2010 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.
This is the final report for project 3655-21000-044-00D to be succeeded by project 3655-21000-053-00D. Much of the work on this project has been directed to understanding the biological mechanisms regulating biosynthesis of a group of anti-oxidants termed ‘avenanthramides’ found uniquely (among food crops) in oat. Laboratory studies are providing increasing evidence for the nutritional benefits of avenanthramides; many of these studies have been facilitated by efforts in this project to provide these metabolites as pure, synthetic compounds. Although avenanthramides are constitutively expressed in oat grain, the levels are highly variable. This variability appears to be strongly influenced by environmental as well as genotypic factors. One study from this project showed a strong correlation between crown rust infection and grain avenanthramide levels in field grown oats. The results showed significant variation between cultivars and, even more revealing, that many cultivars had significantly higher avenanthramide levels (up to 15 fold) when grown under rust pressure versus a rust free environment. More recently we showed that isonicotinic acid and benzothiadiazole (BTH), commercial agrochemicals that elicit the plant systemic acquired resistance (SAR) response, can up-regulate avenanthramide production in oat. This discovery provides an excellent tool to study the biosynthesis of avenanthramides and portends the use of these agrochemicals to stimulate avenanthramide biosynthesis in the oat crop. Efforts to evaluate application methods as well as the dynamics and tissue specific response to these SAR elicitors are continuing. A cDNA library from BTH elicited oat was produced and is currently being screened for genes involved in avenanthramide biosynthesis. During the course of this project a cell culture system to produce avenanthramides from oat callus was also developed.
In collaborative work with nutritional scientists, avenanthramides were shown to effectively inhibit certain benchmarks for atherosclerotic plaque formation. To facilitate these studies we have improved on the published synthetic method for avenanthramides. Using a peptide coupling reagent to activate the reaction, along with a superior deprotection reaction for intermediates in the synthesis, we can now rapidly generate 100s of milligrams of pure compound in a relatively simple synthesis. These synthetic avenanthramides recently allowed a group of researchers at the China Agricultural University in Beijing to demonstrate that avenanthramides could effectively reduce hepatic steatosis (fatty acid deposition) in a cell culture model system.
A microtitre plate method for the calcofluor based beta-glucan analysis was developed in this project. We also provided the beta-glucan analysis for the Barley CAP project and, in addition, analyzed the tocol content from year one and two of the Barley CAP. With over 1800 accessions, this is the largest study for this important nutrient ever conducted on barley. The results show substantial genetic variability in this trait that can likely be enhanced through selective breeding.
Role of avenanthramides in mitigating non-alchohol steatohepatitis. Steatohepatitis refers to the build up of fatty tissue in the liver. It is a liver disorder with emerging importance, especially in regions with high rates of obesity. Avenanthramides, from oat, possess both antioxidant and anti-inflammatory properties. Both these physiological effects can ameliorate steatohepatitis. Thus, selected solvent extracts from oat that are rich in avenanthramides were shown to inhibit steatohepatitis in a cell culture model system. The association with avenanthramides was further substantiated by using pure, synthetic avenanthramides to produce the same effect. The impact of these results is to demonstrate another potential health benefit derived from oat consumption and from oat products.
Survey of the Barley Cooperative Agriculture Project (CAP) year 1 and 2 germplasm for tocols. Although barley is considered a good source of tocochromals (vitamin E) little data exist on the variation in tocol levels in this crop. Both year one and year two of the Barley CAP germplasm, representing over 1800 lines of germplasm, have been analyzed. The results from this study illustrate the feasibility to breed increased levels of tocols in barley, the impact of which will be enhanced nutritional value of barley grain. We have also completed the Barley CAP beta glucan analysis.
Dynamics of benzothiadiazole (BTH) elicitation of avenanthramide production in oat. Avenanthramides are phenolic antioxidants unique to oats that possess desirable nutritional qualities. However, production of avenanthramides in oat is highly variable and is strongly influenced by environmental factors. Thus selective breeding for this phytonutrient is problematic. Recenty, BTH, a commercially available agrochemical, was shown to effectively increase avenanthramide production in oat leaves. Detailed experiments over the past year have provided information on the dynamics and tissue distribution of avenanthramides produced in response to BTH treatment. The impact of these experiments is that the scientific tool developed (chemical manipulation of avenanthramide synthesis) allows acquisition of knowledge on avenanthramide biosynthesis and the signaling mechanism responsible for their production in planta. These data also portend a mechanism to control avenanthramide production through agricultural practice.
Jackson, E.W., Wise, M., Bonman, J.M.,Obert, D.E., Hu, G. and Peterson. D.M.. 2008. QTLs Affecting -tocotrienol, -tocopherol, and total tocopherol concentrations detected in the Ogle/TAM O-301 oat mapping population. Crop Science 48:2141-2152.