Multi-Platform Metabolomic Analyses of Ergosterol-Induced Dynamic Changes in Nicotiana tabacum Cells

Abstract

Metabolomics is providing new dimensions into understanding the intracellular adaptive responses in plants to external stimuli. In this study, a multi-technology-metabolomic approach was used to investigate the effect of the fungal sterol, ergosterol, on the metabolome of cultured tobacco cells. Cell suspensions were treated with different concentrations (0–1000 nM) of ergosterol and incubated for different time periods (0–24 h). Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro-extraction and a general methanol extraction. Chromatographic techniques (GC-FID, GC-MS, GC×GC-TOF-MS, UHPLC-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 analytical techniques. The results showed that ergosterol triggered differential changes in the metabolome of the cells, leading to variation in the biosynthesis of secondary metabolites. PCA scores plots revealed dose- and time-dependent metabolic variations, with optimal treatment conditions being found to be 300 nM ergosterol and an 18 h incubation period. The observed ergosterol-induced metabolic changes were correlated with changes in defence-related metabolites. The ‘defensome’ involved increases in terpenoid metabolites with five antimicrobial compounds (the bicyclic sesquiterpenoid phytoalexins: phytuberin, solavetivone, capsidiol, lubimin and rishitin) and other metabolites (abscisic acid and phytosterols) putatively identified. In addition, various phenylpropanoid precursors, cinnamic acid derivatives and - conjugates, coumarins and lignin monomers were annotated. These annotated metabolites revealed a dynamic reprogramming of metabolic networks that are functionally correlated, with a high complexity in their regulation.

Evaluating plant immunity using mass spectrometry-based metabolomics workflows

“…A similar study characterized the effect of ergosterol on N. tabacum c ells using non-targeted GC- and LC-MS (and other non-MS detection platforms), and found major metabolomic variation after 18 h of incubation with 300 nM ergosterol (Tugizimana et al, 2014). The major metabolic shifts occurred in secondary metabolites (terpenoids, coumarins, lignin precursors) and hormonal signaling molecules (jasmonate, salicylate-glucoside).…”

The penetration of green sample-preparation techniques in comprehensive two-dimensional gas chromatography

“…The DLLME procedure is based on the use of small volumes of an extraction and disperser solvent in aqueous samples. Only a single DLLME-GC×GC experiment has been reported: Tugizimana et al used DLLME-GC×GC-LR-ToF-MS for monitoring ergosterol-induced metabolite changes in Nicotiana tabacum cells [48]. Specifically, metabolic changes were correlated with modifications in defense-related metabolites.…”

Fusarium Mycotoxins and Their Role in Plant–Pathogen Interactions

“…There is limited evidence that secondary metabolites or small molecules from primary metabolism of microbes are also recognized. For ergosterol, a fungi-specific membrane sterol, it has been shown that nanomolar concentrations can elicit defense responses in tomato [22] and tobacco cells [23]. It is unclear whether fungal secondary metabolites also play a role as PAMPs and are recognized by cereal hosts.…”