Ian A. Dubery

Pentacyclic Triterpenoids from the Medicinal Herb, Centella asiatica (L.) Urban

Centella asiatica accumulates large quantities of pentacyclic triterpenoid saponins, collectively known as centelloids. These terpenoids include asiaticoside, centelloside, madecassoside, brahmoside, brahminoside, thankuniside, sceffoleoside, centellose, asiatic-, brahmic-, centellic- and madecassic acids. The triterpene saponins are common secondary plant metabolites and are synthesized via the isoprenoid pathway to produce a hydrophobic triterpenoid structure (aglycone) containing a hydrophilic sugar chain (glycone). The biological activity of saponins has been attributed to these characteristics. In planta, the Centella triterpenoids can be regarded as phytoanticipins due to their antimicrobial activities and protective role against attempted pathogen infections. Preparations of C. asiatica are used in traditional and alternative medicine due to the wide spectrum of pharmacological activities associated with these secondary metabolites. Here, the biosynthesis of the centelloid triterpenoids is reviewed; the range of metabolites found in C. asiatica, together with their known biological activities and the chemotype variation in the production of these metabolites due to growth conditions are summarized. These plant-derived pharmacologically active compounds have complex structures, making chemical synthesis an economically uncompetitive option. Production of secondary metabolites by cultured cells provides a particularly important benefit to manipulate and improve the production of desired compounds; thus biotechnological approaches to increase the concentrations of the metabolites are discussed.

Cell wall reinforcement in cotton hypocotyls in response to a Verticillium dahliae elicitor

Abstract Cotton ( Gossypium hirsutum ) hypocotyl tissue responded with increased lignification following treatment with a protein-lipopolysaccharide elicitor from Verticillium dahliae , the causative agent of vascular wilt disease in cotton. The induction of defence reactions was investigated over a period of 0–35 hr. Following exposure to the elicitor, increased synthesis and deposition of lignin and lignin-like phenolic polymers occurred. The induced phase of active lignification correlated with and was preceded by a transient increase in levels of enzyme activities; phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase and cell wall associated peroxidase. The defence responses in two cultivars of G. hirsutum (cv. OR 19 and Acala 1517-70 , resistant and susceptible to V. dahliae , respectively) were compared. The resistant cultivar exhibited higher and earlier induced levels of enzyme activity and lignin-like polymers compared to the susceptible cultivar. This indicates that the effectiveness of induced defence responses depends on their rapid initiation, development and accumulation and suggests a possible correlation between the timing and intensity of lignin-like polymer accumulation and resistance/tolerance of G. hirsutum seedlings against V. dahliae .

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.

A phytotoxic protein-lipopolysaccharide complex produced by Verticillium dahliae

Abstract A phytotoxic protein-lipopolysaccharide complex (PLPC) was purified to electrophoretic homogenetiy from seven-day-old culture filtrates of Verticillium dahliae by means of acetone precipitation, gel exclusion chromatography, and preparative agarose electrophoresis with a yield of 4.5 mg 1−1 culture filtrate. The PLPC consists of 15.7% protein, 13.0% lipid, 0.4% phosphate and 70% carbohydrate. The isoelectric point is 3.8 and the amino acid composition of the protein fraction was determined. The complex has a Mr of 197 000 and can be dissociated into five protein-containing components, with Mrs of 78 000, 62 000, 48 000, 32 0000 and 28 000. Polygalacturonase and cellulase enzyme activities were identified in the PLPC by means of enzyme staining techniques, and were found to be associated with the 28 000 and 48 000 Mr complexes, respectively. 1,3-β-Glucanase activity was associated with the 32 000 and 28 000 Mr complexes. Treatment of cotton seedlings with concentrations from 2.5 μg ml−1 PLPC resulted in symptoms of wilting and necrosis and was accompanied by inhibition of the H+- ATPase activity of plasma membranes and the rapid transient elicitation of phenylalanine ammonia-lyase activity. Significant differences were found between resistant and susceptible cultivars. When applied to disks of mature cotton leaves, the PLPC elicited the accumulation of pathogenesis-related proteins in the intercellular spaces.

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.

Distinct carbohydrate and lipid-based molecular patterns within lipopolysaccharides from Burkholderia cepacia contribute to defense-associated differential gene expression in Arabidopsis thaliana.

