Terpenes are important specialised metabolites produced by all organisms. Plants produce the greatest diversity of terpenoid compounds, which function in a variety of crucial processes including regulation of growth and development, energy production and plant-insect communication, including pollinator attraction and prevention of herbivore damage. Isopentenyl diphosphate (IPP), the building block for all terpenoid compounds, is synthesised in plants via two unique terpene synthesis pathways located in the plastids and the cytosol, and the regulation of these pathways is still not well understood. The aim of this research was to (1) modify and study the regulation of floral volatile production in Nicotiana tabacum (tobacco) by altering the expression of various enzymes in the terpene biosynthesis pathway and (2) determine the role of specific volatile compounds in floral odour blends in feeding and oviposition behaviours of Helicoverpa armigera, a polyphagous moth of widespread agricultural importance. Expression levels of several enzymes in the terpene biosynthetic pathway were altered by genetic modification in order to modify terpene volatile emissions produced by flowers of N. tabacum. Genes chosen for overexpression were cloned from several species and RNAi hairpins were constructed from gene fragments amplified from tobacco flower cDNA. Transgenic plants were produced by Agrobacterium-mediated transformation, and lines with high levels of transgene expression selected for analysis. The flower-specific Antirrhinum majus chalcone synthase promoter was chosen to control gene expression in transgenic lines in order to avoid the potentially deleterious effects of widespread disruption to terpene biosynthesis. Floral volatiles were sampled using two methods; solid phase microextraction, a highly sensitive technique able to detect even trace levels of volatile compounds in headspace samples, and Tenax sampling, a robust and replicable method to quantify volatile emissions. All floral headspace samples were analysed by gas chromatography-mass spectrometry. Floral volatile analysis determined that wild type Ti68 tobacco flowers emit a simple blend of floral volatiles, with only linalool, a monoterpene, and β-caryophyllene, a sesquiterpene, detected by both sampling methods. Volatile emissions were not subject to temporal regulation, but changes in the floral odour blend were detected during flower development. Overexpression of the plastidic terpene biosynthesis genes 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranyl diphosphate synthase did not affect volatile production, however increased farnesyl diphosphate synthase expression in the cytosol surprisingly caused an increase in linalool emissions, synthesised in the plastids. Downregulation of DXR resulted in an albino phenotype affecting all young leaves, the upper stem and the sepals in the most severely affected lines. A significant three-fold decrease in floral linalool emissions, and a nine-fold reduction of both linalool and β-caryophyllene retained within floral tissues was observed in the transgenic lines. In feeding behaviour tests, male and virgin female H. armigera moths did not discriminate between wild type and DXR knockdown flowers at close-range, despite the significant difference in linalool emissions. Expression of an (E)-β-ocimene synthase gene controlled by the CHS promoter did not result in any transgenic plants emitting the novel monoterpene, (E)-β-ocimene. Significant problems with seed germination suggested that (E)-β-ocimene may cause embryo lethality in these lines. However, overexpression of a heterologous (S)-linalool synthase under control of the constitutive cauliflower mosaic virus 35S promoter resulted in a significant two-fold increase in volatile linalool, and β-glycosidase assays confirmed sequestration of a glycosylated linalool derivative in floral tissues. Oviposition preference tests with mated female H. armigera moths indicated a significant preference for egg-laying on wild type flowers compared to flowers with increased linalool production. The results of this research, and previous studies of volatile production in transgenic tobacco, indicate that IPP precursor exchange occurs predominantly in one direction from the cytosol to the plastids, at least under the stress caused by alterations in pathway flux. Regulation of the cytosolic terpene biosynthetic pathway upstream of IPP synthesis appears to be less strictly controlled than the plastidic pathway. Insect behavioural assays support the findings of recent studies in other moth species, and suggest that close-range feeding attraction of H. armigera may be more strongly influenced by visual cues, whereas odour cues, including contact chemoreception, play a more important role in oviposition preferences. The increase in knowledge of the olfactory contribution toward insect-plant communication demonstrated here, and from future work, will lead to improved management of pest species in agricultural and ecological settings.

