Abstract
M.Sc.
Plants have the ability to continuously respond to various stimuli which alter their
physiology, morphology and development. These stimuli may be abiotic or biotic and
range from essential to toxic in their effects. One of these stimuli is a steroid from
fungal membranes, ergosterol (C28H44O), which does not occur in plants. Ergosterol
acts as a pathogen-associated molecular pattern molecule and triggers defence
mechanisms in plants, characterised by highly regulated and interrelated events that
include the elicitation of the oxidative burst and expression of a number of defencerelated
genes. However, the ergosterol-induced global cellular reprogramming of the
host has not been fully investigated in all aspects. No metabolomic study has
previously been conducted to elucidate, for instance, the effect of ergosterol on plant
metabolism. A clear and broader understanding of the molecular mechanisms involved
in plant : ergosterol interactions is of paramount importance, for it would open up
possibilities of developing novel, more effective and sustainable strategies to control
or eradicate fungal diseases in plants.
In plants, the metabolome is a compilation of all primary and secondary metabolites.
The latter are the final recipients of genetic information, and their levels can influence
gene expression and protein stability. Metabolite patterns reveal the actual cellular
dynamic environment. Hence, qualitative and quantitative measurements of extra- and
intracellular metabolites yield insights into the cellular processes that control the
biochemical phenotype of the cell, tissue or whole organism. Metabolomics, the most
recent of the ‘omics’ approaches, is the holistic analysis of metabolites present within
a biological system under specific physiological conditions.
In the present study a metabolomic approach was used to elucidate and analyse
changes in the metabolism of tobacco (Nicotiana tabacum) cells following ergosterol
treatment. Special attention is given to sesquiterpenoids since the antimicrobial
compounds (phytoalexins) isolated from plants within the Solanaceae are mostly
bicyclic sesquiterpenoids. Suspension of tobacco cells were treated with different
concentrations (0 - 1000 nM) of ergosterol and incubated for different time periods (0
- 24 h). A viability assay, based on the ability of viable cells to reduce 2,3,5-
triphenyltetrazolium chloride (TTC), was used to determine whether cell death
occurred due to ergosterol treatment. No loss of cell viability was observed over the
concentration range and time periods used in this study, indicating that the observed
responses were due to the treatment alone and possible secondary responses due to cell
death could be excluded. Intracellular metabolites were extracted with two methods: a
selective dispersive liquid-liquid micro extraction and a general methanol extraction.
Chromatographic techniques (TLC/HPTLC, GC-FID, GC-MS, GC×GC-TOF-MS,
UPLC-MS) and 1H NMR spectroscopy were used for quantitative and qualitative
analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract
interpretable information from the multidimensional data generated from the
aforementioned techniques.