2011 Annual Report
1a.Objectives (from AD-416)
The first objective of the research is to determine the basis for changes in flour quality that result from high temperatures during wheat grain development. The research will investigate the roles of gluten composition and polymer structure in effects of temperature during grain development on flour quality. The research also will determine the roles of specific non-gluten proteins in effects of temperature during grain development on flour quality. This objective addresses a major concern of millers and bakers and explores two hypotheses:.
1)changes in glutenin polymer amount, size, structure and composition as a result of high temperatures during grain development are responsible for decreases in flour quality and.
2)non-gluten proteins that increase in the grain under high temperature conditions are involved in quality or allergenicity.
The second objective of the research is to identify and characterize wheat proteins responsible for human intolerances and allergies that affect nearly 2% of the U.S. population and to develop methods to detect allergenic proteins in downstream products. This objective will determine whether mass spectrometry (MS) can be used to identify potential wheat allergens in flour and detect these proteins in food ingredients and products.
1b.Approach (from AD-416)
To address the first objective, MS methods coupled with improved methods for protease digestion will be developed so that closely related gluten proteins can be distinguished. Size-exclusion (SE) chromatography and high pressure liquid chromatography (HPLC) will be used to separate glutenin polymers into size classes for determination of subunit composition and key linkages between high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS). Gluten proteins that act as chain terminators in polymer structure will be identified and their roles in polymer structure and size will be evaluated. Polymer composition and size will be measured during grain development under different temperature regimens. The effect of different temperature regimens on accumulation profiles of a specific set of non-gluten proteins and their transcripts during grain development also will be characterized using 2-dimensional polyacrylamide gel electrophoresis (2-DE) and quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). Since many of these proteins may be involved in stabilizing gas bubbles in dough, experiments will be performed to test whether the levels of these proteins increase in dough liquor prepared from flour from grain grown under high temperatures. Tissue localization studies will be performed and the roles that specific proteins play in flour quality and allergenicity will be investigated using transgenic plants in which the corresponding genes are suppressed.
To address the second objective, the allergenic potential of non-gluten proteins that increase under high temperature conditions will be tested using sera from patients with defined wheat allergies. MS will be used to determine mass profiles of protein fractions from wheat flour. These profiles will be examined for signatures of specific allergenic proteins. Methods will be extended to samples from baked products such as bread. Replacing 5325-43000-026-OOD (June/2010).
A major achievement of the project in FY11 was the completion of the first comprehensive map of the flour proteome from the US wheat cultivar Butte 86. The map links the majority of the abundant flour proteins separated by two-dimensional gel electrophoresis (2-DE) with specific gene sequences and provides a critical resource for evaluating the impact of environmental conditions and agronomic inputs on flour protein composition. Using this map, the protein composition of flour from plants grown with or without post-anthesis fertilizer was analyzed. Significant changes in the proportions of 54 unique proteins were observed as a result of the treatment. Most omega-gliadins, HMW-GS, serpins and certain alpha-gliadins and LMW-GS increased with fertilizer. The effect of post-anthesis fertilizer and high temperature on gluten protein accumulation during grain development also was assessed by 2-DE. The relative levels of omega gliadins increased and LMW-GS decreased with high temperature with or without fertilizer. In collaborative research, flour samples from transgenic wheat lines with altered levels of HMW-GS were also analyzed to determine the effect of the transgenes on levels of individual gliadins and glutenins.
Other experiments were directed towards characterizing the glutenin polymer fraction from wheat flour. Glutenin polymers consist of disulfide linked gluten proteins and are particularly difficult to study because they range in size from 100-10,000 kD. A variety of solvent systems were used to extract polymer fractions that then were characterized by size exclusion chromatography. A positive relationship was found between the amount of extractable glutenin polymer and flour quality. Size exclusion chromatography revealed that there was a continuous distribution of polymer sizes. However, gel analysis indicated that the polymer subunit composition was similar across different polymer size classes.