A resistência a doenças em plantas transgênicas tem sido obtida por meio da expressão de genes isolados de bactérias, fungos micoparasitas e plantas. Neste trabalho, relatamos a utilização de um gene do fungo entomopatogênico Metarhizium anisopliae como modo de gerar resistência a doenças fúngicas em plantas. O gene chit1 codifica a quitinase CHIT42 (EC 3.2.1.14), pertencente a uma classe de glicosil-hidrolases capazes de converter quitina em oligômeros de N-acetil-glicosamina (NAcGlc). Quando presentes em tecidos vegetais, supõese que as quitinases ataquem especificamente a parede celular de fungos invasores, provocando danos às hifas e causando a morte por lise das células fúngicas. Deste modo, dois diferentes grupos de plantas transgênicas de Nicotiana tabacum foram produzidos: no primeiro deles, denominado chitplus, os indivíduos possuem o gene chit1 sob o controle do promotor CaMV 35S. O segundo grupo, demoninado chitless, consiste de plantas transformadas com um T-DNA não contendo o gene do fungo. Trinta e quatro plantas transgênicas resistentes à canamicina (17 de cada grupo) foram regeneradas a partir de discos de folhas infectados por Agrobacterium tumefaciens. A produção da quitinase em extratos protéicos de folhas foi analisada por zimogramas em SDS-PAGE contendo glicol-quitina e corados por calcoflúor branco, na forma de um screening dos transgênicos primários. As plantas transgênicas foram testadas, ainda, por meio de ensaios colorimétricos empregando oligômeros sintéticos de NAcGlc como substratos específicos, além de immunoblot e Western blot com soro anti-quitinase. A quantidade de enzima recombinante nas plantas chitplus variou desde nenhuma atividade detectável a elevados níveis de expressão da enzima. A hibridização de Southern blot demonstrou que o número de cópias do gene chit1 integradas no genoma vegetal foi estimado entre uma e quatro. A primeira geração de plantas transgênicas geradas por autofecundação de parentais portadores de duas cópias do transgene foi testada com relação à estabilidade da herança do transgene e em 43 de um total de 67 descendentes, originados de quatro cruzamentos independentes, o padrão de segregação não diferiu das proporções Mendelianas esperadas. Ensaios de resistência, desafiando as plantas transgênicas com o basidiomiceto Rhizoctonia solani foram realizados e uma evidente diminuição da área foliar contendo lesões fúngicas foi observada entre as linhagens transgênicas, embora variações na atividade quitinolítica tenham influenciado o nível de resistência. Nossos resultados sugerem uma relação direta entre a atividade específica de quitinase e ao aumento nos níveis de resistência às lesões causadas pela infecção por R. solani. / Plant resistance in transgenic plants has been obtained by expressing genes isolated from bacteria, mycoparasitic fungi and plants. Here we report the employment of a gene from the entomopathogenic fungus Metarhizium anisopliae as a tool to generate resistance to fungal diseases in plants. The chit1 gene encodes the chitinase CHIT42 (EC 3.2.1.14), belonging to a class of glicosyl-hydrolases able to convert chitin into N-acetyl-glucosamine (GlcNAc) oligomers. When present in plant tissues, chitinases are supposed to disrupt the invading fungal cell wall specifically, causing hyphae damage and leading to cell lysis. Hence two different groups of transgenic Nicotiana tabacum plants were produced. The first group was named chitplus, in which individuals harbour the chit1 gene under the control of the CaMV 35S promoter . The second group, named chitless, carried a T-DNA not containing the fungal gene. Thirty-four kanamicin resistant plants (17 of each group) were regenerated from leaf discs infected with Agrobacterium tumefaciens. Chitinase production in leaf protein extracts was analysed through zymograms in SDS-PAGE containing glycol-chitin and stained by calcofluor white, as a screening of primary transformants. Transgenic plants were also evaluated by colorimetric assays using synthetic GlcNAc oligomers as specific substrates besides immunoblot and Western blot probed with rabbit anti-chitinase sera. The amount of recombinant enzyme in chitplus plants ranged from no detectable chitinase activity to high levels of enzyme expression. Southern blot hybridisation revealed that chit1 copy number inserted into plant genomes varied from one to four. The first self pollinated generation of transgenic lines bearing two copies of the transgene was tested on inheritance stability and in 43 out of 67 descendants, derived from four independent crosses, the segregation pattern was discovered not to differ from the predicted Mendelian ratios. Resistance assays challenging transgenic plants with the basidiomycete Rhizoctonia solani were performed and a clear decrease in the foliar area containing fungal lesions was observed amongst transgenic lines, though variations in chitinase activity also reflected on the resistance level. Our results suggest a direct relationship between chitinase specific activity and the improvement in the resistance to lesions caused by infection.