RP–HPLC and Capillary Electrophoresis of Subunits from Glutenin Isolated by SDS and Osborne Fractionation

The polypeptide subunits present in SDS-unextractable glutenin, the glutenin macropolymer (GMP) and the 70% (v/v) ethanol unextractable protein, the Osborne glutenin fraction, of various cultivars were separated by RP–HPLC and capillary electrophoresis (CE) under denaturing (urea and SDS, respectively) and reducing conditions. In addition, the SDS-extractable protein was separated by CE. HighM_rglutenin subunits were well separated by CE, while the separation of lowM_rglutenin subunits was better by RP–HPLC. HighM_rglutenin subunits separated by RP–HPLC were collected and separated by CE. The subunits were identified unequivocally using the combined information from these two techniques and from SDS–PAGE patterns using the cvs. Spring and Troy Spring. By both RP–HPLC and CE it could be demonstrated for flour from three wheat cvs. (Camp Remy, Obelisk and Rektor) and a blend of flour from two of those cvs. (Camp Remy/Obelisk) that the highM_rglutenin subunit content of the GMP was 29–31%. In contrast, the SDS-extractable protein consisted of 4–6% highM_rglutenin subunits, which accounted for 14–23% of the highM_rglutenin subunits in flour. Interestingly, the SDS-extractable highM_rglutenin subunits consisted mainly (90–96%) of x-type subunits whereas, in the GMP, only 70–75% of the highM_rsubunits were x-type subunits. Although the SDS extractable protein was not separated by RP–HPLC, results similar to those obtained by CE could be inferred from the subtraction of the contents of glutenin subunits of the GMP from the contents in the Osborne glutenin fraction. The results suggest that x- and y-type highM_rglutenin subunits may have a different role in the structure (size and composition) of glutenin polymers.     

Preparative Separation of High and Low Molecular Weight Subunits of Glutenin from Wheat

Wheat flour was washed with Tris-HCl buffer containing 4% Triton X114 before extracting the residual gluten with 70% ethanol. The glutenin extraction with 50% ethanol was performed at various ratios of DTT/protein; a minimum ratio of 0·1 g/g was needed to solubilise the maximum amount of glutenin. An experimental design was used to optimise the extraction conditions to obtain the best yield and purity of lowM_rand highM_rglutenin subunits. The purity of each glutenin subunit fraction was measured by RP-HPLC analysis after reduction and alkylation. Both temperature and protein concentration had an effect on the preparation of these fractions. An increase in the protein concentration enhanced the yield of the highM_rglutenin fraction and simultaneously decreased that of the lowM_rglutenin. Using the Deringer desirability function, conditions giving the optimum separation were determined. The procedure was scaled up and permitted the preparation of 0·96 g of highM_rand 1·64 g of lowM_rglutenin subunits from 5 g of gluten. The purities of these fractions, determined by RP-HPLC, were 90% and 95%, respectively, and their amino acid compositions were similar to those of high and lowM_rsubunits separated by RP-HPLC.     

Salt Sensitivity of Acetic Acid-Extractable Proteins of Wheat Flour

The sensitivities of flour proteins to precipitation by NaCl at acid pH were investigated by extraction with 0·05macetic acid solution containing varying concentrations of salt and by precipitation of the proteins extractable in acetic acid solution by addition of salt to varying concentrations. Flours of two Canadian hard red spring wheat cultivars (Glenlea and Katepwa) were used because of their different dough strengths. Electrophoresis results showed that as the NaCl concentration was raised, higherM_rproteins of gliadins and glutenins were less extractable and were more easily precipitated. This tendency was more evident for the proteins of cv. Glenlea than those of cv. Katepwa, indicating that the former (stronger) is more sensitive to NaCl than the latter. SDS–PAGE results indicated that differences in the molecular size and subunit composition (i.e.relative proportion of high:lowM_rglutenin subunits, and relative proportion of x-:y-type highM_rglutenin subunits) of glutenin polymer contribute to differences in NaCl sensitivity. The differences appear to be related to the baking strength of the flour.     