Lipopolysaccharides are structural components within the cell walls of Gram-negative bacteria. The LPSs as microbe-associated molecular pattern (MAMP) molecules can trigger defense-related responses involved in MAMP-triggered immunity and basal resistance in plants, presumably from an initial perception event. LPS from Burkholderia cepacia as well as two fragments, the glycolipid, lipid A and the polysaccharide (OPS-core) chain, were used to treat Arabidopsis thaliana seedlings to evaluate the eliciting activities of the individual LPS sub-domains by means of Annealing Control Primer-based Differential Display transcript profiling. Genes found to be up-regulated encode for proteins involved in signal perception and transduction, transcriptional regulation and defense – and stress responses. Furthermore, genes encoding proteins involved in chaperoning, secretion, protein–protein interactions and protein degradation were differentially expressed. It is concluded that intact LPS, as well as the two sub-components, induced the expression of a broad range of genes associated with perception and defense as well as metabolic reprogramming of cellular activities in support of immunity and basal resistance. Whilst the lipid A and OPS moieties were able to up-regulate sub-sets of defense-associated genes over the same spectrum of categories as intact LPS, the up-regulation observed with intact LPS was the more comprehensive, suggesting that the lipid A and glycan molecular patterns of the molecule act as partial agonists, but that the intact LPS structure is required for full agonist activity.

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.

Phenylalanine ammonia-lyase from cotton ( Gossypium hirsutum) hypocotyls: properties of the enzyme induced by a Verticillium dahliae phytotoxin

Phenylalanine ammonia-lyase (EC 4.3.1.5), induced by a Verticillium dahliae phytotoxin, has been purified to electrophoretic homogeneity from cotton hypocotyls by differential ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 52%. The enzyme is a tetramer with a molecular weight of 332,000 to 337,000. The isoelectric point is 4.6, and no isoforms were observed. The subunits of the enzyme are unstable and breaks down to fragments with M(r)s of 69,000 and 49,500. The enzyme exhibited only activity with L-phenylalanine as substrate. Deamination was optimal at pH 8.9 and the activation energy was calculated as 100.6 kJ mol-1. Non-Michaelian kinetics were observed with a KmL = 10.0 microM and KmH = 75.0 microM describing the binding of the substrate to the enzyme. Negative cooperative interactions occurred between the substrate binding sites with a Hill coefficient of 0.87. The inhibitors AOPP (S)-2-amino-oxy-3-phenylpropanoic acid), APEP (R)-1-amino-2-phenylethylphosphonic acid) and 2-AIP (2-aminoindan-2-phosphonic acid) strongly inactivated the enzyme, as did various analogues of L-phenylalanine and t-cinnamate. The induced enzyme is also sensitive to inhibition by phenylpropanoid intermediates and precursors involved in lignification such as 4-hydroxycinnamate and 3,4-dihydroxycinnamate.

Proteomic profiling of cellular targets of lipopolysaccharide-induced signalling in Nicotiana tabacum BY-2 cells

Plants constantly monitor for pathogen challenge and utilize a diverse array of adaptive defense mechanisms, including differential protein regulation, during pathogen attack. A proteomic analysis of Nicotiana tabacum BY-2 cells was performed in order to investigate the dynamic changes following perception of bacterial lipopolysaccharides. A multiplexed proteome analysis, employing two-dimensional difference-in-gel-electrophoresis with CyDye DIGE fluors, as well as Ruthenium II tris (bathophenanthroline disulfonate) fluorescence staining and Pro-Q Diamond phosphoprotein-specific gel staining, monitored over 1500 proteins and resulted in the identification of 88 differentially regulated proteins and phosphoproteins responsive to LPS(B.cep.)-elicitation. Functional clustering of the proteins both at the level of their abundance and phosphorylation status, revealed 9 proteins involved in transport, ion homeostasis and signal transduction. A large number of responsive proteins were found to be involved in metabolism- and energy-related processes (36), representing various metabolic pathways. Another abundant category corresponded to proteins classified as molecular chaperones and involved in protein destination/targeting (12). Other categories of proteins found to be LPS(B.cep.)-responsive and differentially regulated include cell structure- and cytoskeletal rearrangement proteins (8) and proteins involved in transcription and translation as well as degradation (11). The results indicate that LPS(B.cep.) induces metabolic reprogramming and changes in cellular activities supporting protein synthesis, -folding, vesicle trafficking and secretion; accompanied by changes to the cytoskeleton and proteosome function. Many of the identified proteins are known to be interconnected at various levels through a complex web of activation/deactivation, complex formation, protein-protein interactions, and chaperoning reactions. The presented data offers novel insights and further evidence for the biochemical action of LPS(B.cep.) as a resistance elicitor, a pathogen-associated molecular pattern molecule and triggering agent of defense responses associated with innate immunity.

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.