Southern Magnolia (Magnolia grandiflora) is a primitive tree species that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Terpenoid constituents were determined from Magnolia leaves and flowers. Magnolia leaves constitutively produced two major terpenoids, andamp;acirc;-cubebene and germacrene A. However, upon wounding Magnolia leaves biosynthesized a significant array of monoand sesquiterpenoids, including andamp;acirc;-pinene, trans-andamp;acirc;-ocimene, andamp;aacute;-gurjunene, andamp;acirc;-caryophyllene and andamp;acirc;-cubebene, along with fatty acid derivatives such as cis-jasmone, for up to 19 hours after treatment. Flowers were also examined for their emission of terpene volatiles prior to and after opening, and also in response to challenge by Japanese beetles. Opened and un-opened flowers constitutively emitted a blend of monoterpenes dominated by andamp;acirc;-pinene and cis-andamp;acirc;-ocimene. However, the emission levels of monoterpenes such as verbenone, geraniol, and citral, and sesquiterpenes such as andamp;acirc;-cubebene, andamp;aacute;-farnesene, and andamp;acirc;-caryophyllene were significantly elevated in the emissions of the beetle-challenged flowers. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young Magnolia leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominantly to andamp;acirc;-cubebene, while Mg17 converted GPP (C5) to andamp;aacute;-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron/exon organizations for the three Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only 4 introns, and Mg25 has only a single intron near the 5 terminus of the gene. Our results suggest that much of the structural diversity observed in the Magnolia TPS genes may have occurred by means other than intron-loss from a common ancestor TPS gene. Costunolide is a sesquiterpene lactone widely recognized for its diverse biological activities, including its bitter taste in lettuces, and as a precursor to the more potent pharmacological agent parthenolide. A lettuce EST database was screened for cytochrome P450 genes that might be associated with sesquiterpene hydroxylation. Five ESTs were selected based on sequence similarity to known sesquiterpene hydroxylases and three of them (Ls7108, Ls3597 and Ls2101) were successfully amplified as fulllength cDNAs. To functionally characterize these cDNAs, they were co-expressed along with a germacrene A synthase and a cytochrome P450 reductase in yeast. Based on product profile comparisons between the three different lines to the control line, only the Ls7108-harboring line produced unique compounds. Neither of the other lines showed a new product peak. The more abundant, polar product generated by the Ls7108-containing line was purified and identified as a 12-acetoxy-germacrene by NMR analysis. In vitro studies using Ls7108 microsomal proteins did not yield the 12-acetoxy-germacrene A, but the putative germacra-1(10),4,11(13)-trien-12-ol intermediate. Catalytic activity of the Ls7108 microsomal enzyme was NADPH, pH and time dependent. Our results demonstrate that Ls7108 is a lettuce cytochrome P450 which catalyzes the hydroxylation of a methyl group of the isopropenyl substituent of germacrene A, generating germacra-1(10),4,11(13)-trien-12-ol, and that when this mono-hydroxylated sesquiterpene is synthesized in yeast, an endogenous yeast enzyme further modifies the germacrenol compound by acetylation of the alcohol group at the C-12 position.