Factors Governing Levels and Composition of the Sodium Dodecyl Sulphate-Unextractable Glutenin Polymers During Straight Dough Breadmaking

The effect of several additives (1·215 μmol KIO₃, 0·892 μmol cysteine, endo-xylanase and 0·5% (w/w) rye-water-extractable arabinoxylans) on changes in the level and glutenin subunit composition of the sodium dodecyl sulphate (SDS)-unextractable protein during breadmaking was investigated. Protein extractability drastically increased during dough mixing and was enhanced both by cysteine and KIO₃. The mixing-induced increase in protein extractability was partly reversed during fermentation. Fermenting doughs containing endo-xylanase had a higher level of SDS-unextractable protein than control doughs, while with KIO₃the amount of SDS-unextractable protein remained very low. During baking most protein became SDS-unextractable. Bread baked from doughs with added KIO₃contained a significantly higher level of SDS-extractable protein. Changes in subunit composition of the SDS-unextractable glutenin polymers, determined with RP-HPLC, coincided with changes in protein extractability during dough processing. Mixing decreased the ratio of high to lowM_rglutenin subunits. Simultaneously, the relative proportions of the different highM_rglutenin subunits in the unextractable glutenin polymers changed. During fermentation changes in subunit composition of the SDS-unextractable glutenin were opposite to those during mixing.     

Specificity of antisera raised against synthetic peptide fragments of highMrglutenin subunits.

In an attempt to detect highM_rglutenin subunits specifically by immunochemical means, antisera were produced against synthetic peptides corresponding to three N-terminal sequences and to two repetitive motifs of highM_rglutenin subunits. The three N-terminal peptides, NT1, NT2 and NT3, differed by a single substitution at the sixth position and correspond, respectively, to the N-termini off Dx subunits, Ax and Bx subunits and By and Dy subunits. The anti-peptide sera did not cross react with gliadins or with lowM_rglutenin subunits, and differed in their ability to recognise highM_rglutenin subunits. The antisera to the repetitive motifs recognised all highM_rglutenin subunits, whereas the antisera to the N-terminal peptides detected only some of the subunits. The antiserum directed against the N-terminal peptide from Dx subunits detected these subunits specifically, whereas the antiserum directed against the N-terminal peptide corresponding to y type subunits did not react with the homologous subunits although it did react with Dx or Bx subunits. Antisera were also produced against internal sequences present in the N-terminal domain specific for x and for y-type subunits, but these antisera did not react with the cognate proteins. The failure of some anti-peptide sera to recognise the homologous highM_rglutenin subunits may be due to differences in conformation between peptides and the corresponding regions in proteins.     

Functional Properties of Wheat Glutenin

The importance of glutenin in bread-making quality has led to a substantial research effort. Studies on glutenin can be grouped into four categories: studies that determine the statistical relationships between the quantity of fractions and quality, studies of reconstitution and fortification, breeding and genetic modification, and those that assess structure–function relationships during processing. Statistical relationships between glutenin, glutenin fractions and glutenin polypeptides and quality have been established. The SDS or acetic acid unextractable glutenin correlated strongly with quality parameters. For highM_rglutenin subunits the relationships with quality are less strong. In some studies it was demonstrated that the presence of some highM_rglutenin subunits is correlated with the quantity of unextractable glutenin. Therefore, subunits are probably indirectly linked with bread-making qualityviathe quantity of unextractable glutenin. Recombination and fortification studies are hampered by changes in functionality of proteins after their separation. Recently, small scale tests have been developed in which small amounts of glutenin fractions can be studied. Controlled breeding studies have demonstrated the importance of highM_rglutenin subunits 5+10 and, to a lesser extent, 1 or 2* for quality. In most of these studies the quantity of unextractable glutenin is not reported. This hampers adequate conclusions on cause–effect relationships. During dough processing large changes occur in the extractability of glutenin. The significance of these changes for dough properties and bread quality still requires investigation.     

Quality-related Epitopes of High Mr Subunits of Wheat Glutenin

Three hundred and eighty four immobilised overlapping nonapeptides, corresponding to the full amino acid sequences of three high M_r subunits of glutenin from bread wheat (Triticum aestivum) grain, were used to determine the linear epitopes recognised by four monoclonal antibodies. These antibodies were selected on the basis of significant and positive correlations between their binding to wheat flour extracts in a two-site ('sandwich') enzyme immunoassay and rheological measures of dough strength, an important aspect of bread wheat quality. The antibodies did not bind to a single, specific sequence but bound a series of related peptides in each high M_r glutenin subunit examined. The sequences recognised were not identical for the four antibodies, but in each case were in the central repeating domain of the high M_r glutenin subunits, and usually comprised regions that overlapped the degenerate repeat nonamer and hexamer sequences. High M_r glutenin subunits that have been associated with greater dough strength, such as the D-genome allelic products 1Dx5 and 1Dy10, displayed an increased number of the epitope sequences. The location of the epitopes in sequences of overlapping β-turns in the repetitive region supports the hypothesis that dough elasticity arises partly from β-turn-forming secondary structure in the repeat regions of the M_r glutenin subunits. Additional β-turn within high M_r subunits may extend their structure to allow increased interaction between the glutenin subunits and with the other proteins of the gluten complex, thus improving dough strength.     

