Comparative analysis of hyoscine in wild-type and in vitro grown Datura innoxia by high performance liquid chromatography

Purpose: To compare the hyoscine contents of Datura innoxia plant grown in the wild and that grown in vitro.Methods: For callus induction from leaf explants, 15 combinations of 2,4-dichlorophenoxy acetic acid, gibberellic acid, benzyl amino purine and kinetin were used. For regeneration of shoots, 8 combinations of BAP, kinetin and indole butyric acid (IBA) were tested. Different concentrations of IBA were used for rooting. The hyoscine contents of the wild and in vitro samples was initially identified by TLC and subsequently quantified by high performance liquid chromatography using hyoscine N-butyl bromide as standard.Results: For callus induction, 2,4-D at 2 mg/L produced maximum calli (96 %). Maximum shoots regeneration was obtained with 2 mg/L BAP and 1 mg/L kinetin. IBA at 0.5 mg/L induced good rooting. Substantial amount of hyoscine was detected in the extracts of in vitro grown plants through TLC and HPLC. Wild root, stem and leaves exhibited higher amounts (approx. 2 μg/mL) of hyoscine than the parts of in vitro grown plants. Green callus grown in vitro contained a maximum concentration of hyoscine (1.01 μg/mL) followed by leaf (0.82 μg/mL) and brown callus (0.432 μg/mL).Conclusion: The callus cultures of D. innoxia are capable of yielding good amounts of hyoscine, and therefore can be exploited to increase hyoscine production using chemical/hormone treatments on a large scale. Thus, this study provides a sustainable and efficient way for the sustainable production of a natural hyoscine product via in vitro-grown D. innoxia cultures.Keywords: Datura innoxia, Hyoscine, Callus, Hyoscine, Explant, Gibberellic acid, Benzyl amino purine, Kineti

Identification et caractérisation fonctionnelle des deux premiers prényltransférases aromatiques impliqués dans la biosynthèse de furanocoumarines et des coumarines prénylés chez deux familles de plantes : Rutaceae et Apiaceae

Furanocoumarins constitute one of the classes of secondary metabolites deriving from the phenylpropanoid biosynthetic pathway. These molecules are described as phytoalexins in plants but are also used by humans for their pharmaceutical properties. A large number of biochemical studies were carried out to understand their biosynthetic pathway but little information was available concerning the genes involved in the pathway. In this study, we focused on the characterization of genes encoding for aromatic prenyltransferases which were described to be involved in this pathway. Prenlyltransferases catalyze the entry step to the linear or angular furanocoumarin pathway. Hence they catalyze the addition of a dimethylallyl pyrophosphate (DMAPP) prenyl moiety to umbelliferone. Using a recently characterized aromatic prenyltransferase (SfN8DT-1) as a probe, we isolated 7 different candidate genes from two plant families (Rutaceae and Apiaceae). As these enzymes were described as membrane bound proteins, we adapted a heterologous expression system made up of Nicotiana benthamian aand we validated its efficiency by using two membrane-associated enzymes: a cytochrome P450 (CYP98A22) and the already described prenyltransferase SfN8DT-1. Subsequently, this system was used to perform the functional characterization of the 7 newly identified proteins. This way we succeeded to characterize the first umbelliferone prenyltransferase of Petroselinum crispum that was able to catalyze both the 6-C and 8-C prenylation of umbelliferone with DMAPP producing demethylsuberosin and osthenol respectively. We made evidence that these reactions occurred both in vitro and in vivo. In addition, in planta studies performed in P.crispum showed a positive relationship between the gene expression level and the content of prenylated umbelliferone. The overexpression of this gene was investigated in Ruta graveolens and we could provide evidences of a link between the enzymatic activity and the disappearance of umbelliferone. We also reported a similar activity for a prenyltransferase isolated from Pastinaca sativa, which makes us assume that the prenylation step is not a rate limiting step in the biosynthetic pathway of angular furanocoumarins since parsley is producing only linear furanocoumarins whereas parsnip is producing both linear and angular furanocoumarins. In addition, using the same N. benthamiana and R. graveolens heterologous expression systems, we identified a second aromatic prenyltransferase able to catalyze the addition of geranyl pyrophosphate (GPP) both to umbelliferone and esculetinLes furocoumarines constituent l'une des classes de métabolites secondaires dérivant de la voie de biosynthèse des phénylpropanoïdes. Elles ont été décrites comme étant des phytoaléxines mais sont également très largement utilisées par l'Homme pour leurs propriétés thérapeutiques. Un certain nombre d'études biochimiques ont été réalisées afin d'en comprendre la biosynthèse mais peu de choses sont connues concernant leur déterminisme moléculaire. Dans cette étude, nous nous sommes concentrés sur la caractérisation fonctionnelle de gènes appartenant aux prényltransférases aromatiques potentiellement impliquées dans cette voie de biosynthèse. Les prényltransférases catalysent la première étape de la voie de biosynthèse des furocoumarines linéaires ou angulaires. Elles permettent l'addition d'un groupement dimethylally pyrophosphate (DMAPP) sur l'umbelliférone. En utilisant SfN8DT-1, une prényltransférase aromatique récemment caractérisée, comme sonde, nous avons identifié 7 gènes candidats chez deux familles de plantes (Rutaceae et Apiaceae). Dans la mesure où il a été décrit que ces enzymes étaient des protéines membranaires, nous avons adapté un système d'expression hétérologue basé sur l'utilisation de N. benthamiana. Ce système a été validé par l'expression de deux protéines membranaires : un cytochrome P450 CYP98A22 et une prényltransférase déjà caractérisée SfN8DT-1. Nous avons ensuite utilisé ce système d'expression pour réaliser l'étude des 7 gènes nouvellement isolés. Ces travaux nous ont permis de caractériser la première umbelliferone prenyltransferase de Petroselinum crispum capable de catalyser in vitro et in vivo la prénylation du carbone 6 ou 8 de l'umbelliférone en présence de DMAPP permettant ainsi la synthèse respectivement de demethylsuberosin et d'osthenol. Par ailleurs, une étude réalisée in planta chez le persil a permis de mettre en évidence une relation positive entre le niveau d'expression du gène et la teneur en umbelliférone prénylée. L'étude de la surexpression du gène chez Ruta graveolens a permis de mettre en évidence un lien entre l'expression du gène et la disparition de l'umbelliférone. Enfin nous avons identifié la même activité pour une prényltransférase de Pastinaca sativa, ce qui nous amène à émettre l'hypothèse que l'étape de prénylation n'est pas une étape limitante dans la biosynthèse des furocoumarines angulaires, étant donné que le persil ne produit que des furocoumarines linéaires, tandis que le panais produit des furocoumarines linéaires et angulaires. L'utilisation de ces mêmes systèmes d'expression hétérologue de N. benthamiana et R. graveolens nous a également permis d'identifier une seconde prényltransférase aromatique capable de catalyser l'addition de géranylpyrophosphate (GPP) sur l'umbelliférone et sur l'esculétin

Identification et caractérisation fonctionnelle des deux premiers prényltransférases aromatiques impliqués dans la biosynthèse de furanocoumarines et des coumarines prénylés chez deux familles de plantes : Rutaceae et Apiaceae