Secondary metabolites are compounds that are not necessary for the survival of the organism, but which are to be related to the organism's interaction with its environment. This work studied compounds of secondary metabolism through the study of chemical and biological extracts and fractions Simaba Maiana Casar. and the involvement of candidate genes (TPS10, TPS14, and CYP71B31 CYP76C3) in the metabolism of monoterpenes in Arabidopsis thaliana. The extracts from roots and stems of Simaba Maiana were tested in antioxidant activity, molluscicide, inhibition of lymphocyte proliferation, inhibition of NO production, anti-Leishmania amazonensis and anti-Trypanosoma cruzi. The four candidate genes (CYP76C3, CYP71B31, TPS10 and TPS14) were selected with the CYPedia, which calculates the coexpression between genes based on the Arabidopsis Affymetrix ATH1 microarray. The extracts and fractions Simaba Maiana showed a lower antioxidant activity by DPPH method, low concentrations of total phenols measured by Folin-Ciocalteu, however, a good antioxidant activity by TBARS method, using three agents of oxidative damage (AAPH, FeSO4 and H2O2). The extract showed cytotoxic activity and molluscicidal concentration of 100 mg/mL. The crude extract of the stem was not active for leishmanicidal and trypanocidal. This extract did not inhibit NO production, but showed a high percentage of inhibition of lymphoproliferation. The skimmianine furoquinoline alkaloid and pellopterin furanocoumarin were isolated from chloroform fraction. The candidate genes showed a similar pattern of expression of the stamen, more specifically the top of the filaments. Heterologous expression in transiently expressed in leaves of Nicotiana benthamiana, in the volatiles of TPS10 TPS14 (alone) were found enantiomers R-(-)-linalool and S-(+)-linalool. In the extraction buffer leaf discs were found that CYP76C3 converts linalool to E-8-hydroxy-linalool and E-8-oxo-linalool, and CYP71B31 in 1,2-epoxy-linalool. The analysis of the methanol extract of the discs incubated in S-(+)-linalool showed the use of this substrate by P450s converting to lilac alcohol for both P450s. Analysis of flowers of Arabidopsis mutants showed minor differences, analyzes with extracts of fresh flowers in UPLC-MS/MS MRM mode, has been found a compound having the same signature as linalool, however with different retention time and may be is an indication of linalool bound. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os metabólitos secundários são compostos que não são necessários para a sobrevivência do organismo, mas que apresentam-se relacionados com a interação do organismo com o seu ambiente. O presente trabalho estudou compostos do metabolismo secundário através do estudo químico e biológico dos extratos e frações Simaba maiana Casar. e a envolvimento dos genes candidatos (TPS10, TPS14, CYP76C3 e CYP71B31) no metabolismo de monoterpenóides em Arabidopsis thaliana. Os extratos das raízes e caule de Simaba maiana foram testadas em ensaios de atividade antioxidante, moluscicida, inibição da linfoproliferação, inibição da produção de NO, anti-Leishmania amazonensis e anti-Trypanosoma cruzi. Os quatro genes candidatos (CYP76C3, CYP71B31, TPS10 e TPS14) foram selecionados com o CYPedia, que calcula a co-expressão entre os genes de Arabidopsis com base no Affymetrix ATH1 microarray. Os extratos e frações de Simaba maiana mostraram uma baixa atividade antioxidante pelo método do DPPH, baixas concentrações de fenóis totais avaliados pelo método de Folin-Ciocalteu, entretanto, uma boa atividade antioxidante pelo método de TBARS, usando três agentes de danos oxidativos (AAPH, FeSO4 e H2O2). Os extratos mostraram atividade moluscicida e citotóxica na concentração de 100 mg/mL. O extrato bruto do caule não foi ativo para as atividades anti-Leishmania e anti-Trypanosoma. Este extrato não inibiu a produção de NO, mas apresentou uma alta porcentagem da inibição da linfoproliferação. O alcalóide furoquinolínico esquiamina e a furanocumarina felopterina foram isolados da fração clorofórmica. Os genes candidatos mostraram um similar padrão de expressão nos estames, mais especificamente na parte superior dos filamentos. Na expressão heteróloga transitoriamente expressa em folhas de Nicotiana benthamiana, nos voláteis de TPS10 e TPS14 (sozinho) foram encontrados os enantiômeros R-(-)-linalol e S-(+)-linalol. No tampão de extração de discos de folhas, verificou-se que CYP76C3 converte linalol em E-8-hidroxi-linalol e E-8-oxo-linalol, e CYP71B31 em 1,2-epoxilinalol. A análise do extrato metanólico dos discos foliares incubados em S-(+)-linalol mostrou a utilização deste substrato por P450s convertendo para lilac álcool para ambos os P450s. Análises de flores em plantas mutantes de Arabidopsis thaliana mostraram pequenas diferenças, em que análises com extratos de flores frescas em UPLC-MS/MS no modo MRM, foi encontrado um composto com a mesma assinatura que linalol, no entanto, com tempo de retenção diferente e pode ser uma indicação de forma ligada do linalol.

