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

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 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

Clonage et caractérisation de gènes corrélés à la biosynthèse des alcaloïdes indoliques monoterpéniques de Catharanthus roseus

TOURS-BU Sciences Pharmacie (372612104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Towards the Microbial Production of Plant-Derived Anticancer Drugs

International audienc

Virus-Induced Gene Silencing: Hush Genes to Make Them Talk

International audienc

A type-B response regulator drives the expression of the hydroxymethylbutenyl diphosphate synthase gene in periwinkle.

International audienceIn plant cytokinin (CK) signaling, type-B response regulators (RRs) act as major players in orchestrating the transcriptome changes in response to CK. However, their direct targets are poorly known. The identification of putative type-ARR1 motifs located within the promoter of the CK-responsive hydroxyl methyl butenyl diphosphate synthase (HDS) gene from the methyl erythritol phosphate (MEP) pathway prompted us to investigate the ability of a previously isolated periwinkle type-B RR (CrRR5) that presents high homologies with ARR1 to interact with the promoter. Electrophoretic mobility shift assays (EMSAs) demonstrated that the CrRR5 DNA-binding domain binds specifically type-ARR1 motifs within the HDS promoter. We also established through yellow fluorescent protein (YFP) imaging the targeting of CrRR5 into cell nucleus in accordance with its putative function of transcription factor. In transient assays performed on periwinkle cells cultivated with CK, overexpression of the full-length CrRR5 or a truncated CrRR5 engineering a constitutive active form (35S:ΔDDK) did not affect the HDS promoter activity that reached a threshold. By contrast, in absence of CK, overexpression of CrRR5ΔDDK enhanced promoter activity up to the threshold level observed in cells grown with CK. Our results strongly suggest that CrRR5 directly transactivates the HDS promoter. CrRR5 is the first identified transcription factor mediating the CK signaling that targets a gene from the MEP pathway involved in isoprenoid metabolism. Moreover, CrRR5 could play a role in a regulatory mechanism controlling CK homeostasis in periwinkle cells

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

International audienceIsopentenyl 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

Synthetic Biology in the Candida (CTG) Clade

International audiencePlant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale