Signaling and analytical strategies

1. Plant lipidomics – signalling
and analytical strategies
Presented by
Divya S
I Ph D Genetics and Plant Breeding

2. Plant lipidomics
• The plant lipidomics is a comprehensive system of all lipids in plants
with respect to cell signalling, membrane architecture,
transcriptional and translational modulation and cell-cell and cell-
protein interactions in response to environmental changes over
time

3. Introduction- Lipids
• Lipids are the fundamental components of biological membranes
and play an important role in biological systems (Wenk 2005 )
• In plants, lipids and lipid-based derivatives fulfil many key functions
like storage of carbon energy, cell compartmentalisation, protection
against pathogens and developmental processes
• Lipids comprise a wide range of functional and regulatory molecules
such as fatty acids, glycerophospholipids, etc., and each cell type
exhibits a different lipid composition and distribution

4. Lipid Signalling in Plants
• Signal transduction is the process in which all cells constantly
receive and act in response to new signals from their environment.
• Many unicellular organisms respond to signalling molecules
secreted by adjacent cells for cell-cell communication
• The signalling molecules may be chemical in nature, for instance,
hormones, pathogen elicitors, mating receptors, ozone and physical
changes (light, temperature and osmotic pressure)
• Cells must continuously monitor their environmental behaviour
and translate this information into an appropriate response via
receptors

5. Contd.,
• The information carried over the plasma membrane into the
cytoplasm will be achieved by initiating specific ion cascade
reactions, receptor kinases or via second messenger-mediated
effector response

6. Receptor-Mediated Signalling
Molecules

7. Regulation of Phospholipid Signalling by G-
Protein-Coupled Receptors
• More than thousands of G-protein-coupled receptors have been
identified in animals and plants induce growth, auxin signalling,
plant defence response
• The breakthrough in G-protein came from mammalian hormones
(such as epinephrine) that regulate the synthesis of cyclic AMP
(cAMP), an important second messenger that mediates cellular
response to a variety of hormones

8. • Later, Martin Rodbell in the 1970s
discovered that the phosphorylation-
dependent GTP is activated followed
by activation of cAMP which is
mediated by adenyl cyclase
• G -protein consists of three subunits
(α, β and γ) commonly called
heterotrimeric G-proteins, and the
activation involves the binding of α-
subunit to guanine nucleotides, which
regulates G-protein activity
• In the quiescent state, α is bound to
GDP (guanine diphosphate) in complex
with β and γ

9. Contd.,
• Both α and βγ subunits can then modulate different target effectors
including phospholipase C, phospholipase D, phospholipase A, PI3K,
adenyl cyclase and ion channels dependent on the specificity of the
subunits
• G-protein activators stimulate phospholipase A 2 (PLA 2 ),
phospholipase C (PLC) and phospholipase D in in vivo studies
(Legendre et al. 1993 ; Munnik et al. 1997 )

10. Phospholipids

11. Phospholipid signalling

12. Phospholipid Response During Cold Stress

13. Lipid Signalling During Plant Stress
• Osmotic stress formed during drought, freezing temperatures, salt-
contaminated soils and water stress hormone; abscisic acid (ABA) seems
to trigger lethal effect on plants
• Both the PLC and the PLD pathways have been extensively implicated in
various plants , addition of salt was found to inhibit PLD activity in tobacco
pollen tubes, due to a subclass of tissue-specific PLD response (Wang et al.
1997; Arisz et al. 2003 )
• Finally, it was revealed that PLDδ is the responsible enzyme involved in
dehydration under drought condition by stimulating PLD activity-mediated
expression of the gene that encodes PLDδ
• Silencing this isoform severely reduced the drought-induced PA response
(Katagiri et al. 2001 )

14. Analytical Strategies in Lipidomics
• ‘Lipidomics Expertise Platform’, an initiative by the European Union in the
year 2005 which offers online resource (http://www.lipidomics.expertise.de)
regarding information relating to institutions involved, lipid databases, lipid
standards and methods
• Advancement in lipidomics from traditional lipid research is focused on two
important points:
(i) to understand the association between lipid metabolic pathways in
biological systems and the metabolic health and
(ii) how the changes in these pathways are related to the disease pathology

15. Techniques used in the identification and
quantification of lipids are broadly classified into
Non-MS-based
techniques
Mass spectrometry
(MS)-based techniques

16. Mass Spectrometry-Based Techniques
• Mass spectrometry is a promising technique in analysing lipid
because of its ability to segregate and characterise charged analytes
in gaseous phase depending on their mass-to-charge ratio (m/z)
• Individual structure can be obtained by fragmenting the lipid ions
using collision-induced dissociation (CID)
Components of MS :

17. Mass analysers
MS- small and volatile lipids were
analysed
Electrospray ionisation (ESI) -
probes produces gas-phase ions
from molecules in a solution and
coupled directly to liquid
chromatography.
Matrix-assisted laser
desorption/ionisation (MALDI) -
intact gas-phase ions are produced
from samples embedded in a dry,
crystalline matrix via laser pulses.
Apart from these mass analysers,
there are also other analysers such
as
 Paul ion trap,
 the linear quadrupole,
 time of flight,
 Fourier transform ion cyclotron
resonance (FT-ICR) and
 orbitrap
These analysers are often coupled
with multistage instruments.
 Commonly coupled instruments
are quadrupole-linear ion trap,
 quadrupole-time-of-flight and
linear ion trap-orbitrap.

