Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily

Background Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as ‘blooms’, providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects.[br/] Results A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ.[br/] Conclusion Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 ‘blooms’ in response to environmental pressures

Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis

Ammi majus L. accumulates linear furanocoumarins by cytochrome P450 (CYP)-dependent conversion of 6-prenylumbelliferone via (؉)-marmesin to psoralen. Relevant activities, i.e. psoralen synthase, are induced rapidly from negligible background levels upon elicitation of A. majus cultures with transient maxima at 9 -10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocoumarin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for (؉)-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via (؉)-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, (؉)-columbianetin failed as substrate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate (؉)-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution. Furanocoumarins are produced by many plants, mostly of the Apiaceae, Rutaceae, Moraceae, or the Coronilla and Psoralea genera of the Fabaceae (1-3). Multiple pharmacological effects have been ascribed to several of these metabolites (4 -6), which were included in clinical screenings but received attention also for their inhibitory effect on monooxygenases involved in drug metabolism (7-9) and potential toxicity (10). The (dihydro)furan-substituted 2H-1-benzopyran-2-one forms the characteristic core structure, and the annulation type distinguishes the linear furanocoumarins or psoralens from the angular furanocoumarin

The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants

International audienceThe production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from Ammi majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae

Etude de la biologie de plantes du genre Psoralea (legumineuses), productrices de furocoumarines a interet pharmaceutique. Essais de cultures in vitro

SIGLEINIST T 71961 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

ETUDE DE LA PRODUCTION DE FUROCOUMARINES PAR LA RUE OFFICINALE (RUTA GRAVEOLENS L.) (CULTURES DE PLANTES AU CHAMP ET CULTURES IN VITRO)

LES FUROCOUMARINES, METABOLITES SECONDAIRES DE PLANTES, PRESENTENT UN INTERET ECONOMIQUE POUR LEUR UTILISATION EN DERMATOLOGIE. LA SYNTHESE CHIMIQUE DES FUROCOUMARINES ETANT TROP ONEREUSE, LEUR APPROVISIONNEMENT DOIT ETRE ASSURE PAR DES VEGETAUX. NOS ETUDES ONT ETE CONDUITES AFIN D'EVALUER LA POSSIBILITE DE PRODUIRE LES FUROCOUMARINES A PARTIR DE RUTA. DEUX TYPES DE CULTURES ONT ETE ETUDIES : DES PLANTES ENTIERES ET DES SUSPENSIONS CELLULAIRES. LA PRODUCTION ET LE PROFIL DE 4 FUROCOUMARINES (PSORALENE, XANTHOTOXINE, ISOPIMPINELLINE ET BERGAPTENE) ONT ETE DETERMINES DANS DIVERSES CONDITIONS DE CULTURES. NOUS AVONS ETUDIE LA TENEUR EN FUROCOUMARINES CHEZ 4 ESPECES DU GENRE RUTA. AGRONOMIQUEMENT, RUTA GRAVEOLENS EST LA PLUS INTERESSANTE : ELLE PRODUIT BEAUCOUP DE MATIERE SECHE, A UNE BONNE RESISTANCE AU FROID ET UNE FORTE TENEUR EN FUROCOUMARINES (DE 4 A 17 MG.G 1 MS DANS LES FEUILLES). LES FRUITS ET LES FEUILLES CONTIENNENT DE 60 A 70% DES FUROCOUMARINES DE LA PLANTE. L'EFFET DE PLUSIEURS FACTEURS (STADE DE DEVELOPPEMENT, COUPES ANNUELLES, DENSITE, FERTILISATION AZOTEE) ONT ETE TESTES SUR LA PRODUCTIVITE EN FUROCOUMARINES. LES PLANTES PRODUISENT UN MAXIMUM DE FUROCOUMARINES AU STADE FRUCTIFICATION. LES COUPES ANNUELLES AMELIORENT LA PRODUCTIVITE. LA FERTILISATION AZOTEE ET LA DENSITE N'ONT PAS EU D'EFFET SIGNIFICATIF DANS NOS CONDITIONS. CES ESSAIS AU CHAMP ONT ETE CONDUITS SUR PLUSIEURS SITES ET ONT MIS EN EVIDENCE UN FORT EFFET TERROIR. ENFIN, DES SUSPENSIONS CELLULAIRES DE RUTA GRAVEOLENS ONT ETE CULTIVEES EN ERLEN ET EN BIOREACTEUR. LEUR TENEUR EN FUROCOUMARINES EST COMPRISE ENTRE 0,3 ET 19 MG.G 1 MS. L'ELICITATION PAR VERTICILLIUM DAHLIAE A AUGMENTE LA PRODUCTION DE FUROCOUMARINES. L'IMMOBILISATION DES CELLULES A FAVORISE LE RELARGAGE DES METABOLITES DANS LE MILIEU. LE PROFIL DES FUROCOUMARINES EST FORTEMENT MODIFIE PAR CES FACTEURS. LES SUSPENSIONS ONT UNE MEILLEURE PRODUCTIVITE QUE LES PLANTES ENTIERES, MAIS ELLES NE SONT PAS ECONOMIQUEMENT RENTABLES.NANCY/VANDOEUVRE-INPL (545472102) / SudocSudocFranceF

