-use quality, yet the consideration of gluten aggregation properties in this context has been limited despite its important role in determining the processing quality. This study has addressed the evaluation of the end-use quality of a collection of 189 Spanish bread wheat (Triticum aestivum L.) landraces. For phenotyping, we utilized the GlutoPeak methodology to assess gluten aggregation traits, the SDS-sedimentation test to measure gluten strength and NIR for grain protein content. In addition to previous genotyping data with numerous DArT-seq markers distributed across the entire wheat genome, we developed ten KASP-based molecular markers designed for GLU-1 loci, which encode high molecular weight glutenins (HMW-GS). The reliability of these markers was thoroughly assessed. Our genome-wide association analysis (GWAS) focused on gluten properties and protein content, revealed 89 marker-trait-associated loci (MTAs). These loci were further grouped into 50 MTA-QTLs across various chromosomes based on linkage disequilibrium. While some MTA-QTLs overlapped with regions identified in other studies, others represented novel genomic regions. Inside these regions, we identified several candidate genes with gluten quality-related annotated functions or grain-specific expression patterns, which represent potential targets for marker development and future wheat end-use quality improvement.

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