No description available.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-04T13:14:56Z No. of bitstreams: 1 diegopandelojose.pdf: 913814 bytes, checksum: 7e149c7c8e0bee3253c4894426a6892c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-04T15:36:35Z (GMT) No. of bitstreams: 1 diegopandelojose.pdf: 913814 bytes, checksum: 7e149c7c8e0bee3253c4894426a6892c (MD5) / Made available in DSpace on 2017-04-04T15:36:35Z (GMT). No. of bitstreams: 1 diegopandelojose.pdf: 913814 bytes, checksum: 7e149c7c8e0bee3253c4894426a6892c (MD5) Previous issue date: 2009-03-03 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / O gênero Lippia pertence à família Verbenaceae, inclusa no clado Asteridaee, ordem Lamiales, compreendendo aproximadamente 175 gêneros e 2800 espécies, onde muitos gêneros apresentam plantas com propriedades medicinais e ornamentais. A espécie Lippia alba, originária da América do Sul, também ocorre no Brasil e é uma das mais estudadas do gênero Lippia. Ela floresce durante o ano todo e recebe grande destaque no gênero, devido às suas inúmeras propriedades medicinais. O óleo essencial de Lippia alba é composto basicamente por sesqui e monoterpenos, que são as substâncias responsáveis por suas propriedades medicinais. O objetivo central do presente trabalho foi clonar e analisar a expressão de dois potenciais genes codificadores de terpeno sintases em Lippia alba. Através do alinhamento de genes codificadores de monoterpeno sintases caracterizadas, primers degenerados foram desenhados dentro de regiões conservadas e utilizados para se obter a clonagem de genes codificadores de terpeno sintases em Lippia alba. Dois potenciais genes codificadores de terpeno sintases foram clonados, LaTPS12 e LaTPS23. Após a clonagem, técnicas de RT-PCR semiquantitativo foram empregadas para análises de expressão desses dois genes em diferentes estágios foliares e em três diferentes quimiotipos de Lippia alba. Os resultados mostraram que em folhas situadas no quarto segmento nodal o gene LaTPS12 apresenta maior nível de expressão. A diferença na expressão do gene LaTPS23 foi menos acentuada nos três quimiotipos analisados em relação ao gene LaTPS12, que apresentou uma expressão diferencial. Análises filogenéticas foram realizadas comparando-se as seqüências desses dois genes com outros genes codificadores de terpeno sintases já caracterizadas de diferentes espécies de plantas. De acordo com essas análises, LaTPS12 e LaTPS23 pertencem à classe TPS-b, que é composta principalmente por monoterpeno sintases de angiospermas. / The genus Lippia belongs to Verbenaceae family, Asteridaee, order Lamiales. This family comprises about 175 genus and 2800 species, and many of them have medicals and ornamentals proprierties. Lippia alba is native from South America, and is also found in Brazil and is the most studied species of the genus Lippia. This plant blooms throughout the year and has great importance due to its medicinal properties. The Lippia alba essential oils are composed by sesquiterpenes and monoterpenes conferring its medicinal properties. The aim of this work was to clone and to analize gene expression of putative terpene synthases genes (TPS) in Lippia alba. Alignment of TPS genes was used to design degenerate primers into conserved domains for cloning of these genes in Lippia alba. We have cloned two putative TPS genes, LaTPS12 and LaTPS23. After cloning, semiquantitative RT-PCR was employed to expression analysis of these two genes in different leaf stages and among three different chemotypes of Lippia alba. The result of expression level showed that LaTPS12 occurred at higher level in leaves located in fourth nodal segment and showed a marked differential expression among the chemotypes. The difference of expression of the LaTPS23 was less prominent comparing the three studied chemotypes. We performed a phylogenetic analysis in order to compare the LaTPS12 and LaTPS23 to others TPS genes in different plant species. The results showed that these LaTPS12 and LaTPS23 belong to the class TPS-b, which comprises mainly angiosperms monoterpene synthases genes.