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Crown gall disease of plants is caused by introduction of foreign DNA into susceptible plant cells by strains of Agrobacterium tumefaciens. The expression of bacterial virulence genes is triggered by chemicals present in plant wound exudates. The exudates contain a number of phenolic compounds which act as chemical signals inducing expression of a number of genes directing the DNA transfer process. These are the virulence or vir genes, and vir::lac reporter gene fusions have been widely used to assay vir gene induction in Agrobacterium tumefaciens strains. Using such strains to monitor vir gene expression, Stachel et al. (1985) isolated from Nicotiana tabacum two active acetophenones: 3,5-dimethoxy-4-hydroxyacetophenone, ("acetosyringone" or AS), and α-hydroxy-3,5-dimethoxy-4-hydroxy-acetophenone, ("hydroxyacetosyringone" or HO-AS). However, in vitro assay results suggested that other more common compounds also exhibited activity (Spencer and Towers, 1988). This analysis of structure-activity relationships of induced vir expression in A. tumefaciens was presented in a previous thesis (Paul Spencer, M.Sc. thesis). The results revealed that a variety of commonly occurring plant phenolic compounds were capable of activating vir genes. In addition to the acetophenones, a variety of benzoic and cinnamic acid derivatives, and even a few chalcones of appropriate ring substitution were active. This thesis reports the isolation and identification of a number of these compounds in plant wound exudates. Some Agrobacterium tumefaciens strains are restricted in host range to certain grapevine cultivars. Subsequent to the development of a convenient and sensitive plate-bioassay method, a strongly active component in grapevine wound exudates was purified. A newly described vir-inducing phenolic compound was isolated from a number of Vitis cultivars using gel filtration, thin layer and high pressure liquid chromatographies. This was identified as syringic acid methyl ester (3,5-dimethoxy-4-hydroxybenzoic acid, methyl ester), using mass spectrometry. However, the presence of this compound in grapevine wound exudates does not provide a simple explanation for host range limitation of grapevine strains since it induces vir gene expression in both limited and wide host range strains of A. tumefaciens. Interestingly, neither AS nor HO-AS were present in grapevine-derived extracts. A convenient polyamide column chromatographic method was subsequently developed to permit rapid purification of plant-derived vir gene inducing mixtures, which were detected using the newly developed plate bioassay. Derivatized polyamide fractions were then analysed by combined gas chromatography-mass spectrometry (GC-MS). GC-MS proved to be an ideal means for the identification of the phenolic components in partially purified extracts. Examination of wound exudates from a range of host and non-host species revealed that the production of the acetophenones is restricted to members of the Solanaceae. Some experiments focussed on the biosynthetic precursors of the acetophenones in Nicotiana species. Wound exudates of the majority of species belonging to other plant families contained benzaldehydes and/or benzoic and cinnamic acid derivatives. The induction of virE gene expression was examined in the related Agrobacterium species, A. rhizogenes. To do this, the virE::lacZ gene fusion plasmid pSM358cd was introduced into A. rhizogenes A4 by triparental mating and the strain "A4/pSM358cd" was used to analyze vir activation. Acetophenones, chalcones, benzaldehydes, and benzoic and cinnamic acid derivatives were found to activate vir genes in A. rhizogenes. / Science, Faculty of / Botany, Department of / Graduate

A invasibilidade do capim-annoni-2 em agroecossistemas tem sido atribuída as suas características biológicas e relações ecológicas. A ecologia da germinação da espécie e a interação alelopática são pouco compreendidas. Este trabalho objetivou determinar a influência de fatores ambientais na germinação, a fitotoxicidade de extratos de capim-annoni-2, identificar e quantificar aleloquímicos fenólicos presentes em suas frações, determinar a influência da degradação da fitomassa e do solo na fitotoxicidade do extrato aquoso, monitorar a degradação dos compostos presentes nos extratos e identificar e quantificar aleloquímicos no solo de área contaminada. Foram realizados experimentos em laboratório utilizando materiais coletados a campo. As sementes germinaram em temperaturas constantes de 15 até 40 °C, mas as percentagens de germinação foram superiores a 94 % apenas em temperaturas alternadas. A exposição à luz aumentou a frequência de plântulas normais. A germinação aumentou com incremento no potencial de água e o número de plantas normais diminuiu com aumento na concentração de cloreto de sódio. A maior taxa de emergência foi observada com as sementes colocadas na superfície do solo, enquanto que nenhuma emergência ocorreu a 4 cm. Foram identificados e quantificados nove compostos fenólicos com potencial aleloquímico nas