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

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

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

Caractérisations histologique, moléculaire et biochimique des interactions compatible et incompatible entre Erwinia amylovora, agent du feu bacterien, et le pommier (Malus x domestica)

Fire blight of Maloideae is caused by the necrogenic gamma-proteobacterium Erwinia amylovora (Ea), whose pathogenicity relies primarily on its ability to inject proteinaceous effectors in the host cells through a type III secretion system. This study aimed at better characterising the Ea-Evereste/MM106 pathosystem, to gain some insight into the mechanisms drivingthe fate of the interaction. The results showed that the bacteria preferentially target leaf spongy parenchyma and stem cortical parenchyma cells and that their progression was accompagnied by a strong repression of the jasmonic acid signalling pathway and a strong induction of the salicylic signalling pathway. On the contrary, several lines of evidence argue for a fast blockade of the bacteria in Evereste infiltrated leaves : (i) an important cell wall lignification takes place under the vascular sclerenchyma, (ii) vascular occlusions appear and (iii) transient modulations of defence gene expression also take place. The fast blockade of the bacteria suggests the existence of constitutive defences. The biochemical study of dihydrochalcones ( DHC), flavonoid compounds that are found in large amounts in apple leaves, suggests that those compounds are unlikely to participate directly in the resistance to fire blight, in spite of their bactericidal and antioxidant properties. However, transformation of the constitutive DHC through enzymatic reactions such as dglucosylation and oxidation might lead to specific compouds, given the intensity of each process. Those compounds might participate to the inducible resistance against Ea.Le feu bactérien des Maloïdées ( Pommier, Poirier, ... ) est causé par la gamma-protéobactérie nécrogène, Erwinia amylovora (Ea), dont le pouvoir pathogène réside essentiellement dans sa capacité d'injection d'effecteurs protéiques dans la cellule hôte par un système de sécrétion de type III. L'objectif de la présente étude était de caractériser le pathosystème Ea-Pommier afin d'identifier les mécanismes régissant le devenir de l'interaction. Les résultats ont permis de montrer que la bactérie attaque principalement, chez un Pommier sensible (génotype MM106), le parenchyme lacuneux des feuilles et le parenchyme cortical des pétioles et tiges, et que cette progression s'accompagne d'une forte répression de la voie de l'acide jasmonique, et d'une forte activation de la voie de l'acide salicylique. A l'inverse, au niveau des feuilles d'un génotype de Pommier résistant (Evereste) plusieurs éléments sont en faveur d'un blocage rapide de la bactérie : (i) lignification importante du sclérenchyme sous-vasculaire dans les nervures, (ii) occlusion du xylème et (iii) modulations transitoires de l'expression de gènes de défense. Ce blocage rapide suggère par ailleurs l'implication de défenses constitutives. L'étude biochimique des dihydrochalcones (DHC), des flavonoïdes présents en très forte quantités dans les feuilles de Pommier, suggère qu'elles ne sont pas impliquées directement dans la résistance constitutive à Ea, malgré leurs propriétés antioxydante et antibactérienne. En revanche, il semble que leur transformation enzymatique, déglucosylation et oxydation, aboutissent, en fonction de leur prépondérance, à des produits qui pourraient jouer un rôle dans l'interaction

Predicting Monoterpene Indole Alkaloid-Related Genes from Expression Data with Artificial Neural Networks

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RNA-seq Analysis of Monoterpene Indole Alkaloid Biosynthetic Pathway Elucidation in Catharanthus roseus

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Chromosome-scale genomes throw light on plant drug biosynthesis

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Computational biotechnology guides elucidation of the biosynthesis of the plant anticancer drug camptothecin

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An Erwinia amylovora inducible promoter for improvement of apple fire blight resistance

International audienceAbstract Key message pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes. Abstract Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora ( Ea ) or moderately constitutive, we focused on polyphenoloxidase genes ( PPO ). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be upregulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 ( pPPO16 ) and MdKFDV02 PPO -like ( pKFDV02 ) for their potential as Ea -inducible and low-constitutive regulatory sequences, respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results does not confirmed pKFDV02 as a constitutive and weak promoter whereas pPPO16 , the first Ea -inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes

The epigenetic regulation of HsMar1, a human DNA transposon

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