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-06-13T16:21:59Z No. of bitstreams: 1 texto completo.pdf: 1075966 bytes, checksum: 047f8bb5c392a3f22d9009ed5f6f71b9 (MD5) / Made available in DSpace on 2016-06-13T16:21:59Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1075966 bytes, checksum: 047f8bb5c392a3f22d9009ed5f6f71b9 (MD5) Previous issue date: 2009-07-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Nesse trabalho foi avaliada a importância do transportador de sacarose especificamente em células guarda (CG) e o papel da sacarose sobre os movimentos estomáticos. Utilizou-se plantas de tabaco transformadas com o antisenso do gene do transportador de sacarose sob controle do promotor KST1, específico de GC. As CG das plantas transgênicas apresentaram menores teores de sacarose, maiores nos de amido e um modesto incremento nos de K + . O menor conteúdo de sacarose nas CG das plantas transgênicas esteve associado com menores valores de condutância estomática (g s ). Essa associação sugere a importância da sacarose no simplasto na manutenção de baixos potenciais osmóticos nas CG. Foi observada uma rápida redução nos teores de amido quando os estômatos estavam se abrindo, fato não observado nas plantas não-transformadas. Nas plantas transformadas, com menor g s , foi possível demonstrar uma restrição difusional (estomática) à fotossíntese (A). As plantas transformadas também apresentaram menor taxa de transpiração (E) e menor concentração de CO 2 na câmara sub-estomática, além de maiores valores da razão de composição isotópica (δ 13 C). Entretanto, maiores valores da razão A/E esteve associado com menores valores de A, conseqüentemente, a uma menor taxa de crescimento, porém não a uma menor eficiência baseada nas taxas de crescimento relativas. Os dados de δ 13 C confirmaram a menor g s e reforçam que esse fenótipo se prolongou pelo desenvolvimento das plantas. Por meio de plantas de tabaco com menor g s foi possível demonstrar que o fenótipo de retardamento à seca foi a principal característica desta transformação, proporcionando as plantas transgênicas um menor consumo de água. Os resultados sugerem que a manipulação do transporte de sacarose em CG foi um mecanismo prático e efetivo na aquisição de plantas mais resistentes à seca. / It was evaluated the importance of guard cell (GC) sucrose transporter and the role of sucrose as osmotic on GC. We transformed tobacco plants with antisense gene construct for sucrose transporter driven by KST1, GC specific promoter. Transgenic plants GC have less sucrose, more starch and modest increase in K + contents. Low sucrose contents in GC of transgenic lines were associated with low stomatal conductance (g s ), suggesting the importance of sucrose transporter and symplastic sucrose in maintaining low osmotic potential on GC. It was observed rapid starch disappearance when the guard cells are swelling, fact not observed in control plants. By means of low g s tobacco plants demonstrated diffusional (stomatal) restriction of photosynthesis (A), low transpiration rate (E) and low sub-stomatal CO 2 concentration, high A/E and higher carbon rate composition (δ 13 C). However, higher A/E was associated with lower A, consequently, a slower crop growth rate, but not smaller “efficiency index” as showed by relative growth rate. The δ 13 C data confirms the low conductance, showing that it represents a common stomata behavior over all plant development. By means of low g s tobacco plants, we got desiccation postponement phenotype as principal feature of this transformation, being high water saving plants. These results suggest that manipulation of sucrose transport in GC may be developed as a practical mechanism for drought avoidance and water conservation during irrigation. These results illustrate the importance of fine tuning of sucrose metabolism transport and metabolism in the fitness of stomatal function in contributing to plant survival or growth under unfavorable water conditions.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-09-23T12:09:04Z No. of bitstreams: 1 texto completo.pdf: 703627 bytes, checksum: 193b0cc47fc6514482df37f3b075f580 (MD5) / Made available in DSpace on 2016-09-23T12:09:04Z (GMT). No. of bitstreams: 1 texto completo.pdf: 703627 bytes, checksum: 193b0cc47fc6514482df37f3b075f580 (MD5) Previous issue date: 2000-07-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os processos que regulam a alocação de carbono para os órgãos vegetais e as sementes em desenvolvimento são muito importantes no desenvolvimento das plantas. Na maioria dos vegetais, o nutriente translocado mais importante é a sacarose, e a compreensão da biologia molecular, da bioquímica e da fisiologia de seu transporte é um problema central na biologia vegetal. Recentemente, foi isolado, em um laboratório da Universidade Federal de Viçosa, Viçosa-MG, um cDNA de uma biblioteca de expressão de sementes de soja que codifica um homólogo da proteína de ligação à sacarose (SBP), denominado S-64. Para analisar a função desse homólogo de SBP, tabacos transgênicos foram obtidos, introduzindo-se genes quiméricos que continham a região codificadora do gene s-64, ligado ao promotor 35S-CaMV, na orientação senso ou anti-senso via transformação mediada por Agrobacterium sp. O acúmulo do homólogo de SBP aumentou em tabacos que superexpressaram o gene s-64, assim como a expressão anti-senso do gene s-64 levou ao decréscimo nos níveis da proteína endógena. As plantas anti-senso desenvolveram sintomas característicos de uma inibição do transporte de sacarose à longa distância e apresentaram crescimento e desenvolvimento vegetal reduzidos. Em contraste, as plantas que continham a construção senso revelaram tendência de crescimento mais rápido e aceleração do desenvolvimento floral. O desempenho no desenvolvimento das plantas transgênicas foi correlacionado com a taxa fotossintética, sob condições normais de irradiância. Enquanto a taxa de fotossíntese nas linhagens anti-senso diminuiu, nas linhagens senso ela aumentou. Além disto, tanto a repressão anti-senso quanto a superexpressão senso do homólogo de SBP alteraram o particionamento de carboidratos em folhas maduras. Na geração seguinte, as plantas transgênicas senso e anti-senso revelaram diferentes padrões de expressão do gene s-64. Enquanto nas plantas senso não foi observado aumento no acúmulo da proteína, nas plantas anti-senso uma forte inibição de S-64/SBP persistiu. Além disto, as plantas transgênicas anti-senso R1 tiveram um reduzido crescimento vegetativo e radicular, acompanhado de um florescimento tardio. Seguindo esse