2011 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.
One of the primary objectives of this research project is to investigate the factors regulating the biosynthesis of avenanthramides in oat. Avenanthramides are secondary metabolites that provide protection to the oat plant against crown rust infection and have shown certain desirable nutriceutical properties in mammals such as reduction of the inflammatory response. During the past 12 months this laboratory has published its finding that benzodiathiazole (BTH), an agrichemical that elicits a disease resistance response in plants, is capable of stimulating the production of avenanthramides in oat leaves (J. Agr. Food Chem., 59:7028-7038(2011)). We are following up on this discovery with laboratory studies with results that show there are significant genotypic differences in the response to BTH and that avenanthramide levels in the filling grain and tissues other than leaf are affected by BTH treatment (with statistically significant increases in cultivars ‘Gem’ and ‘Kame’).
The tocol analysis of four barley mapping populations and one oat tetraploid population (approx 1200 lines) in collaboration with a USDA researcher at Aberdeen, Idaho is complete. This effort has resulted in mapping of several qualitative trait loci for tocol (vitamin E) production in oat and barley. We have also isolated two full length complimentary DNA transcripts of isozymes of phenylalanine ammonia lyase and of hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (the final enzyme in avenanthramide biosynthesis) from an oat leaf complimentary DNA library. A new collaboration with scientists in the Department of Health and Nutritional Sciences, South Dakota State University, to investigate the anti-inflammatory properties of avenanthramides has been initiated.
Analysis of tocol content in oat and barley. Tocopherols and tocotrienols (vitamin E, collectively referred to as tocols) are important nutritional components of both oat and barley. Although there are eight naturally occurring congeners of vitamin E in barley grain, oat usually only produces three or four in significant quantities. Recent studies by ARS researchers at Madison, Wisconsin have shown large variations in tocol content between germplasm, likely based on genotypic differences. There is, however, a marked dearth of information on the quantitative trait loci (QTL) for tocol production in these crops. In collaboration with plant geneticist at the USDA Aberdeen Idaho laboratory, ARS researchers at Madison, Wisconsin have analyzed the tocol content in four mapping populations of barley and a tetraploid population of oat. Results from these experiments will provide data to genetically map the QTLs for tocol production in oat and barley. The impact will be to facilitate breeding oat and barley for superior tocol (vitamin E) content. This might be effected in terms of either higher quantities of tocols in the grain or relative proportion of the various congeners (or possibly quantity and proportion simultaneously).
Genotypic difference in avenanthramide biosynthesis in response to plant defense activators (PDA) in oat. Although PDAs were previously shown to elicit avenanthramide production in one oat cultivar (‘Belle’) the question of whether genotypic differences in the response to chemical PDAs remained unanswered. To investigate whether genotypic differences exist for PDA response, ARS researchers at Madison, Wisconsin have extended their experiments to three additional oat cultivars: ‘Gem’, ‘Ogle’, and ‘Kame’ and have evaluated the effect of BTH treatment on avenanthramide production in leaves as well as in filling grain. The results show there are clear genotypic differences in the response to BTH elicitation. ‘Kame’, for example, shows a much stronger and faster response than the other cultivars. The impact of these studies is the demonstration that plant defense activators are useful selection tools for increasing both the agronomic value (enhanced resistance to crown rust disease) and the human nutritional value (higher levels of grain avenanthramides) of oats leading to the development of better cultivars. These results also portent the utility of preventative treatment with PDAs to enhance rust resistance in the less resistant cultivars.
Guo, W., Nie, L., Wu, D., Wise, M.L., Collins, F., Meydani, S., Meydani, M. 2010. Avenanthramides inhibit proliferation of human colon cancer cell lines in vitro. Nutrition and Cancer. 62(8):1007-1016.
Koenig, R.T., Dickman, J.R., Wise, M.L., Ji, L. 2011. Avenanthramides are bioavailable and accumulate in hepatic, cardiac, and skeletal muscle tissue following oral gavage in rats. Journal of Agricultural and Food Chemistry. 59(12):6438-6443.
Wise, M.L. 2011. Effect of chemical systemic acquired resistance elicitors on avenanthramide biosynthesis in oat (Avena sativa). Journal of Agriculture and Food Chemistry. 59:7028-7038.
Cai, S., Huang, C., Ji, B., Wise, M.L., Zhang, D., Yang, P. 2011. In vitro antioxidant activity and inhibitory effect, on oleic acid-induced hepatic steatosis, of fractions and subfractions from oat (Avena sativa L.) ethanol extract. Journal of Food Science. 124(3):900-905.