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-06-21T14:43:48Z No. of bitstreams: 1 viniciuscariusdesouza.pdf: 3785363 bytes, checksum: 7063d6c5f5cef353643903ad5f125c48 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:09:52Z (GMT) No. of bitstreams: 1 viniciuscariusdesouza.pdf: 3785363 bytes, checksum: 7063d6c5f5cef353643903ad5f125c48 (MD5) / Made available in DSpace on 2017-08-07T19:09:52Z (GMT). No. of bitstreams: 1 viniciuscariusdesouza.pdf: 3785363 bytes, checksum: 7063d6c5f5cef353643903ad5f125c48 (MD5) Previous issue date: 2016-03-03 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Lippia alba, popularmente conhecida por erva-cidreira, é uma espécie vegetal amplamente distribuída pelas Américas e encontrada praticamente em todo o território brasileiro. Esta espécie possui importante uso na medicina tradicional para o tratamento de cólicas, indigestão, náuseas, espasmos, diarreia, disenteria, doenças respiratórias, problemas hepáticos e no tratamento de sífilis e gonorreia. As folhas de L. alba, as quais são preparadas sob a forma de infusão ou decocção e ingeridas por via oral, produzem um óleo essencial rico em moléculas iso-prenóides denominadas terpenóides. Estes compostos não são apenas de interesse farmacológico, mas também industrial já que são usados na confecção de fragrâncias. A composição dos óleos essenciais pode variar em função de diferentes fatores abióticos e genotípicos, como por exemplo nível de ploidia. Neste contexto, os objetivos deste trabalho foram caracterizar o transcriptoma de folha da espécie L. alba e buscar sequencias putativas de enzimas envolvidas na produção de metabólitos secundários. O transcriptoma foi sequenciado pela plataforma Miseq (Illumina) com bibliotecas pairedend de 300 bp. O sequenciamento resultou em um total de 47.498.310 reads paired-end (23.749.155 reads para cada end sequenciado) de 35-308 bp, compreendendo 12.148.327.567 nucleotídeos (-12 Gb). A montagem de novo dos transcritos foi processada a partir do software Trinity que gerou 193.532 transcritos, sendo 128.209 unigenes, com o valor de N50 igual a 1.187 bp. Um total de 86.122 ORF (Open Read Frame) foi obtido e a seguir submetido ao algoritmo de alinhamentos BlastP, o qual encontrou 75.533 sequências com referência no banco de dados NR (Non-Redundant) de proteínas. Aproxima-damente, 78,4% dessas sequências foram anotadas funcionalmente a partir do pipeline utiliza-do pelo software Blast2GO. As análises das sequências anotadas revelaram prováveis enzimas para síntese de terpenóides como geraniol e linalol/nerolidol. Para validação da montagem e anotação, foram realizados ensaios de qPCR para amplificação de sequências de 13 genes para controles endógenos e 4 genes de terpeno sintases. Os resultados obtidos aqui corroboram outros estudos de transcriptoma de espécies não modelo usando tecnologias de sequenciamento de alto-desempenho. / Lippia alba, popularly known as erva-cidreira, is a widely distributed specie in Americas and it is found throughout Brazil. This specie has important using in popular medicine for cramp-ing, indigestion, nausea, diarrhea, dysentery, respiratory diseases, liver disorders treatment and infectious diseases such as syphilis and gonorrhea. The leaves of L. alba, which are pre-pared by infusion or decoction and orally ingested, producing an essential oil rich in terpene compounds. These compounds are of pharmacological and industrial interest, due to their use in fragrance preparation. Interestingly, the composition of essential oils change according to different abiotic factors and genetic variations such as ploidy level. In this context, the aims of this work were to characterize the transcriptome of leaves of L. alba (linalool chemotype) and to search putative enzymes sequences involved