Depolymerisation and Re-polymerisation of Wheat Glutenin During Dough Processing. I. Relationships between Glutenin Macropolymer Content and Quality Parameters

The protein content and the content of protein-related parameters of both flour and dough were related to the maximum resistance (R_max) and extensibility (E) of dough, as determined in a Brabender Extensograph, and to loaf volume. The glutenin macropolymer (GMP) content of flour and dough was more strongly related toR_maxthan protein content or the content of the group of Osborne fractions (glutenin, gliadin, albumin/globulin). Within each group of protein-related parameters, the contents of the Osborne glutenin fractions explained the variation in all quality parameters better than the contents of the gliadin or albumin/globulin fraction. The GMP content of dough after 45 min rest was more strongly related toR_max, whereas the GMP content of flour was more strongly related toEand loaf volume. This demonstrates that, during mixing and resting of dough, changes occur in the GMP that are important forR_maxbut not forEor loaf volume. Although limited numbers of wheat cultivars (15), harvest years (2), flour blends (8) and resting times (3) were investigated, the relationship between the GMP content of dough andR_maxwas independent of these variables.     

Thermal stability of wheat gluten protein: its effect on dough properties and noodle texture

There is a need to develop more sensitive and reliable tests to help breeders select wheat lines of appropriate quality. Gluten thermostability, measured by the viscoelasticity of heated gluten, was assessed for its usefulness in evaluating quality of wheats in breeding programs. Two sets of wheat samples were used: Set I consisting of 20 cultivars and/or breeders' lines (BL), with diverse dough strengths and allelic variations of high M_r glutenin subunits coded at the Glu-A1, Glu-B1 and Glu-D1 loci (N=20) and Set II consisting of 16 near isogenic BL of F7 generation that had been in a quality selection program for three years. Thermostability of the isolated wet gluten was determined by measuring its viscoelastic properties, and was related to noodle texture, flour protein content, protein composition, dough physical properties and other quality predicting tests.Viscoelasticity of heat-treated gluten, isolated with 2% NaCl solution, significantly correlated with most of the tests used to measure dough and/or gluten strength and Chinese white salted noodle texture. The rate of thermal denaturation of proteins depends on M_r and packing density. High ratios of monomeric proteins such as gliadins and low M_r glutenin subunits to high M_r glutenin subunits increase the thermostability of the gluten. The measurement of viscoelasticity of heat-denatured gluten can be a useful test to determine gluten quality. Our study showed that gluten viscoelasticity and most of the tests related to dough and/or gluten strength are independent of allelic variations of the high molecular weight glutenin subunits. This test has been developed for predicting white salted noodle quality.     

Relationships Between Chromosome 1B-encoded Glutenin Subunit Compositions and Bread-making Quality Characteristics of Some Durum Wheat (Triticum turgidum) Cultivars

The high and low M_r glutenin subunit compositions (controlled by the Glu-1 loci and the Glu-B3 locus, respectively) and the bread-making quality characteristics of 26 durum wheat (Triticum turgidum) genotypes were determined. The relationships between quality parameters and Glu-B1 and Glu-B3 controlled glutenin subunit composition were also investigated. The Glu-A1-controlled null allele was present in all the genotypes. High M_r subunits 20, 6 + 8 and 7 + 8 occurred in similar proportions in the cultivars analysed. The Glu-B3 low M_r allelic variants, LMW-1 and LMW-2, were both represented, with LMW-1 being present in lower proportion. Flour protein, SDS-sedimentation volume, dough strength (Alveograph W value), dough mixing time and bread loaf volume varied among the genotypes. Most samples had high Alveograph tenacity/extensibility (P/G) ratios, typical of tenacious gluten character. SDS-sedimentation volume, dough strength, dough mixing time and bread loaf volume were all interrelated. An association with flour protein content was observed only for mixing time, while the Alveograph tenacity/extensibility ratio was not correlated with the other parameters. Comparisons within the Glu-B1 and Glu-B3 loci indicated that the high M_r subunit 7 + 8 and the low M_r subunit LMW-2 had significantly greater beneficial effects on gluten strength and bread-making quality than the high M_r subunits 6 + 8 or 20 and the low M_r subunit LMW-1, respectively. High M_r subunit 6 + 8 had greater beneficial effects on quality than subunit 20.     

