Roger J. Fido

Milling and baking properties of field grown wheat expressing HMW subunit transgenes

Abstract Milling and baking tests were carried out on three transgenic wheat lines and their parental varieties grown in the field at two UK sites. The transgenic and control lines were essentially similar in their milling properties but the subunit 1Ax1 and 1Dx5 transgenes had different effects on breadmaking. The subunit 1Dx5 transgene resulted in a low loaf volume and poor crumb structure when expressed in lines with two or five endogenous HMW subunits, and this was accompanied by a greatly increased elastic modulus of the gel protein fraction. In contrast, the 1Ax1 transgene resulted in improved breadmaking quality and a more modest increase in the gel protein elastic modulus when expressed in the two subunit background. Blending of flour from a line expressing the 1Dx5 transgene with flour from a normal breadmaking wheat variety resulted in decreased breadmaking quality, even at a ratio of 1:9. The difference in the results obtained with the 1Ax1 and 1Dx5 transgenes may relate to the presence of an additional cysteine residue in the protein encoded by the latter, which promotes a more highly cross-linked glutenin network.

Comparative field performance over 3 years and two sites of transgenic wheat lines expressing HMW subunit transgenes

A series of transgenic wheat lines expressing additional high molecular weight (HMW) subunit genes and the corresponding control lines were grown in replicate field trials at two UK sites (Rothamsted Research, approximately 50 km north of London and Long Ashton, near Bristol) over 3 years (1998, 1999, 2000), with successive generations of the transgenic lines (T3, T4, T5) being planted. Four plots from each site were used to determine grain dry weight, grain nitrogen, dough strength (measured as peak resistance by Mixograph analysis) and the expression levels of the endogenous and “added” subunits. Detailed statistical analyses showed that the transgenic and non-transgenic lines did not differ in terms of stability of HMW subunit gene expression or in stability of grain nitrogen, dry weight or dough strength, either between the 3 years or between sites and plots. These results indicate that the transgenic and control lines can be regarded as substantially equivalent in terms of stability of gene expression between generations and environments.

Studies in vivo of ω-gliadins in gluten sensitivity (coeliac sprue disease)

1. Highly purified ω-gliadins from wheat were used to challenge gluten-sensitized individuals. Characteristic responses by mucosal CD3+ and γδ+ lymphocytes were demonstrated. Each lymphocyte subset showed an increase within 8–12 h post-challenge, indicating a specific response by the rectal mucosa to this gliadin species. 2. Available sequence data for the ω-gliadins and homologous proteins from barley and rye indicate a common repeating octapeptide motif (consensus PQQPFPQQ). The results indicate, therefore, that the octapeptide repeat, or a contained sequence such as PQQP, plays an important role in the mucosal immunopathology of gluten sensitivity.

Immunochemical identification of LMW subunits of glutenin associated with bread-making quality of wheat flours

A murine monoclonal antibody (IFRN 0067), one of a library developed against prolamin fractions fromTriticum aestivum, has been characterised using a combination of immunoassay and immunoblotting techniques. The antibody was specific for two glutenin polypeptides which appeared by 2-dimensional electrophoresis to belong to the B group of LMW subunits. From results of antibody-binding studies with material extracted from genetic stocks, it was deduced that the target polypeptides were encoded on the short arm of chromosome 1D. The antibody was used in an immunoassay of bread wheats with a range of anticipated baking scores and for flours of known baking performance. Significant correlations were found between immunoassay and test-bake results. Indeed, correlation of IFRN 0067 binding with loaf volume was equal or better than that provided by alveograph parameters. The results provide evidence that LMW subunits contribute to the bread-making properties of wheat glutenin, as identified by the use of immunological techniques. The use of particular monoclonal antibodies, such as IFRN 0067, in the further development of simple, rapid diagnostic tests for flour quality predictions is discussed.