in production of secondary metabolites. The transcriptome was sequenced by Miseq platform (Illumina) running pair-end libraries 300 bp. The sequencing resulted in 47,498,310 reads (23,749,155 reads for each end sequenced) of 35-308 bp, comprising 12,148,327,567 nucleotides (-12 Gb). The de novo assembly of tran-scripts was processed by Trinity software and generated 193,532 transcripts, in 128,209 uni-genes, with N50 equal to 1,187 bp. 86,122 ORFs (Open Read Frame) were obtained and sub-mitted to BlastP algorithm, finding 75,533 sequences included in NR (Non-Redundant) pro-tein database. Approximately 78.4% of these sequences were functionally annotated using Blast2Go pipeline. Analysis of annotated sequences revealed putative enzymes for synthesis of terpenoids such as geraniol and linalool/nerolidol. For assembly and annotation validation, qPCR assay were realized by amplification of 13 endogenous control genes and 4 terpene synthases genes. The results found here corroborate transcriptome studies in non-model or-ganisms using high-performance sequencing technologies.

Pelargonium sp., appelé aussi « géranium » à odeur de rose ou « Géranium rosat » est l’une des plantes aromatiques et médicinales les plus cultivées au niveau international, essentiellement pour son huile essentielle (HE), utilisée par les industries des cosmétiques et de la parfumerie. Cette essence est extraite des feuilles par distillation vapeur et donne une HE riche de plusieurs centaines de molécules volatiles. Cette complexité est le résultat d’un long processus évolutif et de sélections variétales. Parmi ces composés volatils, les monoterpènes comme le géraniol, le citronellol et l’isomenthone, ou les sesquiterpènes comme le 10- γ-épi-eudesmol et le 6,9-guaiadiène, jouent un rôle prépondérant dans le parfum du Pelargonium. Les proportions relatives de ces différents composés sont d’ailleurs utilisées comme marqueurs de la qualité de l’HE et déterminent la typicité du parfum des différents cultivars et origines (P. cv. ‘rosat Bourbon’, P. cv. ‘rosat Chine’, P. cv. ‘rosat Égypte’ et P. cv. ‘rosat Grasse’). Malgré de très nombreux travaux portant sur la chimie de cette HE, il n’existe aucune information sur les voies de biosynthèse de ces molécules et aucun gène intervenant dans ces voies n’a été isolé. Durant cette thèse, nous avons cloné et caractérisé fonctionnellement par expression et purification des protéines recombinantes chez Escherichia coli des gènes codant les enzymes clés de ces voies de biosynthèse, les terpène synthases. Nous avons ainsi pu caractériser quatre terpène synthases, dont une géraniol synthase mono-produit. Nous avons isolé deux autres monoterpène synthases multi-produits, produisant pour l’une majoritairement du myrcène mais aussi trois autres monoterpènes, et pour l’autre majoritairement du 1,8-cinéole ainsi que 10 autres monoterpènes minoritaires. Enfin, une sesquiterpène multi-produit, la 10-γ-épi-eudesmol synthase, a été caractérisée. Nous avons ensuite analysé l’expression de la géraniol synthase et de la 10-γ-épi-eudesmol synthase dans différentes accessions de Pelargonium par RT-qPCR et nous avons montré la relation entre la capacité de production des différents composés volatils et le niveau d’expression dans les feuilles de ces deux terpène synthases. L’efficacité de la transformation génétique du Pelargonium par Agrobacterium tumefaciens étant élevée, des expériences de transgénèse ont aussi été réalisées afin de compléter la caractérisation fonctionnelle des gènes isolés. Dans une deuxième partie, nous avons réalisé l’analyse des essences produites par 64 espèces et cultivars de Pelargonium d’odeurs très diverses (citron, menthe, rose, abricot, pin, épices…). A l’aide d’analyses statistiques (ACP, analyse discriminante…), nous avons mis en évidence des relations entre la biochimie de ces cultivars, leurs odeurs et leurs proximités génétiques et cela afin de nous donner des pistes sur des croisements potentiellement intéressants. Enfin, un dernier chapitre est consacré à l’amélioration de la production d’HE en Égypte. Grâce à ce programme commencé il y a trois ans, nous améliorons chaque année la qualité et le rendement en HE de plus de 10 Ha de plantation de Pelargonium en Égypte. Un travail d’optimisation de la distillation ainsi que des améliorations des pratiques culturales, nous ont permis de produire une HE de qualité avec un rendement de plus de 60 kg.Ha-1 d’HE. D’autres expériences présentées dans ce chapitre soulignent l’influence de l’environnement et notamment de la température sur le ratio entre le citronellol et le géraniol ainsi que sur la biosynthèse de l’isomenthone, du 10-γ-épi-eudesmol et du 6,9-guaiadiène / Pelargonium sp, also named rose scented « geranium » or « Geranium rosat » is one of the the most cultivated aromatic and medicinal plant worldwide, especially for its essential oil (EO), which is used by cosmetic and perfumery industries. This essence is extracted from leaves by steam distillation and gives an EO containing several hundreds of organic volatile compounds (VOC). This complexity is the result of a long evolutive process and varietal selections. Among these VOC, the monoterpenes like geraniol, citronellol and isomenthone and the sesquiterpenes like 10-γ-epieudesmol and 6,9-guaiadiene, play an important role for the Pelargonium fragrance. The relative proportions of these compounds are used as EO quality markers and determine the different cultivars origins (P. cv. ‘rosat Bourbon’, P. cv. ‘rosat Chine’, P. cv. ‘rosat Egypt’ and P. cv. ‘rosat Grasse’). Despite the important researches on the chemistry of these EO, there is no information on the biosynthesis pathways for these molecules and no genes involved in the pathways have been isolated. During this PhD thesis, we have functionally characterized by recombinant proteins expression and purification in Escherichia coli, four genes, three monoterpene and one sesquiterpene synthases, coding for key enzymes in terpene biosynthesis pathway. The first enzyme is a mono-product geraniol synthase. The second enzyme is a multi-product enzyme with a major peak of myrcene and 3 minor peaks of other monoterpenes. The third enzymes is also a multi-product protein, producing 1,8-cineol as major product and 10 others monoterpenes. The last one is a multi-products sesquiterpene synthase producing mainly the 10-γ-epi-eudesmol and other sesquiterpenes. We have also analyzed the level of expression of the geraniol and 10 γ-epi-eudesmol synthases in several Pelargonium accessions by RT-qPCR and we have demonstrated the relationship between the level of expression of these two terpene synthases and the quantity of the related terpenes produced in leaves. Pelargonium transformation efficiency by Agrobacterium tumefaciens was tested in order to complete the functional characterization of the genes. In a second part, we have analyzed the essence of 64 species and cutivars of Pelargonium having very different fragrances like lemon, mint, rose, apricot, pine, spices… With different statistical tools (PCA, discriminant analysis…), we have highlighted the links between the biochemistry of these species and cultivars, their odors and their phylogenetic relationships. This worked gave us some interesting ideas for some new crossings. Finally, the last chapter concerns the EO production improvements in Egypt. Thanks to these researches, started 3 years ago, we are improving year after year our EO yield and quality in our 10 Ha R&D plantation. An important work was done to optimize the distillation process and improve the agricultural practices which abled us to reach a yield of 60 kg of EO per hectare. Some other experiments show the effect of the environmental factors such as the temperature on the biosynthesis of several important molecules like citronellol and geraniol, 6,9-guaiadiene and 10-γ-epi-eudesmol