2011 Annual Report 1a.Objectives (from AD-416) Nitrogen-containing plant metabolites are an important class of natural products that contribute to quality and utilization and span the range from essential nutrients to phytochemicals that affect mood and mental well being and, in some extreme cases, toxins. Due to complexity and difficulty in detection, past research in this area has targeted very specific compounds, resulting in most nitrogen-containing plant metabolites being largely ignored. Objective 1- Develop new or improve existing methods to detect, identify, and characterize N-containing plant metabolites. Sub-Objective 1.1. Establish extraction methods for nitrogen-containing metabolites from the fruit and leaves of specialty crops (e.g., citrus, grapes, tomatoes, peaches, nectarines and plums) and model plant species. Establish HPLC separation method for the resolution of multiple classes of nitrogen-containing metabolites in a single run. Compare existing methods for the detection and quantification of nitrogen-containing metabolites. Combine the elements of extraction, separation, identification and quantification into an optimized method for the profiling of N-containing plant metabolites. Sub-Objective 1.2. Systematically identify nitrogen-containing metabolites in extracts prepared from the leaves of model plant species (Arabidopsis thaliana, Nicotiana tabacum, and tomato) in preparation to commencing the nitrogen metabolite profiling (NMP) of citrus, grapes, other tomatoes, peaches, nectarines, and plums. Sub-Objective 1.3. Conduct NMP of plant cell cultures from model species (Arabidopsis thaliana, Nicotiana tabacum, and tomato) and determine the culturing conditions that result in nitrogen metabolomes most similar to the profiles to found in leaf and fruit tissues from whole plants (Sub-objective 1.2). Objective 2- Screen specialty crops for their metabolomic profiles with a particular emphasis on nitrogen-containing metabolites. Initial efforts will focus on the fruits and leaves from citrus, grapes, tomatoes, peaches, nectarines, and plums. Sub-Objective 2.1. Commence NMP and systematic identification and quantification of nitrogen-containing metabolites found in leaf and fruit tissues of citrus, grapes, tomatoes, peaches, nectarines and plums using liquid chromatography coupled to mass spectrometer (MS & MS/MS) and nitrogen (CND and/or post column derivatization) detection systems. Sub-objective 2.2. Isolate and/or identify new or novel nitrogen-containing metabolites. Objective 3- Isolate and characterize a SAM-dependent N-methyltransferase in order to increase our knowledge about this class of enzymes in plants and their relationship to nitrogen metabolism. Sub-Objective 3.1. Select a specific SAM-dependent N-methyltransferase for characterization and isolation based upon the results obtained in accomplishing Objectives 1 and 2 and an enzyme activity screen. Sub-objective 3.2. Isolate and characterize the selected SAM-dependent N-methyltransferase. Clone the enzyme and confirm its function in a yeast and plant cell culture system. 1b.Approach (from AD-416) 1) Develop new or improve existing methods to detect, identify, and characterize N-containing plant metabolites. . 2)Screen specialty crops for their metabolomic profiles with a particular emphasis on nitrogen-containing metabolites. Initial efforts will focus on the fruits and leaves from citrus, grapes, tomatoes, peaches, nectarines, and plums. . 3)Isolate and characterize a SAM-dependent N-methyltransferase in order to increase our knowledge about this class of enzymes in plants and their relationship to nitrogen metabolism. 3.Progress Report Part of FY2010 focused on closing out research objectives from the previous in-house project (5325-41430-010-00D). These efforts included analytical work in support of a human trial evaluating the efficacy of limonoid glucoside consumption in reducing cholesterol levels and the evaluation of citrus juice samples for their limonoid content. As part of the analytical work in support of the human study, we continued monitoring the shelf-life stability of limonin glucoside as a beverage component and determined that it was still stable after a year and a half of storage. Citrus juice samples, analyzed for their limonoid content, came from a variety sources including large (Sunkist) and small-scale growers and provided valuable feedback to these growers. Subsequent efforts during the FY have been directed at our current in-house project. Activities related to our project have included the collection of samples and standards and setting up and familiarizing ourselves with the instrumentation needed to conduct analyses directed toward identifying and quantifying nitrogen-containing compounds. This year citrus and tomato samples were collected, processed for preservation, and analyzed for their physicochemical properties. One system consisting of a high pressure liquid chromatrography (HPLC) and in-line nitrogen detector was set up and experiments conducted to characterize the system’s performance factors, including limit of detection, limit of quantification, and run to run variability. 4.Accomplishments 1. Environmental factors affect citrus juice quality. Consumption of citrus fruits and juices has been driven by the US industry’s ability to provide consumers with a high quality, good tasting, and health-promoting product. In order to consistently produce a product desired by consumers, it is essential to identify the environmental factors that contribute to the overall metabolite content and quality of citrus fruits. ARS scientists in the Processed Foods Unit in Albany, CA, partnered with UC Davis scientists in a study to determine how elevation, soil type, root stock differences interact to influence the concentrations of individual compounds found in fruit. From testing juice samples prepared from fruit collected from various locations we found that groves planted at lower elevations (less than 140 m) produced fruit with higher sugar concentrations, while fruit harvested for groves at higher elevation tended to have greater amino acid concentrations. Groves planted on C-35 rootstock in shallow soil produced fruit with significantly higher ethanol concentrations. These results provide important insights that can be used in planning the placement of future citrus groves and improving the fruit quality of existing commercial groves. 2. Plant bioactives. Human health and nutrition can be enhanced by consuming functional foods, these include fresh fruits and vegetables, processed foods, and plant extracts that contain plant bioactive compounds. The presence of functional foods in the marketplace is dependent upon the availability of robust analytical methods to verify the presence and quality of bioactives, as well as sustainable production and manufacturing practices to produce commercial products that contain plant bioactives. ARS chemists and food technologists in the Processed Foods Unit in Albany, CA collaborated together to evaluate the influence of UV-B treatment on enhancing the bioactive content of fruits and vegetables. In the course of this study, it was determined that carrots, among all the crops tested, were particularly responsive to treatment and that UV-B treatment resulted in a significant increase in antioxidant content. Subsequent utilization of a streamlined analytical method resulted in the identification of the compound whose increased concentration contributed to the increased antioxidant content. The process of enhancing the bioactive content of fruits and vegetables through UV treatment and the associated analytical methods provide researchers and producers with tools and methods for bringing consumers value-added health promoting foods. Review Publications Breksa III, A.P., Khan, T., Zukas, A.A., Hidalgo, M., Lee-Yuen, M.S. 2011. Limonoid content of sour orange varieties. Journal of the Science of Food and Agriculture. 91(6): 1789-1794. doi: 10.1002/jsfa.4383. Zhang, X., Breksa Iii, A.P., Mishchuk, D.O., Slupsky, C.M. 2011. Elevation, rootstock, and soil depth affect the nutritional quality of mandarin oranges. Journal of Agriculture and Food Chemistry. 59(1):2672–2679.