Unravelling the metabolomes of plants and their mutants
Application of proton NMR to “crude” plant extracts provides us with a fingerprint of a large range of extractable metabolites. The ability to detect metabolites without complex sample preparations makes the method suitable for high-throughput analysis and screening for mutants with specific metabolic profiles. Conventional NMR has its advantages in the overall qualitative characterization of plant metabolites. Hetero-nuclear NMR methods have developed rapidly and differences in the molecular and composition between wildtypes and mutants can now be easily studied (Fukushima et al, 2002) and the same probably applies to the metabolic composition (Kikuchi et al, 2004).
Stable Isotope Solution: Nuclear Magnetic Resonance (NMR)
A basic concept of NMR-based plant metabolomics has been proposed by Kikuchi et al. (2004). These authors produced uniformly labeled Arabidopsis plants in tissue culture by feeding 13C-labelled glucose and 15N-labelled nitrate as the only C- and N- source, respectively. Stable isotopes like 13C and 15N enable scientists in metabolomics to identify the metabolites of complete metabolomes by combining uniformly labelled metabolites with nuclear magnetic resonance spectroscopy (NMR). For example, in this way the differences in the metabolomes of the wildtype and mutants of Arabidopsis have been studied (Kikuchi et al, 2004). By labelling plants with the stable isotopes 13C and/or 15N during a complete life cycle from seed to maturity, all organs and plant components will contain the isotopes in virtually the same ratio as present in the substrate. When the substrates contain 99% 13C and/or 99% 15N, all plant components will be uniformly labelled at nearly the same 99% enrichment. Subsequently, the components of interest (i.c. metabolites) can be extracted and mixed with samples for analysis by NMR.
Figure 1 illustrates how plants were uniformly labelled with the stable isotopes 13C and 15N (1) and subsequently analysed with hetero-nuclear 2D NMR (2). The stable isotope labeled plants showed a high-level and a high-sensitive separation of metabolite signals (3). Subtraction of spectra (4) of different mutants yielded a quantitative figure of increased and decreased metabolites (5) in the samples (Kikuchi et al, 2004).
Figure 1. Principle of metabolite analysis with NMR.
The authors concluded that NMR will become a key technology in plant metabolomics with the use of stable isotope labeling, “the use of isotope labeling together with newly developed NMR technologies opens a new avenue for plant metabolomics” (Kikuchi et al, 2004).
Fukushima K, J Kikuchi, S Koshiba, Y Kuroda, S Yokoyama. 2002.
Solution structure of the C-terminal domain of DEF45/ICAD. A structural basis for the regulation of apoptotic DNA fragmentation.
Journal of Moleculair Biology 321: 317–326.
Kikuchi J, K Shinozaki, T Hirayama. 2004.
Stable Isotope Labeling of Arabidopsis thaliana for an NMR-Based Metabolomics Approach.
Plant Cell Physiology 45: 1099–1104.