HMW Glutenin Research Articles

Allelic variation of the HMW glutenin subunits in Aegilops tauschii accessions detected by sodium dodecyl sulphate (SDS-PAGE), acid polyacrylamide gel (A-PAGE) and capillary electrophoresis

Variability of high molecular weight glutenin subunits (HMW-GS) was studied in198 accessions of Ae. Tauschii (2n=2x=14, DD) by sodium dodecyl sulphate(SDS-PAGE) and acid polyacrylamide gel electrophoresis (A-PAGE) and capillary electrophoresis (CE). A high allelic variation of HMW-GS, including some novel x- and y-type subunits and variable subunit combinations were observed. One accession(TD159) showed a x-type null form. The results by A-PAGE analysis revealed that the subunits Dx5 t and Dy10 t encoded by Glu-D t 1 locus in Ae. tauschii were different in relative mobilities in comparison with the subunits Dx5 and Dy10 found in bread wheats, whereas they had the same mobilities, respectively, when separated by SDS-PAGE. The higher resolution of Ae. tauschii HMW-GS separated by CE method showed two clear peaks in accordance with x- and y-type subunits, respectively,except the accession TD151 which possessed only subunit Dy12.1*t. The electro elution time of the x-type and y-type subunits were about 13–14 and 7–8minutes, respectively. Characterization of wheat HMW-GS was facilitated by using CE which provides high resolution and increases the speed of analysis in conjunction with the traditional gel electrophoretic methods. A total of 42HMW-GS alleles were identified, among which were several alleles not presently detected in bread wheats. Hence Ae. tauschii is potentially a valuable genetic resource for quality improvement of bread wheat.

Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit (HMW-GS) genes in winter wheat

Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit (HMW-GS) genes in winter wheat

Functional properties of flours from field grown transgenic wheat lines expressing the HMW glutenin subunit 1Ax1 and 1Dx5 genes

The high molecular weight glutenin subunits (HMW-GS) of wheat are major determinants of the viscoelastic properties of gluten and dough. The bread making quality of field grown transgenic lines of bread wheat expressing the HMW-GS 1Ax1 or 1Dx5 genes were evaluated over a two year period. Subunit 1Ax1 represented about 29% and 48% of the total HMW-GS in lines 1-2 and 2-2, respectively, while subunit 1Dx5 represented 65.4% and 62% of the total HMW-GS in transgenic lines 6-2 and 9, respectively. The expression of subunits 1Ax1 or 1Dx5 in transgenic wheat led to corresponding decreases in the proportions of endogenous HMW-GS. HMW-GS 1Ax1 and 1Dx5 had contrasting effects on dough quality determined by the Alveograph and sedimentation test. Subunit 1Ax1 increased the tenacity (P), extensibility (L), deformation work (W), and sedimentation value, with the increase being related to the level of expression. In contrast, subunit 1Dx5 led to a smaller increment in the tenacity (P), but to drastic decrease in both extensibility (L), deformation work (W), and the sedimentation value. Expression of subunit 1Ax1 in transgenic wheat resulted in lines with improved rheological properties whereas the lines expressing subunit 1Dx5 resulted in unsuitable breadmaking-related characteristics.

Quantitative Variation of HMW Glutenin Subunits from Hard Red Spring Wheats Grown in Different Environments

ABSTRACTThe objective of this study was to investigate the quantitative variation of HMW glutenin subunits in relation to glutenin polymers and hence breadmaking quality across different environments. Six genotypes of hard red spring (HRS) wheat were grown at seven locations in North Dakota in 1998 in a randomized complete‐block experimental design with three replicates at each location. Unreduced SDS‐soluble glutenins of flour were fractionated by multistacking SDS‐PAGE into different sized glutenin polymers, followed by SDS‐PAGE and imaging densitometry to determine the quantitative variation of HMW glutenin subunits. SDS‐insoluble glutenin polymers also were examined for their quantitative composition of HMW glutenin subunits. The results showed that the percentage of HMW glutenin subunits was significantly affected by growing locations. The quantity of HMW glutenin subunits in SDS‐insoluble glutenins was significantly and positively correlated with loaf volume. SDS‐insoluble glutenin polymers had a higher percentage of HMW glutenin subunits than did SDS‐soluble glutenins. SDS‐insoluble glutenin polymers in flour were positively and significantly correlated in proportions of both total and individual HMW glutenin subunits in total SDS glutenins. SDS‐insoluble glutenin polymers also were positively and significantly correlated with the combined proportion of HMW glutenin subunits 2* + 5. The results of this study indicated that either subunit 2* or 5 might be more important in forming a greater quantity of larger SDS‐insoluble glutenin polymers than other subunits. SDS‐insoluble glutenin polymers from different cultivars or locations could have different quantities of HMW glutenin subunits in their composition. SDS‐insoluble glutenin polymers with more HMW glutenin subunits might be larger sized than those with less HMW glutenin subunits. Environment significantly influenced the quantitative variation of HMW glutenin subunits, which in turn affected the size distribution of glutenin polymers, and hence breadmaking quality.

