Premium
Measurement of 13 C and 15 N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant–microbe symbiotic association
Author(s) -
Molero Gemma,
Aranjuelo Iker,
Teixidor Pilar,
Araus José Luis,
Nogués Salvador
Publication year - 2011
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4895
Subject(s) - chemistry , isotope ratio mass spectrometry , isotope , mass spectrometry , glycine , amino acid , gas chromatography , isotopes of carbon , stable isotope ratio , isotope analysis , chromatography , biochemistry , environmental chemistry , total organic carbon , ecology , physics , quantum mechanics , biology
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. Copyright © 2011 John Wiley & Sons, Ltd.