Diss. (sammanfattning). Umeå : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Wollongong, School of Biological Sciences. / Typescript. Includes bibliographical references: leaf 269-331.

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Darla J. Sherrier, Dept. of Plant and Soil Science. Includes bibliographical references.

Orientador: Paulo Mazzafera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T23:52:26Z (GMT). No. of bitstreams: 1 Purcino_RubiaPadilha_D.pdf: 1224493 bytes, checksum: 875e464b9ac37fd8cf61d27335a57537 (MD5) Previous issue date: 2006 / Resumo: Xylella fastidiosa (Xf) é uma bactéria fitopatogênica que se desenvolve exclusivamente no xilema de uma grande variedade de plantas cultivadas. Na cultura do citros essa bactéria é conhecida como o agente causal da clorose variega do citros (CVC), doença responsável por prejuízos econômicos consideráveis, desde a década de 80. Embora muitos trabalhos tenham sido realizados com essa bactéria, pouco se conhece sobre o mecanismo de infecção, a composição do meio em que ela se desenvolve, o xilema e o desenvolvimento dos sintomas. Grande parte dos estudos sobre a nutrição da bactéria são realizados in vitro, o que pode não caracterizar a interação entre a bactéria e xilema. Este trabalho é resultado de um subprojeto do genoma funcional, cujo principal objetivo é a caracterização da constituição da seiva do xilema, na tentativa de melhor compreender a nutrição da bactéria e o desenvolvimento da doença. Ao longo da realização dos experimentos e análises dos resultados foram surgindo questões que levaram a necessidade do melhor entendimento do metabolismo do nitrogênio em plantas com CVC. Por isso os resultados gerados foram agrupados em dois capítulos, onde no primeiro são encontrados resultados da caracterização qualitativa e quantitativa das principais substâncias nitrogenadas presentes na seiva do xilema de plantas de citros com CVC, bem como atividade das principais enzimas envolvidas no metabolismo do nitrogênio. A atividade da Redutase do nitrato (RN) nas folhas não sofreu alteração em plantas doentes (PD), porém a atividade da Glutamina Sintetase (GS) foi significativamente maior nos extratos das folhas dessas plantas. Embora a concentração de aminoácidos tenha sido suavemente maior na seiva do xilema de PD, caiu drasticamente no extrato de folhas dessas plantas. O teor de proteínas solúveis também foi menor na seiva do xilema e no extrato de folhas de PD. PD e Planta Sadia (PS) apresentaram o mesmo perfil de aminoácidos em HPLC, contudo em proporções bem diferentes, principalmente dos aminoácidos ASN, GLN e ARG. A poliamina putrescina foi identificada em grande concentração somente em PD. Estes resultados demonstram que PD apresentam alterações no metabolismo de compostos nitrogenados provavelmente em resposta a mudanças na interação dos processos de absorção, assimilação e redistribuirão do nitrogênio. No segundo capítulo são agrupados os dados das análises do metabolismo de açúcares, ácidos orgânicos e investigação das alterações no metabolismo da parede celular. A seiva de PD apresenta menor concentração de glicose, acompanhada de frutose e glicose. Grande quantidade de ácido cítrico também foi determinada nessas plantas. Baseados nesses resultados cultivamos a bactéria em meios com diferentes fontes de carbono e verificamos que o meios que permitiram maior crescimento foram aqueles que a fonte de carbono utilizada foi glicose (I glL), sacarose combinada com citrato nas concentrações de 3g/L de cada fonte de cardono, e citrato na concentração de 3 g/L, o que demonstra que a composição química da seiva do xilema é importante tanto para o processo de agregação como para a formação do biofilme. A infecção com Xi também promoveu modificações nas paredes celulares dos pecíolos. Verificamos por imunocitoquímica em microscópio confocal utilizando os anticorpos 11M 5 e JIM 7, diferenças entre PD e PS nos padrões de esterificação da fração pectina, assim como encontramos no fracionamento da parede celular alterações na composição dos principais carboidratos que constituem a fração hemicelulose / Abstract: Xylellafastidiosa (Xj) is a fastidious bacterium which grows exclusively in the xylem of several plants. In citrus the disease caused by Xf is known as "clorose variegada do citros" (CVC), being responsible for marked loss of productivity since the 80s. Although a meaningful number of reports have been published in the literature about Xf, little is know about the mechanisms controlling infection, the biochemical composition of the xylem fluid where the bacteria develops and how the disease symptoms develop. Most of the studies on the nutritional requirements by the bacteria were carried out with artificial media in vitro, what may not be the exact situation in the xylem. The aim of this thesis was to characterize the xylem sap composition of citrus plants in order to better understand nutritional