padrão, a taxa fotossintética das folhas jovens e maduras diminuiu, o que evidencia uma possível diminuição no carregamento e, ou, descarregamento do floema. Um reduzido peso seco total e radicular, assim como experimentos de enxertia, forneceu evidências de que o gene s-64 está diretamente envolvido no descarregamento do floema. Coletivamente, esses resultados indicam que a proteína S-64 é funcionalmente análoga à proteína de ligação à sacarose e representa um importante componente da via de translocação de sacarose em plantas. / The processes that regulate carbon allocation to various organs in the developing seed is very important in plant development. In most plants, the morg important translocated nutrient is sucrose and, thus, understanding the molecular biology, biochemistry, and physiology of sucrose transport is a central problem in plant biology. A sucrose binding protein (SBP) homologue, designated S-64, was isolated and transgenic tobacco plants were obtained by introducing chimeric genes containing the S-64 coding region linked to the 35S CaMV promoter, either in the sense or antisense orientation, via Agrobacterium tumefaciens- mediated transformation. The accumulation of the SBP homologue was increased in transgenic plants expressing the heterologous sbp gene, whereas those expressing the antisense construct had reduced levels of the protein. The antisense transgenic plants developed symptoms characteristic of an inhibition of sucrose translocation and displayed a reduction in plant growth and development. In contrast, overexpression of the protein accelerated plant growth and the onset of flowering induction. The overall developmental performance of the transgenic plants was correlated with their photosynthetic rate under normal conditions. While photosynthesis in the antisense lines was decreased, in the sense lines photosynthetic rates were increased. Furthermore, both antisense repression and overexpression of the SBP homologue in transgenic lines altered carbohydrate partitioning in mature leaves. On the following generation, both sense and antisense plants revealed different S-64 expression patterns. While the sense plants had no overexpression when compared to control plants, the antisense plants was significantly inhibited by the SBP homologue gene. According to previous results, the antisense plants have a reduced vegetative growth accompanied by a late flowering. The photosynthesis was reduced on young and mature leaves from antisense plants supporting a possible decrease on loading and/or unloading of the phloem. A reduced root growth and dry weight, associated with grafting experiments showed strong evidences that gene is directly involved on phloem unloading. Taken together, these results indicate that S-64 protein is functionally analogous to sucrose binding protein, representing an important component of the sucrose translocation pathway in plants. / Não foi localizado o cpf do autor.

A resistência a doenças em plantas transgênicas tem sido obtida por meio da expressão de genes isolados de bactérias, fungos micoparasitas e plantas. Neste trabalho, relatamos a utilização de um gene do fungo entomopatogênico Metarhizium anisopliae como modo de gerar resistência a doenças fúngicas em plantas. O gene chit1 codifica a quitinase CHIT42 (EC 3.2.1.14), pertencente a uma classe de glicosil-hidrolases capazes de converter quitina em oligômeros de N-acetil-glicosamina (NAcGlc). Quando presentes em tecidos vegetais, supõese que as quitinases ataquem especificamente a parede celular de fungos invasores, provocando danos às hifas e causando a morte por lise das células fúngicas. Deste modo, dois diferentes grupos de plantas transgênicas de Nicotiana tabacum foram produzidos: no primeiro deles, denominado chitplus, os indivíduos possuem o gene chit1 sob o controle do promotor CaMV 35S. O segundo grupo, demoninado chitless, consiste de plantas transformadas com um T-DNA não contendo o gene do fungo. Trinta e quatro plantas transgênicas resistentes à canamicina (17 de cada grupo) foram regeneradas a partir de discos de folhas infectados por Agrobacterium tumefaciens. A produção da quitinase em extratos protéicos de folhas foi analisada por zimogramas em SDS-PAGE contendo glicol-quitina e corados por calcoflúor branco, na forma de um screening dos transgênicos primários. As plantas transgênicas foram testadas, ainda, por meio de ensaios colorimétricos empregando oligômeros sintéticos de NAcGlc como substratos específicos, além de immunoblot e Western blot com soro anti-quitinase. A quantidade de enzima recombinante nas plantas chitplus variou desde nenhuma atividade detectável a elevados níveis de expressão da enzima. A hibridização de Southern blot demonstrou que o número de cópias do gene chit1 integradas no genoma vegetal foi estimado entre uma e quatro. A primeira geração de plantas transgênicas geradas por autofecundação de parentais portadores de duas cópias do transgene foi testada com relação à estabilidade da herança do transgene e em 43 de um total de 67 descendentes, originados de quatro cruzamentos independentes, o padrão de segregação não diferiu das proporções Mendelianas esperadas. Ensaios de resistência, desafiando as plantas transgênicas com o basidiomiceto Rhizoctonia solani foram realizados e uma evidente diminuição da área foliar contendo lesões fúngicas foi observada entre as linhagens transgênicas, embora variações na atividade quitinolítica tenham influenciado o nível de resistência. Nossos resultados sugerem uma relação direta entre a atividade específica de quitinase e ao aumento nos níveis de resistência às lesões causadas pela infecção por R. solani. / Plant resistance in transgenic plants has been obtained by expressing genes isolated from bacteria, mycoparasitic fungi and plants. Here we report the employment of a gene from