Location: Crop Improvement and Genetics Research2011 Annual Report
1a. Objectives (from AD-416)
The first objective of the research is to determine the basis for changes in flour quality that result from high temperatures during wheat grain development. The research will investigate the roles of gluten composition and polymer structure in effects of temperature during grain development on flour quality. The research also will determine the roles of specific non-gluten proteins in effects of temperature during grain development on flour quality. This objective addresses a major concern of millers and bakers and explores two hypotheses: 1) changes in glutenin polymer amount, size, structure and composition as a result of high temperatures during grain development are responsible for decreases in flour quality and 2) non-gluten proteins that increase in the grain under high temperature conditions are involved in quality or allergenicity. The second objective of the research is to identify and characterize wheat proteins responsible for human intolerances and allergies that affect nearly 2% of the U.S. population and to develop methods to detect allergenic proteins in downstream products. This objective will determine whether mass spectrometry (MS) can be used to identify potential wheat allergens in flour and detect these proteins in food ingredients and products.
1b. Approach (from AD-416)
To address the first objective, MS methods coupled with improved methods for protease digestion will be developed so that closely related gluten proteins can be distinguished. Size-exclusion (SE) chromatography and high pressure liquid chromatography (HPLC) will be used to separate glutenin polymers into size classes for determination of subunit composition and key linkages between high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS). Gluten proteins that act as chain terminators in polymer structure will be identified and their roles in polymer structure and size will be evaluated. Polymer composition and size will be measured during grain development under different temperature regimens. The effect of different temperature regimens on accumulation profiles of a specific set of non-gluten proteins and their transcripts during grain development also will be characterized using 2-dimensional polyacrylamide gel electrophoresis (2-DE) and quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). Since many of these proteins may be involved in stabilizing gas bubbles in dough, experiments will be performed to test whether the levels of these proteins increase in dough liquor prepared from flour from grain grown under high temperatures. Tissue localization studies will be performed and the roles that specific proteins play in flour quality and allergenicity will be investigated using transgenic plants in which the corresponding genes are suppressed. To address the second objective, the allergenic potential of non-gluten proteins that increase under high temperature conditions will be tested using sera from patients with defined wheat allergies. MS will be used to determine mass profiles of protein fractions from wheat flour. These profiles will be examined for signatures of specific allergenic proteins. Methods will be extended to samples from baked products such as bread. Replacing 5325-43000-026-OOD (June/2010).
3. Progress Report
A major achievement of the project in FY11 was the completion of the first comprehensive map of the flour proteome from the US wheat cultivar Butte 86. The map links the majority of the abundant flour proteins separated by two-dimensional gel electrophoresis (2-DE) with specific gene sequences and provides a critical resource for evaluating the impact of environmental conditions and agronomic inputs on flour protein composition. Using this map, the protein composition of flour from plants grown with or without post-anthesis fertilizer was analyzed. Significant changes in the proportions of 54 unique proteins were observed as a result of the treatment. Most omega-gliadins, HMW-GS, serpins and certain alpha-gliadins and LMW-GS increased with fertilizer. The effect of post-anthesis fertilizer and high temperature on gluten protein accumulation during grain development also was assessed by 2-DE. The relative levels of omega gliadins increased and LMW-GS decreased with high temperature with or without fertilizer. In collaborative research, flour samples from transgenic wheat lines with altered levels of HMW-GS were also analyzed to determine the effect of the transgenes on levels of individual gliadins and glutenins. Other experiments were directed towards characterizing the glutenin polymer fraction from wheat flour. Glutenin polymers consist of disulfide linked gluten proteins and are particularly difficult to study because they range in size from 100-10,000 kD. A variety of solvent systems were used to extract polymer fractions that then were characterized by size exclusion chromatography. A positive relationship was found between the amount of extractable glutenin polymer and flour quality. Size exclusion chromatography revealed that there was a continuous distribution of polymer sizes. However, gel analysis indicated that the polymer subunit composition was similar across different polymer size classes. A major effort also was aimed at inhibiting the expression of genes encoding allergenic proteins in wheat grain. Central for this work was the development of genetic transformation methods for the commercial US wheat cultivar Butte 86. An RNA interference construct designed to specifically inhibit genes encoding omega-gliadins that are known food allergens was introduced into Butte 86 using biolistics. Stable transformation of Butte 86 plants and transgene inheritance were demonstrated. Analyses of grain protein from transgenic plants revealed that omega gliadins that cause the food allergy wheat-dependent exercise-induced anaphylaxis were absent in grain from the transgenic plants. In addition, RNA interference constructs designed to inhibit the expression of two genes encoding lipid transfer proteins were transformed into the model wheat Bobwhite and transgenic plants were regenerated and confirmed by PCR analysis. These constructs are now being used to transform Butte 86.
Dupont, F.M., Vensel, W.H., Tanaka, C.K., Hurkman II, W.J., Altenbach, S.B. 2011. Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry. Proteome Science. 9(10) available:http://www.proteomesci.com/content/9/1/10.