Fidele Tugizimana

Multi-platform metabolomic analyses of ergosterol-induced dynamic changes in Nicotiana tabacum cells.

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.

Metabolomic Analysis of Methyl Jasmonate-Induced Triterpenoid Production in the Medicinal Herb Centella asiatica (L.) Urban

Centella asiatica is an important source of biologically active pentacyclic triterpenoids. The enhancement of the biosynthesis of the centellosides by manipulation of associated metabolic pathways is receiving much attention. Jasmonates play critical roles in plant metabolism by up-regulating the expression of genes related to secondary metabolites. Here, we investigated the effect of methyl jasmonate (MeJa) in C. asiatica through targeted metabolomic profiling of asiaticoside and madecassoside as well as their aglycones, asiatic acid and madecassic acid. Cell suspensions were treated with 0.2 mM MeJa for 2, 4 and 6 days. Liquid chromatography coupled to mass spectrometry (LC-MS) was used to explore induced changes in metabolite profiles, both qualitatively and quantitatively. Principal component analysis (PCA)-derived scores plots revealed clusters of sample replicates for control and treated samples at 2, 4 and 6 days while loading plots aided in identifying signatory biomarkers (asiatic acid and madecassic acid, as well as asiaticoside and madecassoside) that clearly demonstrate the variability between samples. In addition to increased biosynthesis of the targeted centelloids, other differential changes in the intracellular metabolite profiles reflected the response of the C. asiatica cells to the MeJa-treatment as a reprogramming of the metabolome.

Ergosterol-Induced Sesquiterpenoid Synthesis in Tobacco Cells

Plants have the ability to continuously respond to microbial signals in their environment. One of these stimuli is a steroid from fungal membranes, ergosterol, which does not occur in plants, but acts as a pathogen-associated molecular pattern molecule to trigger defence mechanisms. Here we investigated the effect of ergosterol on the secondary metabolites in tobacco (Nicotiana tabacum) cells by profiling the induced sesquiterpenoids. Suspensions of tobacco cells were treated with different concentrations (0–1,000 nM) of ergosterol and incubated for different time periods (0–24 h). Metabolites were extracted with a selective dispersive liquid-liquid micro-extraction method. Thin layer chromatography was used as a screening method for identification of sesquiterpenoids in tobacco extracts. Liquid chromatography coupled to mass spectrometry was used for quantitative and qualitative analyses. The results showed that ergosterol triggered differential changes in the metabolome of tobacco cells, leading to variation in the biosynthesis of secondary metabolites. Metabolomic analysis through principal component analysis-scores plots revealed clusters of sample replicates for ergosterol treatments of 0, 50, 150, 300 and 1,000 nM and time-dependent variation at 0, 6, 12, 18 and 24 h. Five bicyclic sesquiterpenoid phytoalexins, capsidiol, lubimin, rishitin, solavetivone and phytuberin, were identified as being ergosterol-induced, contributing to the altered metabolome.

Metabolomics-derived insights into the manipulation of terpenoid synthesis in Centella asiatica cells by methyl jasmonate

Centella asiatica is an important medicinal plant with a wide range of bioactivities associated with its secondary metabolites. Using two extraction procedures, metabolomic approaches were used to investigate changes in the metabolome of C. asiatica cells treated with exogenous MeJA. GC–MS and LC–MS platforms were employed for semi-targeted and untargeted analyses, respectively. Multivariate data analyses indicated concentration-dependent changes in the metabolite profiles, indicative of the cellular response to MeJA. Annotation of biomarkers correlated with the treatment indicate differential responses in flavonoid-, phenylpropanoid (cinnamates)- and terpenoid pathways and changes in fatty acid profiles. MeJA treatment triggered the accumulation of bicyclic sesquiterpenoids (aristolochene, deoxy-capsidiol, 15-hydroxysolavetivone, solavetivone, 3-hydroxylubimin) and a tricyclic sesquiterpenoid (phytuberin), indicating the stimulatory effect of MeJA on this branch of the terpenoid pathways. In contrast, flavonoids were mostly negatively correlated with the treatment. The presence of the sesquiterpenoids in MeJA-elicited cells and other tentatively identified metabolites (abscisic acid, fatty acids, phytosterols and metabolites of shikimate–phenylpropanoid pathways) indicates that the changes in the metabolome are associated with a defensive function in response to elicitation by MeJA, rather than just the amplification of existing terpene pathways. These results provide a detailed and comprehensive picture of metabolic changes occurring in C. asiatica cells in response to MeJA elicitation and contribute to the understanding of flexible and controllable aspects of metabolic manipulation.

