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 δ13C and δ15N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R2 = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for δ13C and 0.28‰ for δ15N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N2‐fixing bacteria. Plants were simultaneously labeled over 10 days with 13C‐depleted CO2 (12CO2), which was assimilated through photosynthesis by leaves, and 15N2 fixed via nodules. Subsequently, the C and N isotope compositions (i.e. δ13C and δ15N) 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.

Compound‐specific δ13C and δ15N analysis of amino acids: a rapid, chloroformate‐based method for ecological studies

“…It should be noted that precision may vary considerably between labs, even for the same derivatization method and similar GC/C/IRMS protocols. For example, Molero et al . obtained average δ 15 N value precisions of 0.28‰ while Hofmann et al .…”

“…The derivatization reactions and multiple purification steps of these procedures require hours for completion, and careful temperature control and anhydrous conditions are essential (Table ) . Derivatization via silylation is a simple, single‐step alternative, but silylated derivatives decrease oxidation reactor efficiency and may degrade capillary columns . Furthermore, silylation adds a large amount of non‐analyte carbon – at least six carbons per AA – which is expected to decrease the precision of δ 13 C measurements .…”

Photosynthesis, N2 fixation and taproot reserves during the cutting regrowth cycle of alfalfa under elevated CO2 and temperature

“…So the identification of plant processes conditioning C sinks will be a key point in future plant breeding programs. The application of C and N stable isotopes to research in this area has been described previously (Avice et al, 1996b;Aranjuelo et al, 2008Aranjuelo et al, , 2009bMolero et al, 2011). Specifically, the application of 12 CO 2 labelling in a study conducted in slow growing plants (Aranjuelo et al, 2009b) revealed that the C sink/source imbalance was conditioned by plant growth, the leaf C residence time and a by the capacity to deliver recently fixed C to respiration.…”

Effect of shoot removal on remobilization of carbon and nitrogen during regrowth of nitrogen‐fixing alfalfa

“…Organic acids provide reductant to both the Nase complex and the respiratory chain that fuels Nase with the ATP necessary for nitrogen fixation (Kouchi and Yoneyama , Streeter ). The bacteroid returns ammonium (NH 4 + ) to the host and this is assimilated in the form of amino acids that are partitioned to the rest of the plant through the xylem (Udvardi and Day , Molero et al ). Studies conducted in the past show that, after shoots are removed, nodule functioning is reduced during the first days of regrowth (Boucaud and Bigot , Ta et al ) and is concomitant with an 88% decrease in nitrogen fixation 24 h after cutting (Vance et al ).…”