Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"?

<p>Abstract</p> <p>Background</p> <p>The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine β-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in <it>Catharanthus roseus </it>or ajmaline in <it>Rauvolfia serpentina</it>. However, very few reports focused on the MIA physiological functions.</p> <p>Results</p> <p>In this study we showed that a strictosidine pool existed <it>in planta </it>and that the strictosidine deglucosylation product(s) was (were) specifically responsible for <it>in vitro </it>protein cross-linking and precipitation suggesting a potential role for strictosidine activation in plant defence. The spatial feasibility of such an activation process was evaluated <it>in planta</it>. On the one hand, <it>in situ </it>hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of <it>C. roseus </it>aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both <it>C. roseus </it>and <it>R. serpentina </it>were localised to the vacuole whereas SGD from both species were shown to accumulate as highly stable supramolecular aggregates within the nucleus. Deletion and fusion studies allowed us to identify and to demonstrate the functionality of CrSTR and CrSGD targeting sequences.</p> <p>Conclusions</p> <p>A spatial model was drawn to explain the role of the subcellular sequestration of STR and SGD to control the MIA metabolic flux under normal physiological conditions. The model also illustrates the possible mechanism of massive activation of the strictosidine vacuolar pool upon enzyme-substrate reunion occurring during potential herbivore feeding constituting a so-called "nuclear time bomb" in reference to the "mustard oil bomb" commonly used to describe the myrosinase-glucosinolate defence system in Brassicaceae.</p

Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis

A single gene encodes isopentenyl diphosphate isomerase isoforms targeted to plastids, mitochondria and peroxisomes in Catharanthus roseus

Isopentenyl diphosphate isomerases (IDI) catalyze the interconversion of the two isoprenoid universal C5 units, isopentenyl diphosphate and dimethylally diphosphate, to allow the biosynthesis of the large variety of isoprenoids including both primary and specialized metabolites. This isomerisation is usually performed by two distinct IDI isoforms located either in plastids/peroxisomes or mitochondria/peroxisomes as recently established in Arabidopsis thaliana mainly accumulating primary isoprenoids. By contrast, almost nothing is known in plants accumulating specialized isoprenoids. Here we report the cloning and functional validation of an IDI encoding cDNA (CrIDI1) from Catharanthus roseus that produces high amount of monoterpenoid indole alkaloids. The corresponding gene is expressed in all organs including roots, flowers and young leaves where transcripts have been detected in internal phloem parenchyma and epidermis. The CrIDI1 gene also produces long and short transcripts giving rise to corresponding proteins with and without a N-terminal transit peptide (TP), respectively. Expression of green fluorescent protein fusions revealed that the long isoform is targeted to both plastids and mitochondria with an apparent similar efficiency. Deletion/fusion experiments established that the first 18-residues of the N-terminal TP are solely responsible of the mitochondria targeting while the entire 77-residue long TP is needed for an additional plastid localization. The short isoform is targeted to peroxisomes in agreement with the presence of peroxisome targeting sequence at its C-terminal end. This complex plastid/mitochondria/peroxisomes triple targeting occurring in C. roseus producing specialized isoprenoid secondary metabolites is somehow different from the situation observed in A. thaliana mainly producing housekeeping isoprenoid metabolites.This work was financially supported by the “Ministère de l’Enseignement Supérieur et de la Recherche” (MESR) and by a grant from the University of Tours. Grégory Guirimand and Anthony Guihur were financed by MESR fellowships.Peer reviewe

Triple subcellular targeting of isopentenyl diphosphate isomerases encoded by a single gene

Isopentenyl diphosphate isomerase (IDI) is a key enzyme of the isoprenoid pathway, catalyzing the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors of all isoprenoids. In plants, several subcellular compartments, including cytosol/ER, peroxisomes, mitochondria and plastids, are involved in isoprenoid biosynthesis. Here, we report on the unique triple targeting of two Catharanthus roseus IDI isoforms encoded by a single gene (CrIDI1). The triple localization of CrIDI1 in mitochondria, plastids and peroxisomes is explained by alternative transcription initiation of CrIDI1, by the specificity of a bifunctional N-terminal mitochondria/plastid transit peptide and by the presence of a C-terminal peroxisomal targeting signal. Moreover, bimolecular fluorescence complementation assays revealed self-interactions suggesting that the IDI likely acts as a multimer in vivo.Peer reviewe