Les furocoumarines constituent l'une des classes de métabolites secondaires dérivant de la voie de biosynthèse des phénylpropanoïdes. Elles ont été décrites comme étant des phytoaléxines mais sont également très largement utilisées par l'Homme pour leurs propriétés thérapeutiques. Un certain nombre d'études biochimiques ont été réalisées afin d'en comprendre la biosynthèse mais peu de choses sont connues concernant leur déterminisme moléculaire. Dans cette étude, nous nous sommes concentrés sur la caractérisation fonctionnelle de gènes appartenant aux prényltransférases aromatiques potentiellement impliquées dans cette voie de biosynthèse. Les prényltransférases catalysent la première étape de la voie de biosynthèse des furocoumarines linéaires ou angulaires. Elles permettent l'addition d'un groupement dimethylally pyrophosphate (DMAPP) sur l'umbelliférone. En utilisant SfN8DT-1, une prényltransférase aromatique récemment caractérisée, comme sonde, nous avons identifié 7 gènes candidats chez deux familles de plantes (Rutaceae et Apiaceae). Dans la mesure où il a été décrit que ces enzymes étaient des protéines membranaires, nous avons adapté un système d'expression hétérologue basé sur l'utilisation de N. benthamiana. Ce système a été validé par l'expression de deux protéines membranaires : un cytochrome P450 CYP98A22 et une prényltransférase déjà caractérisée SfN8DT-1. Nous avons ensuite utilisé ce système d'expression pour réaliser l'étude des 7 gènes nouvellement isolés. Ces travaux nous ont permis de caractériser la première umbelliferone prenyltransferase de Petroselinum crispum capable de catalyser in vitro et in vivo la prénylation du carbone 6 ou 8 de l'umbelliférone en présence de DMAPP permettant ainsi la synthèse respectivement de demethylsuberosin et d'osthenol. Par ailleurs, une étude réalisée in planta chez le persil a permis de mettre en évidence une relation positive entre le niveau d'expression du gène et la teneur en umbelliférone prénylée. L'étude de la surexpression du gène chez Ruta graveolens a permis de mettre en évidence un lien entre l'expression du gène et la disparition de l'umbelliférone. Enfin nous avons identifié la même activité pour une prényltransférase de Pastinaca sativa, ce qui nous amène à émettre l'hypothèse que l'étape de prénylation n'est pas une étape limitante dans la biosynthèse des furocoumarines angulaires, étant donné que le persil ne produit que des furocoumarines linéaires, tandis que le panais produit des furocoumarines linéaires et angulaires. L'utilisation de ces mêmes systèmes d'expression hétérologue de N. benthamiana et R. graveolens nous a également permis d'identifier une seconde prényltransférase aromatique capable de catalyser l'addition de géranylpyrophosphate (GPP) sur l'umbelliférone et sur l'esculétineFuranocoumarins constitute one of the classes of secondary metabolites deriving from the phenylpropanoid biosynthetic pathway. These molecules are described as phytoalexins in plants but are also used by humans for their pharmaceutical properties. A large number of biochemical studies were carried out to understand their biosynthetic pathway but little information was available concerning the genes involved in the pathway. In this study, we focused on the characterization of genes encoding for aromatic prenyltransferases which were described to be involved in this pathway. Prenlyltransferases catalyze the entry step to the linear or angular furanocoumarin pathway. Hence they catalyze the addition of a dimethylallyl pyrophosphate (DMAPP) prenyl moiety to umbelliferone. Using a recently characterized aromatic prenyltransferase (SfN8DT-1) as a probe, we isolated 7 different candidate genes from two plant families (Rutaceae and Apiaceae). As these enzymes were described as membrane bound proteins, we adapted a heterologous expression system made up of Nicotiana benthamian aand we validated its efficiency by using two membrane-associated enzymes: a cytochrome P450 (CYP98A22) and the already described prenyltransferase SfN8DT-1. Subsequently, this system was used to perform the functional characterization of the 7 newly identified proteins. This way we succeeded to characterize the first umbelliferone prenyltransferase of Petroselinum crispum that was able to catalyze both the 6-C and 8-C prenylation of umbelliferone with DMAPP producing demethylsuberosin and osthenol respectively. We made evidence that these reactions occurred both in vitro and in vivo. In addition, in planta studies performed in P.crispum showed a positive relationship between the gene expression level and the content of prenylated umbelliferone. The overexpression of this gene was investigated in Ruta graveolens and we could provide evidences of a link between the enzymatic activity and the disappearance of umbelliferone. We also reported a similar activity for a prenyltransferase isolated from Pastinaca sativa, which makes us assume that the prenylation step is not a rate limiting step in the biosynthetic pathway of angular furanocoumarins since parsley is producing only linear furanocoumarins whereas parsnip is producing both linear and angular furanocoumarins. In addition, using the same N. benthamiana and R. graveolens heterologous expression systems, we identified a second aromatic prenyltransferase able to catalyze the addition of geranyl pyrophosphate (GPP) both to umbelliferone and esculeti

Identification et caractérisation fonctionnelle des deux premiers prényltransférases aromatiques impliqués dans la biosynthèse de furanocoumarines et des coumarines prénylés chez deux familles de plantes (Rutaceae et Apiaceae)

