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.     

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.     

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.     

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.     

Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten: Studies on gluten, gliadin and glutenin

The effect of hydrostatic pressure (0.1–800 MPa) in combination with various temperatures (30–80 °C) on the chemical and physical properties of wheat gluten, gliadin and glutenin was studied. Chemical changes of proteins were determined by extraction, reversed-phase high-performance liquid chromatography (HPLC), sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy, thiol measurement and studies on disulphide bonds. Rheological changes were measured by extension tests and dynamic stress rheometry. Treatment of gluten with low pressure (200 MPa) and temperature (30 °C) increased the proportion of the ethanol-soluble fraction (ESF) and decreased gluten strength. The enhancement of both pressure and temperature provoked a strong reduction of the ESF and the thiol content of gluten. Within gliadin types, cysteine containing α- and γ-gliadins, but not cysteine-free ω-gliadins were sensitive to pressure and were transferred to the ethanol-insoluble fraction. Disulphide peptides isolated from treated gluten confirmed that cleavage and rearrangement of disulphide bonds were involved in pressure-induced reactions. Increased pressure and temperature induced a significant strengthening of gluten, and under extreme conditions (e.g. 800 MPa, 60 °C), gluten cohesivity was lost. Isolated gliadin and glutenin reacted differently: solubility, HPLC and SDS-PAGE patterns of gliadin having a very low thiol content were not influenced by pressure and heat treatment; only conformational changes were detected by CD spectroscopy. In contrast, the properties of isolated glutenin having a relatively high thiol content were strongly affected by high pressure and temperature, similar to the effects on total gluten.     

Glutathione and related thiol compounds II. The importance of protein bound glutathione and related protein-bound compounds in gluten proteins

Protein-bound glutathione (PSSG) and protein-bound related thiol compounds, i.e. cysteine (PSSCys), glutamyl-cysteine (PSSGlu-Cys) and cysteinyl-glycine (PSSCys-Gly), were analysed in proteins of Osborne fractions, i.e. gliadin, glutenin and gliadin-, glutenin-subfractions separated by gel filtration chromatography, gel protein and the total gluten proteins separated from wheat varieties with varying breadmaking performances. The results showed that PSSG and some protein-bound related thiol compounds were found in monomeric gliadins, indicating that glutathione and some related thiol compounds are able to form disulphide bonds (SS) with sulphydryl group (SH) of those proteins and the formation of those disulphide bonds may prevent those monomeric proteins from binding to other proteins. It was also observed that a larger amount of PSSG in glutenin proteins was negatively correlated with the molecular weight (M_w) distribution of glutenin polymers, suggesting that PSSG and protein-bound related thiol compounds may play an important role in controlling polymerisation of glutenin. Furthermore, it was found that the level of PSSG in gel protein from flours with poor breadmaking performances was constantly higher and significantly different (p<0.05) from that of flours with good breadmaking performance. The same trend was observed with gluten samples from breadmaking and biscuitmaking flours.     

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.     

Disulphide Bonds in Wheat Gluten Proteins

Disulphide bonds play a key role in determining the structure and properties of wheat gluten proteins. Comparison of the sequences of monomeric gliadins and polymeric glutenin subunits allows the identification of conserved and variant cysteine residues. Direct disulphide bond determination demonstrates that the conserved cysteine residues present in S-rich prolamins (α-type gliadins, γ-type gliadins and LMW subunits) form intra-chain disulphide bonds while additional cysteines residues present only in the LMW subunits form inter-chain bonds with cysteines in HMW subunits and other LMW subunits. Conserved and variant cysteine residues are also present in the HMW subunits but their patterns of disulphide bond formation are less well understood. Further information on the abilities of individual cysteine residues to form intra- and inter-chain disulphide bonds has also been obtained by heterologous expression of wild type and mutant proteins inE. coliand, in the case of the HMW subunits, by examination of the patterns of dimers recovered on partial reduction of glutenin or resulting from the expression of subunits in transgenic tobacco plants. Wheat gluten proteins are folded and assembled within the lumen of the endoplasmic reticulum of the developing endosperm cells, where disulphide bond formation and exchange may be catalysed by the enzyme protein disulphide isomerase. Similarly, disulphide bond reduction, for example to facilitate mobilisation during germination, may be catalysed by thioredoxinh. Understanding the mechanism and specificity of disulphide bond formation in gluten is crucial for the manipulation of its functional properties by genetic engineering or chemical modification.     

