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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

2012 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.


3.Progress Report:
For the current reporting period we initiated our efforts with the development of a method for determining the total soluble nitrogen content in tomato and citrus fruits. The stream-lined method consisted of directly injecting clarified tomato and citrus fruit preparations into a chemiluminescence nitrogen detector (CLND). Since the emphasis of this project is nitrogen-containing small molecules and realizing that the values obtained represented all nitrogen-containing compounds in the preparations, including proteins, polypeptides and small molecules, we sought to refine the method in order to account for the individual contribution of each of these three classes of analytes. To accomplish this we coupled size exclusion chromatography (SEC) with the CLND. For tomato and citrus juices we found that the CLND response during the chromatographic periods in which proteins and polypeptides were to elute did not go above baseline, thus providing evidence that protein and polypeptide concentrations are contributing minimally to the overall nitrogen content determined using the direct injection method.

We also evaluated several analytical techniques for their effectiveness as methods for identifying nitrogen containing metabolites and generating nitrogen metabolome profiles (Objective 1.1). These techniques included nuclear magnetic resonance (NMR), gas chromatography coupled to a mass spectrum detector (GC-MS) and liquid chromatography coupled to an electrochemical detector (LC-EC). Sample types tested using these techniques included citrus, tomato, and cherry fruits, and carrot and parsnip roots. Sample preparation prior to analysis varied from minimal to multi-step preparations that include a solid-phase extraction step. We found that the GC-MS and LC-EC methods could not be used exclusively for the identification of nitrogen containing metabolites, but required the co-analysis of known compounds in order to verify the presence of a nitrogen containing metabolite. In contrast, the NMR method was effective in identifying nitrogen-containing compounds without the use of external standards. However only the most abundant compounds were detected by using NMR. The GC-MS method was more sensitive than NMR, and electrochemical detection was the most sensitive of the methods tested and was capable of detecting concentrations one hundred to a thousand times lower than those detected by NMR.


4.Accomplishments
1. Juice components to be used as diagnostic markers identified. Huanglongbing (HLB), considered the most serious citrus disease in the world, is associated with the bacterium Candidatus Liberibacter asiaticus (Las). While the influence of this disease on complex juice components that contribute to bitterness and aroma have been evaluated, the disease’s influence on the concentration of simpler components such as amino acids and other nitrogen containing metabolites that contribute to the taste and consumer acceptance have been overlooked. In order to evaluate the pathogen's influence on the concentration of these components and to gain further insight into the pathogenesis of the disease, ARS scientists in the Processed Foods Unit in Albany, CA, and at UC Davis partnered together and used nuclear magnetic resonance (NMR) spectroscopy to evaluate citrus juices prepared from fruit from infected and non-infected trees. With the exception of phenylalanine, juices prepared from oranges taken from infected trees had lower concentrations of sugars and amino acids compared to juices prepared from fruit harvested from infection-free trees. The identified metabolite differences may be used diagnostically to determine the disease state of a tree and to identify trees or groves that should be excluded from commercial juice production.


Review Publications
Slisz, A., Breksa III, A.P., Mishchuk, D.O., Mccollum, T.G., Slupsky, C.M. 2012. Metabolomic analysis of citrus infection by Candidatus Liberibacter reveals insight into pathogenicity. Journal of Proteome Research. DOI: abs/10.1021/pr300350x.

Zhang, X., Breksa III, A.P., Mishchuk, D.0., Fake, C., O'Mahony, M.A., Slupsky, C.M. 2012. Fertilization and pesticides affect mandarin orange nutrient composition. Food Chemistry. 134:1020-1024. DOI: 10.1016/j.foodchem.2012.02.218.

Qu, W., Breksa III, A.P., Pan, Z. 2011. Quantitative determination of major polyphenol constituents in pomegranate products. Food Chemistry. 132:1585-1591. DOI: 10.1016/j.foodchem.2011.11.106.

Panthee, D., Cao, C., Debenport, S., Rodriguez, G., Labate, J.A., Robertson, L.D., Breksa Iii, A.P., Van Der Knaap, E., Mcspadden-Gardener, B. 2012. Magnitude of genotype x environment interactions affecting tomato fruit quality. HortScience. 47:721-726.

Last Modified: 10/22/2014
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