Effects of Different Salts on Mixing and Extension Parameters on a Diverse Group of Wheat Cultivars Using 2‐g Mixograph and Extensigraph Methods

ABSTRACTCations of differing chaotropic capacities (LiCl, NaCl, and KCl) were used in small‐scale mixing and extensigraph studies to assess functional changes in dough behavior of wheat cultivars varying in total protein content and HMW glutenin composition. Salt addition, regardless of cationic type, caused an increase in dough strength and stability. The smaller (hydrated) and least chaotrophic cations (Li+<Na+<K+) effected the greatest increase in mixing time (MT) and resistance to extension (Rmax) and produced the most stable resistance breakdown (RBD). The effects of different cations on mixing and extensions indicated strong intercultivar variation; differential responses to salt addition were further shown when the cultivars were grouped according to protein content and Glu‐1D or Glu‐1B genome composition. Increases in dough strength parameters due to the addition of salt were consistently more significant for cultivars showing an overexpression of Bx7 (>12% protein). In the absence of genotypic variation, a significant interactive effect of cultivar type, protein amount, and salt addition was found for all functional dough parameters except extensibility. During mixing, there was a decrease in the amount of apparent unextractable polymeric protein (%UPP) in the dough. This phenomenon was ameliorated by the presence of salt in doughs formed from weaker flours and was most pronounced early on in the mixing process (t = 100–200 sec). Results show the importance of refining 2‐g mixograph studies to include salt in the “flour and water” dough formula.

A Novel y-type High Mr Glutenin Subunit (12·4t) Present in Triticum tauschii

Abstract A large collection of Triticum tauschii accessions was previously characterised according to their high and low molecular weight glutenin subunit composition. As a result, one accession was identified as having a subunit with unusual electrophoretic mobility in SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). This paper describes further analyses of this protein, named 12.4t. RP-HPLC (reversed phase-high performance liquid chromatography) studies enabled this protein to be classified as a high molecular weight (HMV) glutenin subunit of relatively low molecular weight. A more detailed characterisation of this protein by N-terminal sequence analysis was performed, as well as the molecular characterisation of its encoding gene. Deletions of blocks of the repetitive motifs, probably caused by unequal crossing-over, may have given rise to this particularly small y-type HMW glutenin subunit.

Molecular Characterization of Glutenins in Wheat Varieties Differing in Chapati Quality Characteristics

Five wheat (Triticum aestivum) varieties differing in chapati quality characteristics viz. C-306, K-68, HD-2745 and HD-2735 with good and Sonalika with poor chapati quality characteristics, were selected for the characterization or distribution of glutenin genes. Polymorphism was observed when genomic DNA of wheat varieties was hybridized with a HMW glutenin probe [glutenin subunit 10 (Dy10)]. No hybridization was observed in Sonalika. PCR amplification of genomic DNA with the LMW glutenin gene-specific primers did not show any polymorphism. However, with HMW glutenin gene-specific primers a single band of ∼ 650 by was obtained in all the good chapati characteristic wheat varieties.The amplified fragment was sequenced and found to have sequence homology with HMW glutenin subunit Dx5.The deduced protein structure analysis showed that the peptide was made up of N-terminally placed (x-helices and centrally placed repetitive β-turns.