Purification and Characterization of a Glutenin Hydrolysing Proteinase from Wheat Damaged by the New Zealand Wheat Bug, Nysius huttoni

A glutenin hydrolysing enzyme (bug proteinase), present in New Zealand wheat damaged by Nysius huttoni, was purified 50000-fold by anion exchange, hydrophobic interaction, immobilized metal ion affinity and gel filtration chromatography. The enzyme had an apparent M_r of 14·1k as determined by gel filtration chromatography. SDS-PAGE showed a major protein band of M_r 30k and six minor bands of M_r 13·2-28·5k, none of which was a glycoprotein. Isoelectric focusing revealed two major enzyme active bands (pI 9·6 and 9·2) and three minor activity bands (pI 9·9, 8·8 and 8·2). IEF showed no protein contaminants in the most purified sample. The enzymes had optimum activity at pH 8·9 and 45°C. The activity was stable in the pH range 4·5-11 and at 50°C for 20 min at pH 8·9. The bug proteinase was shown to be a serine proteinase by inhibition with phenylmethylsulphonyl fluoride and potato proteinase inhibitors (POT-IC and POT-ID). Thirty other proteinaceous serine proteinase inhibitors did not inhibit the enzyme. Bread baking with partially purified enzyme produced loaves with the poor quality characteristics of loaves made with bug-damage wheat.     

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.     

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.     

Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten II. Studies on the influence of additives

Cysteine, N-ethylmaleinimide, radical scavengers, various salts or urea were added to wheat gluten. After treatment at increasing pressure (0.1–800 MPa) and temperature (30–80 °C) the resulting material was analysed by micro-extension tests and an extraction/HPLC method to measure protein solubility. Furthermore, cysteine was added to isolated gliadin and glutenin prior to high-pressure treatment and protein solubility was determined. The resistance to extension of gluten strongly increased and the solubility of gliadin in aqueous ethanol decreased with increasing pressure and temperature. As compared to experiments without additive the observed effects were much stronger. Isolated gliadin turned largely insoluble in aqueous ethanol when cysteine was added prior to high-pressure treatment. The S-rich α- and γ-gliadins were much more strongly affected than the S-poor ω-gliadins pointing to a disulphide related mechanism. Monomeric gliadin components were completely recovered after reduction of the aggregates with dithioerythritol. In contrast, samples without free thiol groups such as isolated gliadins or with SH groups, which had been blocked by N-ethylmaleinimide, were hardly affected by high-pressure treatment. The addition of radical scavengers to gluten showed no effect in comparison to the control experiment, indicating that a radical mechanism of the high-pressure effect can be excluded. The observed effects can be explained by thiol-/disulphide interchange reactions, which require the presence of free thiol groups in the sample. The addition of salts and urea showed that unfolding of the protein due to weakening of interprotein hydrogen bonds is strongest for ions with a high radius (e.g. thiocyanate). This leads to weakening of gluten at ambient pressure but it facilitates high pressure induced reactions, e.g. of disulphide bonds.     

Salt-Induced Disaggregation/Solubilization of Gliadin and Glutenin Proteins in Water

The effect of salt concentration used in preparing gluten, on the subsequent dissolution of gluten in water, was examined. Flour from a Canadian hard red spring wheat cultivar, Katepwa, was used to prepare glutens using three different solvents, i.e. distilled deionized water (DDW), 0·2% NaCl solution and 2% NaCl solution. The isolated wet glutens were extracted sequentially with DDW, providing four water soluble fractions and an insoluble residue. The amount of protein in each fraction was determined and respective compositions were assessed electrophoretically under reducing and non-reducing conditions. Surprisingly, DDW extracts of gluten prepared with 2% NaCl contained almost all the gliadins, except some ω-gliadin components, and most of the polymeric glutenin. For the gluten prepared with 0·2% NaCl, most of the gliadin, but only a small portion of glutenin, was extracted. For gluten prepared with DDW, only part of the gliadins and almost no glutenin was extractable with water. The DDW solubilities of gluten proteins prepared in DDW, 0·2% NaCl and 2% NaCl were 27, 52, and 85%, respectively, after four sequential extracts with DDW. The large increases in the solubility of gliadin and glutenin proteins in DDW when the gluten is prepared in salt solution (after removal of most of the salt) can be explained on the basis of a salt-induced conformational change of the proteins, which renders water a more effective solvent.     