The distribution of coumarins and furanocoumarins in Citrus species closely matches Citrus phylogeny and reflects the organization of biosynthetic pathways

In 2011, citrus crops represented one of the most important fruit productions in the world with more than 131 million tons. Citrus belong to the Rutaceae family able to produce coumarins which constitute a class of secondary metabolites commonly found in higher plants. In these plants, 7-hydroxycoumarin can undergo a subsequent two step enzymatic modification corresponding firstly to the prenylation at C6 and secondly to the closure of a furan ring, leading to furanocoumarins. In parallel to ecological functions in plants, these molecules can be deleterious for humans. They are potential photosensitizers that can cause photophytodermatitis either after skin contact or ingestion followed by UV A exposure. This photosensitization property is a notable problem with Citrus essential oils, such as bergamot oil, since they are extensively used in perfumes.In this study, coumarin and furanocoumarin contents found in 61 citrus varieties are compared and the chemical diversity is discussed with respect to the genetic diversity. Based on hierarchical ascendant classification, coumarins and furanocoumarins can be separated into 4 groups. Each of these 4 groups is logically organized with respect to the already described plant biosynthetic pathways and allows drawing hypotheses on the doubtful biosynthetic origin of compounds. With reference to Citrus diversity obtained from molecular markers, we highlight the genetic crossings which may have resulted in low coumarin and furanocoumarin content varieties.Citrus varieties identified in this study with low coumarin and furanocoumarin content will constitute invaluable genetic resources to breeding programs, promoting citrus species devoid of these toxic molecules

From bioreactor to entire plants: development of production systems for secondary metabolites

International audienceThe production of secondary metabolites, and more specifically alkaloids, from medicinal plants is still an important objective for many research programs. When natural lead compounds have been discovered and when chemical synthesis cannot be easily performed, the extraction and purification of biomolecules from entire plants is generally the preferred solution. However, it is now established that plant cells and tissue cultures in bioreactors can constitute an alternative solution to this agronomical approach. Our research programs devoted to the production of tropane alkaloids from Datura innoxia and furanocoumarins from Ruta graveolens have shown that hydroponics and aeroponics, techniques situated in-between field and fermentor scales, enable the entire plants to be used as efficient bioreactors. Revisiting scientific advances made in the past decades, the ethical, legal, biological and technological aspects are discussed in the light of the most recent literature, in order to establish a roadmap for further developments of plant secondary metabolite production systems

The distribution of coumarins and furanocoumarins in Citrus species closely matches Citrus phylogeny and reflects the organization of biosynthetic pathways

