15N-labelling of Leaves Combined with GC-MS Analysis as a Tool for Monitoring the Dynamics of Nitrogen Incorporation into Amino Acids

Abstract

Labeling plant material such as detached leaves with ¹⁵NH₄⁺ is a very instrumental method for the characterization of metabolic pathways of mineral nitrogen assimilation and incorporation into amino acids. A procedure of labeling, followed by amino acid extraction, purification, and derivatization for gas chromatography coupled to mass spectrometry (GC/MS) analysis, is presented. The rationale of heavy isotope abundance calculations and amino acid quantification is detailed. This method is adaptable to various plant species and various kinds of investigations, such as elucidating physiological changes occurring as a result of gene mutations (overexpression or inhibition) in natural variants or genetically modified crops, or characterization of metabolic fluxes in genotypes exhibiting contrasted physiological or developmental adaptive responses to biotic and/or abiotic environmental stresses. Furthermore, the benefit of working on detached organs or pieces of organs is to investigate finely the metabolism of species that are not amenable to laboratory work, such as plants growing in natural environments or under agricultural conditions in the field.

Keywords: 15N-labeling; Amino acid; Ammonium; GC-MS; Glutamine synthetase/glutamate synthase (GS/GOGAT); Metabolic flux; Metabolism; Metabolite signaling; Metabolome.