aspects of the bacteria and this can influence the disease development. During the experiments new questions arose and focus was given to the nitrogen metabolism of citrus plants infected with Cvc. Therefore, the thesis has two chapters. The first chapter contains results on the qualitative and quantitative characterization of the xylem sap of health and diseased plants as well as the activity of enzymes ofthe nitrogen metabolism in plants. The activity ofNitrate reductase (NR) in leaves did not change in diseased plant (PD), however, the activity of Glutamine synthethase (GS) was significantly higher in these leaves. Although amino acids concentration was slightly higher in the sap of diseased plants the leveI dropped drastically in the leaves. The protein contents were lower in the sap and in leaves of diseased plants. Diseased and health plants showed the same amino acid profile in HPLC, but different proportions were observed among amino acids, mainly for the amino acids ASN, GLN and ARG. The polyamine putrescine was found in high concentrations only in diseased plants. These results showed that significant changes take place in the nitrogen metabolism of diseased plants, probably as a response to the alterations in the absorption, assimilation and distribution of nitrogen in the plant. In the second chapter the data on carbohydrates, organic acids and cell wall are presented. The xylem sap of diseased plants showed lower concentration of glucose than health plants. High amounts of citric acid were also found in diseased plants. This information was used to design artificial media to grow the bacteria and study its nutritional need. Best growth was observed with media containing glucose (l g/L), sucrose plus citrate both at 3g/L, and citrate alone at 3 g/L, indicating that the composition of the sap might play a role both in the bacterial agregation as well as in the biofilm formation. Infection with XI also promoted changes in the cell wall of leaf peduncle of infected plants. Immunocytochemical analysis with confocal microscopy, using the antibodies 11M 5 and 11M 7, showed marked differences between diseased and health plants regarding the pectin esterification. Additionally the hemicelullose fraction was also affected. / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

Orientadores: Marcos Antonio Machado, Alexandre Morais do Amaral / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T05:44:26Z (GMT). No. of bitstreams: 1 Baptista_JulianaCristina_M.pdf: 1606790 bytes, checksum: 2559d8ff6d67422a6685a6a8d5a31cd6 (MD5) Previous issue date: 2006 / Resumo: O cancro cítrico, causado pela bactéria Xanthomonas axonopodis pv. citri (Xac), e responsável por consideráveis prejuízos para a produção de laranja, no Brasil e no mundo. A doença e altamente agressiva para a planta de citros, provocando, entre outros, lesões em folhas e frutos, com redução tanto na qualidade de frutos quanto na produtividade. Devido a sua importância, recentemente a bactéria teve seu genoma completamente seqüenciado, onde foram identificados, por homologia de seqüência de proteínas, varias ORFs ("open reading frames") associadas ao processo de infecção. Dentre elas, varias seqüências que teoricamente codificam para proteínas envolvidas na degradação da parede celular vegetal, o que pode ser um indicativo de que a bactéria utiliza mecanismos relacionados a este evento durante o seu processo infeccioso e de adaptação. Entretanto, não ha ate o momento informação experimental a respeito da funcionalidade de seqüências em Xac que permitam a degradação da parede celular. O objetivo principal deste trabalho foi identificar funcionalmente, em Xac, através de ensaios in Vectra e in vitro, genes envolvidos na produção ou transporte de proteínas relacionadas a degradação de celulose e com isso analisar a sua relevância na capacidade infectiva ou adaptativa da bactéria. Para tal, foram produzidos mutantes a partir de inserções aleatórias de transposon e sitio-dirigidas, estabelecendo-se uma coleção de linhagens, cujo fenótipo e genótipo foram avaliados por ensaios biológicos e sequenciamento. Verificou-se que o operon xps do sistema de secreção do tipo II e funcional em Xac e que os genes XAC0028, XAC0029, XAC2374, XAC3516 e XAC4231 participam da degradação de celulose / Abstract: The citrus canker, caused by the bacterium Xanthomonas axonopodis pv. citri (Xac), is responsible for considerable losses for the orange production, in Brazil and abroad. The disease is highly aggressive for citrus plants, resulting, among others, in injuries on leaves and fruits, with a reduction in the quality of fruits as much as in the productivity. Due to its importance, recently the bacterium had its genome completely sequenced and a number of open reading frames (ORFs) where identified by homology with protein sequences and annotated as associated to the infection process. Amongst them, some sequences that