Purification and Characterisation of HighMrGlutenin Subunit 20 and its Linked y-type Subunit from Durum Wheat

HighM_rglutenin subunit 20 and its linked y-type subunit, present in the durum wheat cultivar Lira, were purified by preparative reversed-phase high-performance liquid chromatography (RP–HPLC). Amino acid and N-terminal sequence analysis of subunit 20y confirmed that it corresponded to a y-type subunit. Moreover, the number and position of the cysteine residues in subunit 20 were determined by alkylation with the fluorogenic reagent 7-fluoro-4-sulfamoyl-2,1,3,-benzoxadiazole (ABD-F) and subsequent enzymic digestion with trypsin. N-terminal amino acid sequence analysis of the fluorescent peptides showed that subunit 20 had only two cysteine residues, one in the N-terminal region and the other in the C-terminal domain.     

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.     

Functional Properties of LowMrWheat Proteins.III. Effects on Composition of the Glutenin Macropolymer During Dough Mixing and Resting

The effect of lowM_rwheat protein addition on the amount and composition of the glutenin macropolymer (GMP) of dough was investigated for the three wheat cultivars Obelisk (weak), Camp Remy (medium strong) and Rektor (strong). During mixing, the amounts of high and lowM_rglutenin subunit classes, and of the individual subunits decreased. The proportion of highM_rglutenin subunits decreased and that of lowM_rglutenin subunits increased, indicating an inhomogeneous distribution of the two subunit classes within the polymers present in GMP. During resting, the amounts of the glutenin subunit classes and of individual subunits increased. Meanwhile, the proportion of highM_rglutenin subunits in GMP increased. LowM_rwheat protein addition retarded re-polymerisation in that the amounts of glutenin subunit classes and of individual highM_rglutenin subunits in GMP increased less than without addition. The proportion of highM_rglutenin subunits in GMP directly after mixing was also decreased by lowM_rwheat protein addition, and the proportion increased faster during dough resting, compared with the GMP in dough without lowM_rwheat protein addition. Eventually, after 90 or 135 min resting, no differences existed in the proportions in GMP from doughs with and without lowM_rwheat protein addition. LowM_rwheat protein addition had no specific effect on individual highM_rglutenin subunits, nor on the x-type/y-type subunit ratio in the GMP. In contrast, with increasing lowM_rwheat protein addition, a highly significant reduction in the subunit 10 or 12/subunit 9 ratio in GMP was observed. This finding is in line with the decrease in this ratio directly after mixing in GMP of the dough without lowM_rwheat protein addition. Since no specific effects were observed, it can be concluded that the lowM_rwheat protein acts rather unspecifically on the GMP of dough.     

Comparative Studies of HighMrSubunits of Rye and Wheat. I. Isolation and Biochemical Characterisation and Effects on Gluten Extensibility

The structural features of highM_rglutenin subunits of wheat were compared with those of analogous proteins from rye. Subunits of two rye cultivars (Danko and Halo) and of the wheat cultivar Rektor were isolated from defatted flours by extraction with 50% (v/v) aqueous propan-1-ol under reducing conditions at 60°C followed by precipitation using a 60% concentration of propan-1-ol. The yields of dialysed and freeze-dried subunits were 0·33% and 0·32% (w/w of flour), respectively (rye cultivars), and 0·91% (Rektor). SDS–PAGE revealed that the rye cultivars contained at least five subunits with mobilities corresponding to the x-type subunits of wheat. Separation by RP–HPLC indicated that the rye cultivars did not differ in the qualitative composition of subunits, but in their quantitative proportions. The surface hydrophobicities of the rye subunits were significantly lower than those of wheat subunits. The amino acid compositions of single rye subunits were characterised by high contents of Glx, Gly and Pro, and they were closely related to those of wheat subunits, except that the Glx content was generally lower and the Cys content higher. Notable differences between rye and wheat subunits were found in their contributions to gluten strength. Whereas wheat subunits, reoxidised with potassium bromate and mixed with a standard wheat flour, caused a significant increase in gluten strength, reoxidised rye subunits had the opposite effect.     

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).     

Molecular and Biochemical Characterisation of HMW Glutenin Subunits fromT. tauschiiand the D Genome of Hexaploid Wheat

Electrophoretic (urea SDS–PAGE) and chromatographic (RP–HPLC) analysis was performed on 8 allelic variants of HMW glutenin subunits derived fromTriticum tauschiiand from the D genome of a hexaploid wheat species (Triticum macha) and hexaploid landraces. These subunits had been previously identified using SDS–PAGE. The characterisation revealed that subunits Dy10^tand Dy12^tfromT. tauschiicould be differentiated from their bread wheat counterparts using both urea SDS–PAGE and RP–HPLC. In the latter case, theT. tauschiiy-type subunits were clearly more hydrophobic than the Dy type subunits of bread wheat. The characterisation also suggested that subunit Dx5^t, derived from two separateT. tauschiiaccessions, did not contain the extra cysteine residue characteristic of Dx5 from bread wheat. RFLP analysis of the genes encoding the HMW glutenin subunits of interest suggested that the absence of Dx-type HMW glutenins in two hexaploid landraces was due to lack of expression of their encoding genes. The relationship betweenHindIII DNA fragment size and protein subunit size, as measured by electrophoretic mobility, is examined and discussed. Finally, the solubility properties of a HMW protein designated T1 (derived fromT. tauschiiaccession AUS 18913) suggested that it was not a HMW glutenin subunit as was previously thought. Further studies are needed to clarify the identity of this subunit.     

