Comparative Studies of High MrSubunits of Rye and Wheat. II. Partial Amino Acid Sequences

A panel of anti-peptide antibodies specific for each of the different N-terminal sequence types of B- and C-low molecular mass glutenin subunits (L M_rGS) were utilised in immunoblotting studies to identify the chromosomal location of genes encoding different sequences and to characterise the allelic variation of the encoding loci. The MET-type sequences were predominantly found among the B- subunits, while the α- and γ- sequences predominated in the C- subunits. The quantitatively major SHIPGLERPS sequence was found in both the B- and C- mobility regions. Using either biotypes in the cultivar, Aroona or genetic lines containing double rye chromosome 1 substitutions and thus expressing only single LM_r GS alleles, the sequences were determined for most of the major polypeptides expressed by each LM_r GS allele. The L M_rGS from different genomes encoded different numbers of each sequence type. Furthermore, different polypeptides within a particular «block» of subunits encoded by a given allele often had differing N-terminal sequences. However, subunits of similar electrophoretic mobilities encoded by different alleles at each locus usually had identical N-terminal sequences, suggesting that they may instead differ in the number of repeats. In Chinese Spring, genes encoding the SHIPGLERPS and METSHIPGL sequence types were predominantly present on chromosomes 1B and 1D, while the related METSRVPGL sequence was only encoded on 1D. In contrast, the METSCIPGL, α- and γ-sequences were encoded on each of chromosomes 1A, 1B and 1D. Several different electrophoretic and immunoblotting approaches using null lines suggested that some of the α-type L M_rGS may also be encoded by group 6 chromosomes, particularly 6D. The anti- SHIPGLERPS antibody also recognised chromosome 1B encoded β-, γ- and ω-gliadins, while the anti-METSRVPGL antibody recognised 1D encoded α- and β-gliadins. The absence of sequences within the major gliadin families that are highly homologous to the latter two N-terminal L M_rGS sequences may suggest that some monomeric L M_rGS could exist within the electrophoretically-resolved gliadins. These antibodies will provide valuable reagents for the study of the roles of particular L M_rGS families in the structure and function of the glutenin macropolymer, the role of different LM_r GS types in determining the influence of allelic variation of L M_rGS composition on dough properties, and potentially in the development of diagnostics for these flour components.     

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.     

Antibodies to N-terminal Peptides of Low MrSubunits of Wheat Glutenin. II. Detection of Subunits Encoded by Different Loci

A panel of anti-peptide antibodies specific for each of the different N-terminal sequence types of B- and C-low molecular mass glutenin subunits (L M_rGS) were utilised in immunoblotting studies to identify the chromosomal location of genes encoding different sequences and to characterise the allelic variation of the encoding loci. The MET-type sequences were predominantly found among the B- subunits, while the α- and γ- sequences predominated in the C- subunits. The quantitatively major SHIPGLERPS sequence was found in both the B- and C- mobility regions. Using either biotypes in the cultivar, Aroona or genetic lines containing double rye chromosome 1 substitutions and thus expressing only single LM_r GS alleles, the sequences were determined for most of the major polypeptides expressed by each LM_r GS allele. The L M_rGS from different genomes encoded different numbers of each sequence type. Furthermore, different polypeptides within a particular «block» of subunits encoded by a given allele often had differing N-terminal sequences. However, subunits of similar electrophoretic mobilities encoded by different alleles at each locus usually had identical N-terminal sequences, suggesting that they may instead differ in the number of repeats. In Chinese Spring, genes encoding the SHIPGLERPS and METSHIPGL sequence types were predominantly present on chromosomes 1B and 1D, while the related METSRVPGL sequence was only encoded on 1D. In contrast, the METSCIPGL, α- and γ-sequences were encoded on each of chromosomes 1A, 1B and 1D. Several different electrophoretic and immunoblotting approaches using null lines suggested that some of the α-type L M_rGS may also be encoded by group 6 chromosomes, particularly 6D. The anti- SHIPGLERPS antibody also recognised chromosome 1B encoded β-, γ- and ω-gliadins, while the anti-METSRVPGL antibody recognised 1D encoded α- and β-gliadins. The absence of sequences within the major gliadin families that are highly homologous to the latter two N-terminal L M_rGS sequences may suggest that some monomeric L M_rGS could exist within the electrophoretically-resolved gliadins. These antibodies will provide valuable reagents for the study of the roles of particular L M_rGS families in the structure and function of the glutenin macropolymer, the role of different LM_r GS types in determining the influence of allelic variation of L M_rGS composition on dough properties, and potentially in the development of diagnostics for these flour components.     

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.     

