2012 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.
Objective 1 focuses on determining the basis for variations in flour quality that result from environmental conditions during wheat grain development. As part of Subobjective 1A, methods were developed to isolate and characterize gluten polymer from wheat flour. Gluten polymer amount and size-distribution influence wheat flour quality. The effectiveness of acetic acid, sodium iodide in propanol, propanol alone, and Sodium Dodecyl Sulfate (SDS) were compared. Analysis of the soluble and insoluble fractions by two-dimensional gel electrophoresis (2-DE) showed that the most effective protocol was extraction of flour with SDS. Two types of size exclusion chromatography media were evaluated for characterization of the protein fractions using Phenomenix Bio-SEC 4000.
Milestones for Subobjectives 1B and 1C involved growth of plant material under defined temperature and fertilizer regimens for future studies. Because WRRC greenhouse facilities were under renovation for 15 months instead of 3 months as scheduled, it was not possible to produce this material. In the interim, studies on the quantitative analysis of flour proteins in plants produced under different temperature regimens were completed.
In Objective 2, transgenic approaches were used to reduce the levels of proteins that are allergenic in wheat flour in the model wheat Bobwhite S26 and the commercial quality bread wheat Butte 86. Objective 2A focuses on a subset of wheat 9 kDa lipid transfer proteins (LTP) that increase in wheat flour when plants are grown under high temperatures. Because transformation methods for Butte 86 were not available at the time these experiments were initiated, Bobwhite S26 plants were transformed with RNA interference (RNAi) constructs. The constructs utilized either a constitutive promoter from the maize ubiquitin gene or a tissue-specific promoter from the wheat LTP gene and were designed to silence two similar LTP genes in developing grain. Analyses of KCl-soluble, MeOH-soluble proteins from individual grains of transgenic lines by one-dimensional gel electrophoresis suggested that grain from some of the transgenic plants contained reduced levels of proteins in the 9 kDa size range. Since the LTPs overlap with other proteins of a similar size, 2-DE was used to confirm that two protein spots corresponding to the allergenic LTP were absent in grain from plants transformed with the construct containing the ubiquitin promoter. These constructs will be used to transform Butte 86 so that effects of gene silencing on the wheat flour proteome and flour quality can be evaluated in commercial quality wheat.
Objective 2B focuses on a gluten protein associated with the serious food allergy wheat-dependent exercise-induced anaphylaxis. Homozygous lines were generated for each of the six transgenic Butte 86 plants that showed silencing of omega-5 gliadins. Total grain proteins from three of the transgenic lines were analyzed by 2-DE to reveal the specificity of gene silencing. Grain from homozygous plants is being increased in the greenhouse so that sufficient quantities will be available for more detailed studies.
Fertilizer affects allergenic flour proteins in wheat. The application of fertilizer during wheat grain development causes changes in wheat flour proteins that have potential to play an important role in flour quality and immunogenecity. ARS researchers in Albany, California, compared the protein composition of flour from wheat grain produced with and without fertilizer and detected fertilizer-induced increases in specific gluten proteins (glutenins and gliadins) that represent determinants of functional properties of wheat flour. 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 comprised a greater proportion of the flour protein when grain was produced without fertilizer. The work contributes to NP301, Component 04B, Plant Biological and Molecular Processes, Biological Processes that Improve Crop Productivity and Quality.
Altenbach, S.B., Tanaka, C.K., Hurkman Ii, W.J., Whitehand, L.C., Vensel, W.H., Dupont, F.M. 2011. Differential effects of a post-anthesis fertilizer regimen on the wheat flour proteome determined by quantitative 2-DE. Proteome Science. 9:46.
Altenbach, S.B., Vensel, W.H., Dupont, F.M. 2011. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat Butte 86. BMC Research Notes. 4:242.