Improving the end use properties of wheat by manipulating the grain protein composition

The wheat storage proteins account for up to about 60% of the totalgrain proteins. They correspond to the gluten proteins, which form avisco-elastic network that enables dough to be processed into bread, pastaand other products. We are using a range of biochemical, biophysical andmolecular approaches to characterize gluten proteins and to understandtheir role in determining the grain processing properties, focusing on thehigh molecular weight (HMW) subunits of glutenin which are majordeterminants of dough strength. We are also using genetic engineering toexplore the mechanism of glutenin elasticity and to effect improvements,by inserting genes encoding mutant and wild type HMW subunits intomodel lines and commercial cultivars.

Purification of nitrate reductase from spinach (Spinacea oleracea L.) by immunoaffinity chromatography using a monoclonal antibody

Abstract NADH-Nitrate reductase (EC 1.6.6.1) from spinach ( Spinacea oleracea L. v. Noorman) has been purified to apparent homogeneity by immunoaffinity chromatography using a monoclonal antibody linked covalently to Sepharose 4B followed by affinity chromatography. A pre-column of covalently linked non-immune rat γ globulin prevented non-specific binding. The enzyme, released with 1 M KNO 3 , was purified 1550-fold to a specific activity of 24.8 μmol NO 2 − produced min −1 , mg protein −1 with a recovery of 60% of applied NADH-NR activity. Proteolytically ‘nicked’ subunits, detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were removed by 5′-AMP Sepharose chromatography (Fido and Notton, Plant Sci. Lett., 37 (1984) 87).

Novel proteinase inhibitors in seeds of sunflower (Helianthus annuus L.) : polymorphism, inheritance and properties

Abstract A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinases and of the cysteine proteinase ficin in seeds and leaves of sunflower. One major and two minor groups of trypsin inhibitors were identified in seeds, the former having a high pI (@10) and also inhibiting chymotrypsin. Three groups of trypsin/subtilisin inhibitors were also present in seeds, together with three inhibitors of ficin. All groups showed polymorphism between lines of Helianthus annuus, while the trypsin and trypsin/subtilisin inhibitors also varied between wild species of Helianthus, with no apparent relationship to growth type (annual or perennial), genome constitution or ploidy level. Genetic analysis showed that the major trypsin inhibitor and three groups of trypsin/subtilisin inhibitors are each controlled by single Mendelian loci, with the three loci for trypsin/subtilisin inhibitors showing recombination values of 0.23–0.40. Purification by RP-HPLC allowed the Mr of two trypsin inhibitors to be determined by SDS-PAGE to be about 1,500 and 2,500, while the three trypsin/subtilisin inhibitors varied in Mr from about 1,500 to 6,000.

Genotypic variation and polymorphism of 2S albumins of sunflower

Summary2S albumin fractions from seeds of seven inbred lines and two cultivars of sunflower were compared using a combination of electrophoresis (one and two dimensional) and reversed-phase high performance liquid chromatography. Eleven to thirteen major components were present in each genotype with variation in their proportions and absence or presence. All appeared to be single chain proteins with relative molecular masser (Mr) between about 10.000 and 18.000; some had intra-chain disulphide bonds. Preliminary analyses of single F2 seeds indicated that some components were co-inherited, which could result from their being encoded by linked genes or by a single transcript from one gene.

Synthesis of storage reserves in developing seeds of sunflower

Abstract A combination of biochemical analyses and Northern blotting were used to determine the patterns of storage reserve (oil, 2S albumin, helianthinin, oleosin) synthesis during the early stages of embryo development in sunflower. This demonstrated that oleosin biosynthesis is initiated early in development which is consistent with a role in oil body biogenesis. The patterns of synthesis and accumulation of storage compounds were compared in young developing embryos of sunflower. Oil biosynthesis was clearly initiated very early in development with total lipids accounting for about 18% of the dry weight at the earliest stage studied. This was associated with the accumulation of oleosins and the expression of cytochrome b 5 . In contrast, significant amounts of helianthinin (11S globulin) and 2S albumin were only observed in stages 3 and 4, respectively. The early expression of oleosins contrasts with previous studies of other oilseeds and is consistent with the hypothesis that they are intimately associated with the biogenesis of oil bodies.

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.