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.     

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.     

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.     

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.     

Isolation and characterization of a novel variant of HMW glutenin subunit gene from the St genome of Pseudoroegneria stipifolia

The x- and y-type high molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe investigations on the HMW glutenin subunits from several Pseudoroegneria accessions. The electrophoretic mobilities of the HMW glutenin subunits from Pd. stipifolia, Pd. tauri and Pd. strigosa were much faster than those of orthologous wheat subunits, indicating that their protein size may be smaller than that of wheat subunits. The coding sequence of the Glu-1St1 subunit (encoded by the Pseudoroegneria stipifolia accession PI325181) was isolated, and found to represent the native open reading frame (ORF) by in vitro expression. The deduced amino acid sequence of Glu-1St1 matched with that determined from the native subunit by mass spectrometric analysis. The domain organization in Glu-1St1 showed high similarity with that of typical HMW glutenin subunits. However, Glu-1St1 exhibited several distinct characteristics. First, the length of its repetitive domain was substantially smaller than that of conventional subunits, which explains its much faster electrophoretic mobility in SDS-PAGE. Second, although the N-terminal domain of Glu-1St1 resembled that of y-type subunit, its C-terminal domain was more similar to that of x-type subunit. Third, the N- and C-terminal domains of Glu-1St1 shared conserved features with those of barley D-hordein, but the repeat motifs and the organization of its repetitive domain were more similar to those of HMW glutenin subunits than to D-hordein. We conclude that Glu-1St1 is a novel variant of HMW glutenin subunits. The analysis of Glu-1St1 may provide new insight into the evolution of HMW glutenin subunits in Triticeae species.     

Detection, Chromosomal Location and Evaluation of the Functional Value of a Novel High Mr Glutenin Subunit Found in Swedish Wheats

A high M_r glutenin subunit, which has not been described previously, was found in several Swedish wheat (Triticum aestivum L.) breeding lines. The electrophoretic mobility (sodium dodecyl sulphate polyacrylamide gel electrophoresis) of this band was close to the mobility of the subunit that has been referred to as band 21 encoded on chromosome 1B. Reciprocal crosses between wheat materials with and without this band have shown that the synthesis of this subunit is controlled by the locus on chromosome 1A. The new band, called 21^*, is thus allelic to bands 1 and 2^*. The relevance of the novel-subunit to breadmaking quality was investigated by partial-least-square regression analysis. Using this method, the relationship between the electrophoretic patterns of high M_r glutenin subunits and the specific Zeleny volume was determined. The novel glutenin subunit was found in cultivars with a high specific Zeleny volume. Further investigations are needed before it is possible to determined the influence of the new glutenin subunit on baking 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.     

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.     

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.     

中国小麦微核心种质 Glu-A3位点等位变异的分布

低分子量麦谷蛋白亚基在面包和面条食品加工过程中起着十分重要的作用。为了便于对含有LMW-GS优良基因的亲本筛选,选取来源于不同麦区包括地方品种和育成品种在内的208份核心种质为供试材料,用特异PCR方法检测Glu-A3位点LMW-GS基因的等位变异。结果表明,Glu-A3d出现频率(26.4%)明显高于其他等位类型,在地方品种和育成品种中的分布频率分别为26.7%和26.0%;分布频率从大到小依次为Glu-A3d>Glu-A3c>Glu-A3a>Glu-A3b>Glu-A3e>Glu-A3g>Glu-A3f;在来源于冬春兼播麦区的育成品种中未检测出Glu-A3e和Glu-A3f。对面筋强度贡献较大的Glu-A3b在地方品种和育成品种中的分布频率分别为10.7%和18.2%,说明我国小麦总体品质水平有了较大的提升。     

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.     

Relationship Between Gliadin Alleles and Dough Strength in Italian Bread Wheat Cultivars

Dough strength, as determined by Alveograph W, was studied in Italian bread wheat cultivars grown at several locations over 21 years of testing. Broad sense heritability of Alveograph W was found to range between 0·40 and 0·82, variation in this parameter being affected by genotype×year interactions. Standardised Alveograph W values (Wst) across 40 environments (location–year combinations) were computed for 54 cultivars with reference to the long-term control (cv. Mec), and a quality score based on the Wst value was given to each gliadin allele occurring in at least four of the 54 cultivars analysed. Significant differences in Wst values were observed at theGli-B1,Gli-B2andGli-A2loci. AllelesGli-B1b,Gli-B2candGli-A2bwere significantly related to high gluten strength. An overall quality score based on allelic composition at these loci explained 41% of the observed Wst variation in the 54 cultivars used for the score definition, and 23% to 48% of W value variation in three sets of independent data. The combination of this score with a quality score based onGlu-1alleles coding for highM_rglutenin subunits could explain, on average, about 50% of gluten strength variation.