the entomopathogenic fungus Metarhizium anisopliae as a tool to generate resistance to fungal diseases in plants. The chit1 gene encodes the chitinase CHIT42 (EC 3.2.1.14), belonging to a class of glicosyl-hydrolases able to convert chitin into N-acetyl-glucosamine (GlcNAc) oligomers. When present in plant tissues, chitinases are supposed to disrupt the invading fungal cell wall specifically, causing hyphae damage and leading to cell lysis. Hence two different groups of transgenic Nicotiana tabacum plants were produced. The first group was named chitplus, in which individuals harbour the chit1 gene under the control of the CaMV 35S promoter . The second group, named chitless, carried a T-DNA not containing the fungal gene. Thirty-four kanamicin resistant plants (17 of each group) were regenerated from leaf discs infected with Agrobacterium tumefaciens. Chitinase production in leaf protein extracts was analysed through zymograms in SDS-PAGE containing glycol-chitin and stained by calcofluor white, as a screening of primary transformants. Transgenic plants were also evaluated by colorimetric assays using synthetic GlcNAc oligomers as specific substrates besides immunoblot and Western blot probed with rabbit anti-chitinase sera. The amount of recombinant enzyme in chitplus plants ranged from no detectable chitinase activity to high levels of enzyme expression. Southern blot hybridisation revealed that chit1 copy number inserted into plant genomes varied from one to four. The first self pollinated generation of transgenic lines bearing two copies of the transgene was tested on inheritance stability and in 43 out of 67 descendants, derived from four independent crosses, the segregation pattern was discovered not to differ from the predicted Mendelian ratios. Resistance assays challenging transgenic plants with the basidiomycete Rhizoctonia solani were performed and a clear decrease in the foliar area containing fungal lesions was observed amongst transgenic lines, though variations in chitinase activity also reflected on the resistance level. Our results suggest a direct relationship between chitinase specific activity and the improvement in the resistance to lesions caused by infection.

A resistência a doenças em plantas transgênicas tem sido obtida por meio da expressão de genes isolados de bactérias, fungos micoparasitas e plantas. Neste trabalho, relatamos a utilização de um gene do fungo entomopatogênico Metarhizium anisopliae como modo de gerar resistência a doenças fúngicas em plantas. O gene chit1 codifica a quitinase CHIT42 (EC 3.2.1.14), pertencente a uma classe de glicosil-hidrolases capazes de converter quitina em oligômeros de N-acetil-glicosamina (NAcGlc). Quando presentes em tecidos vegetais, supõese que as quitinases ataquem especificamente a parede celular de fungos invasores, provocando danos às hifas e causando a morte por lise das células fúngicas. Deste modo, dois diferentes grupos de plantas transgênicas de Nicotiana tabacum foram produzidos: no primeiro deles, denominado chitplus, os indivíduos possuem o gene chit1 sob o controle do promotor CaMV 35S. O segundo grupo, demoninado chitless, consiste de plantas transformadas com um T-DNA não contendo o gene do fungo. Trinta e quatro plantas transgênicas resistentes à canamicina (17 de cada grupo) foram regeneradas a partir de discos de folhas infectados por Agrobacterium tumefaciens. A produção da quitinase em extratos protéicos de folhas foi analisada por zimogramas em SDS-PAGE contendo glicol-quitina e corados por calcoflúor branco, na forma de um screening dos transgênicos primários. As plantas transgênicas foram testadas, ainda, por meio de ensaios colorimétricos empregando oligômeros sintéticos de NAcGlc como substratos específicos, além de immunoblot e Western blot com soro anti-quitinase. A quantidade de enzima recombinante nas plantas chitplus variou desde nenhuma atividade detectável a elevados níveis de expressão da enzima. A hibridização de Southern blot demonstrou que o número de cópias do gene chit1 integradas no genoma vegetal foi estimado entre uma e quatro. A primeira geração de plantas transgênicas geradas por autofecundação de parentais portadores de duas cópias do transgene foi testada com relação à estabilidade da herança do transgene e em 43 de um total de 67 descendentes, originados de quatro cruzamentos independentes, o padrão de segregação não diferiu das proporções Mendelianas esperadas. Ensaios de resistência, desafiando as plantas transgênicas com o basidiomiceto Rhizoctonia solani foram realizados e uma evidente diminuição da área foliar contendo lesões fúngicas foi observada entre as linhagens transgênicas, embora variações na atividade quitinolítica tenham influenciado o nível de resistência. Nossos resultados sugerem uma relação direta entre a atividade específica de quitinase e ao aumento nos níveis de resistência às lesões causadas pela infecção por R. solani. / Plant resistance in transgenic plants has been obtained by expressing genes isolated from bacteria, mycoparasitic fungi and plants. Here we report the employment of a gene from the entomopathogenic fungus Metarhizium anisopliae as a tool to generate resistance to fungal diseases in plants. The chit1 gene encodes the chitinase CHIT42 (EC 3.2.1.14), belonging to a class of glicosyl-hydrolases able to convert chitin into N-acetyl-glucosamine (GlcNAc) oligomers. When present in plant tissues, chitinases are supposed to disrupt the invading fungal cell wall specifically, causing hyphae damage and leading to cell lysis. Hence two different groups of transgenic Nicotiana tabacum plants were produced. The first group was named chitplus, in which individuals harbour the chit1 gene under the control of the CaMV 35S promoter . The second group, named chitless, carried a T-DNA not containing the fungal gene. Thirty-four kanamicin resistant plants (17 of each group) were regenerated from leaf discs infected with Agrobacterium tumefaciens. Chitinase production in leaf protein extracts was analysed through zymograms in SDS-PAGE containing glycol-chitin and stained by calcofluor white, as a screening of primary transformants. Transgenic plants were also evaluated by colorimetric assays using synthetic GlcNAc oligomers as specific substrates besides immunoblot and Western blot probed with rabbit anti-chitinase sera. The amount of recombinant enzyme in chitplus plants ranged from no detectable chitinase activity to high levels of enzyme expression. Southern blot hybridisation revealed that chit1 copy number inserted into plant genomes varied from one to four. The first self pollinated generation of transgenic lines bearing two copies of the transgene was tested on inheritance stability and in 43 out of 67 descendants, derived from four independent crosses, the segregation pattern was discovered not to differ from the predicted Mendelian ratios. Resistance assays challenging transgenic plants with the basidiomycete Rhizoctonia solani were performed and a clear decrease in the foliar area containing fungal lesions was observed amongst transgenic lines, though variations in chitinase activity also reflected on the resistance level. Our results suggest a direct relationship between chitinase specific activity and the improvement in the resistance to lesions caused by infection.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2018-06-13T17:18:47Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Luis Henrique Konzen.pdf: 646860 bytes, checksum: 511c31d83c08144defe344c571319e35 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-06-14T20:50:11Z (GMT) No. of bitstreams: 2 Luis Henrique Konzen.pdf: 646860 bytes, checksum: 511c31d83c08144defe344c571319e35 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-06-14T20:50:44Z (GMT) No. of bitstreams: 2 Luis Henrique Konzen.pdf: 646860 bytes, checksum: 511c31d83c08144defe344c571319e35 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-14T20:50:50Z (GMT). No. of bitstreams: 2 Luis Henrique Konzen.pdf: 646860 bytes, checksum: 511c31d83c08144defe344c571319e35 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-02 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / O estabelecimento da lavoura de tabaco se dá através do transplante de mudas, e o passo inicial para promover um estande uniforme de plantas e garantir uma boa produtividade da lavoura de tabaco é a utilização de mudas de boa qualidade, para isso, é necessário a utilização de sementes de altíssima qualidade. Neste sentido, os testes de vigor são muito importantes para obtenção de informações adicionais ao teste de germinação e podem auxiliar na tomada de decisão. Assim, objetivou-se com o presente trabalho determinar metodologias do teste de envelhecimento acelerado para avaliação do vigor em sementes de tabaco. Para o estudo, foram utilizados dez lotes de sementes de tabaco. Inicialmente determinou-se a qualidade inicial das sementes de tabaco através dos testes de germinação, primeira contagem de germinação, índice de velocidade de germinação, emergência de plântulas avaliadas aos 7, 14 e 21 dias após semeadura, índice de velocidade de emergência e envelhecimento acelerado com água conduzido conforme proposto pela AOSA. Após a determinação da qualidade inicial das sementes, estudou se o teste de envelhecimento acelerado nas metodologias: envelhecimento acelerado com água, solução salina saturada (40g de NaCl.100mL-1 de água) e solução salina não saturada (11g de NaCl.100mL-1 de água), submetidas a temperatura de 45 e 41⁰C, por períodos de exposição de 24, 48 e 72 horas. O ensaio foi conduzido em delineamento de blocos ao acaso, as médias obtidas por lote, em cada avaliação, foram comparadas pelo teste de Scott-Knott em nível de probabilidade de 5% e foi realizada análise de correlação linear. De acordo com os resultados obtidos, conclui - se que o teste de envelhecimento acelerado com água conduzido sob temperatura de 45ºC combinado com período de exposição de 24 horas mostra-se adequado para avaliação do vigor de sementes de tabaco. / The establishment of tobacco is performed through seedling transplant, and the initial step to promote a uniform plant stand and ensure a good yield of the tobacco crop is the use of good quality seedlings, for this, the use of seeds of the highest quality is necessary. In this sense, vigor tests are very important to obtain additional information to the standard germination test and can assist in decision-making. Thus, the aim of this work was to determine accelerated aging test methodologies for the evaluation of vigor in tobacco seeds. For the study, ten lots of tobacco seeds were used. The initial quality of the tobacco seeds was determined through the germination test, first germination count, germination speed index, emergence of seedlings at 7, 14 and 21 days after sowing, emergence speed index and the accelerated aging with water conducted as proposed by the AOSA. After the determination of the initial quality of the seeds, the accelerated aging test was carry out in the following methods: accelerated aging with water, saturated saline solution (40g NaCl 100mL-1 water) and unsaturated saline solution (11g NaCl 100mL-1 water), submitted to temperature of 45 and 41°C, for periods of exposure of 24, 48 and 72 hours. The assay was conducted in a randomized block designed, the averages obtained per lot at each evaluation were compared by the Scott-Knott test at a 5% probability level and a linear correlation analysis was performed. According to the results, it is concluded that the accelerated aging test with water conducted under a temperature of 45°C combined with a 24 - hour exposure period is adequate for evaluating the vigor of tobacco seeds.

