Title: 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 Authors
Submitted to: Cereal Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 7, 2012
Publication Date: September 5, 2012
Citation: 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. Interpretive Summary: Specific native wheat proteins and novel new ones may be attenuated or enhanced in the grain to produce materials of potential interest in biobased products. In prior research we identified striking and potentially impactful physical property differences in genetically altered wheat lines. In this report we sought to characterize molecular properties of these wheat lines using electrophoretic methods. We identified large and component specific-ethanol solubility differences directly related to hypothesized molecular structure and architectural rigidity and with potential relation to anticipated polymer properties. The results immediately impact understanding of how altered protein composition affects structure and organization and potentially lead to the production of new renewable polymers with unique properties for the biobased product industries.
Technical Abstract: Wheat protein composition and organization play interrelated roles in determining physical properties for technological purposes. In prior research, a number of isogenic wheat lines of Bobwhite that have high levels of expression of the native Dx5 and/or Dy10 high molecular weight subunits (HMW-GS) were examined vis-à-vis physical properties related to separation. In particular, these altered lines were characterized by poor mixing properties, the formation of mixtures in water that could not be separated by conventional mechanical methods, reduced water absorption, unique milling properties, and severely limited development of microscopic fibrils. These attributes suggested inherent organizational differences at submicroscopic and molecular levels among the various lines. Therefore, proteins were fractionated from whole meals using 70% ethanol to elucidate solubility characteristics and compositions and to infer structural properties. Capillary zone electrophoresis and one and two dimensional SDS PAGE revealed striking differences in the protein composition and solubility among these new lines and the Bobwhite from which they were derived. Generally, Bobwhite yielded soluble protein that included not only what would be considered as classical gliadins, but also some of each of the HMW-GS as monomers or polymers with low degrees of polymerization; whereas, the genetically altered lines produced far less total soluble protein and very limited amounts of HMW-GS. In the extreme, high levels of expression of Dx5 subunit not only led to reduced solubility of the HMW-GS, but also limited the solubility of the many other proteins that are normally soluble. In addition, a matrix association factor similar to the classical separation factor of analytical chemistry and chemical engineering was introduced and applied to 2DE data for insoluble and soluble protein to summarize and index relative involvement of specifically enhanced proteins in the insoluble gluten matrix after equilibration with ethanol. Highest relative association was determined for the HMW-GS lines enriched in Dx5 or Dy10 protein and the least for Bobwhite. Greater association was indicated for Dx5 than for Dy10 protein in these lines. The value of the association factor is likely influenced by differences in glutamine/cysteine ratios and differences in altered glutenin chain configurations stemming from high levels of expression of a single or limited number of HMW-GS.