Plant Metabolomics

1. PLANT METABOLOMICS
By,
Dr. Rudresh Gowda B
B.V.Sc and A.H

2. What is Plant Metabolomics..?
• Quantitative and Qualitative analysis, elucidation of all metabolites in Plants under
specific conditions
• Metabolome-Entire complement of small molecules in plant, Ultimate phenotype of cells,
Information of gene expression and modulation of protein function, environmental cues
• Metabolome-Stephen Oliver

3. IMPORTANCE
• Ability to detect vast array of metabolites from single extract, allowing speedy and
precise analysis of metabolites
• Comprehensive view of plant metabolites like small organic compounds, which
participate in different cellular events, representing absolute physiological state of cell
• Valuable tool for advancing our understanding of primary and secondary metabolism
in plants and is revolutionizing field of plant biology

4. LIMITATIONS…
• Enormous complexity and diversity of plant metabolomes and incomplete knowledge of plant
metabolic pathways
• Require wide spectrum of chemistries and instrumentation with wide dynamic range
• Impossible to extract and analyze all metabolites in a plant cell in single analysis metabolome
• Identification and structural elucidation of molecules from analytical detector signals
• Lack of universal metabolite-specific libraries and known reference compounds- major limitation
to definitive identification of metabolites

5. Steps In Plant Metabolomics

6. SAMPLING: FROM WHOLE PLANT TO SINGLE CELL
• Harvesting and quenching
• Plant material harvest- as fast as possible by immediate freezing plant tissue in liquid
nitrogen (i.e. shock freezing) and storing at −80°C
• Quick inactivation of enzymatic reactions and metabolic processes- avoid fluctuations in
the levels of fast turnover metabolites (e.g. glycolytic intermediates)
• Finely homogenized fresh-frozen plant tissues, a process often performed using a pre-
cooled pestle and mortar filled with liquid nitrogen
• Aliquots of fine powdered plant tissue must be rapidly weighed in pre-cooled
polypropylene microfuge tubes to ensure that plant material does not thaw
(Jorge. T et al, 2016)

7. Extraction Of Metabolites
• Chemical stability of metabolites is not affected
• Analytical recoveries of metabolites throughout extraction are complete
• Variability in metabolite concentrations during the extraction is minimized
• Amount of tissue -differ between extraction methods
• Polar organic solvents -both hydrophilic and hydrophobic compounds
• Solvent systems- Combination of polar and Non-polar organic solvents- used to separate
polar phase (hydrophilic metabolites) from a non-polar phase (hydrophobic metabolites-
lipids)
(Jorge. T et al, 2016)

8. Pre-analytical Requirements
• Concentrate metabolite(s) of interest
• Prevent sample carryover in chromatographic systems
• Eliminate interfering components from the plant matrix
• LC and CE allow direct analysis of non-volatile metabolites
• In LC-MS- Plant extract is usually re-dissolved in a solvent with same composition of
initial conditions of LC mobile phase
• CE-MS- Includes very low organic solvent consumption without need for extensive
sample pretreatment
(Jorge. T et al, 2016)

9. ANALYTICAL TECHNOLOGIES
• Separation methods
• Gas chromatography- For analyzing compounds that can be vaporized without
decomposition
• High performance liquid chromatography – Form of column chromatography that
pumps a sample of mixture or analyte in a solvent(mobile phase) at high pressure through
a column with chromatographic packing material(stationary phase)
• Much wider range of analytes can potentially be measured
• Capillary electrophoresis- separates ions based on their electrophoretic mobility with
the use of an applied voltage

10. Detection Methods
• Nuclear Magnetic Resonance Spectroscopy- Only detection technique- does not rely
on separation of analytes, and sample can be recovered for further analysis
• All kinds of small molecule metabolites can be measured simultaneously
• Mass spectrometry- Ionizes chemical species and sorts ions based on their mass to charge
ratio

11. NMR
(Creative Commons)

12. (Wikipedia Commons)

13. • NMR (nuclear magnetic resonance spectroscopy)
• Mass spectrometry (MS)
GC–MS (gas chromatography–MS)
LC–MS (liquid chromatography–MS)
FT- ICR- MS (Fourier transform ion cyclotron resonance–MS)
• High Performance Liquid Chromatography (HPLC)
• Direct Analysis Real Time- Mass Spectrometry (DART/MS)
PLANT METABOLOMICS TECHNIQUES

14. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

16. • Extract – in Room temp. at least 30 min before NMR measurement to avoid bad shimming
• Load NMR tube into the spectrometer, Set sample temp. to 298 K and leave for thermal
equilibration
• H-NMR - metabolomic data of a sample within a relatively short time (5–10 min for 64–
128 scans)
• Tune and match NMR tube and Lock spectrometer frequency to the deuterium resonance
arising from NMR solvents
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

17. • Shim the sample by manual or automated method
• Standard 1H NMR spectroscopy is done
• Convert NMR spectra to a suitable form for further multivariate analysis and
carry out PCA
• Identify as many of metabolites as possible, by comparison with NMR signals
to reference compounds or by 2D NMR spectra

18. Catharanthus roseus leaves
Brassica rapa leaves
C: catharanthine
V: vindoline
I: indole-3- acetic acid
N: neoglucobrassicin
S: trans-sinapoylmalate

