Plant Micrometabolomics: The Analysis of Endogenous Metabolites Present in a Plant Cell or Tissue Sofia Moco,*,† Bernd Schneider,‡ and Jacques Vervoort† Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, and Max-Planck-Institute for Chemical Ecology, Beutenberg Campus, Hans-Kno¨ll-Str. 8, D-07745 Jena, Germany Received November 9, 2008
Identification and quantification of metabolites occurring within specific cell types or single cells of plants and other organisms is of particular interest for natural product chemistry, chemical ecology, and biochemistry in general. The integration of studies at the gene, transcript, protein and metabolite levels in localized regions will provide useful information for the understanding of biology as a system. In this review, we summarize the latest developments in the analysis of metabolites present in small samples, micrometabolomics, dealing with sample preparation methods, with focus on laser-assisted microdissection, and the analytical technologies used. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) are among the most emergent technologies in metabolomics, enabling the shortest route toward metabolite identification. Keywords: mass spectrometry • nuclear magnetic resonance • laser-assisted microdissection • liquid chromatography • hyphenated techniques • micrometabolomics • metabolomics • plant • tissue • single-cell
Introduction The past decade has witnessed a large increase in metabolomics studies, which usually link metabolite profiles or metabolite changes to the holistic phenotype of the organism of interest. Most studies have been conducted at the whole organism level. A detailed understanding of metabolic changes requires the coupling of these processes to tissues or even cells, as the changes in metabolite composition occur at the cellular level. With the recent technological improvements, a new era begins in which analyses on a minute quantity of biological material are feasible.1-32 In plant biology, this opens doors into the characterization of specific organs, tissues, or even single cells, providing detailed information about the localization and identification of endogenous entities such as RNA, proteins, and metabolites. Plants are considered to have about 40 different cell types;33 therefore, the chemical composition of these cell types is destined to be different. For this reason, it is important that the sampling and the analytical data generated is more refined than at the whole organism level, as the information content of the system will increase in resolution and consequently is in a closer proximity to the truth.19,20 This more detailed approach allows not only the detection of potential key metabolites, which by a bulk analysis would not have enough concentration to be detected, but also allows a better understanding of fundamental biotic (e.g., cell differentiation, plant development, signal transduction) and abiotic (e.g., climate * To whom correspondence should be addressed. Phone, +31 317 482620; fax, +31 317 484801; e-mail,
[email protected]. † Wageningen University. ‡ Max-Planck-Institute for Chemical Ecology.
1694 Journal of Proteome Research 2009, 8, 1694–1703 Published on Web 01/29/2009
changes, pathogen attack, mechanical rupture, etc.) phenomena20 that take place in the plant. The simultaneous measurement of a large number of compounds, as performed in metabolomics studies, provides information about chemically related metabolites, some possibly participating in common or related pathways, leading to the attribution of new functions for known metabolites or even the discovery of novel metabolites.19,34-37 So far, metabolomics analyses have been performed mainly on whole plants, organs such as fruits,38-42 leaves,43-46 tubers,47,48 flowers,49,50 and roots,51,52 but few on specific tissues53,54 or even on specific cells.7,8,29 The challenge of performing metabolomics, as well as transcriptomics or proteomics, on specific tissues or even specific cells (micrometabolomics, microgenomics,2,55 and microproteomics4,5) highly relies on the efficacy of the sample preparation. Such methods should ensure the intactness of the tissue be reproducible and robust. Another (not less) important item is the analytical techniques used to perform micrometabolomics, as this should meet a level of performance (in particular the sensitivity, reproducibility, and discrimination of impurities) which can cope with the low amounts of sample. In metabolomics, MS-based36,38,41,42,46,54,56 and NMRbased7,8,36,41,46,51,57,58 analyses are usually performed. The data obtained by NMR and MS is often complementary and, preferably, both techniques should be used when possible. So far, most techniques applied in the chemical analysis of specific tissues or single cells have been focusing on protein and transcript (mRNA) analysis. The application of metabolomics in the analysis of tissues or cells is a challenging task as metabolites (typically
