Transformation of wheat with high molecular weight subunit genes results in improved functional properties.

The high molecular weight (HMW) subunits of wheat glutenin are major determinants of the elastic properties of gluten that allow the use of wheat doughs to make bread, pasta, and a range of other foods. There are both quantitative and qualitative effects of HMW subunits on the quality of the grain, the former being related to differences in the number of expressed HMW subunit genes. We have transformed bread wheat in order to increase the proportions of the HMW subunits and improve the functional properties of the flour. A range of transgene expression levels was obtained with some of the novel subunits present at considerably higher levels than the endogenous subunits. Analysis of T2 seeds expressing transgenes for one or two additional HMW subunits showed stepwise increases in dough elasticity, demonstrating the improvement of the functional properties of wheat by genetic engineering.

Genetic characterisation of dough rheological properties in a wheat doubled haploid population: additive genetic effects and epistatic interactions.

Doubled haploid lines (n = 160) from a cross between wheat cultivars 'Cranbrook' (high dough extensibility) and 'Halberd' (low dough extensibility) were grown at three Australian locations. The parents differ at all high- and low-molecular-weight glutenin loci. Dough rheological parameters were measured using small-scale testing procedures, and quantitative trait locus (QTL) mapping procedures were carried out using an existing well-saturated genetic linkage map for this cross. Genetic parameters were estimated using three software packages: QTLCartographer, Epistat and Genstat. Results indicated that environmental factors are a major determinant of dough extensibility across the three trial sites, whereas genotypic factors are the major determinants of dough strength. Composite interval mapping analysis across the 21 linkage groups revealed that as expected, the main additive QTLs for dough rheological properties are located at the high- and low-molecular-weight glutenin loci. A new QTL on chromosome 5A for M-extensibility (a mixograph-estimated measure of extensibility) was detected. Analysis of epistatic interactions revealed that there were significant conditional epistatic interactions related with the additive effects of glutenin loci on dough rheological properties. Therefore, the additive genetic effects of glutenins on dough rheological properties are conditional upon the genetic background of the wheat line. The molecular basis of the interactions with the glutenin loci may be via proteins that modify or alter the gluten protein matrix or variations in the expression level of the glutenin genes. Reverse-phase high performance liquid chromatography analysis of the molar number of individual glutenin subunits across the population showed that certain conditional epistases resulted in increased expression of the affected glutenin. The epistatic interactions detected in this study provide a possible explanation of the variable genetic effects of some glutenins on quality attributes in different genetic backgrounds and provide essential information for the accurate prediction of glutenin related variance in marker-assisted wheat breeding.

[Some new results from isolation and fractionation of low molecular weight wheat proteins]. / Einige neuere Ergebnisse bei der Isolierung und Fraktionierung von niedrigmolekularen Weizenproteinen

The alcohol-soluble wheat gluten fraction (gliadine) and the petroleum-ether-soluble wheat protein fraction (purothionine) were isolated and analysed. The gliadine fraction was itself fractionated by gel chromatography (Sephadex G-100). The partial hydrolysis of sub-fractions (molecular weight 20000-30000) yielded (after separation by gel and ion-exchange chromatography) several peptide fractions. The amino-acid composition and the N- and C-terminal groups of the gliadine fractions were determined. Characteristic differences were stated between various fractions. By means of gel chromatography, purothionine was separated into 4-6 fractions. The study of the amino-acid composition and of the N-terminal groups of the fractions revealed differences in both properties.

Our obsession with high resolution in gel electrophoresis: does it necessarily give the right answer?

Poor resolution of protein zones in an electrophoretic pattern may not necessarily be the result of poor technique. The example is given of the 'streak material', extracted from wheat flour, now recognised to be aggregated subunits of glutenin. The size distribution of the aggregated glutenin 'streak' is the key to elucidating the functional properties of wheaten dough. A stepped-layer gel technique has been devised to quantitate the proportions of aggregated glutenin in specific size groupings.

Rapid and automated characterisation of seed genotype using Micrograd electrophoresis and pattern-matching software.

New precast microgels are described for use in quickly identifying seed of cereal varieties by determining protein composition within an hour. For example, gliadin proteins are extracted from crushed wheat grain, wheatmeal or flour with ethylene glycol (centrifugation not necessary) and 5 microliters extract is applied to a Micrograd gel (3-15% gel gradient) for ten minutes' electrophoresis at 300 volts in sodium lactate buffer (pH 3.1). Alternatively, precast gels are available for SDS gel electrophoresis for examining a different aspect of grain composition as a means of identification. To further expedite identification, software packages have been developed to match the protein pattern for an unknown sample against those of authentic samples, thus to provide quick and definite identity, based on electrophoretic banding, densitometer scan, HPLC profile, multiple antibody reaction or RFLP pattern (PatMatch program). Furthermore, the program WhatWheat offers advice on the best combination of methods to use for a specific task of identification.

Distinction between genotypes of Lupinus species by sodium dodecyl sulphate-gel electrophoresis and by capillary electrophoresis.

Effective distinction was achieved among a wide range of lupin grain samples by either sodium dodecyl sulphate (SDS)-gel electrophoresis or capillary electrophoresis, based on grain-protein composition. Capillary electrophoresis was faster (< 1 h) and provided slightly greater distinction between the samples. On the other hand, SDS-gel electrophoresis could provide a greater through-put of samples in a 24 h period. Either technique could be used successfully to distinguish between lupin species and cultivars for taxonomic analysis or seed identification.

[Determination of vitamin D3 in premixes and mixed feeds]. / Uber die Bestimmung von Vitamin D3 in Praemixen und Futtermischungen

The authors developed a two-dimensional thin-layer chromatographic method for the qualitative and quantitative determination of vitamin D3 in premixes and mixed feeds. The procedure permits to determine I.U. = 0.128 micrograms of vitamin D3. The method is illustrated by the description of the analyses of 3 premixes and 5 mixed feeds.

Expression of epitope-tagged LMW glutenin subunits in the starchy endosperm of transgenic wheat and their incorporation into glutenin polymers.

The low-molecular-weight (LMW) glutenin subunits are components of the highly cross-linked glutenin polymers that confer viscoelastic properties to gluten and dough. They have both quantitative and qualitative effects on dough quality that may relate to differences in their ability to form the inter-chain disulphide bonds that stabilise the polymers. In order to determine the relationship between dough quality and the amounts and properties of the LMW subunits, we have transformed the pasta wheat cultivars Svevo and Ofanto with three genes encoding proteins, which differ in their numbers or positions of cysteine residues. The transgenes were delivered under control of the high-molecular-weight (HMW) subunit 1Dx5 gene promoter and terminator regions, and the encoded proteins were C-terminally tagged by the introduction of the c-myc epitope. Stable transformants were obtained with both cultivars, and the use of a specific antibody to the c-myc epitope tag allowed the transgene products to be readily detected in the complex mixture of LMW subunits. A range of transgene expression levels was observed. The addition of the epitope tag did not compromise the correct folding of the trangenic subunits and their incorporation into the glutenin polymers. Our results demonstrate that the ability to specifically epitope-tag LMW glutenin transgenes can greatly assist in the elucidation of their individual contributions to the functionality of the complex gluten system.

Bánkúti 1201--an old Hungarian wheat variety with special storage protein composition.

Bánkúti 1201, an old Hungarian wheat variety with special quality traits, was analysed to determine the relationships between its storage protein composition and superior quality-attributes for breadmaking. Based on the storage protein composition, the variety appears to have the nature of a population, containing several genotypes with different gluten protein alleles. Using molecular markers, a new mutant x-type HMW glutenin allele was identified, containing an extra cysteine residue and showing a moderate, positive-effect on gluten properties. In lines possessing subunits Bx7+By8 the overexpression of the Bx-type subunit could be detected, resulting in a higher unextractable polymeric protein (UPP) content and increased dough strength. It was found that the presence or absence of subunit Bx7 has an equilibrating effect on the dough extensibility, which is generally characteristic of the Bánkúti 1201 population. The complex good bread-making quality of the variety, which has strong but highly extensible dough, is probably due to the balance between lines which express subunit Bx7 and those which do not.

Molecular discrimination of Bx7 alleles demonstrates that a highly expressed high-molecular-weight glutenin allele has a major impact on wheat flour dough strength.

High-molecular-weight glutenin subunits (HMW-GS) are important determinants of wheat dough quality as they confer visco-elastic properties to the dough required for mixing and baking performance. With this important role, the HMW-GS alleles are key markers in breeding programs. In this work, we present the use of a PCR marker initially designed to discriminate Glu1 Bx7 and Glu1 Bx17 HMW-GS. It was discovered that this marker also differentiated two alleles, originally both scored as Glu1 Bx7, present in the wheat lines CD87 and Katepwa respectively, by a size polymorphism of 18 bp. The marker was scored across a segregating doubled-haploid (DH) population (CD87 x Katepwa) containing 156 individual lines and grown at two sites. Within this population, the marker differentiated lines showing the over-expression of the Glu1 Bx7 subunit (indicated by the larger PCR fragment), derived from the CD87 parent, relative to lines showing the normal expression of the Glu1 Bx7 subunit, derived from the Katepwa parent. DNA sequence analysis showed that the observed size polymorphism was due to an 18 bp insertion/deletion event at the C-terminal end of the central repetitive domain of the Glu1 Bx 7 coding sequence, which resulted in an extra copy of the hexapeptide sequence QPGQGQ in the deduced amino-acid sequence of Bx7 from CD87. When the DH population was analysed using this novel Bx7 PCR marker, SDS PAGE and RP HPLC, there was perfect correlation between the Bx7 PCR marker results and the expression level of Bx7. This differentiation of the population was confirmed by both SDS-PAGE and RP-HPLC. The functional significance of this marker was assessed by measuring key dough properties of the 156 DH lines. A strong association was shown between lines with an over expression of Bx7 and high dough strength. Furthermore, the data demonstrated that there was an additional impact of Glu-D1 alleles on dough properties, with lines containing both over-expressed Bx7 and Glu-D1 5+10 having the highest levels of dough strength. However, there was no statistically significant epistatic interaction between Glu-B1 and Glu-D1 loci.

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties.

The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an M(r) of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.