theoretically code for proteins involved in the degradation of the plant cell wall, which may indicate that the bacterium uses mechanisms related to this event during the process of adaptation and infection. However, there is no experimental data regarding such sequences to prove their real function during the pathogenesis of Xac. The main objective of this work was to identify functionally, in Xac, through in vitro and in vivo assays, genes involved in the production or transport of proteins related to the degradation of cellulose and, therefore, to analyze its relevance in the capacity of adaptation and infection of the bacterium. During the investigation, a collection of mutant strains of Xac was produced by random and site-directed mutagenesis, which were analyzed through the identification of leaf lesions and their action on degradation of artificial cellulose in plate as well as the growth in leaves. Through tests of mutagenesis and in vitro assays, this study shows that the xps operon that is present in Xac and encodes a type II secretion system machinery is functional and the genes XAC0028, XAC0029, XAC2374, XAC3516 and XAC4231 participate in cellulose degradation. In summary, for the first time it was experimentally demonstrated that Xac is capable in hydrolizing cellulose / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Orientador: Gonçalo Amarante Guimarães Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T09:47:13Z (GMT). No. of bitstreams: 1 Salazar_MarcelaMendes_D.pdf: 4370521 bytes, checksum: 99869d0c9655d47c4e3b2c29de58a274 (MD5) Previous issue date: 2012 / Resumo: A celulose é o polímero mais abundante do planeta e está presente principalmente na parede secundária de células vegetais maduras. O conhecimento dos mecanismos moleculares envolvidos na sua biossíntese, bem como a regulação deste processo é recente e ainda elementar, apesar da sua grande importância. O Eucalyptus é o gênero florestal mais plantado no mundo, especialmente por ser matéria-prima para fabricação de celulose. Investimentos em pesquisas e desenvolvimento estão sendo realizados pelo setor florestal, no intuito de aumentar os ganhos de produtividade de celulose através da pesquisa na área da biologia molecular. Os dados analisados desmostram que a expressão gênica das espécies estudadas (E. globulus, E. grandis e E. urophylla) difere em genes essenciais para a formação dos compostos da parede celular e por fim da madeira. Os dados moleculares são condizentes com dados diferenciais aqui encontrados em relação à composição dos açúcares da parede. Os resultados gerados pela análise de composição da parede por diferentes técnicas, incluindo a recentemente desenvolvida "Perfil Glicômico" mostram uma grande diversidade e quantidade dos carboidratos da parede celular diferentemente distribuídos no xilema das espécies Eucalyptus globulus, grandis e urophylla, bem como em folha. Tais resultados apresentam um grande avanço para o entendimento da composição das paredes celulares de tais espécies. Assim, o objetivo do presente trabalho foi correlacionar os dados moleculares, com dados highthroughput do Perfil Glicômico para entender a composição e arquitetura da parece celular. Espera-se que esses dados possam contribuir para o entendimento da xilogênese e de como os genes trabalham para gerar árvores com características tão distintas, podendo direcionar o melhoramento das mesmas / Abstract: Cellulose is the most abundant polymer in the world and it is present mainly in secondary cell walls of plant mature cells. The knowledge of molecular mechanisms involved in biosynthesis and regulation of this process is recent despite its great importance. The Eucalyptus is the most planted forest genus in the world, especially for being raw material for pulp production. Investments in research and development, especially in the molecular biology field, are being carried out by the forestry sector in order to increase pulp productivity. The data analyzed showed that gene expression of the species the tree species studied here (E. globulus, E. grandis e E. urophylla) differs in essential genes of cell wall compounds formation. Molecular data are consistent with differential data found here in relation to the composition of cell wall sugars. The results generated by the wall composition analysis by various techniques, including the recently developed "Glycome Profiling", showed a wide diversity of carbohydrate of cell wall differently distributed in the xylem of the the Eucalyptus species. These results are a breakthrough in understanding the cell wall composition of these species. The objective of this study was to correlate molecular data with data highthroughput Glycome Profiling to understand the composition and architecture of the cell walls. It is hoped that these data will contribute to the understanding of wood formation and how genes work to generate trees with such different characteristics / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