Field-Based Metabolomics of Vitis vinifera L. Stems Provides New Insights for Genotype Discrimination and Polyphenol Metabolism Structuring

Grape accumulates numerous polyphenols with abundant health benefit and organoleptic properties that in planta act as key components of the plant defense system against diseases. Considerable advances have been made in the chemical characterization of wine metabolites particularly volatile and polyphenolic compounds. However, the metabotyping (metabolite-phenotype characterization) of grape varieties, from polyphenolic-rich vineyard by-product is unprecedented. As this composition might result from the complex interaction between genotype, environment and viticultural practices, a field experiment was setting up with uniform pedo-climatic factors and viticultural practices of growing vines to favor the genetic determinism of polyphenol expression. As a result, UPLC-MS-based targeted metabolomic analyses of grape stems from 8 Vitis vinifera L. cultivars allowed the determination of 42 polyphenols related to phenolic acids, flavonoids, procyanidins, and stilbenoids as resveratrol oligomers (degree of oligomerization 1–4). Using a partial least-square discriminant analysis approach, grape stem chemical profiles were discriminated according to their genotypic origin showing that polyphenol profile express a varietal signature. Furthermore, hierarchical clustering highlights various degree of polyphenol similarity between grape varieties that were in agreement with the genetic distance using clustering analyses of 22 microsatellite DNA markers. Metabolite correlation network suggested that several polyphenol subclasses were differently controlled. The present polyphenol metabotyping approach coupled to multivariate statistical analyses might assist grape selection programs to improve metabolites with both health-benefit potential and plant defense traits

A new path for terpenoid biosynthesis

International audienc

ARResting cytokinin signaling for salt-stress tolerance

International audienc

Social amoebae as a new chassis for drug production

International audienc

Rôle des CaaX-prényltransférases dans la régulation de la biosynthèse des alcaloïdes indoliques monoterpéniques chez Catharanthus roseus

Ce travail relate l'étude de l'implication des prénylations de protéines catalysées par les CaaX-prényltransférases, incluant farnésyltransférase (PFT) et géranylgéranyltransférase de type I (PGGT-I), dans la régulation de la synthèse des d'alcaloïdes indoliques monoterpéniques (AIM) chez Catharanthus roseus. Après clonage des ADNc codant la sous-unité (su) bêta de chaque enzyme et la su alpha commune aux PFT et PGGT-I, une caractérisation biochimique des enzymes recombinantes a été initiée montrant l'effet activateur et inhibiteur de phosphorylations sur l'activité des PGGT-I et PFT respectivement. Par une interférence d'ARN ciblant la su bêta de la PFT ou de la PGGT-I, ces deux activités ont été montrées comme requises à l'expression des gènes associés aux étapes précoces de la synthèse du précurseur terpénique des AIM dans une suspension cellulaire. Ce résultat a été conforté in planta par analyse pharmacologique et par localisation tissulaire des transcrits de la su bêta de la PFT.This work aims to study the involvement of protein prenylation catalysed by CaaX-prenyltransferases including farnesyltransferase (PFT) and type I geranylgeranyltransferase (PGGT-I), in the regulation of the biosynthesis of monoterpenoid indole alkaloid (MIA) in Catharanthus roseus. cDNA encoding the beta subunit of each enzyme and the common alpha subunit of PFT and PGGT-I have been cloned. The biochemical characterisation of recombinant enzymes has been initiated, indicating a stimulating and inhibiting effect of phosphorylation on PGGT-I and PFT activity, respectively. RNA interference targeting the beta subunit of PFT or PGGT-I shows that both activities are essential for the expression of genes involved in the early stages of terpenoid precursor of MIA in cell suspension. This result has been also confirmed in planta by pharmacological analysis and tissular localisation of the transcripts of PFT beta subunit.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF