Location: Crop Improvement and Genetics Research2014 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:
The omega-1,2 gliadins are a group of gluten proteins with highly repetitive sequences that increase in proportion in flour from plants supplied with fertilizer or subjected to high temperatures during wheat grain development. In Subobjective 1A, an RNA interference (RNAi) plasmid targeting the omega-1,2 gliadins was constructed. The construct, consisting of a hairpin structure with an 141 base pair target region from the omega-1,2 gliadin gene, under the control of the High Molecular Weight - Glutenin Subunit (HMW-GS) Dy10 promoter and HMW-GS Dx5 terminator, is being introduced into the spring wheat Butte 86 by particle bombardment and transgenic plants are being regenerated. In Subobjective 1B, grain proteins from transgenic wheat lines in which the omega-5 gliadin genes were silenced by RNA interference were analyzed by quantitative two-dimensional gel electrophoresis (2-DE). Two homozygous lines in which the omega-5 gliadins were significantly reduced or absent with minimal changes in other grain proteins were selected. These plants were grown in triplicate along with a non-transgenic control in a temperature-controlled greenhouse with and without post-anthesis fertilizer. The resulting grain was milled to flour at the ARS Hard Winter Wheat Quality Laboratory in Manhattan, Kansas, and mixing and baking studies were conducted on the biological replicates. Proteomic analysis of the same flour samples was used to determine the protein compositions of grain produced from each of the lines under the two fertilizer regimens. Also as part of Subobjective 1B, a detailed analysis of the protein composition of the large and small gluten polymer fractions was conducted using 2-DE and tandem mass spectrometry (MS/MS). In addition, components of large and small polymer fractions from flour from plants grown with and without post-anthesis fertilizer were analyzed in detail. In Objective 2, the spring wheat Butte 86 was transformed with two different RNAi plasmids designed to silence the wheat 9 kilo Dalton (kDa) lipid transfer protein (LTP), a confirmed food allergen that increases in flour when plants are grown under high temperatures. Numerous transgenic lines were produced and Potassium Chloride (KCl)-soluble, methanol-soluble protein fractions from transgenic grain were evaluated. Only two lines showed high levels of suppression. Homozygous lines are being generated from these lines before detailed analysis by 2-DE. Additionally, constructs were created to silence a subset of alpha gliadin genes that encode proteins containing epitopes involved in celiac disease. These constructs are being introduced into Butte 86 by particle bombardment and transgenic plants are being regenerated. Objective 3 was not addressed in the current fiscal year because a critical vacancy that resulted from an SY retirement in 2012 has not yet been filled.
1. Assessment of transgenic wheat lines with reduced levels of an allergenic protein. Among the wheat gluten proteins, the omega-5 gliadins have been identified as a major source of environmental variability, increasing in proportion in grain from plants that are supplied with fertilizer or subjected to high temperatures during grain development. These proteins also are involved in human food allergies. ARS scientists in Albany, California, used quantitative two-dimensional gel electrophoresis (2-DE) to analyze the protein composition of grain from transgenic wheat lines in which the omega-5 gliadin genes were silenced by RNA interference (RNAi). They demonstrated that six protein spots previously identified as omega-5 gliadins were significantly reduced or absent in grain from the transgenic lines. The study made it possible to select genetic material that can be used to assess the roles of the omega-5 gliadins in flour quality and allergenic potential, and provides insight into how the grain responds to the environment in the absence of these proteins.
2. Protein composition of wheat gluten polymer fractions. There is a strong relationship between the amount of gluten polymers formed by some of the wheat flour proteins and bread making quality, but the protein components of these polymers have not been thoroughly investigated. ARS scientists in Albany, California, separated the gluten polymer into fractions containing large, insoluble polymers and small, soluble polymers and analyzed their protein compositions using 2-dimensional gel electrophoresis combined with tandem mass spectrometry. As expected, both the large and small polymer fractions contained a variety of high molecular weight and low molecular weight glutenin subunits that are able to link together by disulfide bonds. In addition, some proteins that are very similar to gliadins and several types of non-gluten proteins, including serpins, triticins and globulins, were accumulated preferentially in the small polymer fraction. Analysis of the gliadin-like proteins by mass spectrometry revealed that they contain odd numbers of cysteine residues that could allow them to act as terminators of the gluten polymer chains. The data make it possible to formulate hypotheses about how protein composition influences gluten polymer size and structure and provides a foundation for future work aimed at determining how the growth environment affects flour quality.
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