Compound-Specific Isotope Analysis of Amino Acid Labeling with Stable Isotope Nitrogen (15N) in Higher Plants

Abstract

Individual free amino acid δ15N values in plant tissue reflect the metabolic pathways involved in their biosynthesis and catabolism and could thus aid understanding of environmental stress and anthropogenic effects on plant metabolism. In this study, compound-specific nitrogen isotope analysis of amino acid by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was carried out to determine individual free amino acid δ15N values. High correlations were observed between the δ15N values obtained by GC-C-IRMS and elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) determinations, and the mean precision measured was better than 1 ‰. Cation-exchange chromatography was employed to purify the sample, and the difference between prior to and following passage through the resin was within 1 ‰. The amino acid δ15N values of plant leave samples following incubation in 15N-nitrate at different time points were determined. A typical foliar free amino acid 15N-enrichment pattern was found, and glutamine was the most rapidly labeled amino acid; other amino acids derived from the GS-GOGAT cycle were also enriched. The pyruvate family amino acids were labeled less quickly followed by the aromatic amino acids. This study highlighted that amino acid metabolism pathways had a major effect on the δ15N values. With the known amino acid metabolism pathways and δ15N values determined by the presented method, the influence of various external factors on the metabolic cycling of amino acid can be understood well.