Agriculture Reference
In-Depth Information
is gaining importance in plant research in both basic and applied contexts. Metabolomic
studies have already shown how detailed information gained from chemical composition can
help us to understand the various physiological and biochemical changes occurring in the
plants and their influence on the phenotype. The analytical measurement of several hundreds
to thousands of metabolites is becoming a standard laboratory technique with the advent of
“hyphenated” analytical platforms of separation methods and various detection systems.
Separation methods include gas chromatography (GC), liquid chromatography (LC), and
capillary electrophoresis (CE). Different types of mass spectrometry (MS), nuclear magnetic
resonance (NMR), and ultraviolet light spectroscopy (UV/VIS) devices are utilized for
detection. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a
specialized technique often used in direct infusion (DI) mode for metabolomics analyses, as
its high mass accuracy allows a separation solely based on this parameter. Each methodology
offers advantages and disadvantages, and the method of choice will depend on the type of
sample and metabolites to be determined, and the combination of analytical platforms [66].
GC and MS were the first pair of techniques to be combined, delivering high robustness
and reproducibility. GC-MS remains one of the most widely used methods for obtaining
metabolomic data because of its ease of use, excellent separation power, and its reprodu‐
cibility. The main drawback of GC-MS is that only thermally stable volatile metabolites,
or non-volatile compounds that can be chemically altered to make them volatile, can be
detected [67, 68]. NMR spectroscopy is a fingerprinting technique that offers several ad‐
vantages over high-throughput metabolite analyses, such as relatively simple sample
preparation and the non-destructive analysis of samples. NMR can detect different classes
of metabolites in a sample, regardless of their size, charge, volatility, or stability with ex‐
cellent resolution and reproducibility [69]. Labeling of metabolites with isotopes and sub‐
sequent NMR analysis is also useful for metabolic flux analysis and fluxomics as it
allows tracking the selective signal enhancement of isotopologues [70]. Recent advances
with high-throughput approaches using ultra-high-field FT-ICR-MS alone or in combina‐
tion with other tools of 'first pass' metabolome analysis as electrospray ionization mass
spectrometry (ESI-MS) are expected to make inventory of the entire metabolome in a sin‐
gle sample possible in the near future [71, 72].
In metabolomics, the implicit objective is to identify and quantify all possible metabolites in a
cellular system under defined states of stress conditions (biotic or abiotic) over a particular
time scale in order to characterize accurately the metabolic profile [73]. But metabolome studies
have some analytical limitations. It is important to have in mind that from the total amount of
metabolites in a sample, only an informative portion can be reliably identified and quantified.
In addition, metabolic networks in multicellular eukaryotes, specifically in plants, are chal‐
lenging because of the large size of the metabolome, extensive secondary metabolism, and the
considerable variation in tissue-specific metabolic activity [74]. Therefore, experimental design
and sample preparation need to be done with great care because environmental and experi‐
mental variation confer noticeable impact on the resulting metabolic profiles. This has been
demonstrated in legumes in which a high proportion of nutritional and metabolic changes
depend on non-controllable environmental variables [75].
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