Abstract
Labeling plant material such as detached leaves with 15NH4+ is a very instrumental method for the characterization of metabolic pathways of mineral nitrogen assimilation and incorporation into amino acids. A procedure of labeling, followed by amino acid extraction, purification, and derivatization for gas chromatography coupled to mass spectrometry (GC/MS) analysis, is presented. The rationale of heavy isotope abundance calculations and amino acid quantification is detailed. This method is adaptable to various plant species and various kinds of investigations, such as elucidating physiological changes occurring as a result of gene mutations (overexpression or inhibition) in natural variants or genetically modified crops, or characterization of metabolic fluxes in genotypes exhibiting contrasted physiological or developmental adaptive responses to biotic and/or abiotic environmental stresses. Furthermore, the benefit of working on detached organs or pieces of organs is to investigate finely the metabolism of species that are not amenable to laboratory work, such as plants growing in natural environments or under agricultural conditions in the field.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cañas RA, Yesbergenova-Cuny Z, Simons M et al (2017) Exploiting the genetic diversity of maize using a combined metabolomic, enzyme activity profiling, and metabolic modeling approach to link leaf physiology to Kernel Yield. Plant Cell 29:919–943
Cukier C, Lea PJ, Cañas R et al (2018) Labeling maize (Zea mays L.) leaves with with 15NH4+ and monitoring nitrogen incorporation into amino acids by GC/MS analysis. Curr Protoc Plant Biol 3:e20073
Fukushima A, Kusano M (2014) A network perspective on nitrogen metabolism from model to crop plants using integrated 'omics' approaches. J Exp Bot 65:5619–5630
Sheth BP, Thaker VS (2014) Plant systems biology: insights, advances and challenges. Planta 240:33–54
Fernie AR, Geigenberger R, Stitt M (2005) Flux, an important, but neglected, component of functional genomics. Curr Opin Plant Biol 8:174–182
Simons M, Saha R, Guillard L et al (2014) Nitrogen-use efficiency in maize (Zea mays L.): from 'omics' studies to metabolic modelling. J Exp Bot 65:5657–5671
Amiour N, Imbaud S, Clément G et al (2012) The use of metabolomics integrated with transcriptomic and proteomic studies for identifying key steps involved in the control of nitrogen metabolism in crops such as maize. J Exp Bot 63:5017–5033
Urano K, Kurihara Y, Seki M et al (2010) 'Omics' analyses of regulatory networks in plant abiotic stress responses. Curr Opin Plant Biol 13:132–138
Rhee SY, Mutwil M (2014) Towards revealing the functions of all genes in plants. Trends Plant Sci 19:212–221
António C, Päpke C, Rocha M et al (2016) Regulation of primary metabolism in response to low oxygen availability as revealed by carbon and nitrogen isotope redistribution. Plant Physiol 170:43–56
Limami AM, Glévarec G, Ricoult C et al (2008) Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress. J Exp Bot 59:2325–2335
Ricoult C, Cliquet JB, Limami AM (2005) Stimulation of alanine amino transferase (AlaAT) gene expression and alanine accumulation in embryo axis of the model legume Medicago truncatula contribute to anoxia stress tolerance. Physiol Plant 123:30–39
Ricoult C, Echeverria L, Cliquet JB et al (2006) Characterization of alanine aminotransferase (AlaAT) multigene family and hypoxic response in young seedlings of the model legume Medicago truncatula. J Exp Bot 57:3079–3089
Abadie C, Tcherkez G (2019) In vivo phosphoenolpyruvate carboxylase activity is controlled by CO. New Phytol 221:1843–1852
Abadie C, Tcherkez G (2021) C isotope labelling to follow the flux of photorespiratory intermediates. Plants(Basel) 10:427
Kruger NJ, Ratcliffe RG (2015) Fluxes through plant metabolic networks: measurements, predictions, insights and challenges. Biochem J 465:27–38
Freund DM, Hegeman AD (2017) Recent advances in stable isotope-enabled mass spectrometry-based plant metabolomics. Curr Opin Biotechnol 43:41–48
Molero G, Aranjuelo I, Teixidor P et al (2011) 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. Rapid Commun Mass Spectrom 25:599–607
Tcherkez G, Atkin OK (2021) Unravelling mechanisms and impacts of day respiration in plant leaves: an introduction to a Virtual Issue. New Phytol 230:5–10
Amiour N, Décousset L, Rouster J et al (2021) Impacts of environmental conditions, and allelic variation of cytosolic glutamine synthetase on maize hybrid kernel production. Commun Biol 4:1095
Heinemann B, Hildebrandt TM (2021) The role of amino acid metabolism in signaling and metabolic adaptation to stress-induced energy deficiency in plants. J Exp Bot 72:4634–4645
Lea PJ, Miflin BJ (1974) Alternative route for nitrogen assimilation in higher plants. Nature 251:614–616
Acknowledgments
This work was funded by Région des Pays de la Loire, Angers Loire Métropole, University of Angers, and INRAE (Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Limami, A.M., Cukier, C., Hirel, B. (2023). 15N-labelling of Leaves Combined with GC-MS Analysis as a Tool for Monitoring the Dynamics of Nitrogen Incorporation into Amino Acids. In: Couée, I. (eds) Plant Abiotic Stress Signaling. Methods in Molecular Biology, vol 2642. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3044-0_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3044-0_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3043-3
Online ISBN: 978-1-0716-3044-0
eBook Packages: Springer Protocols