A Conversation on Data Mining Strategies in LC-MS Untargeted Metabolomics: Pre-Processing and Pre-Treatment Steps

Untargeted metabolomic studies generate information-rich, high-dimensional, and complex datasets that remain challenging to handle and fully exploit. Despite the remarkable progress in the development of tools and algorithms, the “exhaustive” extraction of information from these metabolomic datasets is still a non-trivial undertaking. A conversation on data mining strategies for a maximal information extraction from metabolomic data is needed. Using a liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomic dataset, this study explored the influence of collection parameters in the data pre-processing step, scaling and data transformation on the statistical models generated, and feature selection, thereafter. Data obtained in positive mode generated from a LC-MS-based untargeted metabolomic study (sorghum plants responding dynamically to infection by a fungal pathogen) were used. Raw data were pre-processed with MarkerLynxTM software (Waters Corporation, Manchester, UK). Here, two parameters were varied: the intensity threshold (50–100 counts) and the mass tolerance (0.005–0.01 Da). After the pre-processing, the datasets were imported into SIMCA (Umetrics, Umea, Sweden) for more data cleaning and statistical modeling. In addition, different scaling (unit variance, Pareto, etc.) and data transformation (log and power) methods were explored. The results showed that the pre-processing parameters (or algorithms) influence the output dataset with regard to the number of defined features. Furthermore, the study demonstrates that the pre-treatment of data prior to statistical modeling affects the subspace approximation outcome: e.g., the amount of variation in X-data that the model can explain and predict. The pre-processing and pre-treatment steps subsequently influence the number of statistically significant extracted/selected features (variables). Thus, as informed by the results, to maximize the value of untargeted metabolomic data, understanding of the data structures and exploration of different algorithms and methods (at different steps of the data analysis pipeline) might be the best trade-off, currently, and possibly an epistemological imperative.

Methylation of the Vitamin D Receptor (VDR) Gene, Together with Genetic Variation, Race, and Environment Influence the Signaling Efficacy of the Toll-Like Receptor 2/1-VDR Pathway

Background The disparity in prevalence of infectious diseases across the globe is common knowledge. Vitamin D receptor (VDR)-mediated toll-like receptor (TLR) 2/1 signaling produces antimicrobial peptides, which is critical as a first line of defense in innate immunity. Numerous studies disclosed the independent role of genetic polymorphisms in this pathway, vitamin D status or season and more recently epigenetics, as factors contributing to infectious disease predisposition. Few studies address the interaction between environment, genetics, and epigenetics. Here, we hypothesized that VDR-mediated TLR2/1 signaling is influenced by a combination of environment, epigenetics and genetics, collectively influencing differential innate immunity. Methods Healthy Black and White South Africans (n = 100) donated blood, while ultraviolet index (UVI) was recorded for the duration of the study. LC-MS/MS supported 25(OH)D3 quantification. Monocyte/macrophage cultures, supplemented with/without 1,25(OH)2D3, were activated with the TLR2/1 elicitor, Pam3CSK4. VDR, cathelicidin antimicrobial peptide, hCAP-18, and 25-hydroxyvitamin D3-24-hydroxylase expression were quantified by RT-qPCR or flow cytometry. Pyrosequencing facilitated VDR methylation analysis and single-nucleotide polymorphism (SNP) genotyping in regions pinpointed through a bioinformatics workflow. Results Season interacted with race showing 25(OH)D3 deficiency in Blacks. UVI correlated with 25(OH)D3 and VDR methylation, likely influencing race differences in the latter. Regarding the TLR2/1 pathway, race differences in SNP genotype distribution were confirmed and functional analysis of VDR-mediated signaling showed interaction between race, season, and 25(OH)D3 status. Multivariate OPLS-DA mirrored several interactions between UVI, 25(OH)D3 status, DNA sequence, and methylation variants. Methylation of the third cytosine-phosphate-guanine dinucleotide (CpG) in the promoter CpG island (CGI) 1062, CGI 1062 CpG 3, significantly discriminated a 5.7-fold above average mean in VDR protein level upon TLR2/1 elicitation, the variation of which was further influenced by 25(OH)D3 status and the VDR SNP TaqI. Conclusion Regulation of VDR-mediated TLR2/1 signaling is multifactorial, involving interaction between environment [UVI and consequent 25(OH)D3 status], epigenetics (VDR methylation at key regulatory sites), and genetics (TLR1, TIRAP, and VDR SNPs).

Highlighting mass spectrometric fragmentation differences and similarities between hydroxycinnamoyl-quinic acids and hydroxycinnamoyl-isocitric acids

BackgroundPlants contain a myriad of metabolites which exhibit diverse biological activities. However, in-depth analyses of these natural products with current analytical platforms remains an undisputed challenge due to the multidimensional chemo-diversity of these molecules, amplified by both isomerization and conjugation. In this study, we looked at molecules such as hydroxyl-cinnamic acids (HCAs), which are known to exist as positional and geometrical isomers conjugated to different organic acids namely quinic- and isocitric acid.ObjectiveThe study aimed at providing a more defined distinction between HCA conjugates from Amaranthus viridis and Moringa oleifera, using mass spectrometry (MS) approaches.MethodsHere, we used a UHPLC–MS/MS targeted approach to analyze isobaric HCA conjugates extracted from the aforementioned plants.ResultsMass spectrometry results showed similar precursor ions and fragmentation pattern; however, distinct differences were seen with ions at m/z 155 and m/z 111 which are associated with isocitric acid conjugates.ConclusionOur results highlight subtle differences between these two classes of compounds based on the MS fingerprints, enabling confidence differentiation of the compounds. Thus, these findings provide a template reference for accurate and confident annotation of such compounds in other plants.