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 location of disulphide bonds in α-type gliadins

Gliadin prepared from gluten of the cultivar Rektor by extraction with 70% (v/v) aqueous ethanol adjusted to pH 5.5 was separated by RP-HPLC. Amongst 23 components obtained, two α-type gliadins (α3- and α8-gliadin) were selected for the determination of disulphide bonds. After both proteins were digested with thermolysin, differential RP-HPLC (chromatography prior to and after reduction of disulphide bonds) was used for the detection of cystine peptides. Two cystine peptides from α3-gliadin and three cystine peptides from α8-gliadin were isolated by RP-HPLC. The resulting peptides were reduced and alkylated with 4-vinylpyridine, separated by RP-HPLC and their amino acid sequences determined. The cystine peptides from both α-type gliadins had similar structures, and the corresponding fragments had homologous sequences. One cystine peptide of each gliadin was composed of three fragments linked by two disulphide bonds. The second cystine peptide consisted of two fragments linked by one disulphide bond. The third cystine peptide derived from α8-gliadin was different from the second peptide in one position of the sequences (glutamic acid instead of glutamine). Comparing complete sequences of α-type gliadins described in the literature, the cystine peptides from α3- and α8-gliadins were identical with corresponding sequences of clones A1235 and A212, respectively¹¹. The structures of the cystine peptides analysed indicate one intramolecular disulphide bond within domain III of α-type gliadins and two disulphide bonds between domains III and V. The linkages found correspond to homologous linkages determined for low M_r subunits of glutenin and glutenin-bound γ-type gliadins⁶. Obviously, these intramolecular disulphide bonds are not linked randomly, but are strongly directed.     

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.     

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.     

Biochemical Characterisation of a Novel Polymeric Protein Subunit from Bread Wheat (Triticum aestivum L.)

A new wheat endosperm protein subunit that was found in accessions belonging to different collections was identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Insoluble in 0·5 M NaCl, 70% ethanol, dimethyl sulphoxide (DMSO) and 50% propan-1-ol, it appeared in the pellet corresponding to the polymeric proteins along with high (HMW) and low molecular weight (LMW) glutenin subunits (GS). In the reduced form, it had an electrophoretic mobility between those two types of glutenin subunits. The apparent M_r of this novel protein was estimated by SDS-PAGE to be 71 000. N-terminal sequence and amino acid analyses indicated a composition similar to the ω-gliadins encoded by genes located on chromosome 1B. This protein can be ascribed to the D-subunits of LMW-GS with at least one cysteine residue that allows it to form part of the polymeric structure of glutenin, as shown by reaction with a fluorogenic reagent specific for sulphydryl groups. Fractions collected after size exclusion high-performance liquid chromatography (SE-HPLC) fractionation and further characterised by SDS-PAGE, confirm that the protein participates in the glutenin polymeric structure. An increase in its concentration was observed in fractions collected within the polymeric peak as elution time increased, implying that a larger amount of this protein is present in small size polymers. The role of this protein in the complex relationship between endosperm proteins and quality parameters is discussed in relation to its likely role as a chain terminator.     

Protein bodies ontogeny and localization of prolamin components in the developing endosperm of wheat caryopses

During caryopsis development, prolamins are initially stored in individual protein bodies, then generate a protein matrix in the ripe caryopsis. The ontogeny of the protein bodies was analyzed by fluorescence and electron microscopy from 7 to 43 days after anthesis (dAA), a period of time from the cellularization of endosperm to its desiccation. A series of antibodies specific to each prolamin type (α/β-, γ-, ω-gliadins, low-molecular weight and high-molecular weight glutenin subunits) made it possible to localize and co-localize the different prolamins in organelles of endosperm cells at different developmental stages. Protein bodies containing prolamins were observed as early as 7 dAA. At the early developmental stages, protein bodies were spherical with diameters around 1–2 μm. Later, around 15 dAA, the PBs enlarged, and aggregation and/or coalescence were prominent at 21 dAA. From 33 dAA, individual PBs were no longer visible, but a protein matrix was confined in the space between starch granules. All prolamins were found in the same protein bodies, without any segregation according to their types. Immunochemical labelling of prolamins failed to reveal in TEM analyses any particular internal organization in protein bodies. Glutenin subunits and gliadins were observed in the Golgi apparatus at the early stages of endosperm development.     

