Lizelle A. Piater

Innate immunity in plants and animals: striking similarities and obvious differences.

Summary:  Innate immunity constitutes the first line of defense against attempted microbial invasion, and it is a well‐described phenomenon in vertebrates and insects. Recent pioneering work has revealed striking similarities between the molecular organization of animal and plant systems for nonself recognition and anti‐microbial defense. Like animals, plants have acquired the ability to recognize invariant pathogen‐associated molecular patterns (PAMPs) that are characteristic of microbial organisms but which are not found in potential host plants. Such structures, also termed general elicitors of plant defense, are often indispensable for the microbial lifestyle and, upon receptor‐mediated perception, inevitably betray the invader to the plants surveillance system. Remarkable similarities have been uncovered in the molecular mode of PAMP perception in animals and plants, including the discovery of plant receptors resembling mammalian Toll‐like receptors or cytoplasmic nucleotide‐binding oligomerization domain leucine‐rich repeat proteins. Moreover, molecular building blocks of PAMP‐induced signaling cascades leading to the transcriptional activation of immune response genes are shared among the two kingdoms. In particular, nitric oxide as well as mitogen‐activated protein kinase cascades have been implicated in triggering innate immune responses, part of which is the production of anti‐microbial compounds. In addition to PAMP‐mediated pathogen defense, disease resistance programs are often initiated upon plant‐cultivar‐specific recognition of microbial race‐specific virulence factors, a recognition specificity that is not known from animals.

Analyses of chlorogenic acids and related cinnamic acid derivatives from Nicotiana tabacum tissues with the aid of UPLC-QTOF-MS/MS based on the in-source collision-induced dissociation method.

BackgroundChlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures.ResultsUsing an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof.ConclusionUsing this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.

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.

Identification of a lipopolysaccharide responsive erk‐like MAP kinase in tobacco leaf tissue

SUMMARY Lipopolysaccharides (LPS) are indispensable cell surface components of Gram-negative bacteria and have diverse roles in plant-microbe interactions. Treatment of Nicotiana tabacum with the LPS of an endophytic strain of Burkholderia cepacia results in an enhanced defensive capacity in the tissue. In this study the rapid and transient phosphorylation of an extracellular signal-regulated (ERK)-like mitogen-activated protein (MAP) kinase in response to LPS from B. cepacia (LPS(B.cep.)) elicitation is reported. Based on in-gel kinase assays it was found that this 43-kDa LPS(B.cep.)-responsive kinase is optimally activated following 7 min elicitation with 100 microg/mL LPS. Its identity as an ERK MAPK was supported by tyrosine-phosphorylated association with induction, immunodetection with pTEpY-specific MAPK antibodies and inhibition of phosphorylation by U0126, an upstream MAPKK inhibitor. The kinase utilized myelin basic protein, but not casein or histone, as substrate. Ca(2+) did not modulate the phosphorylation, nor did wounding. To date, other MAP kinases have been shown to act either independently or upstream from reactive oxygen intermediates produced during the oxidative burst. It was found that hydrogen peroxide is either not generated in leaf tissue in response to LPS elicitation or, if generated, does not trigger the phosphorylation of the kinase. Physicochemical characterization of the ERK-like MAPK indicated a molecular mass of 43 kDa and a pI of 6.3; two-dimensional gel analysis indicated two charge isomers. This is the first demonstration of such an LPS-responsive MAP kinase phosphorylation in plants.

Multivariate statistical models of metabolomic data reveals different metabolite distribution patterns in isonitrosoacetophenone-elicited Nicotiana tabacum and Sorghum bicolor cells

Isonitrosoacetophenone (INAP, 2-keto-2-phenyl-acetaldoxime) is a novel inducer of plant defense. Oxime functional groups are rare in natural products, but can serve as substrates depending on existing secondary pathways. Changes in the metabolomes of sorghum and tobacco cells treated with INAP were investigated and chemometric tools and multivariate statistical analysis were used to investigate the changes in metabolite distribution patterns resulting from INAP elicitation. Liquid chromatography combined with mass spectrometry (UHPLC-MS) supplied unique chemical fingerprints that were generated in response to specific metabolomic events. Principal component analysis (PCA) together with hierarchical cluster analysis (HCA) and Metabolic Trees were used for data visualization. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) and shared and unique structure (SUS) plots were exploited in parallel to reveal the changes in the metabolomes. PCA indicated that the cells responded differentially to INAP through changes in the metabolite profiles. Furthermore, HCA and Metabolic Trees showed that INAP induced metabolic perturbations in both cell lines and that homeostasis was re-established over time. OPLS-DA-based shared and unique structure (SUS) plots confirmed the results and revealed differences in the metabolites distribution patterns between tobacco and sorghum cells. Chemometric analyses of metabolomic data offers insight into changes in metabolism in response to chemical elicitation. Although similar, the response in sorghum cells was found to be more consistent and well-coordinated when compared to tobacco cells, indicative of the differences in secondary metabolism between cyanogenic and non-cyanogenic plants for oxime metabolism.

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.

Phenylpropanoid Defences in Nicotiana tabacum Cells: Overlapping Metabolomes Indicate Common Aspects to Priming Responses Induced by Lipopolysaccharides, Chitosan and Flagellin-22.

