Robust Method for Investigating Nitrogen Metabolism of ¹⁵N Labeled Amino Acids Using AccQ•Tag Ultra Performance Liquid Chromatography-Photodiode Array-Electrospray Ionization-Mass Spectrometry: Application to a Parasitic Plant–Plant Interaction

Abstract

An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of ¹⁵N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie <i>Brassica napus</i> (rapeseed) and the parasitic weed <i>Phelipanche ramosa</i> (broomrape). This method was developed and validated using amino acid standard solutions containing ¹⁵N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of ¹⁵N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as ¹⁵N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to ¹⁵N₂-glutamine. ¹⁵N₂-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of <i>Phelipanche ramosa</i>