Embriões somáticos de Ocotea catharinensis foram utilizados como modelo para a investigação da via biossintética para a formação de neolignanas com esqueleto benzofurânico, através do uso de precursores marcados com 13C e análise dos produtos por RMN de 13C. Isotopômeros de L-[13C]-fenilalanina administrados aos embriões, foram incorporados às neolignanas 5\'-metoxiporosina e armenina B . Entre os precursores intermediários, o acetato de [8-13C]-coniferila, preparado a partir da condensação de Knoevenagel seguido de várias etapas, foi incorporado na neolignana 5\'-metoxiporosina. No ensaio de bioconversão utilizando frações proteicas das culturas dos embriões de O. catharinensis, o acetato de coniferila foi convertido em isoeugenol que, junto com o eugenol, seriam os precursores hipotéticos para a formação dessas neolignanas. A análise filogenética de diversas espécies de Lauraceae indicou a proximidade entre espécies do gênero Ocotea, produzindo como principais metabólitos lignanas e neolignanas. Foi obtida a seqüência parcial do gene de proteína dirigente em O. catharinensis e O. macrophylla e a análise filogenética baseada na homologia das seqüências de proteína dirigente de Ocotea e de espécies de diferentes gêneros, indicaram um clado distinto formado para espécies de Ocotea. Estes dados dão suporte para a proposta de que neolignanas di-hidrobenzofurânicas seriam oriundas do acoplamento oxidativo entre unidades de E-isoeugenol e 5-metóxi-eugenol, com possível participação de proteína dirigente na regio- e estereoespecificidade das reações de dimerização . Baseando-se nestes resultados foi possível propor algumas etapas envolvidas na biossíntese de neolignanas. / Somatic embryos of Ocotea catharinensis were used as model to investigate the biosynthetic pathway of benzofuran neolignans formation by means of feeding precursors followed by analysis with 13C NMR. Isotopomers of L-[13C]-phenylalanine were administered to embryos and incorporated into di-hydrobenzofuran neolignans. Among the intermediate precursors, [8-13C]-coniferyl acetate, prepared by Knoevenagel and several steps, was incorporated in the neolignan 5\'-methoxyporosin. The studies of bioconversion using protein fraction obtained from the embryogenic cultures, the precursor coniferyl acetate was converted into isoeugenol, which together eugenol, is one of the putative precursors to the neolignans formation. Phylogenetic analysis of several species of Lauraceae indicated the proximity of Ocotea species producing lignans and neolignans as the main metabolites. A partial sequence of the dirigent protein gene from O. catharinensis and O. macrophylla were obtained and the phylogenetic analysis based on homology of dirigent protein sequences from Ocotea and from different plant genus indicated a distinct clade formed by Ocotea species. These findings support the hypothesis that the dihydrobenzofuran neolignans would be derived from the oxidative coupling between E- isoeugenol and 5- methoxy-eugenol and the dirigent protein would be involved in the regio- and stereospecificity of the dimerization reactions. Thus, it was possible to suggest some of the steps involved in the biosynthesis of neolignans.

