Project Number: 5325-41430-011-00
Project Type: Appropriated

Start Date: Jun 13, 2010
End Date: Jun 12, 2015

Objective:
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 High Pressure Liquid Chromatograpy (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.

Approach:
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. Replacing 5325-41430-010-00D 05/2010.