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Labeling Maize (Zea mays L.) Leaves with 15 NH 4 + and Monitoring Nitrogen Incorporation into Amino Acids by GC/MS Analysis

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  • C. Cukier
  • P.J. Lea
  • R. Cañas
  • A. Marmagne
  • A.M. Limami
  • B. Hirel
Article numbere20073
<mark>Journal publication date</mark>12/09/2018
<mark>Journal</mark>Current protocols in plant biology
Issue number3
Publication StatusPublished
Early online date10/09/18
<mark>Original language</mark>English


The human body contains approximately 3.2% nitrogen (N), mainly present as protein and amino acids. Although N exists at a high concentration (78%) in the air, it is not readily available to animals and most plants. Plants are however able to take up both nitrate (NO3- ) and ammonium (NH4+ ) ions from the soil and convert them to amino acids and proteins, which are excellent sources for all animals. Most N is available as the stable isotope 14 N, but a second form, 15 N, is present in very low concentrations. 15 N can be detected in extracts of plants by gas chromatography followed by mass spectrometry (GC/MS). In this protocol, the methods are described for tracing the pathway by which plants are able to take up 15 N-labeled nitrate and ammonium and convert them into amino acids and proteins. A protocol for extracting and quantifying amino acids and 15 N enrichment in maize (Zea mays L.) leaves labeled with 15 NH4+ is described. Following amino acid extraction, purification, and separation by GC/MS, a calculation of the 15 N enrichment of each amino acid is carried out on a relative basis to identify any differences in the dynamics of amino acid accumulation. This will allow a study of the impact of genetic modifications or mutations on key reactions involved in primary nitrogen and carbon metabolism. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.