A major effort also was aimed at inhibiting the expression of genes encoding allergenic proteins in wheat grain. Central for this work was the development of genetic transformation methods for the commercial US wheat cultivar Butte 86. An RNA interference construct designed to specifically inhibit genes encoding omega-gliadins that are known food allergens was introduced into Butte 86 using biolistics. Stable transformation of Butte 86 plants and transgene inheritance were demonstrated. Analyses of grain protein from transgenic plants revealed that omega gliadins that cause the food allergy wheat-dependent exercise-induced anaphylaxis were absent in grain from the transgenic plants. In addition, RNA interference constructs designed to inhibit the expression of two genes encoding lipid transfer proteins were transformed into the model wheat Bobwhite and transgenic plants were regenerated and confirmed by PCR analysis. These constructs are now being used to transform Butte 86.
Transformation methods facilitate alteration of flour protein composition in a U.S. wheat cultivar. Although the genetic transformation of wheat was reported nearly two decades ago, transformation methods remain genotype-dependent and are efficient for only a few wheat varieties. Genetic transformation methods were developed for the well-characterized bread wheat Butte 86, a commercial US cultivar in which the majority of flour proteins have been associated with specific gene sequences. The ability to transform Butte 86 coupled with the detailed knowledge of genes expressed in developing grain facilitates targeted efforts to alter flour composition in transgenic plants by gene silencing. This research makes it possible to test hypotheses about the roles of specific gluten proteins in glutenin polymer formation and flour quality and to reduce the immunogenic potential of wheat flour for patients with celiac disease and food allergies.
Silencing of genes encoding food allergens in grain from transgenic wheat plants. Wheat is among the top eight foods responsible for IgE-mediated food allergies. An RNA interference construct was designed to silence genes encoding a subset of omega gliadins that trigger wheat-dependent exercise-induced anaphylaxis (WDEIA), a serious food allergy that occurs in certain individuals when the ingestion of wheat is followed by physical exercise. The construct was introduced into the commercial US wheat cultivar Butte 86 using biolistics and plants were regenerated. Protein analysis revealed that the omega-5 gliadins were absent in grain from transgenic plants. The elimination of omega-5 gliadins from wheat flour should reduce the risks involved in eating wheat-based products for individuals sensitive to WDEIA.
A comprehensive proteome map links the majority of wheat flour proteins to specific gene sequences. Determining the effects of environment on the accumulation of wheat flour proteins is essential to minimize the variations in flour quality that create problems for millers and bakers. However, these analyses are challenging because the major gluten proteins consist of many similar proteins and very small changes in the sequences of individual proteins can be important for flour quality. A two-dimensional electrophoresis (2-DE) map of the wheat flour proteome was developed in which 234 proteins were identified by tandem mass spectrometry (MS/MS) and associated with specific gene sequences from the US wheat Butte 86. This proteome map is a major achievement in wheat quality research, making it possible to measure the precise effects of environmental factors on flour protein composition, including subunit composition of the glutenin polymer and prevalence of proteins that contain celiac epitopes or are food or occupational allergens. Thus far, the map has been used to delineate the precise effects of fertilizer on the entire array of wheat flour proteins.
Dupont, F.M., Vensel, W.H., Tanaka, C.K., Hurkman II, W.J., Altenbach, S.B. 2011. Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry. Proteome Science. 9(10) available:http://www.proteomesci.com/content/9/1/10.
Hurkman Ii, W.J., Wood, D.F. 2010. High temperature during grain fill alters the morphology of protein and starch deposits in the starchy endosperm cells of the developing wheat (Triticum aestivum L.) grain. Journal of Agricultural and Food Chemistry. 59(9):4938-4946. DOI: 10.21/jfl102962t.
Depaolo, R.W., Abadie, V., Tang, F., Fehlner-Peach, H., Hall, J.A., Wang, W., Marrieta, E.V., Kasarda, D.D., Waldmann, T.A., Murray, J.A., Semrad, C., Kupfer, S.S., Belkaid, Y., Guandalini, S., Jabri, B. 2011. Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens. Nature. 471(7337):220-224.
Altenbach, S.B., Allen, P.V. 2011. Transformation of the US bread wheat Butte 86 and silencing of omega-5 gliadin genes. GM Crops. 2(1):67-74.
Vensel, W.H., Dupont, F.M., Altenbach, S.B., Sloane, S.M. 2011. Effect of Cleavage Enzyme, Search Algorithm and Decoy Database on Mass Spectrometric Identification of Wheat Gluten Proteins. Phytochemistry. 72(10):1154-1161.