Les acides chlorogéniques (CGAs) représentent une famille d'esters formés d'un dérivé de l'acide cinnamique conjugué à l'acide quinique ou shikimique. Ces métabolites secondaires produits par la voie des phénylpropanoides sont largement répandus chez les végétaux terrestres et sont une source majeure d'antioxydants alimentaires. Les esters hydroxycinnamoyl-CoA sont les précurseurs des CGAs et d'autres composés phénoliques tels que les lignines. Ces intermédiaires activés sont synthétisés à partir d'un acide hydroxycinnamique et du coenzyme A par la 4-coumarate CoA ligase (4CL) appartenant à la superfamille des enzymes formant des adénylates. Nicotiana tabacum 4CL2 a été utilisée pour la production d'esters et sa structure a été résolue par remplacement moléculaire. Deux gènes codant pour des hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransférases chez Coffea canephora ont été clonés. CcHCT et CcHQT, qui appartiennent à la superfamille des acyltransférases acyl-CoA-dépendantes, ont été surexprimées dans E. coli et purifiées à homogénéité. L'analyse par diffraction aux rayons X de cristaux de CcHCT a permis de déterminer sa structure par remplacement moléculaire. Un modèle a été dérivé par homologie de séquence pour CcHQT afin de proposer les déterminants de la préférence pour l'acide quinique ou shikimique. Des modélisations moléculaires ont été réalisées afin d'identifier les résidus potentiellement impliqués dans les intéractions enzyme-substrat. L'analyse par chromatographie liquide haute performance des réactions enzymatiques ont montré que ces enzymes sont capables de synthétiser l'acide 5-O-caféoylquinique mais aussi le diester 3,5-O-dicaffeoylquinique, qui est un composé majeur du grain de café avant mûrissement. La production de variants par mutagenèse dirigée a permis l'identification de résidus importants pour la catalyse des réactions de mono- et de diacylation. L'approche combinée de la biologie structurale et de l'enzymologie s'avère particulièrement utile pour mieux comprendre le rôle de HCT et HQT. / Chlorogenic acids (CGAs) represent a family of esters formed between a cinnamic acid derivative and quinic or shikimic acid. CGAs are secondary metabolites produced via the phenylpropanoid pathway by higher plants and are a major source of dietary antioxidants. Hydroxycinnamoyl-CoA esters are the precursors for CGAs and other phenolic compounds such as lignins. These activated intermediates are synthesized from a hydroxycinnamic acid and coenzyme A by 4-coumarate CoA ligase (4CL), which belongs to the adenylate-forming enzyme superfamily. Nicotiana tabacum 4CL2 was used to produce hydroxycinnamoyl-CoA esters and its structure was solved by molecular replacement. Two genes encoding hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferases from Coffea canephora were cloned. CcHCT and CcHQT, which belong to the acyl-CoA-dependent acyltransferase superfamily, were overexpressed in E. coli and purified to homogeneity. X-ray diffraction analysis of CcHCT crystals resulted in a structural solution by molecular replacement. A homology model was derived for CcHQT in order to propose some determinants of the preference for quinic or shikimic acid. Docking experiments were carried out in order to identify potential residues involved in enzyme-substrate interactions. High performance liquid chromatography analysis of enzymatic reactions showed that these enzymes are capable of synthesizing 5-O-caffeoylquinic acid but also the diester 3,5-O-dicaffeoylquinic acid, which is a major component of the coffee grain before ripening. The production of variants by site-directed mutagenesis enabled the identification of residues important for catalysis of the mono- and diacyltransfer reactions. The combined approach of structural biology and enzymology provides molecular insights into the role of HCT and HQT in CGA biosynthesis.

No description available.

Industrial hemp (Cannabis sativa) is a highly productive crop that is well suited to cultivation in Canada. To better understand the development of bast (phloem) fiber secondary walls and to facilitate reverse genetics screening for improved germplasm, I undertook two sets of microarray experiments. The first compared transcript expression in stem peels at three positions along the length of the stem. The second set of microarray experiments compared transcript expression in adjacent tissue layers along the radial axis of the stem. The transcripts that were enriched in fiber-producing tissues in both studies were consistent with a dynamic program of cell wall deposition. Detailed qRT-PCR analysis of specific lignification genes identified the best targets for reverse genetics. Finally, as a first step towards establishing a virally induced gene silencing (VIGS) system, I identified viruses that produced visual symptoms of infection, although qRT-PCR failed to confirm the infection / Plant Biology

No description available.

