2009 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 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.
In the past year we have discovered that plants treated with benzothiadiazole (BTH), a commercially available agrochemical used to elicit phytoalexins in various crops, respond by producing avenanthramides in oat leaf tissue. Preliminary results also show that the oat grain may, likewise, demonstrate enhanced avenanthramide production. This finding is important for a number of reasons: (1) Previously it has only been possible to "turn-on" the avenanthramide biosynthetic machinery by either infecting live plants with crown rust spores or by treating separated leaves with elicitors or by using suspension cultures treated with elicitors. (2) The treatment regime involves treating the oat roots with the BTH, demonstrating that there is a systemic signaling mechanism at work (in contradiction to previously published reports). (3) This treatment regime will allow investigation on the dynamics of avenanthramide biosynthesis using whole live plants. At present, for example, we are generating a cDNA library from BTH treated leaf tissue that should provide enhanced levels of elicited genes involved in the entire avenanthramide biosynthetic pathway. If the BTH effect on grain is proven, we will also be able to generate a cDNA library from elicited grain tissue as well.
We have cloned the full coding sequence for two enzymes involved in the biosynthesis of avenanthramides: an oat phenylalanine ammonia lyase (PAL) and a hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl Transferase (HHT). These gene sequences were captured using the Rapid Amplification of cDNA ends (RACE) technique. Work is continuing to clone these two enzymes as full length transcripts and getting them functionally expressed. These data will prove valuable in seeking additional isozymes of these critical elements in the avenanthramide biosynthetic pathway and, when functionally expressed, provide useful tools for the investigation of the biosynthesis of these metabolites.
We have also completed the analysis of the Barley CAP year 3 germplasm for beta-glucan content and the year 1 and 2 germplasm for tocol (vitamin E) content. The analysis of the germplasm for tocol content represents the largest such survey for these phytonutrients in barley and the results show a dramatic variation in tocol levels. These results suggest that the tocol levels of barley can be readily manipulated through selective breeding.
Survey of the Barley CAP year 1 germplasm for tocols. Although barley is considered a good source of tocochromals (vitamine E) no data exist on the natural variation in tocol levels in this crop. This examination of the tocol content in the 960 germplasm lines from the first year of the Barley CAP is, by far, the largest to date and revealed a dramatic range of tocol levels both within and between breeding programs. Year two germplasm is currently being analyzed. These results illustrate the feasibility to breed increased levels of tocols in barley, the impact of which will be enhanced nutritional value of grain. The results are being presented as a poster at the 2009 American Society of Plant Biologist Meeting.
Elicitation of avenanthramide biosynthesis with BTH. No means of ensuring a consistent induction of avenanthramide synthesis has been reported for oats. We have discovered that treatment of oats with the systemic acquired resistance (SAR) elicitor benzothiadiazole (BTH) results in a dramatic up-regulation of avenanthramide production in the leaf tissue of oats and may increase oat grain levels as well. This technique will impact research by providing a tool to investigate the dynamics of avenanthramide biosynthesis in whole plants under controlled conditions. BTH might also provide a means to regulate avenanthramide production in the oat crop and enhance crown rust disease resistance in the field. Results from this study will be submitted for publication in the fall of 2009.
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Wise, M.L., Sreenath, H.K., Skadsen, R.W., Kaeppler, H.F. 2009. Biosynthesis of Avenanthramides in Suspension Cultures of Oat (Avena sativa). Plant Cell Tissue And Organ Culture. 97:81-90.
Ji, L., Koenig, R., Wise, M.L. 2008. Oat Avenanthramides: A Novel Antioxidant. In: Mine, Y., Miyashita, K., Shahidi, F., editors. Nutrigenomics and proteomics in Health and Disease. Ames, IA: Wiley-Blackwell. p. 239-249.
Wise, M.L., Doehlert, D.C., Mcmullen, M.S. 2008. Association of Avenanthramide Concentration in Oat (Avena sativa) Grain with Crown Rust Incidence and Genetic Resistence. Cereal Chemistry. 85(5)639-641.
Doehlert, D.C., Simsek, S., Wise, M.L. 2009. The Green Oat Story: Possible Mechanisms of Green Color Formation in Oat Products During Cooking Color Changes in Cooked Oat Products. Journal of Food Science. 74(6):S226-S231