Variation in HMW glutenin subunits of different species of wheat

Method ISTA SDS-PAGE was used for separation, detection and evaluation of high molecular weight glutenin subunits (HMW) in the different wheat species. The relation has been studied between the HMW glutenin subunit alleles and the bread-making quality of 25 world wheat cultivars and 21 regional varieties common wheat varieties (Triticum aestivum L.), 17 winter spelt wheat (Triticum spelta L.), 3 durum wheat cultivars (Triticum durum DESF.), 9 cultivars of Triticum turgidum L. and 5 cultivars of Triticum polonicum L. The highest frequency of occurrence of HMW glutenin subunits 2*, 13 + 16 and 5 + 10 were found in world wheat cultivars. In Slovak wheat varieties were analysed subunits 0, 7 + 9 and 5 + 10, 2 + 12. The HMW subunits 0, 7 + 8 with Glu-score 4 were determined in Triticum durum DESF. Three electrophoretical profile groups of different HMW glutenin subunits were found in Triticum turgidum L. and Triticum polonicum L. and six electrophoretical profile groups were determined in Triticum spelta L. The verified correlations between bread-making quality and specific HMW subunits of glutenin can be utilised by wheat breeders using SDS-PAGE of proteins as a screening test for the prediction of bread-making quality of wheat.

Allelic variation of HMW and LMW glutenin subunits, HMW secalin subunits and 75K gamma-secalins of hexaploid triticale

Although the endosperm storage protein of hexaploid triticale have alreadybeen analysed, the allelic diversity of glutenins and secalins remains to bedescribed. Analysis, by SDS-PAGE, of about one thousand F2 seeds fromten triticale crosses allowed the inheritance of these storage proteins to bestudied in order to determine their allelic forms and to assign them toparticular chromosomes. Two new alleles encoding HMW subunits ofglutenin and five new alleles encoding HMW subunits of secalin weredetermined at Glu-B1 and Glu-R1 loci respectively. In additionto the three allelic forms of 75K gamma-secalins encoded at Gli-R2and previously reported, a null form was found. A nomenclature for theseproteins and their corresponding alleles was suggested. The composition ofB-LMW glutenin subunits of hexaploid triticale was described and allelicforms were determined: Five alleles were encoded at Glu-A3 andseven at Glu-B3 including one and two new allelic formsrespectively.

Quality (End-Use) Improvement in Wheat

Abstract Wheat provides nutrients and the raw materials for industrialized food production. Recent global economic trends and increases in urban population growth have led to an increased demand for wheat-based convenience foods (fast, ready-to-eat, frozen foods, etc.) and for new wheat-based products. These factors have resulted in a greater emphasis than ever on the end-use quality of wheat. This paper reviews grain compositional aspects influencing the processing and quality attributes of the main foods produced with wheat, as well as the breeding strategies and methodologies used to achieve germplasm with desirable end-use quality. Common wheat (Triticum aestivum) is used in bread (leavened, flat, and steamed), noodles, biscuits, and cakes. Durum wheat (T. turgidum L. var. durum) is used globally in alimentary pasta and regional foods (flat breads, couscous, and burghoul) in North Africa and West Asia. Grain characteristics (grain hardness, protein content/quality, enzymatic activity, etc.) play a moderate to important role in the processing and end-use quality of wheat-based products. Among these, gluten strength and extensibility, which are determined by glutenin (HMW and LMW) and gliadin composition, are two of the main factors that determine quality. The complex and generally additive nature of inheritance of most quality traits has led to the development of several indirect tests used in early and advanced generations to increase the frequency of high yielding lines with desirable quality attributes. Additionally, characterization of HMW and LMW glutenins and gliadins allows breeders to combine protein content and quality more effectively. The use of molecular-marker-assisted selection and genetic transformation is expected to accelerate the tailoring of new wheat varieties to meet specific end-use quality requirements. Accumulating desirable quality genes will help reduce genotype X environment effects on quality-presently among the major challenges confronting breeders.

