Final published version
Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Labeling Maize (Zea mays L.) Leaves with 15 NH 4 + and Monitoring Nitrogen Incorporation into Amino Acids by GC/MS Analysis
AU - Cukier, C.
AU - Lea, P.J.
AU - Cañas, R.
AU - Marmagne, A.
AU - Limami, A.M.
AU - Hirel, B.
PY - 2018/9/12
Y1 - 2018/9/12
N2 - 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.
AB - 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.
KW - 15N labeling
KW - amino acid
KW - ammonium
KW - fluxes
KW - GC/MS
KW - Zea mays
U2 - 10.1002/cppb.20073
DO - 10.1002/cppb.20073
M3 - Journal article
VL - 3
JO - Current protocols in plant biology
JF - Current protocols in plant biology
SN - 2379-8068
IS - 3
M1 - e20073
ER -