Measurement of C-13 and N-15 isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association

作者：Molero Gemma^*; Aranjuelo Iker; Teixidor Pilar; Luis Araus Jose; Nogues Salvador

来源：Rapid Communications in Mass Spectrometry, 2011, 25(5): 599-607.

DOI：10.1002/rcm.4895

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 delta C-13 and delta N-15 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 parts per thousand for delta C-13 and 0.28 parts per thousand for delta N-15 (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 C-13-depleted CO2 ((CO2)-C-12), which was assimilated through photosynthesis by leaves, and N-15(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. delta C-13 and delta N-15) 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 gamma-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.

• 出版日期2011-3-15