M.Sc. (Biochemistry) / Plants respond to various stress stimuli by activating a broad-spectrum of defence responses that can be expressed locally at the site of pathogen infection (hypersensitive response-HR) as well as systemically in uninfected tissue (systemic acquired resistance-SAR). The ability to continuously respond to both abiotic and biotic stimuli leads to changes in the plants’ physiology, morphology and development. Therefore, there is a need to define and understand the mechanism of the plant defence system, including the mode of recognition, activation of signalling pathways and subsequent defence. In so doing, a long lasting and effective protection against various pathogens may be established. In the current study, the transcriptome status of cultured cells of Nicotiana tabacum was investigated using annealing control primer (ACP)-based differential display (DD) since it is an improved technology to compare patterns of gene expression in RNA samples, isolated from tissue / cells under different biological conditions, using a novel priming system. Here, ACP-DDRT-PCR was used in combination with a next-generation sequencing technology, namely 454 pyro-sequencing, which is the only technique that generates longer reads which are suitable for de novo assembly and annotation of non-model plants like tobacco of which the genome is not yet published in Genbank. SAR occurs following induction by biotrophic or necrotising pathogens. However, it can also be manifested artificially after chemical treatment. In this study, isonitrosoacetophenone (INAP), a novel compound that was originally isolated from extracts of citrus peel undergoing oxidative stress, was used as a chemical inducer and it was hypothesised that this compound induces defence-related responses in plants. In order to investigate this, tobacco cell suspensions were elicited with 1 mM INAP, followed by ACP-DDRT-PCR and subsequent identification of differentially expressed genes using pyro-sequencing.

Les histones désacétylases de type 2 (HD2) ont été caractérisées chez le tabac comme étant des régulateurs négatifs de la mort cellulaire associée à la réponse hypersensible induite par la cryptogéine, un éliciteur protéique.Les principaux objectifs de cette thèse sont d’accroître nos connaissances générales sur les HD2 et de caractériser leur rôle dans la voie de signalisation induite par la cryptogéine.Nous nous sommes tout d’abord intéressé à l’évolution des HD2 au sein des Viridiplantae et nous avons défini deux groupes de HD2. Nous nous sommes ensuite focalisés sur l’évolution des HD2 dans le genre Nicotiana. Nous avons identifié chez le tabac six gènes codant sept isoformes de HD2. Les quatre isoformes de HD2 de Gr1 seraient redondants fonctionnellement.Pour mieux comprendre le mode d’action des HD2 à l’échelle moléculaire et étant donné que nous n’avons pas pu détecter d’activité enzymatique chez les HD2, nous avons tenté d’identifier les gènes cibles et les partenaires protéiques potentiels des HD2.Des expériences de puces à ADN nous ont permis d’identifier des gènes régulés par la cryptogéine de manière HD2 dépendante, parmi lesquels ERF3 qui pourrait constituer un gène majeur impliqué dans l’initiation de la mort cellulaire.Des expériences de co-immunopurification combinées à des analyses par spectrométrie de masse nous ont permis d’identifier des partenaires protéiques des HD2 dont l’histone H4.De manière générale, les résultats obtenus au cours de cette thèse soulèvent la question de savoir si les HD2 possèdent une activité HDAC intrinsèque et nous ont permis de mieux comprendre le rôle des HD2 dans la signalisation nucléaire induite par la cryptogéine. / Type 2 histone deacetylases (HD2s) have been characterized in Nicotiana tabacum as negative regulators of hypersensitive response (HR)-associated cell death induced by cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea.The main objectives of this thesis are to increase our general knowledge about HD2s and to characterize their role in the signaling pathway induced by cryptogein.We first examined the evolution of HD2s in green plants and defined two groups of HD2s. We then focused on the evolution of HD2s in the genus Nicotiana. Six genes coding seven isoforms of HD2s were identified in N. tabacum. The four Gr1 HD2 isoforms are functionally redundant in the response to salt stress.To better understand the mode of action of HD2s at the molecular level, and since we – as well as other research groups – were unable to detect any enzymatic activity from HD2s, we tried to identify target genes and potential protein partners of HD2s.Microarray experiments allowed us to identify genes that are regulated by cryptogein in a HD2-dependant fashion. Among these genes, ERF3, coding an ethylene-responsive factor, could be a major gene involved in the initiation of HR-associated cell death.Co-immunopurification experiments combined with mass spectrometry analyses permitted us to identify protein partners of HD2s such as histone H4.Globally, the results obtained during this thesis call into question whether HD2s do have an HDAC activity themselves, or are associated into complexes containing HDACs from the RDP3/HDA1 family. These results also permitted us to better understand the role of HD2s in the nuclear signaling pathway induced by cryptogein in tobacco.