Les furocoumarines constituent l'une des classes de métabolites secondaires dérivant de la voie de biosynthèse des phénylpropanoïdes. Elles ont été décrites comme étant des phytoaléxines mais sont également très largement utilisées par l'Homme pour leurs propriétés thérapeutiques. Un certain nombre d'études biochimiques ont été réalisées afin d'en comprendre la biosynthèse mais peu de choses sont connues concernant leur déterminisme moléculaire. Dans cette étude, nous nous sommes concentrés sur la caractérisation fonctionnelle de gènes appartenant aux prényltransférases aromatiques potentiellement impliquées dans cette voie de biosynthèse. Les prényltransférases catalysent la première étape de la voie de biosynthèse des furocoumarines linéaires ou angulaires. Elles permettent l'addition d'un groupement dimethylally pyrophosphate (DMAPP) sur l'umbelliférone. En utilisant SfN8DT-1, une prényltransférase aromatique récemment caractérisée, comme sonde, nous avons identifié 7 gènes candidats chez deux familles de plantes (Rutaceae et Apiaceae). Dans la mesure où il a été décrit que ces enzymes étaient des protéines membranaires, nous avons adapté un système d'expression hétérologue basé sur l'utilisation de N. benthamiana. Ce système a été validé par l'expression de deux protéines membranaires : un cytochrome P450 CYP98A22 et une prényltransférase déjà caractérisée SfN8DT-1. Nous avons ensuite utilisé ce système d'expression pour réaliser l'étude des 7 gènes nouvellement isolés. Ces travaux nous ont permis de caractériser la première umbelliferone prenyltransferase de Petroselinum crispum capable de catalyser in vitro et in vivo la prénylation du carbone 6 ou 8 de l'umbelliférone en présence de DMAPP permettant ainsi la synthèse respectivement de demethylsuberosin et d'osthenol. Par ailleurs, une étude réalisée in planta chez le persil a permis de mettre en évidence une relation positive entre le niveau d'expression du gène et la teneur en umbelliférone prénylée. L'étude de la surexpression du gène chez Ruta graveolens a permis de mettre en évidence un lien entre l'expression du gène et la disparition de l'umbelliférone. Enfin nous avons identifié la même activité pour une prényltransférase de Pastinaca sativa, ce qui nous amène à émettre l'hypothèse que l'étape de prénylation n'est pas une étape limitante dans la biosynthèse des furocoumarines angulaires, étant donné que le persil ne produit que des furocoumarines linéaires, tandis que le panais produit des furocoumarines linéaires et angulaires. L'utilisation de ces mêmes systèmes d'expression hétérologue de N. benthamiana et R. graveolens nous a également permis d'identifier une seconde prényltransférase aromatique capable de catalyser l'addition de géranylpyrophosphate (GPP) sur l'umbelliférone et sur l'esculétineFuranocoumarins constitute one of the classes of secondary metabolites deriving from the phenylpropanoid biosynthetic pathway. These molecules are described as phytoalexins in plants but are also used by humans for their pharmaceutical properties. A large number of biochemical studies were carried out to understand their biosynthetic pathway but little information was available concerning the genes involved in the pathway. In this study, we focused on the characterization of genes encoding for aromatic prenyltransferases which were described to be involved in this pathway. Prenlyltransferases catalyze the entry step to the linear or angular furanocoumarin pathway. Hence they catalyze the addition of a dimethylallyl pyrophosphate (DMAPP) prenyl moiety to umbelliferone. Using a recently characterized aromatic prenyltransferase (SfN8DT-1) as a probe, we isolated 7 different candidate genes from two plant families (Rutaceae and Apiaceae). As these enzymes were described as membrane bound proteins, we adapted a heterologous expression system made up of Nicotiana benthamian aand we validated its efficiency by using two membrane-associated enzymes: a cytochrome P450 (CYP98A22) and the already described prenyltransferase SfN8DT-1. Subsequently, this system was used to perform the functional characterization of the 7 newly identified proteins. This way we succeeded to characterize the first umbelliferone prenyltransferase of Petroselinum crispum that was able to catalyze both the 6-C and 8-C prenylation of umbelliferone with DMAPP producing demethylsuberosin and osthenol respectively. We made evidence that these reactions occurred both in vitro and in vivo. In addition, in planta studies performed in P.crispum showed a positive relationship between the gene expression level and the content of prenylated umbelliferone. The overexpression of this gene was investigated in Ruta graveolens and we could provide evidences of a link between the enzymatic activity and the disappearance of umbelliferone. We also reported a similar activity for a prenyltransferase isolated from Pastinaca sativa, which makes us assume that the prenylation step is not a rate limiting step in the biosynthetic pathway of angular furanocoumarins since parsley is producing only linear furanocoumarins whereas parsnip is producing both linear and angular furanocoumarins. In addition, using the same N. benthamiana and R. graveolens heterologous expression systems, we identified a second aromatic prenyltransferase able to catalyze the addition of geranyl pyrophosphate (GPP) both to umbelliferone and esculetinMETZ-SCD (574632105) / SudocNANCY1-Bib. numérique (543959902) / SudocNANCY2-Bibliotheque electronique (543959901) / SudocNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

