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

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

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

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

Background information: Plant Priming

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

Background information: Metabolomics

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

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

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.)

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

Materials and methods: MS parameters

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

Results and interpretation:

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

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.

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

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, 524-531. Conrath U., (2009). Priming of induced plant defense responses. Advances in Botanical Research, 51, 362-395. 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, 155-171. Goellner K., and Conrath U., (2008). Priming: it’s all the world to induced disease resistance. European Journal of Plant Pathology, 121, 233-242. Gust A. A., Brunner F. and Thorsten Nurnberger T. (2010). Biotechnological concepts for improving plant innate immunity. Current Opinion in Biotechnology, 21, 204-210. 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

Thank You