Affinity Chromatography with Immobilised Endoxylanases Separates TAXI- and XIP-type Endoxylanase Inhibitors from Wheat (Triticum aestivum L.)

An affinity-based purification procedure allowed the resolution of two distinct groups of endoxylanase inhibitors with different molecular structures and endoxylanase specificities from wheat wholemeal. The first group comprises the so-called Triticum aestivum L. Endoxylanase inhibitor (TAXI)-type proteins which are of approx. M_r 40 000 and occur in two different molecular forms. These inhibitors were removed from a concentrated cation exchange chromatography fraction from wheat wholemeal on a Bacillus subtilis endoxylanase affinity column. The second group of structurally different endoxylanase inhibitors, the so-called xylanase inhibiting protein (XIP)-type, of approx.M_r 29 000–32 000, with pI values varying between 8·8 and 9·2, was purified from the unbound fraction from the B. subtilis endoxylanase affinity column by chromatography on an Aspergillus niger endoxylanase affinity column followed by gel permeation chromatography. The XIP-type inhibitors are not active against the B. subtilis endoxylanase and were consequently not retained on the B. subtilis endoxylanase column. Further analysis of the XIP-type proteins by high-resolution cation exchange chromatography, SDS-PAGE and iso-electrofocusing, revealed several forms. They had similar endoxylanase specificities and N-terminal amino acid sequences.     

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.     

The Role of Dextrins in the Stickiness of Bread Crumb made from Pre-Harvest Sprouted Wheat or Flour Containing ExogenousAlpha-Amylase

Dextrins were extracted in water from bread made from pre-harvest sprouted wheat or standard flour supplemented with exogenousalpha-amylases. The dextrins were separated by gel permeation chromatography and the dextrin content (% of crumb weight) determined for different degree of polymerisation (DP) size classes; DP 1–2, DP 3–10, DP 11–50, DP 51–200 and DP >200. There were significant correlations between the dextrin content in each size class and crumb stickiness (r=0·84–0·91, 22 df ). The most significant correlation (r=0·96) was between total dextrin content and crumb stickiness. Addition of dextrins of various DP ranges from various sources to standard flour produced bread with sticky crumb. Again, the degree of stickiness was generally related to the amount of total dextrin in the crumb and not to size distribution of dextrins. In this instance, extensive enzymic hydrolysis of starch was not necessary to produce sticky crumb; the dextrins caused crumb stickiness directly. Addition of dextrins to reconstituted gluten–starch flour produced bread with unexpectedly low dextrin levels and correspondingly low stickiness scores. It is concluded that, to produce sticky crumb, high levels of dextrin of any size are necessary in the crumb; a sticky mass is produced when dextrins dissolve in the excess «free» water that is normally «bound» to starch, gluten and other insoluble components of bread crumb.     

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.     

Protein Inhibitors ofAlpha-amylase in Mature and Germinating Grain of Rye (Secale cereale)

The activities of endogenous (R-type) and exogenous acting (D-type) protein inhibitors ofalpha-amylase and the activities ofalpha- and total amylase were determined in milling fractions of rye. High D-type amylase inhibitor activities were detected in the embryo (255 IU/g) and in the endosperm fraction (64·9 IU/g), low inhibitor activities were found in the aleurone layer fraction (25·9 IU/g). The highest R-typealpha-amylase inhibitor activity was found in the aleurone layer fraction (32·6 IU/g), and the lowest value in the epidermis containing fraction (5·0 IU/g). The D- and R-typealpha-amylase inhibitor activities varied with growing conditions. D-type amylase inhibitor activities were found to be high in those samples which grew under drought conditions and low in samples cultivated under wet and cool weather. Higher R-typealpha-amylase inhibitor activities were found in rye genotypes cultivated under wet conditions and lower values under dry weather. There were small variations inalpha-amylase inhibitor activities between sprout-stable and sprout-sensitive rye genotypes. The D- and R-typealpha-amylase inhibitor activities of all varieties were stable during 72 h of germination. Similar soil conditions will therefore lead to differentialalpha-amylase inhibitor activities depending on weather conditions during growth.     

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.     

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.