2007 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. 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. 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 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. 3.Progress Report (1) Cloning of oat phenylalanine ammonia lyase (PAL) isozymes. Preliminary Southern analysis of oat genomic DNA indicates there are at least 3 PAL isozymes and 4 HHT isozymes. Two oat PAL fragments have been cloned, one represents >90% of the sequence with some 3’-UTR, the other fragment is about 500 bp of coding sequence. (2) Over 3371 oat and barley samples have been processed for oil, protein, beta-glucan and avenanthramide content. These include 980 genotypes analyzed for beta-glucans as part of the Barley Coordinated Agriculture Project (Barley CAP). A manuscript describing an oat suspension cell system producing avenanthramides is in peer review for publication (Phytochemistry); another manuscript describing the environmental effect of crown rust on oat avenanthramide is in preparation. 4.Accomplishments Oat Suppression, Subtractive Hybidization (SSH) Library. This project is principally intended to identify enzymes (and their specific isozymes) as well as other gene products associated with avenanthramide biosynthesis and transport. An SSH library from oat suspension cultures has been produced from chitin elicited vs non-elicited cells. The library contains 89 unique gene products that identify oat genes selectively expressed in response to chitin elicitation (a molecular mimic of fungal infection). The gene fragments will also provide genetic probes to analyze expression of these oat genes under various stress conditions and to identify the enzymes encoded. In alignment with NP 302, Plant Biological and Molecular Processes, Component 2: Biological processes that improve crop productivity and quality. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 4