19. • Unbiased, rapid, non-destructive technique- requires little sample preparation and lessens
chance of sample loss
• In this, No analyte separation process involved- provide selectivity without separation,
and does not require sample derivatisation prior to analysis
• NMR spectrum of a multicomponent extract is result of superposition of collective
spectra of all NMR-visible individual compounds present in sample under study
• NMR analysis- global view of all metabolites (primary and secondary) in a sample
Advantages

20. ELECTRO-SPRAY IONIZATION
• ESI- soft ionization technique extensively used for production
• of gas phase ions (without fragmentation) of thermally labile large supramolecules

21. GAS CHROMATOGRAPHY- MASS SPECTROMETRY
• Separation of analytes -depends on analyte interactions with stationary face and boiling
point
• Only Volatile compounds- separated on GC column, non-volatile metabolites must be
derivatize polar compounds containing functional groups such as –OH, –SH or –NH
• Portion of sample is introduced into inlet of GC instrument
• Inlet temperature is higher than 250°C, at which many metabolites are evaporated

22. • Whole sample is introduced onto high resolution capillary column(10- 60 m)
• Metabolites eluting from GC- ionized by Electron-impact (EI), Here vaporized
metabolites impacted by a beam of electrons with sufficient energy to fragment
and ionize molecule
• EI results in molecular ion fragmentation, which is of great importance for
structural interpretation of the metabolites
• To identify compounds, databases of ion fragmentation patterns of molecules, as
NIST are used

23. Advantages
• Capable of analyzing volatile compounds and requires extensive chemical derivatization
procedures to increase volatility and thermo-stability
• To profile a wide range of metabolites with different physico-chemical properties in a
single plant extract, while providing structural information of detected metabolites
• GC-MS- highly reproducible technique than LC-MS, due to electron ionization (EI)
method, in which gas-phase molecules interact with kinetically activated electrons at
accepted average standard energy of 70 eV

25. Allwood. W et al, 2011

26. LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY
• LC-MS- To profile a small set of known metabolites or compound classes of the plant
metabolome, an approach referred to as target metabolite analysis
• Ionization method- Electrospray ionization (ESI)- introduces little internal energy and
gives little information on structure because few fragments are generated
• Provides additional structural information- aid in identification of new or unusual
metabolites or in characterization of known metabolites in cases where ambiguity exists

28. FOURIER-TRANSFORM ION CYCLOTRON-RESONANCE
MASS SPECTROMETRY
• For determining mass to charge ratio based on cyclotron frequency of ions in a fixed
magnetic field, provides mass accuracy
• Water(W) and Chloroform(C) fractions- reconstituted in methanol/ water (1:1)
• Methanol(M) and Acetonitrile(A) fractions suspended in respective pure solvent. For
metabolites analysis, all fractions diluted 1000-fold in appropriate solvent
• M and A fractions diluted in same solvent for positive and negative-ion mode analysis
• W and C fractions diluted in methanol in methanol/water (1:1) for ESI
(Maia. M et al, 2016 )

29.  Standard leucine encephalin added to all samples at concentration of 0.5 mg/ml, and was
used as standard for control and quality assessment of particular analytical precision
Extracted metabolites were analyzed by direct infusion in FTICR-MS with preset flow
rate

30. (Maia. M et al, 2016)

31. DIRECT ANALYSIS IN REAL TIME- MASS SPECTROMETRY
• It enables rapid analyzing of solids, liquids, and gases at atmospheric pressure without sample
preparation
• Helium is conducted via an axial tube and supports a corona discharge- engenders ions, electrons,
and excited atoms
• Helium passes through other two chambers, where electrons are removed, passing into atmospheric
reaction zone- include only electronically excited substances
• Released atoms in tube cause environmental gas to undergo gas-phase reaction ionization cascade
•

32. • These ions- chemical ionizing reagents near surface of analyzed sample, resulting
in analyte ions finally getting transferred to mass analyzer
• Penning ionization is most important step in DART-MS
• Flow rate of carrier gas and temperature are two major factors that affect DART
ionization performance
• Prominent features of DART- high throughput, minor cross-contamination, and
simplicity
• Typically used to analyze small molecular compounds with m/z of 50–1200

35. • Advantages
• Samples can be analyzed directly without the extraction process
• Low sample consumption
• Sample analysis cycle was sharply shortened
• Disadvantages
• Polar compounds are difficult to ionize
• Ion suppression

36. REFERENCES
• J. WILLIAM ALLWOOD, RIC C. H. DE VOS, ANNICK MOING, CATHERINE DEBORDE,
ALEXANDER ERBAN, JOACHIM KOPKA, ROYSTON GOODACRE, AND ROBERT D.
HALL, 2011, Plant Metabolomics and Its Potential for Systems Biology Research:
Background Concepts, Technology, and Methodology, Methods in Enzymology, 500: 299-
336.
• Gullberg, J. 2005, Metabolomics: A Tool for Studying Plant Biology, Swedish University of
Agricultural Sciences, 1-61.
• TIAGO F. JORGE, ANA T. MATA, AND CARLA ANTÓNIO, 2016, Mass spectrometry as a
quantitative tool in plant metabolomics, Philos. Trans. Math. Phys. Eng. Sci, 374(2079): 1-34.
• ROBERT VERPOORTE, 2010, NMR-based metabolomic analysis of plants, Nat. Protocol,
237, 1-34.
• JUN-LING REN, AI-HUA ZHANG, LING KONG AND XI-JUN WANG, 2018, Advances in mass
spectrometry-based metabolomics for investigation of metabolites, 40, 1-31.

37. THANK YOU..