1. Metabolomic analysis of the effect of priming agents on plant cells
Msizi Mhlongo, Paul Steenkamp, Lizelle Piater, Edwin Madala and Ian Dubery
Department of Biochemistry
Auckland Park Kingsway Campus
UJ Biochem. Dept. Research day – November 2014

2. Outline: Problem statement
Background information: Plant immunity, Plant priming and Metabolomics
Present study and objectives
Hypothesis
Materials and methods
Results and interpretation
Conclusion
Acknowledgements

3. Problem statement:
Food and nutrition library 2.2
2020 Global population ≤ 8 billion
90% in developing countries (Asia 1.5 billion and Africa 1.2 billion)
“ enough food be produced sustainably to meet the food needs for every person in the world and everyone must have economic and physical access to sufficient food”
intensive agriculture and horticulture for food production
International food policy and institute; Liu et al., 2013

4. Background Information: Plant immunity
Plant defence: Physical barrier and immune response
Immune response is specific, self-tolerance and memorized
Plant immune vs vertebrate immune

5. Background information: Plant Priming

6. Background Information: Metabolomics
Newly emerging field of 'omics' research
Comprehensive and simultaneous systematic determination of metabolite levels in the metabolome and their changes over time as a consequence of stimuli
Metabolome
Refers to the complete set of small-molecule metabolites in a living biological system
Reflects dynamic changes to environmental stimulus

7. Background information: Metabolomics

8. Present Study and Objectives
FLG = Bacterial flagellin
LPS = Bacterial lipopolysaccharides
CHIT = Fungal chitosan
SA = Salicylic acid SA
FLG
LPS
CHIT

9. Hypothesis:
Metabolomics can be used to decipher the underlying secondary metabolic changes due to different chemical and biological priming agents.

10. Materials and methods:
Sample preparation
Plant cells (Tobacco)
Treated with various elicitors / priming agents
Extraction with MeOH
Data acquisition and mining
Chromatographic separation UHPLC-qTOF-MS/MS ( HSS T3 column (1.7 µm, 2.2 mm X 150 mm)
Using chemometric methods i.e. Multivariate Data analysis (SIMCA-P13) : PCA and OPLS-DA and XCMS online
Compound identification
MS-based identification (accurate mass)
From metabolomics databases (e.g. PantCyc, AraCyc, Dictionary of Natural Products, etc.)

11. Materials and methods:
UPLC parameters
Waters Acquity PDA: Sampling rate: 20 points/sec
Range: 200 nm – 500 nm (resolution : 1.2 nm)

12. Materials and methods:
MS parameters

19. Results and interpretation:
Mhlongo et al., 2014, Mhlongo et al., 2014

20. Results and interpretation:

21. Results and interpretation:
PHENYLPROPANOID
DEFENCES
Mhlongo et al., 2014

22. Conclusion
Metabolomic approach was successful to study metabolites associated with primed state
High definition MS in combination with multivariate statistics helped to identify biomarkers associated with priming
Simca and XCMS adds to the data complementarity
First study of CGAs as biomarkers for plant priming.

23. Acknowledgements: UJ and the NRF for financial support
Dr Madala and Mr Tugizimana for sharing their knowledge
Prof Dubery and Dr Piater for their guidance and manuscript preparation
Prof Steenkamp for sample analysis and method development

24. References:
Boubakri H., Poutaraud A., Wahab M. A., Clayeux C., Baltenweck-Guyot R., Steyer D., Marcic C., Mliki A., and Soustre-Gacougnolle I. (2013) Thiamine modulates metabolism of the phenylpropanoid pathway leading to enhanced resistance to Plasmopara viticola in grapevine. BMC Plant Biology, 13, 1-30.
Conrath U. (2011). Molecular aspects of defense priming. Trend in Plant Cell Science, 16,
Conrath U., (2009). Priming of induced plant defense responses. Advances in Botanical Research, 51,
Edreva A., (2004). A novel strategy for plant protection: Induced resistance. Journal of Cell and Molecular Biology 3, 61-69,
Fiehn O., (2002). Metabolomics – the link between genotypes and phenotypes. Plant Molecular Biology, 48,
Goellner K., and Conrath U., (2008). Priming: it’s all the world to induced disease resistance. European Journal of Plant Pathology, 121,
Gust A. A., Brunner F. and Thorsten Nurnberger T. (2010). Biotechnological concepts for improving plant innate immunity. Current Opinion in Biotechnology, 21,
Mhlongo M. I., Piater L. A., Steenkamp P. A., Madala N. E., and Dubery I. A. (2014) Metabolomic fingerprinting of primed tobacco cells provide the first evidence for the biological origin of cis-chlorogenic acid. Biotechnology letter. In press.
Mhlongo M. I., Piater L. A., Steenkamp P. A., Madala N. E., and Dubery I. A. (2014) Priming agents of plant defence stimulate the accumulation of mono- and di-acylated chlorogenic acids in cultured tobacco cells. Physiological and Molecular Plant Pathology. Submitted

25. Thank You