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 CD(3)OD:KH(2)PO(4) buffer in D(2)O), NMR analysis (standard (1)H, J-resolved, (1)H-(1)H 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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Sample preparation in plant metabolomics is a fundamental and critical step with important consequences for the accuracy of results. Depending on the analytical tools and the metabolites of interest, sample preparation has to be decided. However, the various methods reported in the literature have many steps in common and consequently the practical considerations concerning the pros and cons are similar. In this review, each step of the sample preparation - harvesting, drying, extraction and purification - will be discussed in detail.

A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected by 10 types of phytoplasmas was carried out using one-dimensional and two-dimensional NMR spectroscopy followed by principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it. With a combination of these techniques, we were able to identify those metabolites that were present in different levels in phytoplasma-infected C. roseus leaves than in healthy ones. The infection by phytoplasma in C. roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the 1 H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Based on the NMR and PCA analysis, it might be suggested that the biosynthetic pathway of terpenoid indole alkaloids, together with that of phenylpropanoids, is stimulated by the infection of phytoplasma.

Root exudates influence the surrounding soil microbial community, and recent evidence demonstrates the involvement of ATPbinding cassette (ABC) transporters in root secretion of phytochemicals. In this study, we examined effects of seven Arabidopsis (Arabidopsis thaliana) ABC transporter mutants on the microbial community in native soils. After two generations, only the Arabidopsis abcg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared with the wild type using automated ribosomal intergenic spacer analysis. Similarly, root exudate profiles differed between the mutants; however, the largest variance from the wild type (Columbia-0) was observed in abcg30, which showed increased phenolics and decreased sugars. In support of this biochemical observation, whole-genome expression analyses of abcg30 roots revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated, while some sugar transporters were down-regulated compared with genome expression in wild-type roots. Microbial taxa associated with Columbia-0 and abcg30 cultured soils determined by pyrosequencing revealed that exudates from abcg30 cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria (i.e. plant-growth-promoting rhizobacteria and nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, we report how a single gene mutation from a functional plant mutant influences the surrounding community of soil organisms, showing that genes are not only important for intrinsic plant physiology but also for the interactions with the surrounding community of organisms as well.

Tobacco mosaic virus (TMV) infection of tobacco is a well-known and extensively studied model system for which a number of genes and proteins involved in the systemic acquired resistance (SAR) have been characterized. Little is known about the metabolic changes connected with the infection and SAR. Here we describe the use of NMR spectroscopy in combination with multivariate data analysis to study the metabolic changes. Particularly 2-D NMR methods, such as 2-D J-resolved spectra and their projected spectra, are shown to be powerful tools in the metabolomic studies. The macroscopic view of the metabolomes obtained by NMR spectroscopy of crude extracts enabled the identification of a series of totally different metabolites that seem connected with resistance, such as the clearly increased 5-caffeoylquinic acid, alpha-linolenic acid analogues, and sesqui- and diterpenoids in the infected plant parts.

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The metabolomic analysis of 12 Cannabis sativa cultivars was carried out by 1H NMR spectroscopy and multivariate analysis techniques. Principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by principal component 1 (PC1) and principal component 3 (PC3) in cannabinoid fraction. The loading plot of PC value obtained from all 1)H NMR signals shows that Delta9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) are important metabolites to differentiate the cultivars from each other. The discrimination of the cultivars could also be obtained from a water extract containing carbohydrates and amino acids. The level of sucrose, glucose, asparagine, and glutamic acid are found to be major discriminating metabolites of these cultivars. This method allows an efficient differentiation between cannabis cultivars without any prepurification steps.