Abstract The efforts to improve forest trees could be accelerated by means of genetic engineering. Thus, the performance and effects of genetically modified (GM) trees have been investigated in numerous studies, which have generally concluded that GM trees have similar effects on environment and/or other organisms as do conventionally bred trees. In the present study, GM silver birch (Betula pendula Roth) and hybrid aspen (Populus tremula L. × tremuloides Michx.) lines were utilized to study the influence of transgenes to the transcription of related endogenous genes and to the production of soluble phenolic compounds in relation to ectomycorrhizal symbiosis or herbivory. The GM silver birch lines had altered lignin composition, whereas the hybrid aspen lines produced the hemoglobin of Vitreoscilla sp. (VHb). The Pt4CL1a lines were generated using biolistic transformation and monitored under greenhouse conditions for three growing seasons. The Pt4CL1a and PtCOMT silver birch lines, with altered lignin syringyl/guaiacyl ratio, had also reduced transcript levels of endogenous genes, Bp4CL1 and BpCOMT, respectively. This indicates that these members of the 4CL and COMT multigene families are likely to contribute to the monolignol biosynthesis pathway of silver birch. No unintended effects were detected in the PtCOMT or Pt4CL1a lines in relation to ECM symbiosis or performance of insect larvae. Moreover, in soluble phenolic compounds, alterations were found mainly in cinnamic acid derivatives, a group of compounds involved in the biosynthesis of monolignols. In addition, the responses of the studied hybrid aspen lines that were exposed to herbivory for 24 hours were found to be comparable. Furthermore, the proportional weight gain of lepidopteran larvae was alike when fed with leaves of the VHb and non-transgenic hybrid aspen lines. Taken together, no unintended changes were found in the GM silver birch lines with altered lignin composition or in the VHb hybrid aspen lines. However, it is acknowledged that these short-term studies that were conducted under controlled conditions have certain limitations. / Tiivistelmä Puiden ominaisuuksia on mahdollista muuttaa geenitekniikkaa käyttämällä huomattavasti perinteistä jalostusta nopeammin. Geneettisen muuntamisen vaikutuksia puiden ominaisuuksiin ja vuorovaikutussuhteisiin on selvitetty useissa tutkimuksissa geenitekniikkaan liitettyjen riskien arvioimiseksi. Muunnettuja kohdeominaisuuksiaan lukuun ottamatta geneettisesti muunnettujen (GM) puiden ei ole yleisesti ottaen tutkimuksissa havaittu eroavan ympäristövaikutuksiltaan perinteisellä jalostuksella tuotetuista puista. Tässä työssä tutkittiin siirrettyjen geenien vaikutuksia GM-rauduskoivun (Betula pendula Roth) sekä hybridihaavan (Populus tremula L. × tremuloides Michx.) endogeenisten geenituotteiden ja liukoisten fenoliyhdisteiden määriin. Lisäksi työssä tarkasteltiin ligniinirakenteeltaan muunnettujen rauduskoivulinjojen ektomykorritsasymbioosia sekä ligniinimuunnettujen ja Vitreoscilla sp. -bakteerin hemoglobiinia (VHb) tuottavien hybridihaapalinjojen lehtien laatua perhostoukkien ravintona. Biolistisella geeninsiirrolla tuotetuista Amerikan haavan 4-kumaraattikoentsyymi A-ligaasi -geeniä (Pt4CL1) ilmentävistä rauduskoivulinjoista yhdessä havaittiin ligniinin syringyyli- ja guaiasyyliyksikköjen suhteessa muutos. Havaittu muutos aiheutui todennäköisesti koivun Bp4CL1-geenituotteiden määrän vähenemisestä. Myös kaffeaatti/5-hydroksylaatti O-metyylitransferaasi -geeniä (PtCOMT) ilmentävissä, ligniinirakenteeltaan muunnetuissa rauduskoivulinjoissa havaittiin endogeenisen BpCOMT-geenin tuotteiden määrän väheneminen. Tulokset viittaavat siihen, että Bp4CL1- ja BpCOMT-geenien tuottamat entsyymit toimivat rauduskoivun monolignolien biosynteesissä. Ligniiniominaisuuksiltaan muunnettujen rauduskoivujen liukoisista fenoliyhdisteistä