220 Identification of a novel HMW glutenin subunit and compari-son of its amino acid sequence with those of homologous sub-units

Aegilops tauschii is the donor of the D genome of common wheat (Triticum aestivum). Genetic variation of HMW glutenin subunits encoded by the Glu-1Dtlocus of Ae. tauschii has been found to be higher than that specified by the Glu-1D locus in common wheat. In the present note, we report the identification of a novel HMW glutenin subunit, Dy13t, from Ae. tauschii. The newly identified subunit possessed an electrophoretic mobility that was faster than that of the Dyl2 subunit of common wheat. The complete ORF of encoding the Dy13t subunit contained 624 codons (excluding the stop codons). The amino acid sequence deduced from the Dy13tgene ORF was the shortest among those of the previously reported subunits derived by the D genome. A further comparison of Dy13t amino acid sequence with those of the subunits characterized from the A, B, D, R genomes of Triticeae showed that the smaller size of the Dy13t subunit was associated with a reduction in the size of its repetitive domain.

Characterisation of high- and low-molecular weight glutenin subunits associated to the D genome of Aegilops tauschii in a collection of synthetic hexaploid wheats

Synthetic hexaploid wheats (2n=6x=42, AABBDD) involving genomes from Triticum turgidum (2n= 4x=28, AABB) and Aegilops tauschii (2n=2x=14, DD) have been produced as a means for introducing desirable characteristics into bread wheat. In the present work we describe the genetic variability present at the Glu-D t 1 and Glu-D t 3 loci, encoding high- (HMW) and low-molecular-weight (LMW) glutenin subunits respectively, derived from Ae. tauschii, using electrophoretic and chromatographic methods, in a collection of synthetic hexaploid wheats. A wide variation both in mobility and surface hydrophobicity of HMW glutenin subunits was observed between different accessions of Ae. tauschii used in the production of the synthetic hexaploids. A combination of electrophoretic and chromatographic methods improves the identification of HMW glutenin subunits; in fact subunits with identical apparent mobility were revealed to have a different surface hydrophobicity by reversed-phase high performance liquid chromatography. None of the Dx5t subunits present in Ae. tauschii showed the presence of the extra cysteine residue found in the HMW glutenin subunit Dx5 of Triticum aestivum, as revealed by selective amplification with polymerase chain reaction (PCR). The wide variability and the high number of subunits encoded by the Glu-D t 3 locus suggests that Ae. tauschii may be a rich source for enhancing the genetic variability of glutenin subunits in bread wheat and improving bread-making properties.

Separation and Quantification of HMW Glutenin Subunits by Capillary Electrophoresis

ABSTRACTThe use of capillary electrophoresis in SDS (SDS‐CE) for separation and quantification of HMW glutenin subunits (HMW‐GS) was investigated. HMW‐GS were precipitated with 40% acetone from 50% 1‐propanol extract of flour under reducing conditions after removal of monomeric proteins with 50% 1‐propanol. Poly (ethylene oxide) was used in the running buffer (3% w/v) for SDS‐CE. The results indicated that HMW‐GS could be well separated by SDS‐CE, including subunits 7+8, 7+9, 2+12, 5+10, and 17+18. However, HMW‐GS showed delayed migration times compared with molecular weight protein standards. Some HMW‐GS were reversed in their mobilities in SDS‐CE compared with their mobility and molecular weights by SDS‐PAGE. Therefore, the SDS‐CE was unsuitable for MW determination of HMW‐GS. A linear response was obtained from SDS‐CE of a plot of the concentration of HMW‐GS of the 40% acetone precipitate versus corrected areas for absorbance at 214 nm. Quantification of HMW‐GS for the two biotypes (subunits 5+10 vs. 2+12) of an Australian wheat cultivar Warigal confirmed the differences between the two biotypes in their quantity of HMW‐GS. Therefore, the technique could be used to quantify HMW‐GS in conjunction with SDS‐PAGE.

Segregation for isozymes and HMW glutenins in a doubled‐haploid cross of winter wheat carrying 1BL.1RS and 5DL.5RS translocations from rye

AbstractThe doubled haploid (DH) wheat line ‘dh 5841’ carrying two translocations from rye, 5DL.5RS and 1BL.1RS, has been crossed to the subline of wheat cultivar ‘Amadeus 7143’ with a 1BL.1RS translocation. The resulting F1 hybrid IJ 98 with a heterozygous 5DL.5DS‐5DL.5RS chromosome pair has been used to produce doubled haploids. A total of 57 DH lines were obtained from plantlets regenerated in anther culture after successful colchicine treatment and seed set. These lines were identified regarding the constitution of chromosome 5D (5DL.5DS or 5DL.5RS) by means of isoenzyme marker analysis. Thirty DH lines possessed the 5DL.5DS chromosome, while the remaining 27 lines carried the 5DL.5RS translocation. For some of these lines, the 5DL.5RS chromosome was cytologically confirmed by C‐banding. Furthermore, the DH lines were evaluated for their high molecular weight glutenin subunit composition. All possible combinations for the four independent loci —Skdh, Glu‐Al, Glu‐B1 and Glu‐D1— were detected in only 57 DH lines and no segregation distortion was observed.