Embriões somáticos de Ocotea catharinensis foram utilizados como modelo para a investigação da via biossintética para a formação de neolignanas com esqueleto benzofurânico, através do uso de precursores marcados com 13C e análise dos produtos por RMN de 13C. Isotopômeros de L-[13C]-fenilalanina administrados aos embriões, foram incorporados às neolignanas 5\'-metoxiporosina e armenina B . Entre os precursores intermediários, o acetato de [8-13C]-coniferila, preparado a partir da condensação de Knoevenagel seguido de várias etapas, foi incorporado na neolignana 5\'-metoxiporosina. No ensaio de bioconversão utilizando frações proteicas das culturas dos embriões de O. catharinensis, o acetato de coniferila foi convertido em isoeugenol que, junto com o eugenol, seriam os precursores hipotéticos para a formação dessas neolignanas. A análise filogenética de diversas espécies de Lauraceae indicou a proximidade entre espécies do gênero Ocotea, produzindo como principais metabólitos lignanas e neolignanas. Foi obtida a seqüência parcial do gene de proteína dirigente em O. catharinensis e O. macrophylla e a análise filogenética baseada na homologia das seqüências de proteína dirigente de Ocotea e de espécies de diferentes gêneros, indicaram um clado distinto formado para espécies de Ocotea. Estes dados dão suporte para a proposta de que neolignanas di-hidrobenzofurânicas seriam oriundas do acoplamento oxidativo entre unidades de E-isoeugenol e 5-metóxi-eugenol, com possível participação de proteína dirigente na regio- e estereoespecificidade das reações de dimerização . Baseando-se nestes resultados foi possível propor algumas etapas envolvidas na biossíntese de neolignanas. / Somatic embryos of Ocotea catharinensis were used as model to investigate the biosynthetic pathway of benzofuran neolignans formation by means of feeding precursors followed by analysis with 13C NMR. Isotopomers of L-[13C]-phenylalanine were administered to embryos and incorporated into di-hydrobenzofuran neolignans. Among the intermediate precursors, [8-13C]-coniferyl acetate, prepared by Knoevenagel and several steps, was incorporated in the neolignan 5\'-methoxyporosin. The studies of bioconversion using protein fraction obtained from the embryogenic cultures, the precursor coniferyl acetate was converted into isoeugenol, which together eugenol, is one of the putative precursors to the neolignans formation. Phylogenetic analysis of several species of Lauraceae indicated the proximity of Ocotea species producing lignans and neolignans as the main metabolites. A partial sequence of the dirigent protein gene from O. catharinensis and O. macrophylla were obtained and the phylogenetic analysis based on homology of dirigent protein sequences from Ocotea and from different plant genus indicated a distinct clade formed by Ocotea species. These findings support the hypothesis that the dihydrobenzofuran neolignans would be derived from the oxidative coupling between E- isoeugenol and 5- methoxy-eugenol and the dirigent protein would be involved in the regio- and stereospecificity of the dimerization reactions. Thus, it was possible to suggest some of the steps involved in the biosynthesis of neolignans.

A análise filogenética e reconstrução dos caracteres químicos em espécies de Piper indicou clados especializados na produção de ácidos benzoicos/cromenos, amidas e lignóides. Os perfis químicos das espécies foram determinados pela análise dos extratos brutos por RMN de 1H combinados com análise de componente principal (PCA). Destacou-se a ocorrência de lignanas dibenzilbutirolactônicas e furofurânicas no clado Macrostachys, enquanto que para Schilleria foi observado o acúmulo preferencial de fenilpropanóides, lignanas tetraidrofurânicas e benzofurânicas. Espécies de localidades distintas classificadas como Piper solmsianum apresentaram diferentes perfis químicos e características morfológicas. A análise da variabilidade química e genética por PCA e microssatélites, respectivamente, indicou a segregação das populações de acordo com o local de coleta e apontou o potencial para o uso conjunto das técnicas para distinguir os espécimens de P. solmsianum. As sequências dos genes acetiltransferase, propenilfenol sintase e proteína dirigente forneceram as primeiras informações sobre os genes envolvidos na biossíntese de lignanas/fenilpropanoides em P. solmsianum. / The phylogenetic analysis and reconstruction of chemical characters in Piper species distinguished clades specialized in the production of benzoic acids/chromenes, amides and lignoids. The chemical profiles of these species were determined by analysis of crude extracts by 1H NMR combined with principal component analysis (PCA). It is remarkable that lignans of dibenzylbutyrolactone and furofurans type occur in Macrostachys while Schilleria contains dimers of propenylphenols such as tetrahydrofurans lignans and benzofuran neolignans. Piper solmsianum specimens collected from distinct sites showed differences in their morphology and chemical profiles. The assessment of chemical and genetic variability by PCA and microsatellites, respectively, indicated the segregation of populations according to the sampling site and pointed out to the potential use for the combined approaches to distinguish P. solmsianum specimens. The sequences of the genes acetyltransferase, propenylphenol synthase and dirigent protein provided the first information about the genes involved in the biosynthesis of lignans/phenylpropanoids in P. solmsianum.

