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Exploitable results
Following the development of a theory of dough elasticity, a new understanding of the role of sequence details of the High Molecular Weight subunits has been developed. This will enable a route to the rational design of these proteins for the control of dough rheology.
A 150 amino acid section of the repetitive domain of HMWDx15 was subcloned and ligated so as to produce sections of various length containing repeat consensus sequences. Small angle x-ray scattering, sedimentation analysis and diffusion measurements indicated the central domain poses on elongated shape. Extensive small angle neutron scattering studies in water and deuterium oxide were combined with the previous data and modeled. A self-consistent picture of the repetitive domains resulted which differs from one obtained with any single technique. Repeating the measurements on units of different length, confirms that length is not an important factor in determining the folding properties of the protein. In contrast to models based on a single type of measurement, these combined data clearly show that the dynamics of the protein is best modeled with a worm-like chain model.
A new chromatographic method was developed to purify HMW subunits by dye-ligand chromatography. HMW subunits recognized Cibacron Blue F3GA dye immolized on a chromatographic support and were separated by using their different ability to bind the dye.
The results indicated that it is possible to purify HMW subunits from a mixture, without preliminary purification, by dye-ligand chromatography in a single step.
Pairs of near isogneic lines with HMW glutenin subunits 1Dx5+1Dy10 or 1Dx2+1Dy12 have been grown during two seasons at two nitrogen levels in Sweden, UK and France.
The grain samples have been analysed for protein quantity and quality, rheological properties and baking quality. In winter wheats the 5+10 lines have shown stronger dough characteristics and usually better breadmaking performance, especially at more intensive dough treatments.
In the tested spring wheat pairs, with better genetic background, the 2+12 lines show higher baking quality.
The different nitrogen fertilization levels showed that low protein contents tend to give overstrong doughs, especially in 5+10 lines.
The winter wheat pairs with the 1B.1R rye chromosomes segment had very weak dough properties and low baking quality when grown in Sweden and UK, but in the French sunny weather the quality was acceptable.
The spring wheat pair with subunit 1Ay21* had very good quality in combination with both subunits 5+10 and 2+12.
The folding and aggregation of the HMW DX5 domains studied at various temperatures shows that none of the domains is compactly folded so that there is little hysteresis at elevated temperatures. Aggregation does occur at temperatures above 50oC. Therefore, intermolecular interactions, which develop at elevated temperatures, play a larger role in determining the mesoscopic properties of the system than do intramolecular interactions. Characterization of other HMW proteins for these properties should help develop a self-consistent set of rules enabling one to screen for and select properties which will be usable in developing desired baking properties.
Pairs of near isogenic lines with HMW glutenin subunits 1Dx5+1Dy10 or 1Dx2+1Dy12 have been grown during two seasons in Sweden, France and UK.
The material with reasonable protein content was tested for gluten content and quality and rheological performance with Alveograph. The so-called "Berilla index" was calculated. This index was constructed for the evaluation of the pasta quality of durum wheat but was used here for bread wheats.
In both winter wheats and spring wheats the 5+10 lines had better pasta quality than the 2+12 sister lines.
If bread wheats are to be used for pasta, the stronger gluten in 5+10 lines seems to be more suitable than the weaker 2+12 lines.
The strong dough properties should be combined with high carotenoid content and yellow flour.
The thermal behaviour of the HMW glutenin subunits 1Dx5, 1Bx7, 1Dx2, 1Bx20, 1Dy12, 1Bx6, 1Dy10 and of a 58,000 peptide was investigated using differential scanning calorimetry and thermogravimetric analysis. The plasticizing effect of water on each protein was investigated. When submitted to the calorimetric analysis, the HMW glutenin subunits exhibited a glass transition phenomenon that was affected by the water content.
13C NMR techniques enable the extent of "plasticisation" of wheat subunits by hydration to be estimated. This was done for the individual subunits 1Dx5, 1Dy10, 1Dx2 and 1Dy12 as well as for the 1Dx5/1Dy10 and 1Dx2/1Dy12 pairs. Upon hydration, hydrogen bonds between glutamines are broken and about 50% of the protein is mobilized, the remaining protein remaining held rigid by strong hydrogen bonds. This behaviour is observed for all subunits.
The behaviour of the 1Dx5/1Dy10 and 1Dx2/1Dy12 pairs upon hydration was compared with that of the single subunits and it was seen that 1) the 5+10 pair behaves identically to the "sum behaviour" of the single 1Dx5 and 1Dy10 subunits and 2) the 2+12 pair behaves differently from the "sum behaviour" of the single subunits.
Therefore, a closer/stronger molecular interaction seems to be established between the molecules of 1Dx2 and 1Dy12 subunits. This may be at the basis of the different technological performances of the two subunit pairs.
13C NMR techniques enable the extent of "plasticisation" of wheat subunits by hydration to be estimated. This was done for the individual subunits 1Dx5, 1Dy10, 1Dx2 and 1Dy12 as well as for the 1Dx5/1Dy10 and 1Dx2/1Dy12 pairs. Upon hydration, hydrogen bonds between glutamines are broken and about 50% of the protein is mobilized, the remaining protein remaining held rigid by strong hydrogen bonds. This behaviour is observed for all subunits.
The behaviour of the 1Dx5/1Dy10 and 1Dx2/1Dy12 pairs upon hydration was compared with that of the single subunits and it was seen that 1) the 5+10 pair behaves identically to the "sum behaviour" of the single 1Dx5 and 1Dy10 subunits and 2) the 2+12 pair behaves differently from the "sum behaviour" of the single subunits.
