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United States Department of Agriculture

Agricultural Research Service

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Research Project: Improved Utilization of Ag. Products Through Identification of Nitrogen-Containing Bioactive Components Important to Quality & Human Health

Location: Healthy Processed Foods Research

2013 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 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.


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


3.Progress Report:
Efforts for the current reporting period focused on the successful development of an HPLC method suitable for the detection of nitrogen containing metabolites using a chemiluminescence nitrogen detector (CLND) and are directly related to fulfilling Objective 1 (Develop new or improved existing methods to detect, identify, and characterize N-containing plant metabolites). The resulting method is equally compatible with other detection methods, including mass spectrometer (MS), evaporative light scattering (ELS), and is more environmentally friendlier than other methods. Utilizing this method, we evaluated the response of the multiple detectors listed above to a series of nitrogen containing metabolites, including amino acids, caffeine and glycoalkaloids present in tomatoes, eggplants, and potatoes. Although not as sensitive as mass spectrometery, we found detection by chemiluminescence superior in its linearity of response. Additionally, we found that caffeine could be used as a surrogate for quantification, thus relieving the need to have analytical standards for each compound under analysis. The developed method has been applied to the analysis of tomato, citrus, and potato samples (Objective 2: Screen specialty crops for their metabolic profiles with particular emphasis on nitrogen-containing metabolites). For tomato samples, the emphasis was on the quantification of Esculeoside A, a glycoalkaloid reported to contain potential health promoting properties. In evaluating tomatoes available as canned and fresh products, we found that cherry and grape type tomatoes had the highest concentrations of Esculeoside A, and whereas for canned products glycoalkaloid concentrations were below the level of detection of the method.


4.Accomplishments
1. Vintage tomato varieties evaluated for their nutritional properties. Tomatoes are one of the most highly consumed fruits in the United States and throughout the world. Consumers, as well as producers of tomatoes, are looking for varieties with improved nutritional properties and tomato breeders are seeking existing varieties that can be used to generate new varieties to meet consumer needs. In an effort to provide key nutritional information on varieties found in the USDA’s germplasm collection, ARS scientists in the Processed Foods Unit in Albany, California, and Plant Genetic Resources Unit in Geneva, New York, partnered together to evaluate over 50 varieties for their agricultural and nutritional properties. Information gathered from this collaborative project will provide researchers and breeders with much needed data for the generation of future tomato varieties.


Review Publications
Panthee, D., Labate, J.A., Mcgrath, M., Francis, D., Breksa III, A.P., Robertson, L.D. 2013. Genotype and environmental interaction for fruit quality traits in vintage tomato varieties. Euphytica. 193:169-182.

Last Modified: 9/21/2014
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