Deciphering the influence of column chemistry and mass spectrometry settings for the analyses of geometrical isomers of L-chicoric acid

Resolving the chemo-diversity of plant extract samples is an essential step for in-depth analyses of natural products which often exhibit promising biological activities. One of the challenges in this endeavor has been the confident differentiation of geometrical isomers. In this study, we investigated these aspects in chromatography (column chemistry and mobile phase composition) and mass spectrometry settings with regards to better differentiation of geometrical isomers. A standard of a hydroxycinnamic acid (HCA) derivative, L-chicoric acid (L-CA) - a di-acylated caffeoyltartaric acid ester found in a number of plant families - was used. Geometrical isomers of L-CA were formed by exposing the compound to ultraviolet (UV) radiation, to mimic the natural environment. The high performance liquid chromatography photo-diode array (HPLC-PDA) and ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) platforms were used to analyze the trans and cis geometrical isomers of L-CA. The HPLC-PDA results confirmed the generation of two cis geometrical isomers following UV exposure of the authentic trans-L-CA standard. Furthermore, the HPLC-PDA analyses demonstrated that the changes in both column chemistry (reverse-phase: C18, biphenyl, phenyl-hexyl and pentafluorophenyl propyl) and mobile phase composition (aqueous acetonitrile and aqueous methanol) affect the chromatographic elution profiles of the L-CA isomers. The MS results, on the other hand, revealed undisputed fragmentation differences between the geometrical isomers of L-CA. Thus, this study demonstrates that the identification of the L-CA isomers can be achieved more efficiently and confidently with good chromatography coupled to well-optimized mass spectrometry conditions, a requirement which has been proven impossible with other types of HCA derivatives. Moreover, differences in the binding modes of L-CA geometrical isomers to the HIV type 1 integrase enzyme were observed, suggesting a synergistic anti-HIV-1 activity of these isomers.

Metabolomics in Plant Priming Research: The Way Forward?

A new era of plant biochemistry at the systems level is emerging, providing detailed descriptions of biochemical phenomena at the cellular and organismal level. This new era is marked by the advent of metabolomics—the qualitative and quantitative investigation of the entire metabolome (in a dynamic equilibrium) of a biological system. This field has developed as an indispensable methodological approach to study cellular biochemistry at a global level. For protection and survival in a constantly-changing environment, plants rely on a complex and multi-layered innate immune system. This involves surveillance of ‘self’ and ‘non-self,’ molecule-based systemic signalling and metabolic adaptations involving primary and secondary metabolites as well as epigenetic modulation mechanisms. Establishment of a pre-conditioned or primed state can sensitise or enhance aspects of innate immunity for faster and stronger responses. Comprehensive elucidation of the molecular and biochemical processes associated with the phenotypic defence state is vital for a better understanding of the molecular mechanisms that define the metabolism of plant–pathogen interactions. Such insights are essential for translational research and applications. Thus, this review highlights the prospects of metabolomics and addresses current challenges that hinder the realisation of the full potential of the field. Such limitations include partial coverage of the metabolome and maximising the value of metabolomics data (extraction of information and interpretation). Furthermore, the review points out key features that characterise both the plant innate immune system and enhancement of the latter, thus underlining insights from metabolomic studies in plant priming. Future perspectives in this inspiring area are included, with the aim of stimulating further studies leading to a better understanding of plant immunity at the metabolome level.

Untargeted metabolomics analysis reveals dynamic changes in azelaic acid- and salicylic acid derivatives in LPS-treated Nicotiana tabacum cells

To counteract biotic stress factors, plants employ multilayered defense mechanisms responsive to pathogen-derived elicitor molecules, and regulated by different phytohormones and signaling molecules. Here, lipopolysaccharide (LPS), a microbe-associated molecular pattern (MAMP) molecule, was used to induce defense responses in Nicotiana tabacum cell suspensions. Intracellular metabolites were extracted with methanol and analyzed using a liquid chromatography-mass spectrometry (UHPLC-qTOF-MS/MS) platform. The generated data were processed and examined with multivariate and univariate statistical tools. The results show time-dependent dynamic changes and accumulation of glycosylated signaling molecules, specifically those of azelaic acid, salicylic acid and methyl-salicylate as contributors to the altered metabolomic state in LPS-treated cells.