Allelic variation of the HMW glutenin subunits in Spanish accessions of spelt wheat (Triticum aestivum ssp. spelta L. em. Thell.)

Spelt wheat (Triticum aestivum ssp. spelta L. em. Thell.) is a hulled wheat of Germanic origin that survives at marginal areas in Asturias (Spain). The HMW glutenin subunit composition of 403 accessions of spelt wheat from Spain has been analysed by SDS-PAGE. Three allelic variants were detected for Glu-A1. For the Glu-B1 locus, two of seven alleles detected have not been found before; while four of nine alleles detected for the Glu-D1 are not previously described. Considering the three loci, twenty five combinations were found among all the evaluated lines. This wide polymorphism could be used to transfer new quality genes to wheat, and widen the genetic basis of them.

Variation in the HMW and LMW glutenin subunits from Spanish accessions of emmer wheat (Triticum turgidum ssp. dicoccum Schrank)

Emmer wheat (Triticum turgidum ssp. dicoccum Schrank) is hulled wheat that survives in marginal areas of the Mediterranean Region. The HMW and LMW glutenin subunit composition of 97 accessions of emmer wheat from Spain have been analysed by SDS-PAGE. For the HMW glutenin subunits, four allelic variants were detected for the Glu-A1 locus; one of them has not been previously described. For the Glu-B1 locus, three of the nine alleles detected have not been found before. A high degree of variation was evident for the LMW glutenin subunits, and up to 23 different patterns were detected for the B-LMW glutenin subunits. Considering both types of proteins (HMW and LMW), 30 combinations were found between all the evaluated lines. This wide polymorphism can be used to transfer new quality genes to wheat, and to widen its genetic basis.

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide ( M r 22 005) in the crude cell extracts and both this and the R3 peptide ( M r 12 005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar β-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.

MOLECULAR MODELING OF TWO HEXAPEPTIDE REPEAT MOTIFS OF HMW GLUTENIN SUBUNITS

The three-dimensional structures of two hexapeptide repeat motifs (PGQGQQ and SGQGQQ, in one letter code) in the repetitive central domain of HMW glutenin subunits are investigated by using the multicanonical simulation procedure. Ramachandran plots were prepared and analyzed to predict the relative occurrence probabilities of β-turn and γ-turn structures and helical state. Structural predictions of PGQGQQ repeat motif indicated the presence of high level of β-turns and considerable level of γ-turns. Simulations of the repeat motifs in the repetitive central domain of HMW glutenin subunits indicated that these structures take important part in the three-dimensional structures of repeat motifs.

Effect of Multiple Substitution of Glutenin and Gliadin Proteins on Flour Quality of Canada Prairie Spring Wheat

ABSTRACTThe 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.

Identification, Cloning and Characterisation of a HMW-Glutenin Gene from an Old Hungarian Wheat Variety, Bánkúti 1201

Despite its good functional properties, the variety Bankuti 1201 has been found to possess 2+12 or 3+12 allele composition on chromosome 1D. In order to determine the reason for its quality traits a gene-specific PCR technique was applied in preliminary experiments to examine the HMW glutenin allele composition of the variety. In the course of the analysis a fragment characteristic of Bankuti 1201 was identified and the nucleotide sequence was determined. This showed the presence of a 1Ax2* gene variant which, despite near homology, differed from the original 1Ax2* gene at one important point. At 1181 bp of the 1Ax2* sequence nucleotide exchange was observed which is the middle nucleotide of the TCT-TGT base triplet, involving the exchange of serine for cysteine. The gene was designated 1Ax2*B. The presence of an extra sulphydryl group, like that of the extra cysteine in the 1Dx5 gene, facilitates the formation of further disulphide bonds, might lead to an improvement in gluten quality characters.