todettiin muutoksia ensisijaisesti kanelihappojohdannaisissa, jotka liittyvät läheisesti monolignolien biosynteesireittiin. Ektomykorritsasymbioosissa tai perhostoukkien kasvunopeudessa ei havaittu kasvien geneettisestä muuntamisesta johtuvia eroja. Merkitseviä eroja ei todettu myöskään hybridihaapalinjojen herbivoria-vasteissa. On kuitenkin otettava huomioon, että kaikki tutkimuksen kokeet suoritettiin kasvihuoneissa käyttäen vasta juveniilivaiheessa olevia kasveja. Jotta abioottisten ja bioottisten ympäristötekijöiden sekä GM-puiden vuorovaikutusta olisi mahdollista arvioida kokonaisvaltaisesti, puita pitäisi tutkia pitkäaikaisissa kenttäkokeissa.

No description available.

Teak tree (Tectona grandis L.f.) has a high value in the timber trade for fabrication of woody products due to its extraordinary qualities of color, density and durability. Despite the importance of this species, genetic and molecular studies available are limited. Also, the lack of molecular information about secondary xylem and tree maturation has hindered genetic exploration of teak. Therefore, gene expression studies and transcriptomic profiling are essential to explore wood formation and lignin biosynthesis through the development and aging of vascular plants. Aiming the gene expression studies, it was essential to identify and clone reference genes for teak. Eight genes were tested, commonly used in qRT-PCR, including TgRP60S, TgCAC, TgACT, TgHIS3, TgSAND, TgTUB, TgUBQ and TgEF1a. Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem). Stability validation by NormFinder, BestKeeper, geNorm and Delta Ct programs showed that TgUBQ and TgEF1a are the most stable genes to use as qRT-PCR reference genes in teak in the conditions tested. Due to the availability of 12- and 60-year-old teak trees, RNA-seq was performed in diferent organs (seedlings, leaves, flowers, root, stem and branch secondary xylem). A total of 462,260 transcripts were obtained by assembling with \"Trinity\" software. Also, 1,502 and 931 genes differentially expressed were identified for stem and branch secondary xylem, respectively, using DESeq program, and MYB transcription factors, which were characterized. TgMYB1 amino acid sequence displayed a predicted coiled-coil (CC) motif while TgMYB2, TgMYB3 and TgMYB4 showed R2R3-MYB domain. All of them were phylogenetically grouped with several gymnosperms and flowering plants. High expression of TgMYB1 and TgMYB4 in lignified tissues of 60-year-old trees was observed. In this work, the Cinnamyl Alcohol Dehydrogenase (CAD) gene family was also studied. One complete (TgCAD1) and three partial (TgCAD2 to TgCAD4) members were characterized. The four enzymes presented residues for catalytic and structural zinc action, NADPH binding and substrate specificity, consistent with the mechanism of alcohol dehydrogenases. TgCAD3 and TgCAD4 were highly expressed in young and mature sapwood and seem to be duplicated and highly related with lignin biosynthesis. Tree genetic improvement, marker-assisted selection and plant transformation seem to be promising lines of research for the data obtained from this research. This is the first study addressing gene characterization and expression, phylogeny and transcriptomic profiling in teak. / A árvore de teca (Tectona grandis L.f.) tem alto valor no comércio de madeira para a fabricação de produtos lenhosos, devido às suas qualidades extraordinárias de cor, densidade e durabilidade. Apesar da importância desta espécie, são poucos os estudos genéticos e moleculares disponíveis. Também, a falta de informação molecular sobre xilema secundário e maturação da árvore tem dificultado a exploração genética de teca. Assim, estudos de expressão gênica e perfis transcricionais são relevantes para explorar a formação da madeira e a biossíntese de lignina durante o desenvolvimento e envelhecimento das