frações dos extratos de capim-annoni-2: ácido caféico, ácido cumárico, ácido ferúlico, ácido gálico, ácido vanílico, catequina, epicatequina, resveratrol e rutina. A ordem decrescente de fitotoxicidade geral dos extratos brutos no desenvolvimento de plântulas de trigo e alfafa foi acetato de etila, metanólico, aquoso e hexânico. Foi identificada correlação inversamente proporcional entre concentração de ácido vanílico e comprimento de radícula e total de plântulas de alfafa. A simulação da degradação da fitomassa e do solo ocasionou aumento na fitotoxicidade dos extratos sobre a germinação e o desenvolvimento de plântulas, comprovando a liberação dos aleloquímicos e a influência do solo no aumento de sua fitotoxicidade. Extratos etéreos da fitomassa e do solo incubados apresentaram modificações em seus respectivos cromatogramas do primeiro até o sétimo dia de incubação, enquanto a mistura de fitomassa + solo apresentou maiores modificações apenas entre o primeiro e o terceiro dia de degradação. Foram identificados quatro compostos fenólicos com potencial aleloquímico no solo da área infestada com capim-annoni-2: ácido cumárico, ácido ferúlico, catequina e epicatequina. A concentração destes compostos foi maior na superfície do solo e horizontalmente próximo às plantas de capim-annoni-2, evidenciando associação entre as concentrações dos aleloquímicos com a proximidade da planta. / The invasiveness of South African lovegrass in agroecosystems has been attributed to its biological characteristics and the ecological relations. The ecology of species germination and allelopathic interaction are poorly understood. The objective of this work was to determine the influence of environmental factors on germination, phytotoxicity of South African lovegrass extracts, to identify and quantify phenolic allelochemicals present in their fractions, to determine the influence of phytomass and soil degradation on the phytotoxicity of the aqueous extract, to monitor the degradation of the compounds present in the extracts and to identify and quantify allelochemicals in a infested soil. Experiments were carried out in the laboratory using materials collected in the field. Seeds germinated at constant temperatures of 15 to 40 ° C, but the percentages of germination were greater than 94 % only at alternating temperatures. Exposure to light increased the frequency of normal seedlings. Germination increased with increasing water potential and the number of normal plants decreased with increasing sodium chloride concentration. The highest emergence rate was observed with the seeds placed on the soil surface, whereas no emergence occurred at 4 cm. A total of nine phenolic compounds with allelochemical potential were identified in the fractions of extracts of South African lovegrass: caffeic acid, coumaric acid, ferulic acid, gallic acid, vanillic acid, catechin, epicatechin, resveratrol and rutin. The general decreasing order of general phytotoxicity of crude extracts in the development of wheat and alfalfa seedlings was ethyl, methanolic, aqueous and hexane acetate. An inversely proportional correlation was identified between vanillic acid concentration and root and total alfalfa seedlings length. The simulation of phytomass and soil degradation caused an increase in the phytotoxicity of the extracts on germination and seedling development, proving the release of the allelochemicals and the influence of the soil in the increase of its phytotoxicity. The ether extracts of the straw and soil showed changes in their chromatograms from the first to the seventh day of incubation, while the mixture of straw + soil showed greater changes only between the first and third day of degradation. Four phenolic compounds with allelochemical potential were identified in the South African lovegrass infested soil: coumaric acid, ferulic acid, catechin and epicatechin. The concentration of these compounds was higher at the soil surface and horizontally near the South African lovegrass plants, showing an association between concentrations of allelochemicals and plant distance.

CAPES / A procura por novas formas de conservação de alimentos é uma alternativa para minimizar o uso abusivo de conservantes, que em muitos casos são prejudiciais à saúde. Os compostos fenólicos, principalmente os antioxidantes naturais, tem um papel em destaque na nutrição humana. A casca da semente de pinhão (Araucária angustifólia (Bertol.) Kuntze) é um resíduo que pouco aproveitado e que apresenta em sua constituição compostos com atividade antioxidante. A proposta do presente trabalho foi o estudo da extração destes compostos através de um Delineamento Composto Central Rotacional (DCCR) envolvendo a proporção entre a casca e o solvente (X1), proporção entre os solventes de extração (água e etanol, X2) e a temperatura de extração (X3). Foi utilizado o método de extração assistida por Ultra-Turrax (sistema rotor-estator) e os resultados foram avaliados quanto à atividade antioxidante frente ao radical DPPH (equivalente em Trolox, ET), a compostos fenólicos totais (CFT, equivalente em ácido gálico, EAG) e