Project Number: 3655-21000-040-00
Project Type: Appropriated

Start Date: Mar 29, 2004
End Date: Aug 31, 2006

Objective:
Isolate, clone and characterize the final enzyme (HHT) in the avenanthramide biosynthetic pathway and evaluate genetic, biotic and abiotic factors regulating the content, biosynthesis and metabolic profile of avenanthramides (antioxidants found uniquely in oat). Investigate the metabolic fate of avenanthramides (and related antioxidants) in oat suspension cultures and developing seeds. Evaluate various cultivars and wild species of oat and barley for the presence of novel phytochemicals or markedly high expression levels of known phytochemicals. 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. With collaborating with plant breeders, develop improved oat germplasm by enhancing for higher concentrations of specific phytochemicals. Determine physiological effects of oat, barley, and components of these grains in humans, model animals, and tissue cultures through collaborative research.

Approach:
The metabolic profile of avenanthramides and their intermediates will be evaluated under various elicitation regimes by HPLC and LC/MS of extracts from suspension cultures, germinating seeds, and developing leaves and grain. Intermediates will be identified by HPLC retention time, UV spectra and mass spectral identity with known compounds. Unidentified metabolites will be isolated for NMR analysis. Efforts will be made to identify the signaling pathway using specific inhibitors of the oxidative burst and direct monitoring of hydrogen peroxide generation. Jasmonic acid and salicylic acid production in response to elicitor treatment will be measured. cDNA libraries will be constructed from elicited and non-elicited cultures, and screened for HHT, using sequence data from a similar enzyme in carnation. The native HHT will be isolated from elicited suspension cells by standard protein purification procedures. N-terminal sequencing of the native enzyme, compared with the cloned enzyme will indicate whether the gene contains a signaling or transit peptide sequence. Oat lines of diverse genotype will be grown in diverse environments, and harvested grain will be analyzed for avenanthramides, tocols, and other phytonutrients. Multivariate analysis techniques will indicate influences and interactions. Recombinant lines from specific crosses will be grown and analyzed for concentrations of various constituents to map QTL. Cell cultures and developing seeds will be fed radioisotope-labeled avenanthramide precursors to trace their fate. Their incorporation, under conditions for optimal avenanthramide production, will be determined by scintillation counting of the chromatographically isolated avenanthramides. Seeds and leaf tissues of various cultivars and wild species of oat and barley will be screened for antioxidant activity using an HPLC with diode array detection and in-line antioxidant analysis. Promising metabolites will be isolated and further characterized by LC/MS and NMR. Barley and oat samples from the National Small Grains Collections will be analyzed for protein, oil and beta-glucan. The data will be entered into the GRIN database. Oat groat samples from the uniform nurseries will be analyzed for protein, oil, and beta-glucan. We are analyzing oat samples from lines introgressed with Avena macrostachya for protein, oil, beta-glucan, tocols, and avenanthramides. We are assisting in breeding improved lines of oat and barley by analyzing breeding lines for protein, oil and beta-glucan. Synthetic avenanthramides will be prepared and used in collaborative studies on their effects on indices of atherosclerosis and inflammation in cell culture systems and subsequently with animal models. We will measure the bioavailability of avenanthramides in rats following a single mega-dose of the radiolabeled compound, and their metabolism in various tissues will be determined.