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NMR-based metabolomic analysis of plants

Abstract

Nuclear magnetic resonance (NMR)-based metabolomics has many applications in plant science. Metabolomics can be used in functional genomics and to differentiate plants from different origin, or after different treatments. In this protocol, the following steps of plant metabolomics using NMR spectroscopy are described: sample preparation (freeze drying followed by extraction by ultrasonication with 1:1 CD3OD:KH2PO4 buffer in D2O), NMR analysis (standard 1H, J-resolved, 1H–1H correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC)) and chemometric methods. The main advantage of NMR metabolomic analysis is the possibility of identifying metabolites by comparing NMR data with references or by structure elucidation using two-dimensional NMR. This protocol is particularly suited for the analysis of secondary metabolites such as phenolic compounds (usually abundant in plants), and for primary metabolites (e.g., sugars and amino acids). This procedure is rapid; it takes not more than 30 min for sample preparation (multiple parallel) and a further 10 min for NMR spectrum acquisition.

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Figure 1: Representative 1H-nuclear magnetic resonance (NMR) spectra of several plant extracts.
Figure 2: 1H-nuclear magnetic resonance (NMR) spectra of two different accessions of Arabidopsis thaliana.
Figure 3: J-resolved nuclear magnetic resonance (NMR) spectra of methyl jasmonate (MJ)-treated Brassica rapa leaves.
Figure 4: Heteronuclear multiple bond correlation (HMBC) spectrum of aromatic moiety of phenylpropanoids of Brassica rapa leaves in the range of δ 6.60–δ 7.30 of 1H and δ 100–δ 150 of 13C.
Figure 5: Heteronuclear single quantum coherence spectroscopy (HSQC) spectra of Nicotiana tabacum leaves.
Figure 6: Experimental procedures for sample preparation.
Figure 7: Metabolites changes of Brassica rapa leaves after methyl jasmonate (MJ).
Figure 8: 1H-nuclear magnetic resonance (NMR) spectra of methyl jasmonate (MJ)-treated Brassica rapa leaves (a,b).
Figure 9: Metabolites changes of Nicotiana tabacum leaves after tobacco mosaic virus (TMV) infection.
Figure 10: Proposed metabolomic alterations in the Nicotiana tabacum leaves infected by tobacco mosaic virus.

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Acknowledgements

We thank Ms. E.G. Wilson for reviewing the manuscript and providing helpful comments. We also thank Dr. A. Meissner, Mr. C. Erkelens and Mr. A.W.M. Lefeber for their kind help in setting up NMR parameters. This research has received funding from the European Community′s Seventh Framework Programme [FP7/2007-2013] under Grant Agreement No 217895.

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All authors discussed all the steps of the protocol, its implications and applications. H.K.K. wrote the manuscript, and Y.H.C. and R.V. revised it.

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Correspondence to Robert Verpoorte.

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Kim, H., Choi, Y. & Verpoorte, R. NMR-based metabolomic analysis of plants. Nat Protoc 5, 536–549 (2010). https://doi.org/10.1038/nprot.2009.237

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