Essa pesquisa objetivou avaliar métodos para estimar o vigor das sementes de fumo (Nicotiana tabacum L.) variedade Virgínia, cultivar CSC 439, nuas e peletizadas representadas por cinco lotes de sementes. Essas sementes foram submetidas aos seguintes testes de vigor: condutividade elétrica (0,5; 0,8; e 1,0 g e 2,5; 4,0 e 5,0 g de sementes nuas e peletizadas, respectivamente, hidratadas por 2, 4, 6, 8 e 24h em 25 mL de água destilada à 25 °C), envelhecimento acelerado (41 °C e 43 °C por 12 e 24h) com água (100% UR) e com solução salina de NaCl saturada (76% UR) e deterioração controlada (graus de umidade de 20% e 24% para sementes nuas e 8% e 12% para peletizadas, a 40 °C e 43 °C por 24 e 48h). As avaliações foram realizadas aos 7, aos 10 e aos 16 DAS (dias após a semeadura). Adicionalmente foi determinado o grau de umidade e realizados os testes de germinação, de primeira contagem de germinação e, a emergência da plântula e a velocidade de emergência da plântula. O delineamento experimental foi o inteiramente casualizado com quatro repetições. Os dados foram submetidos separadamente à análise de variância e a comparação das médias pelo teste de Tukey a 5% de probabilidade. Todas as análises foram repetidas uma vez. Conclui-se que o teste de condutividade elétrica não é eficiente para ordenar os lotes de semente de fumo, nuas e peletizadas, em diferentes níveis de vigor. Para o teste de envelhecimento acelerado as condições mais adequadas são 41 ºC por 12 horas de exposição com avaliação aos 7 dias após a semeadura, utilizando água (100% UR) para as sementes nuas e solução salina de NaCl (76% UR) para as sementes peletizadas. Para o teste de deterioração controlada, as combinações mais adequadas para as sementes nuas são 24% de água, exposição a 43 °C por 24h e avaliação aos 7 dias após a semeadura e para as sementes peletizadas 8 % de água a 43 °C por 48h e avaliação aos 16 dias. / The objective of this research was to evaluate methods for estimating the physiological quality of tobacco seeds (Nicotiana tabacum L.) \'Virginia variety, CSC 439\' cultivar. For this, five original seeds lots and five coated seed lots were used. The seed vigor were evaluated by electrical conductivity test (0.5, 0.8, and 1.0 g and 2.5, 4.0 and 5.0 g of original and coated seeds, respectively, hydrated for 2, 4, 6, 8 and 24 hours in 25 mL of distilled water at 25 ° C), accelerated aging test (41 ° C and 43 ° C for 12 and 24 hours) with water and saturated salt solution (NaCl) and controlled deterioration test (moisture content 20% and 24% for original seeds and 8% and 12% for coated seeds at 40 ° C and 43 ° C for 24 and 48). The evaluations were performed at 7, 10 and 16 DAS (days after sowing). Additionally, it was determined the seed the moisture content, germination test, first counting, seedling emergence and speed of seedling emergence. The experimental design was a completely randomized and the means were compared by Tukey test (5%). In conclusion, the electrical conductivity test is not efficient to sort lots of original and coated tobacco seeds in different levels of vigor. On the accelerated aging test the most adequate conditions are observed at 41 ºC for 12 hours of exposition and evaluations performed at 7 days after sowing, by using water (100% HR) for the original seeds and NaCl saturated salt solution (76% HR) for coated seeds. On the controlled deterioration test for the tobacco seeds the most adequated conditions are observed with the combinations of 24% of moisture content for the original seeds at 43 °C for 24 hours on evaluations performed at 7 days after sowing and 8% moisture content at 43 °C for 48 hours of exposition for coated seeds.