plantas vasculares. Visando os estudos de expressão gênica, foi essencial identificar e clonar genes de referencia para a teca. Foram testados oito genes comumente usados em qRT-PCR, TgRP60S, TgCAC, TgACT, TgHIS3, TgSAND, TgTUB, TgUBQ e TgEF1a. Perfis de expressão destes genes foram avaliados por qRT-PCR em seis amostras de tecidos e órgãos (folhas, flores, plântulas, raiz, xilema secundário de caule e ramo). A validação da estabilidade pelos programas NormFinder, BestKeeper, geNorm e Delta CT mostrou que TgUBQ e TgEF1a são os genes mais estáveis para usar como genes de referência em teca nas condições testadas. Em virtude da disponibilidade de árvores de teca de diferentes idades, entre 12 e 60 anos, foi realizado o RNAseq de diferentes órgãos (plântulas, folhas, flores, raiz, ramos e caules de árvores de 12 e 60 anos). Obteve-se um total de 462.260 transcritos pela montagem com o software \"Trinity\". Foram identificados 1.502 e 931 genes diferencialmente expressos para xilema secundário de caule e ramo, respectivamente, utilizando o programa DESeq e fatores de transcrição MYB, que foram posteriormente caracterizados. A sequência de aminoácidos do TgMYB1 exibiu um motivo \"coiled-coil\" (CC), enquanto TgMYB2, TgMYB3 e TgMYB4 mostraram domínio R2R3-MYB. Todos eles foram filogeneticamente agrupados com várias gimnospermas e angiospermas. Observou-se alta expressão do TgMYB1 e TgMYB4 em tecidos lignificados de árvores de 60 anos de idade. Neste trabalho também foi estudada a família gênica Cinamil álcool desidrogenase (CAD). Foi caracterizado um membro completo (TgCAD1) e três parciais (TgCAD2 a TgCAD4). As quatro enzimas apresentaram resíduos de ação catalítica e estrutural de zinco, de ligação ao NADPH e de especificidade de substrato, em conformidade com o mecanismo conservado de álcool desidrogenases. TgCAD3 e TgCAD4 foram altamente expressos no alburno jovem e maduro e parecem estar duplicados e relacionados com a biossíntese de lignina. O melhoramento genético de árvores, a seleção assistida utilizando marcadores moleculares e a transformação de plantas parecem ser linhas promissoras de pesquisa, a partir dos dados obtidos nesta pesquisa. Este é o primeiro estudo sobre caracterização e expressão gênica, filogenia e perfis transcricionais em teca.

Teak tree (Tectona grandis L.f.) has a high value in the timber trade for fabrication of woody products due to its extraordinary qualities of color, density and durability. Despite the importance of this species, genetic and molecular studies available are limited. Also, the lack of molecular information about secondary xylem and tree maturation has hindered genetic exploration of teak. Therefore, gene expression studies and transcriptomic profiling are essential to explore wood formation and lignin biosynthesis through the development and aging of vascular plants. Aiming the gene expression studies, it was essential to identify and clone reference genes for teak. Eight genes were tested, commonly used in qRT-PCR, including TgRP60S, TgCAC, TgACT, TgHIS3, TgSAND, TgTUB, TgUBQ and TgEF1a. Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem). Stability validation by NormFinder, BestKeeper, geNorm and Delta Ct programs showed that TgUBQ and TgEF1a are the most stable genes to use as qRT-PCR reference genes in teak in the conditions tested. Due to the availability of 12- and 60-year-old teak trees, RNA-seq was performed in diferent organs (seedlings, leaves, flowers, root, stem and branch secondary xylem). A total of 462,260 transcripts were obtained by assembling with \"Trinity\" software. Also, 1,502 and 931 genes differentially expressed were identified for stem and branch secondary xylem, respectively, using DESeq program, and MYB transcription factors, which were characterized. TgMYB1 amino acid sequence displayed a predicted coiled-coil (CC) motif while TgMYB2, TgMYB3 