Plants have evolved both constitutive and inducible defence strategies to cope with different biotic stimuli and stresses. Exposure of a plant to a challenging stress can lead to a primed state that allows it to launch a more rapid and stronger defence. Here we applied a metabolomic approach to study and compare the responses induced in Nicotiana tabacum cells by microbe-associated molecular pattern (MAMP) molecules, namely lipopolysaccharides (LPS), chitosan (CHT) and flagellin-22 (FLG22). Early response metabolites, extracted with methanol, were analysed by UHPLC-MS/MS. Using multivariate statistical tools the metabolic profiles induced by these elicitors were analysed. In the metabolic fingerprint of these agents a total of 19 cinnamic acid derivatives conjugated to quinic acids (chlorogenic acids), shikimic acid, tyramine, polyamines or glucose were found as discriminant biomarkers. In addition, treatment with the phytohormones salicylic acid (SA), methyljasmonic acid (MJ) and abscisic acid (ABA) resulted in differentially-induced phenylpropanoid pathway metabolites. The results indicate that the phenylpropanoid pathway is activated by these elicitors while hydroxycinnamic acid derivatives are commonly associated with the metabolic response to the MAMPs, and that the activated responses are modulated by both SA and MJ, with ABA not playing a role.

Subcritical Water Extraction of Biological Materials

Extraction is a vital prerequisite in most scientific studies involving the isolation and analysis of compounds from biological/environmental systems. The use of large quantities of organic solvents in performing both conventional and modern methods of extraction of these substances stirs issues of safety, environmental concern and cost-effectiveness. Subcritical water extraction (SWE) offers a suitable, safe, cost-effective and environmentally safe alternative compared to other methods as it takes advantage of the special properties of supercritical water under high temperature and pressure conditions (100–374 °C, >50 bar) to extract non-polar analytes. This review presents a critical appraisal of the principles and dynamics of SWE, and the current applications as a viable tool in the extraction of compounds from various biological matrices. Although more research is needed to improve full SWE applications, its adoption in the extraction of phytochemicals as well as other bioactive molecules including mycotoxins from both plant and animal components seems promising and needs to be properly exploited.

Metabolomic analysis of isonitrosoacetophenone-induced perturbations in phenolic metabolism of Nicotiana tabacum cells

Plants have developed biochemical and molecular responses to adapt to different stress environments. One of the characteristics of the multi-component defence response is the production of defence-related metabolites. Plant defences can be triggered by various stimuli, including synthetic or naturally occurring molecules, especially those derived from pathogens. In the current study, Nicotiana tabacum cell suspensions were treated with isonitrosoacetophenone (INAP), a subcomponent of a plant-derived stress metabolite with anti-fungal and anti-oxidant properties, in order to investigate the effect thereof on cellular metabolism. Subsequent metabolomic-based analyses were employed to evaluate changes in the metabolome. UPLC-MS in conjunction with multivariate data analyses was found to be an appropriate approach to study the effect of chemical inducers like INAP on plant metabolism in this model system. Principal component analysis (PCA) indicated that INAP is capable of inducing time-dependent metabolic perturbations in the cultured cells. Orthogonal projection to latent structures discriminant analysis (OPLS-DA) revealed metabolites of which the levels are affected by INAP, and eight of these were tentatively annotated from the mass spectral data and online databases. These metabolites are known in the context of plant stress- and defence responses and include benzoic- or cinnamic acid derivatives that are either glycosylated or quinilated as well as flavonoid derivatives. The results indicate that INAP affects the shikimate-, phenylpropanoid- and flavonoid pathways, the products of which may subsequently lead to an anti-oxidant environment in vivo.

Profiling of Altered Metabolomic States in Nicotiana tabacum Cells Induced by Priming Agents

Metabolomics has developed into a valuable tool for advancing our understanding of plant metabolism. Plant innate immune defenses can be activated and enhanced so that, subsequent to being pre-sensitized, plants are able to launch a stronger and faster defense response upon exposure to pathogenic microorganisms, a phenomenon known as priming. Here, three contrasting chemical activators, namely acibenzolar-S-methyl, azelaic acid and riboflavin, were used to induce a primed state in Nicotiana tabacum cells. Identified biomarkers were then compared to responses induced by three phytohormones—abscisic acid, methyljasmonate, and salicylic acid. Altered metabolomes were studied using a metabolite fingerprinting approach based on liquid chromatography and mass spectrometry. Multivariate data models indicated that these inducers cause time-dependent metabolic perturbations in the cultured cells and revealed biomarkers of which the levels are affected by these agents. A total of 34 metabolites were annotated from the mass spectral data and online databases. Venn diagrams were used to identify common biomarkers as well as those unique to a specific agent. Results implicate 20 cinnamic acid derivatives conjugated to (i) quinic acid (chlorogenic acids), (ii) tyramine, (iii) polyamines, or (iv) glucose as discriminatory biomarkers of priming in tobacco cells. Functional roles for most of these metabolites in plant defense responses could thus be proposed. Metabolites induced by the activators belong to the early phenylpropanoid pathway, which indicates that different stimuli can activate similar pathways but with different metabolite fingerprints. Possible linkages to phytohormone-dependent pathways at a metabolomic level were indicated in the case of cells treated with salicylic acid and methyljasmonate. The results contribute to a better understanding of the priming phenomenon and advance our knowledge of cinnamic acid derivatives as versatile defense metabolites.