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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Analysis of Effects of Environment on Wheat Flour Quality and Allergenic Potential

Location: Crop Improvement & Utilization Research

2013 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).

3.Progress Report:
This project terminated in September of 2013 and was replaced with project 5325-43000-028-00D, "Molecular Analysis of Proteins Involved in Wheat Flour Quality and Allergenic Potential in Response to Environmental and Nutritional Stress". This is the final report for this project.

A proteomic approach provided new insights into the effects of post-anthesis fertilizer and high temperature on genes and proteins involved in wheat flour quality and allergenic potential in the US wheat Butte 86 (Objective 1). Using a detailed proteomic map in which most flour proteins were linked to specific gene sequences, the proportions of individual gluten proteins were determined in flour from plants grown with and without post-anthesis fertilizer. Of the gluten proteins, the omega gliadins showed the greatest change with fertilizer, increasing 144%. High-molecular-weight glutenin subunits and a few alpha gliadins increased to a lesser extent while several low-molecular-weight glutenin subunits decreased. Proportions of alpha gliadins containing epitopes involved in the food intolerance celiac disease were not altered. One important type of food allergen, the omega-5 gliadins, increased with fertilizer while a number of non-gluten proteins that are potential food allergens were more abundant in flour produced without fertilizer. Additional analyses demonstrated that high temperatures had surprisingly similar effects on gluten protein accumulation. However, a number of non-gluten proteins increased with high temperature, including several proteins that are food allergens. A proteomic approach also was used to determine the precise protein compositions of gluten polymer fractions (Objective 1). Flour proteins were fractionated on the basis of solubility in 0.5% SDS, further separated into monomeric and polymeric fractions by size exclusion chromatography, and analyzed by quantitative two-dimensional gel electrophoresis (2-DE) followed by tandem mass spectrometry. The analysis revealed the presence of gliadins containing an odd number of cysteine residues in polymer fractions, supporting their role as chain-terminators of the gluten polymer. In addition, several types of non-gluten proteins were found in fractions containing small polymers. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure. Progress also was made in developing molecular approaches to reduce the allergenic potential of wheat flour (Objective 2). Genetic transformation methods were developed for the US commercial wheat cultivar Butte 86. RNA interference was used to produce transgenic plants in which genes encoding two different types of food allergens, omega-5 gliadins and lipid transfer proteins (LTPs), were silenced. Omega-5 gliadins increase in response to both fertilizer and high temperature, while LTPs increase in response to high temperature. Quantitative 2-DE was used to determine the precise effects of the genetic modifications on the proteome in homozygous plants showing suppression of omega-5 gliadins. These analyses identified the best genetic material for further analyses of flour quality and allergenic potential.

1. High temperatures and fertilizer have similar effects on the wheat gluten proteins. The wheat gluten proteins determine the functional properties that make it possible to produce bread, noodles and a wide variety of products from wheat flour. ARS researchers at Albany, California, used comparative proteomics to examine the accumulation of individual gluten proteins in flour from grain produced under different fertilizer and temperature regimens. Although fertilizer and high temperature have very different effects on grain development and yield, the same set of gluten proteins responded to both treatments. The data suggest that high temperature conditions mimic high nitrogen conditions by shortening the period of time in which nitrogen is remobilized to the grain. The study provides new insight into how environmental conditions during wheat grain development affect the functional properties of the flour.

Review Publications
Denery-Papini, S., Boninier, M., Pineau, F., Triballeau, S., Tranquet, O., Adel-Patient, K., Moneret-Vautrin, Bakan, B., Marion, D., Mothes, T., Mameri, H., Kasarda, D.D. 2011. Immunoglobulin-E-binding epitopes of wheat allergens in patients with food allergy to wheat and in mice experimentally sensitized to wheat proteins. Journal of Clinical and Experimental Allergy. DOI:10.1111.j.1365-2222.2011.03808.x.

Kasarda, D.D., Adelsteins, E., Lew, E., Lazo, G.R., Altenbach, S.B. 2013. Characterization of a novel wheat endosperm protein belonging to the prolamin superfamily. Journal of Agricultural and Food Chemistry. 61:2407-2417.

Altenbach, S.B. 2012. New insights into the effects of high temperature, drought and post-anthesis fertilizer on wheat grain development. Journal of Cereal Science. 56:39-50.

Hurkman II, W.J., Tanaka, C.K., Vensel, W.H., Thilmony, R.L., Altenbach, S.B. 2013. Comparative proteomic analysis of the effect of temperature and fertilizer on gliadin and glutenin accumulation in the developing endosperm and flour from Triticum aestivum L. cv. Butte 86. Proteome Science. doi:10.1186/1477-5956-11-8.

Robertson, G.H., Hurkman II, W.J., Anderson, O.D., Tanaka, C.K., Cao, T., Orts, W.J. 2012. Differences in alcohol-soluble protein from genetically altered wheat using capillary zone electrophoresis, one- and two-dimensional electrophoresis and a novel gluten matrix association factor analysis. Cereal Chemistry. 90(1):13-23. DOI: 10.1094/cchem-10-11-0123.

Boggess, M.V., Lippolis, J.D., Hurkman II, W.J., Fagerquist, C.K., Briggs, S.P., Gomes, A.V., Righetti, P., Bala, K. 2013. The need for agriculture phenotyping: “Moving from genotype to phenotype”. Journal of Proteomics. doi:

Robertson, G.H., Cao, T., Gregorski, K.S., Hurkman II, W.J., Tanaka, C.K., Chiou, B., Glenn, G.M., Orts, W.J. 2013. Modification of vital wheat gluten with phosphoric acid to produce high free solution capacity. Journal of Applied Polymer Science. DOI: 10.1002/app.39440.

Last Modified: 7/24/2014
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