Plant metabolome analysis

The study of the composition of a mixture is an extremely common problem in analytical and bioanalytical chemistry. While chromatography and solvent extraction are commonly employed to simplify the analysis prior to characterization of the constituents, NMR has provided a series of tools that help in unravelling the components of complex samples, when a previous separation of the pure compounds is not feasible or complete. Thus, TOCSY, NMR diffusometry (DOSY, among all) and heteronuclear correlation experiments are widely used to this purpose, for example, for the characterization of small molecules in biologically relevant samples, such as in metabolomics,1 plant extracts analysis,2 food quality control,3 4 to name a few cases. 

The increasing accessibility of bench-top LC-MS systems to researchers of all disciplines, combined with the tandem and high-resolution mass spectrometry capabilities of such instruments, will only increase the number of applications to which LC-MS can be directed. The examples documented in this chapter illustrate some of the diversity and power of the techniques, including analytical applications for known analytes in various matrices, metabolomic analysis, the tentative structural identification of novel compounds, and the screening of extracts for minor, and perhaps novel, components of the alkaloidal profile of plants. 

It was then necessary to validate that NP-1 targeted the same enzyme in higher plants. A metabolomic analysis showed that metabolites in the target pathway that... 

Until recently, this approach has focused mainly on the stndy of human diseases, pharmaceuticals, plant metabolism analysis, and toxicology. However, nowadays it is being extended to food science for food component analysis, food quality and authenticity, and food consumption monitoring in order to improve consumer health and confidence. Because metabolomics allows the simultaneous characterization of large numbers of molecules, this approach has been recently introduced by food and nutrition scientists to obtain a comprehensive fingerprint of food and nutrition. In this context, Cifuentes [2] introduced the term foodomics as a discipline that stndies food and nutrition domains through the application of advanced omics technologies. 

Probe electrospray ionization (PESI) and TOF-MS were used for direct profiling of phytochemicals in different parts of a fresh tulip bulb [80], which emphasized the possibility of conducting in-vivo MS analysis of less sensitive biological matrices such as plant tissues. Recently, Pan et al. [81] demonstrated single-probe MS which can conduct metabolomic analysis of individual living cells in real time. The diameter of this probe is < 10 pm which makes the device compatible with eukaryotic cells. Atmospheric pressure ion sources are particularly suitable for analysis of live biological specimens. Cellular metabolism does not need to be quenched before analysis. For instance, in laser ablation electrospray ionization (LAESI)-MS, cells are irradiated by a laser beam in order to extract small amounts of cytosolic components, and to transfer them to the ESI plume [82]. 

Fiehn O, Wohlgemuth G, Scholz M, et al. (2008) (Quality control for plant metabolomics reporting MSl-compliant studies Plant J 53 691-704 Frenzel T, Miller A, Engel K H (2002) Metabolite profiling - A fractionation method for analysis of major and minor compounds in rice grains. Cereal Chem 79 215-221... 

George C, Ridley WP, Obert JC, et al. (2004) Composition of grain and forage from com root-worm-protected com event MON 863 is equivalent to that of conventional com (Zea mays L.). J Agric Food Chem 52 4149-4158 Grata E, Boccard J, Guillarme D, et al. (2008) UPLC-TOF-MS for plant metabolomics A sequential approach for wound marker analysis in Arabidopsis thaliana. J Chrom B (2008) 871 261-270... 

L. W. Summer, E. Urbanczyk Wochniak, and C. D. Broekling, Metabolomics data analysis, visualization, and integration, in Plant Bioinformatics Methods and Protocols, D. Edwards, ed., Humana Press, Towota, NJ, 2007, pp. 409 436. 

Toghe T, Nishiyama Y, Hirai MY, Yano M, Nakajima J, Awazuhara M, Inoue E, Takahashi H, Goodenowe DB, Kitayama M, Noji M, Yamazaki M, Saito K. 2005. Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant J42 218-235. 

Fourier transform ion-cyclotron (FT-ICR-MS) provides the highest mass resolution and accuracy, and enables the determination of the elemental compositions of metabolites, which facilitates annotation procedures for unknown compounds (95). Direct infusion analysis of plant extract without a previous separation and/or derivatization can be achieved however, its use is very restricted due to the equipment cost, the difficulties in hardware handling, and the extremely large amount of data generated. Takahashi et al. applied this technique to elucidate the effects of the overexpression of the YK1 gene in stress-tolerant GM rice (96). More than 850 metabolites could be determined, and the metabolomics fingerprint in callus, leaf, and panicle was significantly different from one another. 

In what many consider to be a landmark publication on metabolomics, Fiehn et al. (2000) state it is crucial to perform unbiased (metabolite) analyses in order to define precisely the biochemical function of plant metabolism. The authors argue that for metabolomics/metabolite profiling to become a robust and sensitive method suited to automation, a mature technology such as gas chromatography-mass spectrometry (GC-MS) is required as an analytical technique. The authors go on to describe a simple sample preparation and analysis regime that allowed for the detection and quantification of more than 300 compounds from a single-leaf sample extract. 

The combined genomics and chemical approaches to plant terpenoid research are not restricted to the few plant species for which more or less complete genome sequences are now available. The discovery of many of the genes and enzymes for the formation of terpenoids such as menthol and related monoter-penes in peppermint Mentha x piperita) (15), artemisinin in Artemisia annua (16), Taxol in the yew tree (Taxus) (17), or conifer diterpene resin acids in species of spmce (Picea ) and pine (Pinus) (18) have been possible on the foundation of highly specialized efforts of EST and full-length cDNA sequencing combined with characterization of recombinant enzymes and analysis of the terpenoid metabolome of the target plant species. 

NMR metabolomics has also been applied to investigate potential MOA. Plant researchers from BASF generated more than 400 1H NMR spectra from plants treated with different herbicides. Statistical analysis of the NMR spectra accurately clustered the extracts based on the metabolic action of the herbicides.41 NMR metabolomics may therefore offer the natural product researcher new ways to investigate MOA for compounds where activity is demonstrated but MOA is undetermined. 

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