Therefore, a closer/stronger molecular interaction seems to be established between the molecules of 1Dx2 and 1Dy12 subunits. This may be at the basis of the different technological performances of the two subunit pairs.
A total of 31 independently transformed lines containing various engineered and native HMW glutenin genes were generated in two model wheat backgrounds: L88-31 and L88-6.
The presence of transgenes in all T0 plants was confirmed by PCR. The expression of the HMW glutenin proteins was confirmed by SDS-PAGE, showing good correlation with PCR analysis. Many of these lines now exist in a homozygous state in the T3 generation.
Five lines were also generated containing a HMW:GUS promoter: reporter fusion to confirm endosperm-specific expression of transgenes. These showed that the HMW promoter directed expression specifically to the endosperm in the L88 wheat background.
Analysis of these lines will contribute to our understanding of the relationships between HMW subunit structure and functional properties.
Provide an overview of the result which gives the reader an immediate impression of the nature of the result, its relevance and its potential; Briefly describe the current status/applications of the result (if appropriate) with non confidential information on entities potentially involved.
Glutens extracted from four near-isogenic lines differing in their HMW subunits were analyzed. Rheological properties were studied by dynamic assay in shear and creep and recovery. Size distribution of prolamin was determined by sequential extraction and Size Exclusion High Performance Liquid Chromatography (SE-HPLC). Assays carried out at 20 ºC provided mechanical spectra over a broad angular frequency range. They were typical of a transient network. Gluten viscoelasticity was determined by large glutenin polymers. Their amounts in the glutens depended on the HMW subunit composition of the lines. The deletion of HMW subunit genes was shown to cause a collapse of gluten viscoelasticity and subunit alleles differed in their "viscoelastic potential", expressed through polymers and aggregates. Glutens were submitted to heating/cooling cycles, in the presence or in the absence of SH-blocking agent. In the 20-40ºC temperature range, no irreversible change of mechanical properties occurred. Weakening of gluten connectivity was in agreement with disruption of intermolecular H bonds and increased chain mobility upon heating. The necessity of covalent crosslinking to build viscoelastic protein material was demonstrated by studying the effects of heating gluten above 40ºC as well as the effects of limited disulphide reduction on gluten mechanical properties
The role of the terminal domains on the interfacial properties of HMW-GS 1Dx5 was investigated by a comparison with the 58K central repetitive domain of subunit 1Dx5. The effect of ionic strength was also investigated. It was found that the 58K peptide adsorbed to a solid hydrophobic surface and was insensitive to the ionic strength. Further, the amount adsorbed showed only a weak dependence on the bulk concentration. This contrasts to the adsorption of subunit 1Dx5 that was more sensitive to both the ionic strength and the bulk concentration. The conclusions were: i) The terminal domains are responsible for dependence of ionic strength on the interfacial properties of subunit 1Dx5 ii) Subunit 1Dx5 can give a higher adsorbed amount than the 58K peptide due to a preferential attachment of the terminal domain(s) and hence a more vertical orientation.
Tensiometric measurements showed that the 58K peptide had a very low ability to reduce surface tension. Subunit 1Dx5, on the other hand, showed a significant reduction of surface tension, even at adsorption from low bulk concentrations.
This suggests that in situations where the interfacial behaviour of HMW-GS is of interest, the focus should be on the properties of the terminal domains.
Provide an overview of the result which gives the reader an immediate impression of the nature of the result, its relevance and its potential; Briefly describe the current status/applications of the result (if appropriate) with non confidential information on entities potentially involved.
Results show that hydrating wheat subunits leads to the formation of an insoluble network where crosslinked proteins segments form large pockets of water. The "stiffness" of the network, as measured by molecular mobility, depends on several structural factors including the presence of SS bonds, primary structure, heterogeneity, the mixture of different subunits and residual lipids). It is concluded that:
1. the presence of SS bonds induces higher network flexibility
2. higher number of consecutive glutamine residues induces less network flexibility
3. higher heterogeneity induces less network flexibility
It is the combined action of 1, 2 and 3 that determines the final molecular flexibility of the subunits. Hence, careful control of these factors may be used to tailor the properties of the network. These properties may potentially be correlated to the technological properties of wheat subunits.
Crosses have been made, involving several bread wheat cultivars and lines, in order to produce isogenic wheat lines differing in number and type of HMW subunits. In particular, lines differing in number of subunits (from three up to six) and size of the repetitive domain have been generated in the bread wheat cultivar Pegaso. Such lines are being used to develop novel germplasm to improve the breadmaking properties of flour as well as to provide basic information on the relationship between HMW subunits and gluten functionality.
Glutenin aggregation and mixing properties of transgenic lines of wheat, in which different HMW glutenin subunits were inserted, were studied. Glutenin aggregation depended on the number of HMW subunits expressed by the lines. Insertion of 1Ax1 subunit increased glutenin aggregation, but no insoluble aggregate was formed. Insertion of 1Dx5 subunit resulted in the formation of large amounts of highly covalently cross-lined aggregates. This difference was attributed to the presence of an additional cysteine residue in the 1Dx5 subunit, available for intermolecular cross-linking. The expression of inserted 1Ax1 subunit improved significantly dough mixing properties. On the other hand expression of 1Dx5 subunit resulted in abnormal mixing behaviour: no homogenous dough was formed with any mixing time tested. This result suggested that over expression of subunit 5 in the absence of additional subunit 10 resulted in extensive restructuring of glutenin polymers. The results demonstrated that transformation by insertion of HMW glutenin subunits permits to induce new technological properties in relationship with subunit specific structure.
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