title: 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 (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-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 Phelipanche ramosa.
authors.affiliation: EA 1157, Laboratoire de Biologie et de Pathologie Végétales (LBPV), Université de Nantes , SFR 4207 QUASAV, 44322 Nantes, France.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Gaudin
authors.forename: Delphine
authors.initials: D
authors.lastname: Cerveau
authors.forename: Nathalie
authors.initials: N
authors.lastname: Marnet
authors.forename: Alain
authors.initials: A
authors.lastname: Bouchereau
authors.forename: Philippe
authors.initials: P
authors.lastname: Delavault
authors.forename: Philippe
authors.initials: P
authors.lastname: Simier
authors.forename: Jean-Bernard
authors.initials: JB
authors.lastname: Pouvreau
mesh_terms: D000641:Ammonia / Q000032:analysis* / Q000378:metabolism; D029688:Brassica napus / Q000737:chemistry / Q000378:metabolism* / Q000469:parasitology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005973:Glutamine / Q000032:analysis* / Q000378:metabolism; D057230:Limit of Detection; D009584:Nitrogen / Q000032:analysis* / Q000378:metabolism; D009587:Nitrogen Isotopes; D031672:Orobanche / Q000737:chemistry / Q000378:metabolism*; D018517:Plant Roots / Q000737:chemistry / Q000378:metabolism* / Q000469:parasitology; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D009587:Nitrogen Isotopes; D005973:Glutamine; D000641:Ammonia; D009584:Nitrogen