2012 Annual Report
1a.Objectives (from AD-416):
Objective 1: Identify the key isozymes involved in avenanthramide biosynthesis and evaluate their role in determining the levels and types of avenanthramides produced in planta.
Objective 2: Determine the physiological effect of avenanthramides in mammals by producing pure compounds for collaborative research with nutrition scientists.
Objective 3: Evaluate oat and barley germplasm for antioxidants and other phytonutrients.
1b.Approach (from AD-416):
The overarching rationale for these experiments is to determine the role of specific isozymes of phenylalanine ammonia lyase (PAL) and / or 4-coumaryl CoA ligase (4-CL) in avenanthramide biosynthesis, and their relation to the biosynthesis of specific forms of avenanthramides. Although, a number of plant DNA sequences corresponding to both PAL and 4-CL are found in GenBank, currently there are none from oat. It is expected that, like most plants, oat will possess multiple isozymes of PAL and 4-CL, thus it is important to determine how many genes are present in oat and to obtain DNA sequence information for these isozymes. These data will allow development of isozyme specific probes to evaluate expression of the target genes over the course of seed maturation and in different plant organs in field grown oats. Although a route to the synthesis of avenanthramides is available, this method is cumbersome and time-consuming. We have found the use of the peptide coupling reagent benzotriazol-1-yloxytris(dimethylamino) phosphonium hexaflurophosphate (BOP) to be effective in the synthesis of avenanthramides. We will also explore the use of other peptide coupling reagents for their utility in avenanthramide synthesis. The synthesized avenanthramides are being used, in collaboration with nutrition scientists at the USDA Jean Mayer Laboratory of Human Nutrition (Tufts University) and at the University of Wisconsin, Department of Kinesiology, to evaluate the effects of avenanthramides in mammalian systems. Oat and barley germplasm will be evaluated for the content of other phytochemical constituents that may have physiological effects, and for unusually high concentrations of known phytochemicals. Entries from the National Small Grains Collections, elite nurseries, and selections from collaborating plant breeders will be analyzed for various constituents, including protein, oil, beta-glucan, and phytochemicals.
During the past year this laboratory explored new areas of research in the function of phytochemicals in oat and barley. We investigated the possible role of small molecules in oat cold hardiness in collaboration with other United States Department of Agriculture research groups. Some winter oat cultivars with enhanced cold hardiness produce a distinct fluorescent layer of tissue in their root crowns in response to freezing. This laboratory was able to demonstrate, by High-performance liquid chromatography analysis using fluorescent detection, that solvent extracts of these cold hardy root crowns, subsequent to freezing, showed a distinct enhancement in several fluorescent metabolites compared to non-cold hardy root crowns. These results warrant further investigation to specifically identify these metabolites.
Preliminary field trials to enhance oat crown rust resistance through application of Actigard (benzodiathiazole), a commercial plant defense activator, were initiated in collaboration with oat breeders. Results have not been fully analyzed.
A new collaboration with scientists in the Department of Health and Nutritional Sciences, South Dakota State University, to investigate the anti-cancer properties of avenanthramides has demonstrated that avenanthramides inhibit HeLa (human cervical cancer) cell proliferation in vitro. These results were presented as a poster at the Ubiquitin, Protein Quality Control, and Molecular Pathogenesis Symposium, Deadwood, South Dakota, June 13-15, 2012 and will soon be submitted for peer-review publication.
Relationship of tocol content to winter hardiness in barley. There is a considerable interest in developing germplasm for winter barley varieties. Recent studies with Arabidopsis have shown a relationship between tocol biosynthesis and enhanced cold tolerance. This project is designed to investigate whether barley lines with enhanced winter hardiness demonstrate a clear quantitative or qualitative difference in their tocol content. This project is being conducted in collaboration with barley breeders at Oregon State University and other ARS scientists at Madison, Wisconsin and is still in its preliminary stages. Environmental conditions are being explored to differentiate cold tolerant barley seedlings from non-tolerant lines. These experiments will determine if tocol content can be used as a phenotypic marker associated with winter hardiness in barley. Tocol content has been analyzed for the plants planted in the fall, post winter tissue still requires analysis.
The Cooperative Oat Research Enterprise (CORE) project, funded by Agriculture and Food Research Initiative aims to expand the genomic information on oat genotypes in order to improve molecular marker assisted breeding technique. As a collaborator on this project this laboratory is responsible for analyzing tocol (Vitamin E) and avenanthramide content of approximately 1000 germlines per year for two years. We have completed the tocol analysis of year one and are currently analyzing year one avenanthramide content.
Analysis of tocol content in oat. Tocopherols and tocotrienols (vitamin E, collectively referred to as tocols) are important nutritional components of both oat and barley. Oat produces three or four of the eight naturally occurring tocols, and the amounts and proportions of these can be highly variable largely due to genotypic influence. A better understanding of the genetic basis for this variation will lead to germplasm with more desirable vitamin E production. As collaborators with the Cooperative Oat Research Enterprise (CORE) we have completed the analysis of the year one germplasm for tocols (analysis of avenanthramide content is in progress). The impact will be to facilitate breeding oat for superior tocol (vitamin E) content, thus providing consumers with a more nutritious product.