From Lab to Pharmacy Shelves: The Story of a Plant Derived Anticancer Drug, Paclitaxel

Paclitaxel (Taxol®) is a highly praised anticancer drug, known for its efficiency in treating different cancers. It belongs to a class of compounds called taxanes that are derived from the plants of the genus Taxus. This drug is now FDA approved, but there was a time when the name Taxol was less known to the scientific world. Taxol, being a unique molecule in its structure, properties and mechanism of action, has undergone more than 30 years of trials, and faced a lot of hurdles to leave the bench of a laboratory and reach the shelves of a pharmacy. This review focuses on the story of Taxol, how it began and how it achieved the status of Paclitaxel

Specific functions for Mediator complex subunits from different modules in the transcriptional response of Arabidopsis thaliana to abiotic stress

Adverse environmental conditions are detrimental to plant growth and development. Acclimation to abiotic stress conditions involves activation of signaling pathways which often results in changes in gene expression via networks of transcription factors (TFs). Mediator is a highly conserved co-regulator complex and an essential component of the transcriptional machinery in eukaryotes. Some Mediator subunits have been implicated in stress-responsive signaling pathways; however, much remains unknown regarding the role of plant Mediator in abiotic stress responses. Here, we use RNA-seq to analyze the transcriptional response of Arabidopsis thaliana to heat, cold and salt stress conditions. We identify a set of common abiotic stress regulons and describe the sequential and combinatorial nature of TFs involved in their transcriptional regulation. Furthermore, we identify stress-specific roles for the Mediator subunits MED9, MED16, MED18 and CDK8, and putative TFs connecting them to different stress signaling pathways. Our data also indicate different modes of action for subunits or modules of Mediator at the same gene loci, including a co-repressor function for MED16 prior to stress. These results illuminate a poorly understood but important player in the transcriptional response of plants to abiotic stress and identify target genes and mechanisms as a prelude to further biochemical characterization

Pseudomonas syringae infectivity correlates to altered transcript and metabolite levels of Arabidopsis mediator mutants

Rapid metabolic responses to pathogens are essential for plant survival and depend on numerous transcription factors. Mediator is the major transcriptional co-regulator for integration and transmission of signals from transcriptional regulators to RNA polymerase II. Using four Arabidopsis Mediator mutants, med16, med18, med25 and cdk8, we studied how differences in regulation of their transcript and metabolite levels correlate to their responses to Pseudomonas syringae infection. We found that med16 and cdk8 were susceptible, while med25 showed increased resistance. Glucosinolate, phytoalexin and carbohydrate levels were reduced already before infection in med16 and cdk8, but increased in med25, which also displayed increased benzenoids levels. Early after infection, wild type plants showed reduced glucosinolate and nucleoside levels, but increases in amino acids, benzenoids, oxylipins and the phytoalexin camalexin. The Mediator mutants showed altered levels of these metabolites and in regulation of genes encoding key enzymes for their metabolism. At later stage, mutants displayed defective levels of specific amino acids, carbohydrates, lipids and jasmonates which correlated to their infection response phenotypes. Our results reveal that MED16, MED25 and CDK8 are required for a proper, coordinated transcriptional response of genes which encode enzymes involved in important metabolic pathways for Arabidopsis responses to Pseudomonas syringae infections

Molecular evolution of parsnip (Pastinaca sativa) membrane-bound prenyltransferases for linear and/or angular furanocoumarin biosynthesis

International audienceIn Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different prenyltransferases, Pastinaca sativa prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin

Molecular Cloning and Characterization of a Geranyl Diphosphate-Specific Aromatic Prenyltransferase from Lemon

International audiencePrenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species