In 2011, citrus crops represented one of the most important fruit productions in the world with more than 131 million tons. Citrus belong to the Rutaceae family able to produce coumarins which constitute a class of secondary metabolites commonly found in higher plants. In these plants, 7-hydroxycoumarin can undergo a subsequent two step enzymatic modification corresponding firstly to the prenylation at C6 and secondly to the closure of a furan ring, leading to furanocoumarins. In parallel to ecological functions in plants, these molecules can be deleterious for humans. They are potential photosensitizers that can cause photophytodermatitis either after skin contact or ingestion followed by UV A exposure. This photosensitization property is a notable problem with Citrus essential oils, such as bergamot oil, since they are extensively used in perfumes.In this study, coumarin and furanocoumarin contents found in 61 citrus varieties are compared and the chemical diversity is discussed with respect to the genetic diversity. Based on hierarchical ascendant classification, coumarins and furanocoumarins can be separated into 4 groups. Each of these 4 groups is logically organized with respect to the already described plant biosynthetic pathways and allows drawing hypotheses on the doubtful biosynthetic origin of compounds. With reference to Citrus diversity obtained from molecular markers, we highlight the genetic crossings which may have resulted in low coumarin and furanocoumarin content varieties.Citrus varieties identified in this study with low coumarin and furanocoumarin content will constitute invaluable genetic resources to breeding programs, promoting citrus species devoid of these toxic molecules

Distribution of phenolic compounds across contrasting citrus cultivars using a UPLC-MS approach (phenolic compound landscape in citrus)

organisateurs : Bellon Stéphane, Bertin Nadia Favery Armelle Girerd-Potin Caroline Jouinot Simon Penvern Servane Simon Sylvaine Urban Laurent Warlop françoisAccession Number : Agritrop : 579096Label : F60; F30; U40The polyphenol family encompasses several classes of molecules corresponding to different branching points of the biosynthetic pathway. Citrus plants produce large amounts of phenolic compounds, especially flavanones, flavones and coumarins. Some of them are specific to Citrus. In Citrus, these bioactive compounds are involved in fruit quality as well as in plant defense and therefore are a target of breeding programs with some compounds being desired and some others avoided. Many studies have characterized their composition in Citrus. However, few studies reported several classes of polyphenols in a single analysis. In addition, although phenolic compounds vary strongly with developmental and environmental conditions, these factors are often omitted thus preventing comparative analyses. To overcome these problems, we took care to collect plant organs experiencing similar environmental conditions. This step is essential to link biochemical data to the key steps of the pathway involved in genetic variability or shifts in metabolite biosynthesis in different tissues. Then, we developed a high-throughput procedure that permitted rapid reconstruction of the distribution of main classes of soluble polyphenols in contrasting tissues and genotypes. We evidence that the combination of datasets from contrasting tissues improved cultivar classification based on polyphenol contents. Drawing a map of the distribution of phenolic compounds across cultivars highlights potential regulatory nodes of the biosynthetic pathway. In addition to the steps catalyzed by rhamnosyltransferases, we proposed that the steps catalyzed by phenylalanine ammonia-lyase, the step conducing to 2',4'-dihydrocinnamic acid from p-coumaric acid and the step involving flavone synthase were important regulatory nodes in in “Clementine” and “Star Ruby” grapefruit

Les plantes, sources d'acides gras essentiels omega 3

National audienceElonging to the omega-3 fatty acid group, alpha -linolenic acid (ALA) is considered an essential fatty acid since it can not be produced by the body and must be provided by diet. Plants are important sources of ALA, from fruits and seeds to green vegetables. This survey showed the relative importance of plant material as ALA sources.L'acide α-linolénique (ALA) fait partie de la famille des acides gras dits oméga 3. Il est dit essentiel parce qu'il ne peut pas être synthétisé dans notre organisme et doit donc être fourni par l'alimentation. Les plantes, sous forme de fruits, de légumes verts, de graines ou d'huiles issues de ces graines constituent une source majeure d'ALA. Cette synthèse a pour but de rappeler l'importance relative des végétaux quant à leur capacité à nous fournir des acides gras essentiels. Elle vise à faire le point sur les différentes voies développées par l'industrie agroalimentaire et à envisager les développements futur