Location: Crop Improvement and Genetics Research2015 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 continued to focus on generating transgenic plants that could be used to establish links between flour quality and the quantitative changes in the flour proteome that occur in response to temperature or fertilizer. The omega gliadins are flour proteins that show some of the largest quantitative changes when wheat plants are grown under different temperature or fertilizer regimens. Transgenic wheat lines have been established in which the omega-5 gliadins, a subset of omega gliadins, were substantially reduced or eliminated in the flour. These lines were grown under different fertilizer regimens, and changes in flour protein composition and end-use quality were assessed. In collaboration with ARS scientists in Manhattan, Kansas, they demonstrated that were improved dough mixing properties were demonstrated in flour from transgenic lines compared to their non-transgenic parent. Moreover, quantitative changes in proteins other than omega gliadins were similar in both the transgenic and the control lines in response to fertilizer, suggesting that the transgenic lines may have more consistent flour quality when grown under varying environmental conditions. The omega-1,2 gliadins are a group of gluten proteins with highly repetitive sequences that show some of the largest increases in flour when plants are supplied with fertilizer or subjected to high temperatures during wheat grain development. Wheat plants were genetically transformed with an RNA interference (RNAi) plasmid designed to silence genes encoding the omega-1,2 gliadins. Transgenic lines were regenerated and grains from the resulting plants were screened by one-dimensional gel electrophoresis for the absence of the corresponding proteins. Three lines were identified in which the omega-1,2 gliadins were absent. Homozygous lines were generated for each and grain protein composition was evaluated by two-dimensional gel electrophoresis (2-DE). In one line the suppression was very specific and there were very few changes in the levels of proteins other than the omega-1,2 gliadins. Two other lines showed considerable off-target suppression of alpha, gamma and omega-5 gliadins as well as low molecular weight glutenin subunits (LMW-GS). Seed from these lines is being increased in the greenhouse for further characterization. In addition, efforts are underway to create and identify additional transgenic lines with the desired changes in protein composition. Variability in wheat flour quality is a major concern for end-users. Much of this variability is due to the environmental conditions under which the crop is grown. Research to address Subobjective 1B focused on analyzing the structure of the glutenin polymer. Methods were developed to characterize disulfide cross-linkages between gluten proteins by mass spectrometry (MS). Various conditions during sample preparation of the glutenin polymer - including the maintenance of the proteins at low pH and the use of inhibitors of disulfide scrambling - were assessed to determine whether or not the existing disulfide bonds linking the gluten proteins could be preserved. Because of the complexity of the samples, peptides from thermolytic digests of the polymer fraction were first separated by hydrophilic interaction liquid chromatography (HILIC). Forty fractions from this separation were then individually introduced into the mass spectrometer by nanoflow reverse phase high pressure liquid chromatography (RP-HPLC), each utilizing 30-minute solvent gradients. This two-step procedure maximized peptide separation and efficient identification. An initial MS analysis of the soluble glutenin polymer yielded the identities of many peptides. However, only a few of these were linked by disulfide bonds. Research under Objective 2 focused on reducing the levels of immunogenic proteins in wheat flour through biotechnology. The omega-5 gliadins are the major sensitizing allergens in the serious food allergy, wheat-dependent exercise-induced anaphylaxis (WDEIA). In collaboration with scientists from France, the reactivity of flour proteins from control and two transgenic lines in which omega-5 gliadins were silenced by RNAi was evaluated by two-dimensional immunoblotting using sera from a collection of 11 patients with confirmed cases of WDEIA. Sera from seven patients showed greatly reduced levels of IgE reactivity to omega-5 gliadins in both transgenic lines. However, the study also revealed the complexity of the immunological responses in this group of patients and suggested that, despite the elimination of a major allergen, flour from the transgenic lines would not be suitable for individuals already diagnosed with WDEIA. Nonetheless, the introduction of wheat lacking omega-5 gliadins could reduce the number of people that become sensitized to these proteins and thereby decrease the overall incidence of this serious food allergy. In addition, two transgenic lines were created in which a lipid transfer protein (LTP), another known food allergen, was silenced by RNAi. These lines were grown in the greenhouse to generate enough material for studies of end-use quality. The effects of the genetic modifications on the compositions of three protein fractions from the grain (total flour proteins, KCl-soluble proteins and KCl-soluble/methanol soluble proteins) were analyzed in detail by 2-DE. Additionally, the identities of a number of proteins from the transgenic lines were confirmed by mass spectrometry. Other research efforts under Objective 2 were directed towards producing transgenic wheat with reduced levels of alpha gliadins that are immunogenic or toxic for celiac patients. A transgenic wheat line was identified with reduced levels of alpha gliadins. Because the RNAi construct was designed to target only those proteins containing celiac epitopes, some alpha gliadins were still present in the grain from the transgenic line. These proteins are being identified by mass spectrometry to determine the specificity of gene silencing. The transgenic line will be grown in the greenhouse to provide enough flour for detailed proteomic studies and end-use quality analysis. Efforts are underway to identify additional lines that show suppression of these genes. Non-gluten proteins from wheat flour may play roles in celiac disease. It is well-known that celiac disease is triggered by the ingestion of gluten proteins from wheat and related cereals. However, the roles of other wheat proteins in the pathogenesis of celiac disease and the related condition dermatitis herpetiformis have not been investigated. In collaboration with scientists at Columbia University, two-dimensional immunoblotting combined with mass spectrometry was used to identify non-gluten proteins from wheat flour that reacted with sera from 70 individuals with confirmed cases of celiac disease or dermatitis herpetiformis. Several types of proteins showed strong reactions with the patient sera, but did not react with sera from control patients. These proteins include serpins, alpha-amylase/protease inhibitors, purinins, globulins and farinins. The data suggest that specific non-gluten proteins, in addition to many well-known gluten proteins, contribute to inflammatory processes that result in mucosal damage in patients with celiac disease. Collaborative research under the Rural Development Administration-Agricultural Research Service Virtual Laboratories (RAVL) program was also initiated in FY15. The research focuses on relationships between specific low molecular weight glutenin subunits (LMW-GS) and wheat flour quality. Genetic transformation experiments are in progress to overexpress specific LMW-GS genes in transgenic plants. Additionally, LMW-GS from a variety of standard wheat cultivars as well as from a Korean bread wheat cultivar were excised from 2-D gels and identified by mass spectrometry. Finally, as part of an informal collaboration with scientists at the University of California-Berkeley, select proteins from transgenic sorghum lines overexpressing the enzyme thioredoxin were identified by mass spectrometry.
Moeller, S., Tanaka, C.K., Green, P.H., Zone, J.J., Vensel, W.H., Kasarda, D.D., Briani, C., Altenbach, S.B., Alaedini, A. 2014. Specific nongluten proteins of wheat are novel target antigens in celiac disease humoral response. Journal of Proteome Research. 14:503-511.
Altenbach, S.B., Tanaka, C.K., Seabourn, B.W. 2014. Silencing of omega-5 gliadins in transgenic wheat eliminates a major source of environmental variability and improves dough mixing properties of flour. Biomed Central (BMC) Plant Biology. 14:393 doi: 10.1186/s12870-014-0393-1.