title: Measurement of 13C and 15N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association.
abstract: We have developed a method based on a double labeling with stable isotopes and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses to study amino acid exchange in a symbiotic plant-microbe association. Isotopic precision was studied for 21 standards including 15 amino acid derivatives, three N-protected amino acid methyl esters, three amines and one international standard. High correlations were observed between the δ(13)C and δ(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for δ(13)C and 0.28‰ for δ(15)N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. δ(13)C and δ(15)N) of free amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed the pattern of C and N exchange between leaves and nodules, highlighting that γ-aminobutanoic acid and glycine may represent an important form of C transport from leaves to the nodules. The results confirmed the validity, reliability and accuracy of the method for assessing C and N fluxes between plants and symbiotic bacteria and support the use of this technique in a broad range of metabolic and fluxomic studies.
authors.affiliation: Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Spain. gemma.molero@ub.edu
authors.forename: Gemma
authors.initials: G
authors.lastname: Molero
authors.forename: Iker
authors.initials: I
authors.lastname: Aranjuelo
authors.forename: Pilar
authors.initials: P
authors.lastname: Teixidor
authors.forename: José Luis
authors.initials: JL
authors.lastname: Araus
authors.forename: Salvador
authors.initials: S
authors.lastname: Nogués
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000704:Analysis of Variance; D002138:Calibration; D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D000455:Medicago sativa / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000032:analysis / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism; D053204:Root Nodules, Plant / Q000737:chemistry / Q000378:metabolism; D016962:Sinorhizobium meliloti / Q000378:metabolism*; D013559:Symbiosis / Q000502:physiology*
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes