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

Diversity and Evolution of Sensor Histidine Kinases in Eukaryotes

Histidine kinases (HKs) are primary sensor proteins that act in cell signaling pathways generically referred to as "two component systems" (TCSs). TCSs are among the most widely distributed transduction systems used by both prokaryotic and eukaryotic organisms to detect and respond to a broad range of environmental cues. The structure and distribution of HK proteins are now well documented in prokaryotes but information is still fragmentary for eukaryotes. Here, we have taken advantage of recent genomic resources to explore the structural diversity and the phylogenetic distribution of HKs in the prominent eukaryotic supergroups. Searches of the genomes of 67 eukaryotic species spread evenly throughout the phylogenetic tree of life identified 748 predicted HK proteins. Independent phylogenetic analyses of predicted HK proteins were carried out for each of the major eukaryotic supergroups. This allowed most of the compiled sequences to be categorised into previously described HK groups. Beyond the phylogenetic analysis of eukaryotic HKs, this study revealed some interesting findings: (i) characterisation of some previously undescribed eukaryotic HK groups with predicted functions putatively related to physiological traits; (ii) discovery of HK groups that were previously believed to be restricted to a single kingdom in additional supergroups and (iii) indications that some evolutionary paths have led to the appearance, transfer, duplication, and loss of HK genes in some phylogenetic lineages. This study provides an unprecedented overview of the structure and distribution of HKs in the Eukaryota and represents a first step towards deciphering the evolution of TCS signaling in living organisms

Vers la bioproduction de métabolites anticancéreux par les levures

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De novo production of the plant-derived alkaloid strictosidine in yeast

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Design, Synthesis and Evaluation of New Marine Alkaloid-Derived Pentacyclic Structures with Anti-Tumoral Potency

This work describes the synthesis and biological evaluation of a new heterocyclic hybrid derived from the ellipticine and the marine alkaloid makaluvamine A. Pyridoquinoxalinedione 12 was obtained in seven steps with 6.5% overall yield. 12 and its intermediates 1–11 were evaluated for their in vitro cytotoxic activity against different cancer cell lines and tested for their inhibitory activity against the human DNA topoisomerase II. The analysis by electrophoresis shows that the pentacycle 12 inhibits the topoisomerase II like doxorubicine at 100 µM. Compound 9 was found to have an interesting profile, having a cytotoxicity of 15, 15, 15 and 10 μM against Caco-2, HCT-116, Pc-3 and NCI cell lines respectively, without any noticeable toxicity against human fibroblast

Genetic Manipulation of Meyerozyma guilliermondii

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Prenylated Proteins Are Required for Methyl-Jasmonate-Induced Monoterpenoid Indole Alkaloids Biosynthesis in Catharanthus roseus

In Catharanthus roseus, monoterpenoid indole alkaloids (MIA) result from the condensation of the indole precursor tryptamine with the terpenoid precursor secologanin, which is derived from the plastidial methyl-d-erythritol 4-phosphate (MEP) pathway. Nevertheless, inhibition of the classical so-called mevalonate pathway leads to inhibition of MIA biosynthesis, suggesting that there is some cross regulation between these two pathways. The purpose of this chapter is to outline a new function for protein prenylation. Our results suggest that prenylated proteins, apparently mevalonate pathway end products, act as part of the regulatory mechanism coordinating the exchange of metabolites between compartmentalized metabolic pathways and that this process is governed by methyl jasmonate. Methyl jasmonate is a major inducer of alkaloid biosynthesis through enhancing MEP pathway gene expression. In C. roseus cells, inhibition of protein prenylation leads to the down-regulation of methyl-jasmonate-induced expression of MEP pathway genes and thus abolishes MIA biosynthesis. Jointly, failure of protein prenylation also inhibits the methyl-jasmonate-induced expression of the transcription factor ORCA3 which acts as a central regulator of MIA biosynthesis. Furthermore, the specific silencing of protein prenyltransferases in C. roseus cells mediated by RNA interference shows that inhibition of type I protein geranylgeranyltransferase down-regulates the expression of ORCA3. These data point to a specific role of protein geranylgeranylation in jasmonate signalling leading to MIA formation

Identifying Missing Biosynthesis Enzymes of Plant Natural Products

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