Bread making quality of durum wheat genotypes with some novel glutenin subunit compositions

Durum wheat genotypes with some novel high M_r (high molecular weight, HMW) and low M_r (low molecular weight, LMW) glutenin subunits were grown in Sicily for two years of testing in order to compare their rheological and baking properties with respect to commercial durum wheat cultivars. Good bread making quality, as measured by Alveograph W and P/L, Farinograph and Mixograph parameters, and loaf volume was observed in genotypes combining high M_r subunits 2⁺, 1 or 11 encoded at the Glu-A1 locus with the so-called LMW-2 subunit group encoded at the Glu-B3 locus. The cultivar Avanzi, which carries high M_r subunit 2⁺ and LMW-2-like subunits, and the cultivars Dritto and Keops, which contain novel high and low M_r subunits, gave higher loaf volumes than control cultivars. The LMW-2 group subunits were found to be the main factor in determining dough strength (Alveograph W). The increase in the amount of high M_r subunits in genotypes with one expressed Glu-A1 gene may account for their improved rheological and baking properties.     

Characterisation of polyclonal and monoclonal anti-peptide antibodies specific for some lowMrsubunits of wheat glutenin and their use in the detection of allelic variants atGlu-3loci

Polyclonal and monoclonal antibodies (Mabs) were produced against the major type ofN-terminal amino acid sequence of lowM_rglutenin subunits. The reactivities of these antibodies were determined using glutenin extracts of several bread wheat cultivars of known allelic composition. Analyses were performed by immunoblotting after one or two-dimensional electrophoresis. One Mab (Mab 6x1) was found to react with lowM_rglutenin subunits encoded by chromosomes 1B and 1D but not with subunits controlled by chromosome 1A. Only some of the subunits encoded at theGlu-D3locus were recognised. In contrast, this Mab reacted with all the subunits controlled by theGlu-B3locus. After single dimension SDS–PAGE, we observed significant differences between immunoblot patterns of cultivars expressing different lowM_rglutenin subunits from chromosome 1B. Mab6 x1 is a useful reagent for analysing the allelic composition at theGlu-B3locus.     

Polymorphism of High MrGlutenin Subunits in Triticum tauschii: Characterisation by Chromatography and Electrophoretic Methods

A large collection of accessions of the wild wheat progenitor Triticum tauschii, the donor of the D genome of Triticum aestivum, was evaluated for the variability of high molecular weight (M_r) glutenin subunits by electrophoretic and chromatographic methods. A large range of allelic variation at theGlu-D^t1 locus was found in this collection and some novel subunits were observed in both x- and y-type glutenin subunits, including x- or y-type null forms. A few accessions showed three bands in the high M_rglutenin subunit region. However, only two subunits were observed when monomeric proteins were removed before SDS-PAGE analysis of polymeric proteins. The presence of monomeric proteins in this region is discussed. Characterisation of these subunits was also carried out by reversed phase-high performance liquid chromatography (RP-HPLC). Very different surface hydrophobicities were observed between x- and y-type subunits and in some cases it was possible to identify glutenin subunits with the same apparent molecular weight but different surface hydrophobicity. Differences in elution times that were detected when the same subunit was either reduced or reduced and alkylated were related to the number of cysteine residues present in each glutenin subunit. The newGlu-D^t1 glutenin subunits have the potential to enhance the genetic variability available for improving the quality of bread wheat (T. aestivum).     

Detection of y-type Subunit at theGlu-A1Locus in Some Swedish Bread Wheat Lines

Electrophoretic and reversed phase high performance liquid chromatographic (RP–HPLC) analyses were performed on gluten proteins extracted from flours milled from two different Swedish bread wheat lines; these lines have been reported to possess a novel highM_rglutenin subunit controlled by a gene at theGlu-A1locus, referred to as 21*. Although RP–HPLC indicated that subunit 21* has a surface hydrophobocity similar to that of the commonly occurring allelic subunits 1 or 2*, it differs from them in isoelectric point, being more basic when analysed by two dimensional gel electrophoresis (IEF/SDS–PAGE). RP–HPLC separations of highM_rglutenin subunits showed the presence of an additional peak, the behaviour of which was similar to that of y-type subunits encoded by genes at theGlu-A1ylocus and present only in wild wheatsT. urartu(AA) orT. dicoccoides(AABB). Based on chromatographic results and on the tight linkage observed with subunit 21*, it is suggested that the additional component (indicated as 21*y), present in the breeding lines analysed, corresponds to the y-type subunit encoded at theGlu-A1locus. Genes encoding the subunits 21* and 21*y were also analysed by polymerase chain reaction (PCR). Contrary to what was observed for the polypeptide itself, the gene corresponding to subunit 21* was similar in size to that encoding subunit 2* and shorter than that corresponding to subunit 1. Moreover, the amplification product corresponding to the active 21*y gene was shorter than that of the allelic inactive gene present in the bread wheat cultivar Cheyenne. As reported for other highM_rglutenin subunits, gene size differences observed were due to a different length of the repetitive region. Because cultivated polyploid wheats have been shown to have only the x-type subunit at theGlu-A1locus, it is speculated that the new combination, with both x- and y-type subunits expressed, might have been introgressed during breeding processes from the wild wheat progenitorsT. urartuorT. dicoccoides, which have genotypes expressing both types of subunits.     

Polymorphism of Low Mr Glutenin Subunits in Triticum tauschii

A collection of 173 Triticum tauschii accessions was analysed to evaluate the variability of low molecular weight (M_r) glutenin subunits. These proteins were analysed by one-step one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were divided into B-, C- and D-subunits in accordance with their electrophoretic mobility. Extensive polymorphism, both in the number and electrophoretic mobility, was detected in lowM_r glutenin subunits present in T. tauschii. Thirty different patterns for B-subunits and forty-three for C-subunits were identified, some of which were with identical electrophoretic mobility than those observed in hexaploid wheat. Glutenin subunits with the same electrophoretic mobilities of low M_r D-glutenin subunits as well as subunits encoded at the Glu-D4 and Glu-D5 loci, were also detected in accessions of T. tauschii. These results provide new basic knowledge regarding the genetics variability of the low M_r glutenin subunits, as well as their potential to create novel germplasm for the improvement of wheat quality in breeding programs.     

Antibodies to N-terminal Peptides of Low MrSubunits of Wheat Glutenin. I. Characterisation of the Antibody Response

The relationship between allelic composition of the low molecular mass glutenin subunits (LM_r GS) and dough properties is poorly understood. Differentiating the L M_rGS on the basis of their N-terminal sequence type may provide an important alternative in understanding the relationship. Polyclonal and/or monoclonal antibodies were produced using synthetic peptides corresponding to each of the seven N-terminal amino acid sequence types of the B- and C- L M_rGS, namely SHIPGLERPS-, METSHIPGL-, METSRVPGL-, METSCIPGL-, METRCIPGL-, NMQVDPSGQVQ- (γ-type) and VRVPVPQLQP- (α-type). Each of the polyclonal antisera recognised both the corresponding peptide and LM_r GS. For monoclonal antibodies, the proportion of hybridoma clones that produced antibody which recognised either the peptide or L M_rGS varied between 1 and 88%. However, antibodies from only 4% of antibody-secreting stable cell lines recognised both the peptide immunogen and intact L M_rGS. Using ELISA, the majority of the antibodies cross-reacted with related synthetic peptides corresponding to more than one N-terminal LM_r GS sequence, although several of these bound small groups of L M_rGS on immunoblots. Different polyclonal antisera prepared to a given immunogen exhibited similar patterns of subunit recognition on immunoblots. Monoclonal antibodies prepared to the same immunogen exhibited a variety of patterns, although each of the antibodies specific for a particular peptide or combination of peptides on ELISA recognised a similar pattern of L M_rGS on immunoblots. For each sequence type, polyclonal or monoclonal antibodies specific for individual N-terminal sequences were identified. These probes may be useful tools to determine whether the type and amount of each N-terminal sequence is correlated with dough properties.     

A Null Gli-D1 Allele with a Positive Effect on Bread Wheat Quality

The French wheat cultivar Darius (Da) has very good bread-making quality, even though it possesses the high M_r glutenin subunit combination 2, 7 and 12, which is associated with poor quality, and a null allele at the Gli-D1 locus. Darius was crossed with three cultivars, Corin (Cor), Capitole (Cap) and Courtot (Cou), of poor, medium, and good quality, respectively. The three progenies (Cor × Da, Cap × Da and Da × Cou) were used to investigate the genetic basis of the good quality of Darius. Gliadin and glutenin compositions were analysed by acid polyacrylamide gel electrophoresis (A-PAGE) and SDS-PAGE, respectively, from half F2 grains, and the quality was evaluated using six technological criteria for the corresponding plants. The high M_r glutenin subunit alleles of Darius produced a negative effect on quality. The null allele of Darius, characterised by the absence of the Gli-D1 encoded ω-gliadins, was associated significantly with higher dough tenacity P , and strength W (up to 40% in the Cor × Da progeny). Darius had a higher amount of B zone low M_r glutenin subunits than the three other cultivars. The null allele of Darius reduced the dough extensibility in the two first progenies, and probably increased the ratio of aggregated glutenin to unaggregated gliadins. These results demonstrated that using only one locus breeders can improve particular quality traits.     

The effect of mixing on glutenin particle properties: aggregation factors that affect gluten function in dough

Previously we reported that the SDS insoluble gel-layer: the Glutenin Macro Polymer (GMP) can be considered as a gel consisting of protein particles. These glutenin particles have a size of about 10⁻¹–10² μm and consist of HMW-GS and LMW-GS only. In GMP isolates from flour, the particles are spherical. In isolates from dough, glutenin particles have lost this shape. This seems relevant, since mixing disrupts the particles and the mixing energy required for dough development correlated with the glutenin particle size in flour. The question studied in this paper is how changes at a glutenin particle level affected the subsequent process of gluten network formation during dough rest and if this could be used to explain resulting dough rheological properties. To this end, we studied how various mixing regimes affected the dough properties during and after resting (elasticity). We cannot fully explain the differences in the final dough properties observed using parameters such as the quantity of GMP in flour, the quantity of re-assembled GMP in dough and the size of re-assembled glutenin particles. However, other parameters were found to be important: (1) the Huggins constant K′ reflecting the tendency of glutenin particles to interact at level II of the Hyperaggregation model; (2) the composition of glutenin particles affecting the potential to form smaller or larger particles and (3) for over-mixed dough, covalent re-polymerisation at the so-called level I of hyperaggregation. Using these parameters we can better explain dough viscoelasticity after resting.     

High Mr Glutenin Subunits of Indian Wheat Cultivars: Association of Subunits 5 + 10 with the 1BL/1RS Wheat-Rye Translocation

The seed proteins of 110 commercially-released Indian wheat cultivars were fractionated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to determine their high M_r glutenin subunit compositions. Amongst the cultivars, three alleles were indentified for the Glu-A1 locus (subunits 1, 2^* and the null phenotype), eight alleles for the Glu-B1 locus (7, 7 + 8, 7 + 9, 6 + 8, 20, 13 + 16, 17 + 18 and a new allele) and two for the Glu-D1 locus (2 + 12 and 5 + 10). Nine of the cultivars were heterogeneous and possessed two or more 'biotypes' with respect to high M_r subunits. The cultivars were also analysed for the presence of the 1BL/1RS wheat-rye translocation by SDS-PAGE of unreduced prolamins and hybridisation of DNA dot blots with a rye-telomere-specific repetitive DNA probe, pAW-161. Both methods revealed that the majority of newly-released Indian wheat cultivars carry this translocation, thus confirming the agronomic superiority of these lines. While most of the normal wheat cultivars possessed high M_r subunits 2 + 12, 14 of the 18 translocation cultivars had the allelic subunits 5 + 10, even though no selection was made for these subunits during the breeding process. This suggests that the subunits 5 + 10 may play a compensating role for the loss of dough strength associated with the 1BL/1RS translocation.     

The primary structure of wheat glutenin subunit 1Dx2 revealed by electrospray ionization mass spectrometry

The high molecular weight glutenin subunits (HMW-GS) play a key role in end-use quality of wheat. Their particular primary structure is mostly derived from DNA sequencing, which gives no information on potential post-translational modifications. This paper reveals the primary structure of HMW-GS 1Dx2 by proteomic analysis. For this purpose, HMW-GS were first isolated from wheat flour (cv. Contra). The relative molecular mass (M_r) of subunit 1Dx2 present in the HMW-GS mixture was then very accurately determined with high-performance liquid chromatography–electrospray ionization-mass spectrometry using a quadrupole-time-of-flight mass analyzer (HPLC–ESI-QTOF-MS). The obtained M_r value (87,105) differed from the value derived from its protein sequence in the NCBI database (87,007). The subunit was further purified by preparative reversed-phase HPLC and partially hydrolyzed with chymotrypsin. The resulting 1Dx2 peptides were then analyzed by HPLC–ESI-MS/MS and the MS data were compared to amino acid sequences in protein databases. The discrepancy between the calculated and the measured M_r of 1Dx2 was explained by a missing proline in the 1Dx2 amino acid sequence from the database and not by any post-translational glycosylation.