2013 Annual Report
A proteomic approach provided new insights into the effects of post-anthesis fertilizer and high temperature on genes and proteins involved in wheat flour quality and allergenic potential in the US wheat Butte 86 (Objective 1). Using a detailed proteomic map in which most flour proteins were linked to specific gene sequences, the proportions of individual gluten proteins were determined in flour from plants grown with and without post-anthesis fertilizer. Of the gluten proteins, the omega gliadins showed the greatest change with fertilizer, increasing 144%. High-molecular-weight glutenin subunits and a few alpha gliadins increased to a lesser extent while several low-molecular-weight glutenin subunits decreased. Proportions of alpha gliadins containing epitopes involved in the food intolerance celiac disease were not altered. One important type of food allergen, the omega-5 gliadins, increased with fertilizer while a number of non-gluten proteins that are potential food allergens were more abundant in flour produced without fertilizer. Additional analyses demonstrated that high temperatures had surprisingly similar effects on gluten protein accumulation. However, a number of non-gluten proteins increased with high temperature, including several proteins that are food allergens. A proteomic approach also was used to determine the precise protein compositions of gluten polymer fractions (Objective 1). Flour proteins were fractionated on the basis of solubility in 0.5% SDS, further separated into monomeric and polymeric fractions by size exclusion chromatography, and analyzed by quantitative two-dimensional gel electrophoresis (2-DE) followed by tandem mass spectrometry. The analysis revealed the presence of gliadins containing an odd number of cysteine residues in polymer fractions, supporting their role as chain-terminators of the gluten polymer. In addition, several types of non-gluten proteins were found in fractions containing small polymers. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure. Progress also was made in developing molecular approaches to reduce the allergenic potential of wheat flour (Objective 2). Genetic transformation methods were developed for the US commercial wheat cultivar Butte 86. RNA interference was used to produce transgenic plants in which genes encoding two different types of food allergens, omega-5 gliadins and lipid transfer proteins (LTPs), were silenced. Omega-5 gliadins increase in response to both fertilizer and high temperature, while LTPs increase in response to high temperature. Quantitative 2-DE was used to determine the precise effects of the genetic modifications on the proteome in homozygous plants showing suppression of omega-5 gliadins. These analyses identified the best genetic material for further analyses of flour quality and allergenic potential.
Denery-Papini, S., Boninier, M., Pineau, F., Triballeau, S., Tranquet, O., Adel-Patient, K., Moneret-Vautrin, Bakan, B., Marion, D., Mothes, T., Mameri, H., Kasarda, D.D. 2011. Immunoglobulin-E-binding epitopes of wheat allergens in patients with food allergy to wheat and in mice experimentally sensitized to wheat proteins. Journal of Clinical and Experimental Allergy. DOI:10.1111.j.1365-2222.2011.03808.x.
Kasarda, D.D., Adelsteins, E., Lew, E., Lazo, G.R., Altenbach, S.B. 2013. Characterization of a novel wheat endosperm protein belonging to the prolamin superfamily. Journal of Agricultural and Food Chemistry. 61:2407-2417.
Altenbach, S.B. 2012. New insights into the effects of high temperature, drought and post-anthesis fertilizer on wheat grain development. Journal of Cereal Science. 56:39-50.
Hurkman II, W.J., Tanaka, C.K., Vensel, W.H., Thilmony, R.L., Altenbach, S.B. 2013. Comparative proteomic analysis of the effect of temperature and fertilizer on gliadin and glutenin accumulation in the developing endosperm and flour from Triticum aestivum L. cv. Butte 86. Proteome Science. doi:10.1186/1477-5956-11-8.
Robertson, G.H., Hurkman II, W.J., Anderson, O.D., Tanaka, C.K., Cao, T., Orts, W.J. 2012. Differences in alcohol-soluble protein from genetically altered wheat using capillary zone electrophoresis, one- and two-dimensional electrophoresis and a novel gluten matrix association factor analysis. Cereal Chemistry. 90(1):13-23. DOI: 10.1094/cchem-10-11-0123.
Boggess, M.V., Lippolis, J.D., Hurkman II, W.J., Fagerquist, C.K., Briggs, S.P., Gomes, A.V., Righetti, P., Bala, K. 2013. The need for agriculture phenotyping: “Moving from genotype to phenotype”. Journal of Proteomics. doi: http://dx.doi.org/10.1016/j.jptot.2013.3.021.
Robertson, G.H., Cao, T., Gregorski, K.S., Hurkman II, W.J., Tanaka, C.K., Chiou, B., Glenn, G.M., Orts, W.J. 2013. Modification of vital wheat gluten with phosphoric acid to produce high free solution capacity. Journal of Applied Polymer Science. DOI: 10.1002/app.39440.