A análise filogenética e reconstrução dos caracteres químicos em espécies de Piper indicou clados especializados na produção de ácidos benzoicos/cromenos, amidas e lignóides. Os perfis químicos das espécies foram determinados pela análise dos extratos brutos por RMN de 1H combinados com análise de componente principal (PCA). Destacou-se a ocorrência de lignanas dibenzilbutirolactônicas e furofurânicas no clado Macrostachys, enquanto que para Schilleria foi observado o acúmulo preferencial de fenilpropanóides, lignanas tetraidrofurânicas e benzofurânicas. Espécies de localidades distintas classificadas como Piper solmsianum apresentaram diferentes perfis químicos e características morfológicas. A análise da variabilidade química e genética por PCA e microssatélites, respectivamente, indicou a segregação das populações de acordo com o local de coleta e apontou o potencial para o uso conjunto das técnicas para distinguir os espécimens de P. solmsianum. As sequências dos genes acetiltransferase, propenilfenol sintase e proteína dirigente forneceram as primeiras informações sobre os genes envolvidos na biossíntese de lignanas/fenilpropanoides em P. solmsianum. / The phylogenetic analysis and reconstruction of chemical characters in Piper species distinguished clades specialized in the production of benzoic acids/chromenes, amides and lignoids. The chemical profiles of these species were determined by analysis of crude extracts by 1H NMR combined with principal component analysis (PCA). It is remarkable that lignans of dibenzylbutyrolactone and furofurans type occur in Macrostachys while Schilleria contains dimers of propenylphenols such as tetrahydrofurans lignans and benzofuran neolignans. Piper solmsianum specimens collected from distinct sites showed differences in their morphology and chemical profiles. The assessment of chemical and genetic variability by PCA and microsatellites, respectively, indicated the segregation of populations according to the sampling site and pointed out to the potential use for the combined approaches to distinguish P. solmsianum specimens. The sequences of the genes acetyltransferase, propenylphenol synthase and dirigent protein provided the first information about the genes involved in the biosynthesis of lignans/phenylpropanoids in P. solmsianum.

Dans la famille des protéines dirigeantes, AtDIR6, oriente le couplage de radicaux phénoxyles afin de former un lignane optiquement pur : le (-)-pinorésinol. Comme de nombreux lignanes, le pinorésinol est un produit d’intérêt avec des propriétés anti-cancéreuse et anti-fongique. Avec pour objectif à long terme de mettre au point de nouveaux biocatalyseurs énantiosélectifs, nous nous sommes intéressés au mécanisme d’action d’AtDIR6.Dans un premier temps, la production d’AtDIR6 et les conditions de bioconversion ont été optimisées. L’effet dose-dépendant de la protéine sur la formation du (-)-pinorésinol a été confirmé. Les études spectroscopiques entreprises afin de préciser la nature du « substrat » pris en charge par AtDIR6 ont permis de confirmer que l’alcool coniférylique est un mauvais substrat. De plus, par RPE nous avons pu mettre en évidence pour la première fois qu’AtDIR6 augmente la durée de vie de la forme radicalaire de l’AC. Par ailleurs, le greffage covalent d’une sonde radicalaire sur AtDIR6 nous a permis de marquer une tyrosine localisée dans la cavité. Avec AtDIR6 marquée la régiosélectivité de la réaction est affectée. Par ailleurs, la reconnaissance de molécules substrats par AtDIR6 semble être naturellement limitée à un motif 2-methoxy-phénol. Nous montrons pour la première fois que le motif propényle peut être modifié comme en témoigne les produits de bioconversion de l’acétate d’AC pour lesquels une régiosélectivité dépendante de la présence d’AtDIR6 est observée. L’ensemble de nos résultats ouvre des perspectives dans l’utilisation de protéines dirigeantes pour la formation contrôlée de nouveaux produits de couplage. / The family of the leading proteins gathers proteins widely spread within the plant world. In this family, AtDIR6, one of the first characterized proteins, directs the coupling of phenoxyl radicals to form an optically pure lignan: the (-)-pinoresinol. Like many lignans, pinoresinol is a product of interest with anti-cancer and anti-fungal properties. With the long-term goal of developing new enantioselective biocatalysts, we are interested in the mechanism of action of AtDIR6.At first, AtDIR6 production and bioconversion conditions were optimized. The dose-dependent effect of the protein on (-)-pinoresinol formation was confirmed. Spectroscopic studies undertaken to clarify the nature of the "substrate" accommodated by AtDIR6 have confirmed that coniferyl alcohol is a poor substrate. Furthermore, by EPR we have been able to highlight for the first time that AtDIR6 increases the life of the radical form of coniferyl alcohol. Moreover, the covalent grafting of a radical probe on AtDIR6 allowed us to tag a tyrosine located in the cavity. With the tagged protein the regioselectivity of the reaction is affected. On the other hand, the recognition of substrate molecules by AtDIR6 seems to be naturally limited to AC or its radical, apparently recognized because of the 2-methoxyphenol motif. We show for the first time that the propenyl unit can be modified as evidenced by the bioconversion products of AC acetate for which a regioselectivity dependent on the presence of AtDIR6 is observed. All of our results open up prospects for the use of dirigent proteins for the controlled formation of new coupling products.