and TgMYB4 showed R2R3-MYB domain. All of them were phylogenetically grouped with several gymnosperms and flowering plants. High expression of TgMYB1 and TgMYB4 in lignified tissues of 60-year-old trees was observed. In this work, the Cinnamyl Alcohol Dehydrogenase (CAD) gene family was also studied. One complete (TgCAD1) and three partial (TgCAD2 to TgCAD4) members were characterized. The four enzymes presented residues for catalytic and structural zinc action, NADPH binding and substrate specificity, consistent with the mechanism of alcohol dehydrogenases. TgCAD3 and TgCAD4 were highly expressed in young and mature sapwood and seem to be duplicated and highly related with lignin biosynthesis. Tree genetic improvement, marker-assisted selection and plant transformation seem to be promising lines of research for the data obtained from this research. This is the first study addressing gene characterization and expression, phylogeny and transcriptomic profiling in teak. / A árvore de teca (Tectona grandis L.f.) tem alto valor no comércio de madeira para a fabricação de produtos lenhosos, devido às suas qualidades extraordinárias de cor, densidade e durabilidade. Apesar da importância desta espécie, são poucos os estudos genéticos e moleculares disponíveis. Também, a falta de informação molecular sobre xilema secundário e maturação da árvore tem dificultado a exploração genética de teca. Assim, estudos de expressão gênica e perfis transcricionais são relevantes para explorar a formação da madeira e a biossíntese de lignina durante o desenvolvimento e envelhecimento das plantas vasculares. Visando os estudos de expressão gênica, foi essencial identificar e clonar genes de referencia para a teca. Foram testados oito genes comumente usados em qRT-PCR, TgRP60S, TgCAC, TgACT, TgHIS3, TgSAND, TgTUB, TgUBQ e TgEF1a. Perfis de expressão destes genes foram avaliados por qRT-PCR em seis amostras de tecidos e órgãos (folhas, flores, plântulas, raiz, xilema secundário de caule e ramo). A validação da estabilidade pelos programas NormFinder, BestKeeper, geNorm e Delta CT mostrou que TgUBQ e TgEF1a são os genes mais estáveis para usar como genes de referência em teca nas condições testadas. Em virtude da disponibilidade de árvores de teca de diferentes idades, entre 12 e 60 anos, foi realizado o RNAseq de diferentes órgãos (plântulas, folhas, flores, raiz, ramos e caules de árvores de 12 e 60 anos). Obteve-se um total de 462.260 transcritos pela montagem com o software \"Trinity\". Foram identificados 1.502 e 931 genes diferencialmente expressos para xilema secundário de caule e ramo, respectivamente, utilizando o programa DESeq e fatores de transcrição MYB, que foram posteriormente caracterizados. A sequência de aminoácidos do TgMYB1 exibiu um motivo \"coiled-coil\" (CC), enquanto TgMYB2, TgMYB3 e TgMYB4 mostraram domínio R2R3-MYB. Todos eles foram filogeneticamente agrupados com várias gimnospermas e angiospermas. Observou-se alta expressão do TgMYB1 e TgMYB4 em tecidos lignificados de árvores de 60 anos de idade. Neste trabalho também foi estudada a família gênica Cinamil álcool desidrogenase (CAD). Foi caracterizado um membro completo (TgCAD1) e três parciais (TgCAD2 a TgCAD4). As quatro enzimas apresentaram resíduos de ação catalítica e estrutural de zinco, de ligação ao NADPH e de especificidade de substrato, em conformidade com o mecanismo conservado de álcool desidrogenases. TgCAD3 e TgCAD4 foram altamente expressos no alburno jovem e maduro e parecem estar duplicados e relacionados com a biossíntese de lignina. O melhoramento genético de árvores, a seleção assistida utilizando marcadores moleculares e a transformação de plantas parecem ser linhas promissoras de pesquisa, a partir dos dados obtidos nesta pesquisa. Este é o primeiro estudo sobre caracterização e expressão gênica, filogenia e perfis transcricionais em teca.