Proteomic analysis of wheat recombinant inbred lines: Variations in prolamin and dough rheology

To investigate the impact of the 1BL.1RS translocation on dough strength and to understand how 1BL.1RS genotypes may overcome the loss of Glu-B3 and Gli-B1, proteomic profiles of 16 doubled haploid (DH) lines of similar glutenin composition but of different strength, as measured by Chopin's alveograph, were compared. The results showed that 32 spots, mainly prolamins, were differentially expressed and that five others were specific to high-strength DH lines. The identification and quantification of the prolamin fractions on the two-dimensional (2D) electrophoresis gels demonstrated that the high-molecular weight glutenin sub-unit (HMW-GS) were up-regulated by 25% in 1BL.1RS DH lines, even though the corresponding genes were not located on the missing 1BS chromosome. The γ-gliadins were also up-regulated (by 36%) in such lines to counterbalance, to some extent, the loss of LMW-GS of Glu-B3. The polymeric prolamin fractions also accumulated in high-tenacity lines and decreased in high-extensibility lines confirming the role of the inter-chain disulfide bonds in resistance to deformation. In contrast, the monomeric fraction of α-gliadin favored extensibility and decreased tenacity by increasing the accumulation (+12%) of α-gliadins in high-extensibility lines; the Gli-A1 allele of the parent Toronit was found to be more abundant when compared to the Gli-A1 allele of parent 211.12014.     

Influence of sulphur fertilisation on quantities and proportions of gluten protein types in wheat flour

Although different supplies of sulphur (S) during wheat growth are known to influence the quantitative composition of gluten proteins in flour, an effect on the amount and on the proportions of single protein types has yet not been determined. Therefore, wholemeal flours of the spring wheat ‘Star’ grown on two different soils and at four different levels of S fertilisation (0, 40, 80, 160 mg S per container) were analysed in detail using an extraction/HPLC procedure. The results demonstrated that the amount of total gluten proteins as well as of the crude protein content of flour was little influenced, whereas amounts and proportions of single protein types were strongly affected by the different S fertilisation. The changes were clearly dependent on the Cys and Met content of each protein type. The amount of S-free ω-gliadins increased drastically, and that of S-poor high-molecular-weight (HMW) glutenin subunits increased moderately in the case of S deficiency. In contrast, the amounts of S-rich γ-gliadins and low-molecular-weight (LMW) glutenin subunits decreased significantly, whereas the amount of α-gliadins was reduced only slightly. S deficiency resulted in a remarkable shift of protein proportions. The gliadin/glutenin ratio increased distinctly; ω-gliadins became major components, and γ-gliadins minor components, whereas the ratio of HMW to LMW glutenin subunits was well-balanced.     

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.     

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.     

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.     

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.     

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.     

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.     

Functional Properties of Wheat Gliadins. II. Effects on Dynamic Rheological Properties of Wheat Gluten

The effects of addition of purified total gliadin and its subgroups (α-, β-, γ- and ω-gliadins) on the dynamic rheology of gluten were investigated. The frequency sweeps of gluten with added α-, β-, γ- and ω2-gliadins showed unexpected increases in the magnitude of G′ and G′′, suggesting stiffening of the native gluten. Conversely, a reduction in the magnitude of G′ and G′′ occurred upon addition of the total gliadin fraction and the ω1-gliadin, implying softening of the gluten. Addition of individual gliadin fractions increased the values of slope log G′ vs log frequency, suggesting increased concentrations of uncrossed-linked material compared with the native gluten. There were significant differences in the slope values for individual gliadin fractions. The increasing order of slopes for different gliadins was: β- >γ- >α- =ω1>ω2, indicating that glutens containing ω- and α- gliadins are relatively less crossed-linked than those containing β- and γ-gliadins. The dynamic moduli, G′ and G′′, of cv. Hereward gluten showed significant positive relationships with Mixograph parameter peak dough resistance (PDR), and loaf volume for gliadin subgroups added to cv. Hereward flour.     

Disulphide structure of high-molecular-weight (HMW-) gliadins as affected by terminators

This study aimed at elucidating SS-bonds of HMW-gliadins (HGL) from wheat with the focus on terminators of glutenin polymerisation. HGL from wheat flour extracts non-treated or treated with the S-alkylation reagent N-ethylmaleinimide (NEMI) were compared. HGL from wheat flour Akteur were isolated, hydrolysed with thermolysin and the resulting peptides pre-separated by gel permeation chromatography and analysed by liquid chromatography/mass-spectrometry using alternating electron transfer dissociation/collision-induced dissociation. Altogether, 22 and 28 SS-peptides from samples without and with NEMI treatment, respectively, were identified. Twenty-six peptides included standard SS-bonds of α- and γ-gliadins, high-molecular-weight and low-molecular-weight glutenin subunits. Eleven SS-bonds were identified for the first time. Fifteen peptides unique to HGL contained cysteine residues from gliadins with an odd number of cysteines (ω5-, α- and γ-gliadins). Thus, gliadins with an odd number of cysteines, glutathione and cysteine had acted as terminators of glutenin polymerisation. Decisive differences between samples without and with NEMI treatment were not obvious showing that the termination of polymerisation was already completed in the flour. The two HGL samples, however, were different in the majority of ten peptides that included disulphide-linked low-molecular-weight (LMW) thiols such as glutathione and cysteine with the former being enriched in the non-treated HGL-sample.