Location: Crop Improvement and Genetics Research2017 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 were directed towards establishing links between wheat flour quality and quantitative changes in the flour proteome that occur in response to temperature or the application of fertilizer during grain development. The Low Molecular Weight Glutenin Subunits (LMW-GS) are a complex group of more than 20 closely-related proteins that link together with High Molecular Weight (HMW)-GS to form large protein polymers. These polymers are essential for the functionality of wheat flour because they contribute elasticity to wheat flour dough. In previous studies, a subset of LMW-GS, referred to as S-type LMW-GS because they begin with an N-terminal serine residue, were found to decrease in flour in response to the application of post-anthesis fertilizer while other LMW-GS showed no changes. Gene silencing was used to assess the roles of the S-type LMW-GS in flour quality and the response of the grain to post-anthesis fertilizer. A Ribonucleic acid (RNA) interference (RNAi) plasmid containing a 56 base pairs (bp) sequence identical to the 5’ end of the coding regions of S-type LMW-GS genes was constructed and used to transform the U.S. wheat Butte 86. A number of transgenic plants were recovered and fractionated proteins from the resulting grain were analyzed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). None of the transgenic plants showed significant suppression of LMW-GS. As a result, a second RNAi plasmid was constructed that included a 101 bp region adjacent to the sequences used in the first construct. Transgenic plants were recovered and a number of lines showed suppression of LMW-GS in SDS-PAGE. Flour proteins from five of these lines were further assessed by 2-dimensional electrophoresis (2-DE). While these showed complete suppression of the S-type LMW-GS, most also showed partial suppression of other LMW-GS. In addition, partial suppression of HMW-GS was evident in some of the lines. These lines will be grown in greenhouses in larger quantities for evaluations of flour quality. Research efforts under Objective 2 focused on the analysis of transgenic lines in which alpha gliadins were eliminated from the flour by gene silencing, thereby significantly reducing the load of epitopes that trigger celiac disease. The specificity of the silencing was evaluated in two homozygous lines by quantitative 2-DE combined with tandem mass spectrometry (MS/MS). Within the alpha gliadin region of the 2-D gel, some spots were completely absent in the transgenic lines while others were significantly reduced. Protein spots within the alpha gliadin region from both the control and transgenic lines were identified by MS/MS. Surprisingly, some 2-DE spots identified as alpha gliadins in the non-transgenic line were identified as either gamma or delta gliadins in the transgenic lines, suggesting that either these proteins were unmasked by the silencing of the alpha gliadins or increased in the transgenic lines to partially compensate for the loss of the alpha gliadins. Small reductions in some HMW-GS were also noted. Seed increase in the greenhouse and assessment of flour quality in these lines are underway. Also under Objective 2, additional transformation experiments were conducted to obtain transgenic plants in which the omega-1,2 gliadins were silenced. The omega-1,2 gliadins show some of the largest responses to post-anthesis fertilizer and high temperature and are known to contain immunodominant epitopes involved in celiac disease. One line was obtained in which the omega-1,2 gliadins were completely suppressed without other effects on the proteome. This line was grown with and without post-anthesis fertilizer and the resulting flour samples are being evaluated by quantitative 2-DE. Interestingly, the same RNAi construct also yielded lines in which all gliadins and LMW-GS were suppressed as well as one line in which the omega-5 gliadins, gamma gliadins and LMW-GS were suppressed. Assessments of immunogenic potential and quality of some of these transgenic lines are underway. Collaborative research that focuses on relationships between specific flour proteins and wheat quality continued in 2017 under the Rural Development Administration-Agricultural Research Service Virtual Laboratories Program (RAVL). 2-DE combined with MS/MS was used to develop a proteomic map of a gliadin fraction from a Korean wheat cultivar. The work contributes to international efforts to better understand these complex gene families in a collection of cultivars that contain different versions of gliadins. The work also facilitates future studies on proteins that trigger wheat allergies and celiac disease.
Lee, J., Kang, C., Beom, H., Jang, Y., Altenbach, S.B., Lim, S., Kim, Y., Park, C. 2016. Characterization of a wheat mutant missing low-molecular-weight glutenin subunits encoded by the B-genome. Journal of Cereal Science. 73:158-164.
Jang, Y., Beom, H., Altenbach, S.B., Lee, M., Lim, S., Lee, J. 2017. Improved method for reliable HMW-GS identification by RP-HPLC and SDS-PAGE in common wheat cultivars. Molecules. 22(7):1055. doi:10.3390/molecules22071055.
Altenbach, S.B. 2017. Proteomics of wheat flour. In: Colgrave, M.L., editor. Proteomics in Food Science: From Farm to Fork. 1st edition. London, England:Academic Press. p. 57-73.