The extension of frozen-thawed epidermal strips prepared from the first centimetre below the hypocotyl hook of six day old dark grown Phaseolus vulgaris seedlings while immersed in various buffers and under various tensions was characterized. This was done in an attempt to determine if the acid wall loosening phenomenon, which according to the Acid-growth theory (Taiz, 1984) is thought to mimic part of the auxin mechanism of action, is mediated by unspecified wall loosening enzymes. Epidermal strips were found to be significantly loosened by media pH 6.0 to pH 2.6 (0.05M citric acid-O.lOM disodium phosphate) relative to pH 7.5. A minimum stress between 1.6 and 7.6 grams was required for the acid-extension of strips 4.5±0.5 mm wide. Regardless of tension, extension by tissues in an acid medium was largely transient For example, tissues tensioned by a 16.0 gram load reached a maximum extension rate of 6.18 ±1.37% of initial length per hour (L°/hr) between 4 and 6 minutes after immersion in pH 4.8. The rate was 1.29±0.17% L°/hr between 55 and 60 minutes and 1.05±0.14% L°/hr between 220 and 240 minutes. Total acid-extension over four hours was 4.24±0.57% L°. The extension response was found to be stable; newly harvested tissues whether frozen or not performed similarly to strips aged up to 15 days at -12°C before being extended. The performance of strips immersed in unbuffered solutions indicated that tissues were self-buffering at an acid pH probably because of the fixed carboxyls within the wall. The capacity for acid-extension by epidermal strips was lost in mature tissues harvested 4-5 cm below the hypocotyl hook. Temperature coefficients from extension rates were determined at several pHs. The results were highly variable. The acid-extension of strips boiled 15 minutes in ethanol or extracted in 3M NaCl for 4 hours at 4°C or 6M LiCl for 8 hours was determined in several pHs. The impact of the treatments was largely a suppression of the initial burst of acceleration. Extension rates following the initial surge were relatively unaffected. Glycosidase activities in untreated, ethanol-boiled, or salt extracted strips were determined. β-glucosidase was found to be most active in untreated strips with lesser levels of β-galactosidase and β-xylosidase and a trace of α-galactosidase being detected. Ethanol-boiling and LiCl-extraction removed or deactivated all four activities from the strips and NaCl-extraction lowered all four activities 70-80%. NaCl proved to have solubilized most of the missing β-glucosidase and β-galactosidase when the extraction solution was assayed following desalting and concentration. LiCl solubilized most of β-xylosidase. It was concluded that glycosidases and any other similarly soluble enzyme cannot be responsible for long term acid wall loosening in bean epidermis. If an enzyme is involved, it must be extremely stable and tightly bound to the wall. The acid-extension performance of frozen-thawed longitudinally halved hypocotyl sections in comparison to epidermal strips, as well as other evidence was considered support for another hypothesized mechanism of acid wall loosening, the displacement of calcium bridges. / Science, Faculty of / Botany, Department of / Graduate

The position of the cell division plane in plants is determined by the position of the preprophase band. The pre prophase band (PPB) is a ring of microtubules centered around the nucleus on the inner side of plasma membrane that establishes the cortical division site. The PPB forms at the end of G2 and breaks down at the end of prophase leaving behind protein markers of its position that are collectively called the cortical division site. During cytokinesis the phragmoplast expands towards the cortical division site and mediates the fusion of the new cell plate with the mother cell at that position. Several proteins necessary for PPB formation in plants have been identified, including maize DCD1 and ADD1 and Arabidopsis TON2, which are all type 2A protein phosphatase (PP2A)B" regulatory subunits. DCD1, ADD1, and TON2 localize to the PPB and the cortical division site through metaphase. The PP2A subunits each have two EF-hand domains, which are predicted to bind calcium ions. Since calcium ions are important for some aspects of cell division, we designed a series of constructs to test if TON2 binds calcium. TON2 protein was cloned into expression vectors, pET42a, and expression of TON2 protein was confirmed via Western blotting and immunodetection using a GST antibody. Site directed mutagenesis was used to mutate the TON2 EF-hand domains and mutated cDNAs were also cloned into expression vectors. These were then expressed in bacterial systems. Finally, the GST tagged proteins were purified. In the future, wild-type and mutated proteins TON2 proteins will used in calcium binding assays to determine if TON2 binds calcium.