Plante de grande culture aux multiples applications, le lin accumule dans ses graines des métabolites spécialisés appelés lignanes aux propriétés phytooestrogène et antioxydante, bénéfiques en santé humaine et, dans ses feuilles, des lignanes de nature toxique. Pour l’utilisation et l’étude de ces composés phénoliques issus de la voie des monolignols, une technique d’éco-extraction assistée par les ultrasons a été développée et appliquée à un criblage de différentes variétés pour leur contenu en lignanes conférant une haute valeur ajoutée aux extraits et produits. Les potentiels verrous métaboliques de la voie de biosynthèse de ces composés ont fait l’objet d’investigations au niveau de la régulation transcriptionnelle des gènes responsables de la formation des énantiomères opposés de sécoisolaricirésinol dans la graine et les parties aériennes. La famille multigénique des protéines dirigeantes (DIR) du lin, premiers acteurs de cette voie, a été criblée au niveau génomique et transcriptionnel afin de déterminer des candidates pour fonctionner avec les pinorésinol laricirésinol réductases (PLR), enzymes bifonctionnelles catalysant la formation du sécoisolaricirésinol. L’étude de la régulation fine de l’expression des deux isoformes des gènes PLR a mis en évidence des profils d’inductibilité très contrastés et a conduit à l’identification d’éléments cis-régulateurs ainsi que de facteurs de transcription impliqués dans ces voies de régulation. L’ensemble des résultats convergent vers un rôle de défense des lignanes in planta et a permis de construire une vue d’ensemble des mécanismes complexes de régulation de leur biosynthèse et enfin de proposer des pistes pour l’amélioration de la production de ces molécules naturelles par une stimulation de leur accumulation et l’augmentation des rendements d’extraction par chimie verte. / As a crop with multiple purposes, flax accumulates lignans, specialized metabolites with health benefits, known for their phytoestrogenic and antioxidant properties in its seed, and toxic lignans in its leaves. In order to use and study these phenolic compounds derived from monolignols, an eco-friendly method based on ultrasound-assisted extraction was developed and applied to the screening of cultivars for their lignan content which confers high value to extracts and flaxseed by-product. Regulation of the lignan biosynthetic pathway was investigated at the transcriptional level for the genes responsible for the formation of secoisolariciresinol in seeds and aerial parts. The multigene family of dirigent proteins (DIR), first actors of this pathway, was explored by genomic and transcriptional approaches in order to select candidates to operate with pinoresinol lariciresinol reductases (PLR), bifunctional enzymes catalyzing secoisolariciresinol biosynthesis. The study of the transcriptional regulation of PLR genes evidenced very contrasting expression profiles and led to the identification of transcription factors acting as master regulators of this biosynthesis and their cis-regulatory target elements. Results allowed first to reinforce the hypothesis of the lignans defensive role in planta, second to afford an overall view of complex mechanisms occurring in the regulation of lignan natural production and finally to suggest leads to improve lignan yield by a stimulation of natural production and an enhancement of extraction yield using green chemistry methods.