METABOLISM AND ANALYSIS OF CEREAL PHYTOCHEMICALS
Location: Cereal Crops Research
Project Number: 3655-21000-044-00
Start Date: Mar 14, 2007
End Date: Sep 30, 2010
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. Avenanthramides are polyphenolic alkaloids found uniquely in oat. They result from conjugation of one of three major phenylpropanoids and 5-hydroxy-anthranilic acid. The composition and quantities of avenanthramides in the oat seed tends to be highly variable within cultivars and between growing environments. Some of this variability likely results from differential expression of isozymes of key enzymes in the phenylpropanoid biosynthetic pathway. Objective 2. Determine the physiological effect of avenanthramides in mammals by producing pure compounds for collaborative research with nutrition scientists. Studies on the nutritional effects of avenanthramides can require hundreds of milligrams of pure authentic compound. Synthesis of these natural products is the only practical means to provide these quantities free from other naturally occurring metabolites. We are exploring innovative methods to facilitate laboratory scale synthesis and purification of the avenanthramides. Objective 3. Evaluate oat and barley germplasm for antioxidants and other phytonutrients. Determine the contents of protein, oil, beta-glucan, and certain phytochemicals in oat and barley germplasm from the National Small Grains Collections and from collaborating researchers. This objective is part of a larger ARS objective to characterize the national collections so that they will become more useful to researchers. Develop improved oat and barley germplasm by enhancing for higher concentrations of specific phytochemicals. This will be done by collaborating with plant breeders, who do not have the capability for measuring these compounds in large numbers of samples.
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 isozyme. 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.