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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Research Project #425203

Research Project: Molecular Analysis of Proteins Involved in Wheat Flour Quality and Allergenic Potential in Response to Environmental and Nutritional Stress

Location: Crop Improvement and Genetics Research

2018 Annual Report

1a. Objectives (from AD-416):
The quality of US wheat flour varies considerably depending upon the growth conditions of the crop during grain development. Mitigation of effects on end-users requires fundamental understanding of the influence of plant nutrition and environmental stress on flour protein composition, quality and allergenic potential. The project builds upon a portfolio of research accomplishments in proteomics, molecular biology and plant biotechnology using the US spring wheat Butte 86. Specifically, the research combines both transgenic and proteomic approaches to explore relationships between wheat flour quality and individual proteins that were shown previously to respond to high temperature or fertilizer during grain development. Objective 1 focuses on specific gluten proteins that are likely to play a role in flour quality while Objective 2 focuses on both gluten and non-gluten proteins that are known food allergens. A final component of the research investigates the effects of drought on the flour proteome thereby refining the picture of environmental impacts on flour quality and allergenic potential. Objective 1: Establish links between flour quality and quantitative changes in the flour proteome that occur in response to temperature or plant nutrition. Subobjective 1A. Create transgenic wheat lines in which genes encoding specific proteins that respond to temperature or fertilizer have been silenced. Subobjective 1B. Determine the effects of gene silencing on the flour proteome, glutenin polymer distribution and flour quality. Objective 2: Develop transgenic approaches to reduce the immunogenic potential of wheat grown under changing environmental conditions. Objective 3: Determine the molecular basis for variations in flour quality that occur in response to drought during grain fill.

1b. Approach (from AD-416):
Two hypotheses will be tested in Objective 1. Subobjective 1A will test the hypothesis that the silencing of genes in transgenic Butte 86 plants will provide genetic material for elucidating the roles of specific proteins in flour quality and in the response of the grain to post-anthesis nitrogen or high temperatures. RNA interference will be used to create transgenic lines suppressing omega-5 gliadins, omega-1,2 gliadins or s-type LMW-GS. The precise effects of the genetic modifications on the flour proteome will be evaluated by quantitative 2-dimensional gel electrophoresis (2-DE). Subobjective 1B will test the hypothesis that reductions in omega-5 gliadins, omega-1,2 gliadins or s-type LMW-GS alter glutenin polymer distribution and flour quality in transgenic plants grown under different post-anthesis nitrogen regimens. Transgenic lines will be grown under a moderate temperature regimen with and without post-anthesis nitrogen to determine how the grain responds to nitrogen in the absence of omega-5 gliadins, omega-1,2 gliadins or s-type LMW-GS. Quantitative 2-DE will be used to compare the amounts of individual flour proteins from control and transgenic plants grown under the two regimens. The proportions of individual gluten proteins in extractable and unextractable glutenin polymer fractions also will be determined. Proteomic results will be correlated with analyses of flour quality in the same samples. Objective 2 will determine whether food allergens that increase in the developing grain in response to temperature or fertilizer can be eliminated without major impacts on grain development or flour quality. RNA interference will be used to reduce or eliminate two confirmed food allergens, the omega-5 gliadins and the 9 kDa lipid transfer proteins, in grain from transgenic wheat plants. The extent of suppression will be examined by quantitative 2-DE of flour proteins and quality of the transgenic flour will be assessed using mixing and baking studies. Allergenic potential of transgenic lines lacking specific proteins will be tested by immunoblot analysis using sera from patients with confirmed wheat allergies. Objective 3 will determine whether drought results in quantitative changes in some of the same flour proteins that are affected by temperature and fertilizer. Wheat plants will be grown in greenhouses under controlled temperature, fertilizer and water regimens and quantitative 2-DE will be used to identify specific gluten and non-gluten flour proteins that respond to drought. The effects of drought on glutenin polymer distribution and composition and flour quality also will be determined. The studies will expand the understanding of the effects of environment on the wheat grain and identify new targets for genetic modification.

3. Progress Report:
This is the final report for this project that was replaced on March 12, 2018 by 2030-21430-014-00D, “New Genetic Resources for Breeding Better Wheat and Bioenergy Crops”. For additional information, see the report for the new project. Significant progress was made on all three objectives over the four and a half years of the project despite the retirement, December 2012, and death, February 2016, of two ARS scientists assigned to the project. Most of the work utilized the U.S. hard red spring wheat, Butte 86, a well-studied commercial cultivar for which a detailed map of the flour proteome was previously developed. This wheat cultivar also can be genetically transformed. Objective 1: Work focused on three major groups of wheat gluten proteins previously demonstrated to show quantitative changes in response to the application of fertilizer or high temperatures during grain development, the omega-5 gliadins, the omega-1,2 gliadins and a subset of low molecular weight glutenin subunits (LMW-GS), referred to as s-type LMW-GS. In Sub-objective 1A, transgenic lines were created in which genes encoding each group of proteins were silenced using RNA interference. Homozygous lines in which the targeted proteins were significantly reduced or eliminated were identified. In Sub-objective 1B, the precise effects of the genetic modifications on the flour proteome were assessed in transgenic lines by quantitative 2-dimensional gel electrophoresis (2-DE). Plants producing flour in which the omega-5 gliadins or the omega-1,2 gliadins were significantly reduced with few other changes on the flour proteome were selected for further study. In quality analyses, flour from transgenic lines missing the omega-5 gliadins were found to have increased mix times and mix tolerances, suggesting that omega-5 gliadins have a negative effect on the functional properties of the flour. Thus, increases in the levels of these proteins that occur when plants are supplied with post-anthesis fertilizer or subjected to high temperatures may be responsible for negative effects on flour quality. Similar analyses of lines in which the omega-1,2 gliadins were silenced are underway. In addition, both control and transgenic lines were grown in greenhouses with and without post-anthesis fertilizer and quantitative 2-DE analyses indicated that the transgenic lines had more stable protein compositions. In comparison, quantitative analyses of flour from most of the lines in which the s-type LMW-GS were targeted showed inhibition of other LMW-GS as well as compensatory effects on other flour proteins. Transgenic lines exhibiting different protein profiles were grown in greenhouses in sufficient quantities for quality analyses. These analyses will provide insight into how the proportions of protein types in the flour influence quality. The sizes and amounts of glutenin polymers are important for flour functionality. In Sub-objective 1B, the precise protein compositions of fractions containing large, insoluble gluten polymers and small, soluble gluten polymers were determined in flour produced with and without post-anthesis fertilizer. Both large and small polymer fractions contained both high molecular weight glutenin subunits (HMW-GS) and LMW-GS. However, certain LMW-GS, referred to as s-type and m-type, were more abundant in large polymer fractions while LMW-GS referred to as i-type were more abundant in small polymer fractions. This suggests that very similar LMW-GS proteins play different roles in the structure of the gluten polymer. Gliadins containing odd numbers of cysteine residues also were accumulated preferentially in the small polymer fraction, supporting the hypothesis that these proteins serve as terminators of polymer chains and reduce polymer size. Several types of non-gluten proteins, particularly those classified as serpins, triticins and globulins, also were found in polymer fractions, suggesting that changes in the levels of these proteins may influence quality by affecting polymer structure. Objective 2: The goal was to develop approaches to reduce the immunogenic potential of wheat flour. The omega-5 gliadins are the major sensitizing proteins in the serious food allergy wheat-dependent-exercise-induced anaphylaxis (WDEIA). In collaboration with scientists from the French National Institute for Agricultural Research (INRA) in Nantes, France, transgenic lines in which omega-5 gliadins were silenced were evaluated by 2-dimensional immunoblot analysis using sera from patients with confirmed cases of WDEIA. The data demonstrated that the transgenic lines had reduced allergenic potential. In a collaborative project with scientists from South Korea, a breeding approach was also used to reduce the allergenic potential of wheat flour. A mutant line missing omega-5 gliadin was identified among a double haploid population produced from two Korean wheat cultivars. Detailed characterization of this line by 2-DE, combined with tandem mass spectrometry (MS/MS), revealed that several LMW-GS and gamma gliadins were also missing due to a deletion of a region of the 1B chromosome of at least 5.8 Mb. While immunoblot analysis using sera from WDEIA patients showed that the mutant line had reduced allergenic potential, there were two minor protein spots that reacted strongly with WDEIA patient sera. Surprisingly, subsequent analysis by MS/MS indicated that these two proteins were omega-5 gliadins encoded by the 1D chromosome. The data suggest that breeding approaches to reduce the omega-5 gliadins are complicated by the linkage of genes encoding omega-5 gliadins, gamma gliadins and LMW-GS and the finding that some omega-5 gliadins are encoded on chromosomes other than 1B. Transgenic lines were also produced in which proteins involved in celiac disease were reduced by ribonucleic acid (RNA) interference. Several lines were created in which alpha gliadins, the major proteins that trigger celiac disease, were significantly reduced. The omega-1,2 gliadins also contain immunodominant epitopes involved in celiac disease. The immunoreactivity of transgenic lines in which the omega-1,2 gliadins were silenced are currently being evaluated in collaboration with scientists at Columbia University. Finally, transgenic lines were created in which a 9 kilodalton (kDa) lipid transfer protein (LTP) was silenced by RNA interference. This LTP is both a known food allergen and an inhalation allergen involved in a respiratory allergy called baker’s asthma. Other work related to Objective 2 focused on the identification of immunogenic proteins in flour from the U.S. wheat, Butte 86. In collaborative studies with scientists at Columbia University, 2-D immunoblot analysis combined with tandem mass spectrometry (MS/MS) was used to identify novel proteins that contribute to inflammatory processes in celiac disease. Serpins, amylase/protease inhibitors, purinins, globulins and farinins were among the non-gluten proteins that reacted with sera from a collection of 70 patients with celiac disease. On-going studies are aimed at identifying both gluten and non-gluten proteins that play roles in non-celiac wheat sensitivities (NCWS) as well as identifying the specific epitopes that are immunoreactive. This work has been facilitated by the creation of a proteomic map of the non-gluten protein fraction from Butte 86 wheat flour that complements the proteomic map of total flour protein published in 2011. The results described above serve as the foundational work for using genome editing to reduce the immunogenic potential of wheat flour as outlined in Sub-objective 1A of the new project, 2030-21430-014-00D. Additionally, proteomic maps of Butte 86 wheat flour as well as transgenic lines containing defined changes in specific flour proteins will facilitate the development and validation of gel-free targeted mass spectrometry methods that can be used to measure the levels of peptides corresponding to specific quality-related or immunogenic proteins in wheat flour as outlined in Sub-objective 2C of the new project. Objective 3: The effects of drought on the accumulation of proteins in wheat flour were evaluated by quantitative 2-DE. Plants were grown in the greenhouse under controlled regimens in which both fertilizer levels and water were varied. Flour samples from triplicate sets of plants were analyzed by quantitative 2-DE. Although the drought treatment had notable effects on seed size and yield, there were surprisingly few significant effects on protein composition, regardless of the fertilizer level. In the same experiment, fertilizer was shown to have a major effect on flour protein composition, consistent with previously published results. The experiments suggest that plant nutrition and high temperatures are more important than water levels for influencing flour protein composition and quality.

4. Accomplishments
1. Proteomic profiling and epitope analysis of wheat flour from a commercial bread wheat. The wheat gliadins contain epitopes that trigger celiac disease and the serious food allergy, wheat-dependent exercise-induced anaphylaxis. ARS scientists in Albany, California, in collaboration with scientists from the National Institute of Agricultural Science in Jeonju, Korea, used 2-dimensional gel electrophoresis combined with tandem mass spectrometry to determine the gliadin composition of a commercial wheat cultivar. The relative amounts of individual proteins accumulated in the flour and the distribution of epitopes for celiac disease and wheat allergy were assessed. Detailed knowledge about the complement of gliadins accumulated in different commercial wheat cultivars provides the background necessary to design strategies to alter protein composition of the flour using breeding or biotechnology approaches and to improve the functionality and healthfulness of wheat.

Review Publications
Lee, J., Jang, Y., Beom, H., Altenbach, S.B., Lim, S., Lee, C.K. 2017. Allelic analysis of low molecular weight glutenin subunits using 2-DGE in Korean wheat cultivars. Breeding Science. 67:398-407.
Huo, N., Dong, L., Zhang, S., Wang, Y., Zhu, T., Mohr, T.J., Altenbach, S.B., Liu, Z., Dvorak, J., Anderson, O.D., Luo, M., Wang, D., Gu, Y.Q. 2017. New insights into structural organization and gene duplication in a 1.75-Mb chromosomal region harboring the alpha-gliadin gene family in Aegilops tauschii. Plant Journal. 92(4):571–583.