18. Principle of mass spectrometry
In mass spectrometry, organic molecules are bombarded with a
beam of energetic electrons (70 eV) in gaseous state under pressure
between 10-7 to 10-5 mm of Hg, using tungsten or rhenium filament
 Molecules are broken up into cations and many other fragments

19. These cations (molecular or parent ion) are formed due to loss of an
electron usually from n or π orbital from a molecule, which can further
break up into smaller ions (fragment ions or daughter ions).
All these ions are accelerated by an electric field, sorted out according to
their mass to charge ratio by deflection in variable magnetic field and
recorded.
The output is known as mass spectrum. Each line upon the mass spectrum
indicates the presence of atoms or molecules of a particular mass.
The most intense peak in the spectrum is taken as the base peak. Its
intensity is taken as 100 and other peaks are compared with it.

20. Ion Trap Mass Spectrometer
• Commonly used and coupled with HPLC through the ESI interface
• Based upon the user-selected time, ion trap MS can capture ions and
trapped ions are subjected to MS
• Use of ion trap MS in lipidomics began by Larsen and co-workers (Larsen et
al. 2001 ) to characterise phospholipids up to MS
• Apart from benefits, few disadvantages are low mass accuracy, limited
resolving powers and low dynamic range
• The development of a ‘linear’ or a ‘two-dimensional ion trap’, linear trap
quadrupole (LTQ) or linear ion trap (LIT) can partly expand dynamic range
and increase resolution

21. Quadrupole mass analyzer
• A typical quadrupole mass analyzer consists of four rods with a hyperbolic cross
section that are accurately positioned parallel in a radial array
• The quadrupole rods are typically constructed using molybdenum alloys because of
their inherent inertness and lack of activity
• Very high degrees of accuracy and precision
• Quadrupole mass spectrometers ~QMSs’ are widely used in both industry and
research for fast accurate analysis of gas and vapors
• The QMS contains basically three elements; i) ion source, ii) mass filter, and iii) ion
detector
• Quadrupole mass analyzer is one type of
mass analyzer used in mass spectrometry

22. Imaging MS by MALDI-TOF
• One of the most promising techniques in the field of lipidomics is MALDI-TOF
which is used mainly for imaging lipids in tissues.
• The sample is coated with a solid matrix (any aromatic compound) with a
specific absorption spectrum
• Within the vacuum chamber, the sample is introduced and a pulsed laser which
emits light at a particular wavelength (depending on the solid matrix) is focused
on the region of interest
• The matrix is then vaporised and the analytes (lipids) are carried

23. Contd.,
• After this process, lipids are charged and these ions are accelerated over
a short distance by means of electric field
• All the ions, based on their mass, receive identical kinetic energy and
travel at different velocities
• These ions are then introduced into a TOF mass spectrometer consisting
of a long-field free flight-tube maintained under sufficiently high vacuum
so that no ion collisions with background gas molecules can occur
• Because no external forces (via electric fields) are applied, the lipid ions
travel through the flight-tube with the mass-dependent velocities that
they acquired during the brief initial acceleration.

24. Contd.,
• Therefore, by measuring the time
required for the lipid ions to
traverse this tube, their m/z
values can be deduced.
• In applications devoted to
mapping the lipid profiles across
a tissue slide, the process is then
repeated by moving the laser
beam across the slide.
Image source : https://www.sigmaaldrich.com/technical-documents/articles/biology/custom-
dna-oligos-qc-analysis-by-mass-spectrometry.html

25. High-Resolution and High Mass Accuracy Mass Spectrometer
• Lipids are identified based upon their mass using Fourier transform mass
spectrometer (FTMS) which is one of the most accurate instruments
available because it has the ability to trap ions in a strong magnetic field
under high vacuum
• For analysing lipid mixture, FTMS is coupled with HPLC-ESI
Direct-Infusion ESI-Based MS Technologies
• ESI-MS helps in the detection of lipid species at femtometre amounts (Gross
and Han 2006 )
• In spite of certain advantages, ESI is not widely used in lipidomics because
during ESI process, phospholipids can either acquire positive or negative
charge which can be studied only using positive or negative ESI-MS/MS

26. Non-MS-Based Techniques
• Nuclear Magnetic Resonance
• High-Throughput Molecular Lipidomics
• Bioinformatics for Lipidomics
• Lipid Analysis Softwares
 Simplified Molecular Line Entry Specification (SMILES)
 LIPID MAPS, Lipid Navigator, TriglyAPCI and Spectrum extraction from
chromatographic data (SECD)
 Lipid Profiler (developed by MDS Sciex) combined with the software called Analyst
help in identifying and quantifying lipids detected by multiple precursor ion scanning
(MPIS).

29. Future prospects
• Lipidomics in association with genomics, proteomics and
metabolomics will contribute in delineating disease mechanism and
provide insights into molecular mechanisms of lipid action.
• Mapping plant lipid content helps in revealing the exact plant
defence mechanisms in response to various stresses
• Further improvement in analytical methods and universal
bioinformatics databases has to be developed to fuel lipid research in
an effective way.

30. References
• Namasivayam, E., Kowsalya, R., Padarthi, P. K., Manigandan, K.,
Jayaraj, R. L., Johnravindar, D., & Jagatheesh, K. (2015). Plant
Lipidomics: Signalling and Analytical Strategies. In PlantOmics: The
Omics of Plant Science (pp. 331-356). Springer, New Delhi
• Li, L., Wang, F., Yan, P., Jing, W., Zhang, C., Kudla, J., & Zhang, W.
(2017). A phosphoinositide‐specific phospholipase C pathway elicits
stress‐induced Ca2+ signals and confers salt tolerance to rice. New
Phytologist, 214(3), 1172-1187
• Munnik, T., & Meijer, H. J. (2001). Osmotic stress activates distinct
lipid and MAPK signalling pathways in plants. FEBS letters, 498(2-3),
172-178

31. THANK YOU