2013 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.
This is a new project which continues the research from 5325-43000-027-00D, "Molecular Analysis of Effects of Environment on Wheat Flour Quality and Allergenic Potential". Please see the report for 5325-43000-027-00D for more information.