Location: Crop Improvement and Genetics Research2016 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:
Research efforts under Objective 1 combine biotechnology and proteomics approaches to establish links between flour quality and quantitative changes in the flour proteome that occur in response to temperature or plant nutrition. The omega-1,2 gliadins are a group of gluten proteins that show some of the largest increases in flour when plants are supplied with fertilizer or subjected to high temperatures during wheat grain development. This group includes proteins that are present in the flour as monomers as well as proteins that contain a single cysteine and are linked into the glutenin polymer. The omega-1,2 gliadins also contain immunodominant epitopes that trigger celiac disease. A transgenic wheat line, in which all omega-1,2 gliadins were suppressed, was created by ribonucleic acid (RNA) interference and grown in triplicate in the greenhouse under different fertilizer and water regimens. The resulting flour samples are being analyzed by quantitative 2-DE to determine how environmental conditions affect the flour proteome in the absence of the omega-1,2 gliadins. In addition, the complement of low-molecular-weight glutenin subunit (LMW-GS) genes expressed in Butte 86 developing grains were characterized and a trigger for an RNA interference construct that will specifically silence a subset of LMW-GS encoded by the B-genome was designed. Also referred to as the s-type LMW-GS, these are some of the most abundant LMW-GS in Butte 86 flour and among the few LMW-GS proteins that respond to post-anthesis fertilizer. These experiments will make it possible to assess the role of the s-type LMW-GS in flour quality. As part of Subobjective 1B, the protein components of large and small glutenin polymers from flour produced with and without post-anthesis fertilizer were analyzed using quantitative 2-dimensional electrophoresis (2-DE) and mass spectrometry. Changes in the proportions of certain proteins, including chain-terminating LMW-GS and serpins, indicate that these proteins would be good targets for future translational proteomic studies of flour quality. Research efforts under Objective 2 were directed towards producing transgenic wheat with reduced levels of immunogenic proteins. Transgenic lines in which the 9000 dalton lipid transfer proteins, known food allergens, were suppressed by RNA interference were further characterized. In conjunction with collaborators from France, the allergenic potentials of non-transgenic and transgenic lines are being assessed. Additionally, two transgenic lines were identified in which most alpha gliadins were eliminated, thereby significantly reducing the load of epitopes that trigger celiac disease in the flour. The specificity of the silencing is being evaluated by quantitative 2-DE combined with mass spectrometry. The transgenic lines also are being grown in the greenhouse in sufficient quantities for studies of flour quality. To address Objective 3, Butte 86 wheat plants were grown in the greenhouse in triplicate under well-watered and drought conditions with and without post-anthesis fertilizer. Two separate growth experiments were conducted. Grain samples were harvested and effects of the treatments on grain size and yield were determined. Flour from the resulting grain is being analyzed by quantitative 2-DE to determine the precise effects of the treatments on the flour proteome. Collaborative research under the Rural Development Administration-Agricultural Research Service Virtual Laboratories Program (RAVL) that focuses on relationships between LMW-GS and wheat flour quality continued in FY16. A comprehensive analysis of LMW-GS genes and proteins was carried out in a Korean bread wheat cultivar. Since both the Korean cultivar and the U.S. cultivar Butte 86 have two of the same LMW-GS alleles, these gene sequences were valuable in efforts to characterize the Butte 86 LMW-GS. In addition, wheat flour proteins from several Korean wheat cultivars, including one that is missing LMW-GS encoded by the B-genome, were characterized using 2-DE and mass spectrometry. Genetic transformation experiments also were conducted to overexpress specific LMW-GS genes in transgenic wheat plants.
1. Reduced-allergen wheat. Wheat flour is one of eight foods responsible for 90% of the food allergies in the U.S. ARS scientists in Albany, California, created transgenic wheat lines in which the omega-5 gliadins, the major sensitizing allergens in a severe food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA), were significantly reduced in the flour without adverse effects on flour quality. In collaboration with scientists at the French National Institute for Agricultural Research (INRA), Nantes, France, the allergenic potentials of these lines were evaluated using sera from a collection of WDEIA patients. Most patients showed strong reactivity to the omega-5 gliadins in flour from the non-transgenic control and little or no reactivity to omega-5 gliadins in the transgenic lines, indicating that the transgenic lines could be considered reduced-allergen. However, low levels of reactivity with other gluten proteins were also observed in the transgenic lines. While flour from the transgenic lines would not be suitable for individuals diagnosed with WDEIA, introduction of wheat lacking omega-5 gliadins could reduce the number of consumers who become sensitized to these proteins and decrease the overall incidence of this food allergy.
5. Significant Activities that Support Special Target Populations:
Altenbach, S.B., Tanaka, C.K., Pineau, F., Lupi, R., Drouet, M., Beaudouin, E., Morisset, M., Denery-Papini, S. 2015. Assessment of the allergenic potential of transgenic wheat (Triticum aestivum) with reduced levels of omega-5 gliadins, the major sensitizing allergen in wheat-dependent exercise-induced anaphylaxis. Journal of Agricultural and Food Chemistry. 63:9323-9332.
Altenbach, S.B., Tanaka, C.K., Whitehand, L.C., Vensel, W.H. 2015. Effects of post-anthesis fertilizer on the protein composition of the gluten polymer in a US bread wheat. Journal of Cereal Science. 68:66-73.
Lee, J., Beom, H., Altenbach, S.B., Lim, S., Kim, Y., Kang, C., Yoon, U., Gupta, R., Kim, S., Ahn, S., Kim, Y. 2016. Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety. Functional and Integrative Genomics. 16:269-279.
Blechl, A.E., Beecher, B.S., Vensel, W.H., Tanaka, C.K., Altenbach, S.B. 2016. RNA interference targeting rye secalins alters flour protein composition in a wheat variety carrying a 1Bl.1RS translocation. Journal of Cereal Science. 68:172-180.