o rendimento em volume da extração (% em relação ao volume de solvente de extração). A última resposta foi considerada com finalidade de avaliar a retenção de extrato pela matriz sólida, devido ao possível inchamento das fibras da casca de pinhão. Os resultados obtidos foram de até 354,21 µmolET.g-1 para DPPH (X1 = 8,75gcasca.100mL-1, X2 = 38,07 %etanol e X3 = 42,5°C), 7304,64 mgEAC.100g-1 para CFT (X1 = 5gcasca.100mL-1, X2 = 50 %etanol e X3 = 55°C) e 80% de rendimento (X1 = 2,44gcasca.100mL-1, X2 = 67,5 %etanol e X3 = 42,5°C). Os modelos preditivos para as respostas foram significativos (R2adj> 0,78), não apresentando falta de ajuste. Para a resposta de CFT foi possível verificar significância (p < 0,05) dos fatores X1, X2, X22 e X3 sem interações significativas (p > 0,05). Já para o modelo de DPPH os fatores que apresentaram significância foram X12, X2, X3 e a interação entre X2X3. O rendimento em volume apresentou influência significativa de X1 e X2 bem como da interação entre X2X3. Os resultados dos modelos demonstraram que o rendimento em volume foi potencializado com o aumento do etanol na solução extratora, sendo este comportamento distinto das respostas de caráter antioxidante. A otimização foi realizada visando a maximização das variáveis respostas, utilizando a função de desejabilidade para unificar os modelos das mesmas, possibilitando a utilização do algoritmo simplex sequencial para determinar a condição ótima. Dentre as condições avaliadas, com a condição de mesma importância para as variáveis (desejabilidades individuais 1-1-1), foi possível maximizar todas as respostas utilizando X1 = 2,45gcasca.100mL-1, X2 = 44,75 %etanol e X3 = 63,51°C. Os resultados foram validados experimentalmente sendo iguais a 7777,84 mgEAG.100g-1 para CFT, 378,66 µmolET.g-1 para DPPH e de 68% para o rendimento em volume. O inchamento da matriz vegetal foi verificado por imagens de Microscopia Eletrônica de Varredura, sendo observado o dilaceramento da matriz vegetal por atuação do sistema rotor-estator que proporciona elevado cisalhamento, além de acentuado significativamente pelo aumento na concentração de água e elevação da temperatura de extração, conforme demonstrado pelos efeitos significativos determinado pelo modelo de resposta do DCCR. Os extratos obtidos com a aplicação das condições experimentais ótimas do DCCR foram avaliados por HPLC-UV-VIS/MS quanto aos compostos fenólicos presentes. O composto identificado em maior concentração foi a + catequina com 13,13 m.g-1 para a condição experimental onde X1 = 12,5 gcasca.100mL-1, X2 = 30 %etanol e X3 = 30°C. O processo de otimização foi um sucesso, podendo ser aplicável a outros compostos. / The search for new food preservation methods is an alternative to minimize the abusive use of conservatives, which in many cases present negative health effects. Phenolic compounds mainly natural antioxidants play an important role in human nutrition. Pinhão seed (Araucária angustifólia (Bertol.) Kuntze) shells is a solid waste which is underestimated while presenting constituents with antioxidant properties. The objective of this work was to study the extraction of such compounds from pinhão shells using the Rotational Central Composite Design (RCCD) with the following variables: shell:solvent proportion (X1), solvent composition (water and ethanol proportion, X2) and extraction temperature (X3). Extraction was carried out with a high energy Ultra-Turrax stirrer and the evaluated responses were DPPH antioxidant activity (Trolox equivalent, TE), total phenolic content (TPC, acid galic equivalent, AGE) and extraction volumetric yield. The later response was considered in order to evaluate the extract retention in the fibrous matrix due to the shell fibers swelling. Results obtained were 354.21 µmolET.g-1 para DPPH (X1 = 8.75gshell.100mL-1, X2 = 38.07 %ethanol e X3 = 42.5°C), 7304.64 mgAGE.100g-1 para TPC (X1 = 5gshell.100mL-1, X2 = 50 %ethanol e X3 = 55°C) e 80% yield (X1 = 2.44gcasca.100mL-1, X2 = 67.5 %ethanol e X3 = 42.5°C). Predictive models to the responses were statistically significant (R2 adj > 0,78) without lack of fit. The significant effects for TPC were X1, X2, X22 e X3, with no significant interaction with each other. For the DPPH model the significant effects were X12, X2, X3 as well as the interaction between X2 and X3. For the extraction yield model the significant effects were X1 e X2 as well as the interaction between X2 and X3. Also, model showed that extraction yield is higher for high ethanol to water proportions, with the opposite effect on the other responses. Optimization was carried out to maximize the evaluated responses using a desirability function to unify all three models and thus using a sequential simplex algorithm to find the optimized condition. Considering the same importance to all variables (individual desirability 1-1-1) it was possible to maximize them using the following conditions: X1 = 2.45gshell.100mL-1, X2 = 44.75 %ethanol e X3 = 63.51°C. For these conditions, the responses were experimentally validated as being 7777.84 mgGAE.100g-1, 378.66 µmolTE.g-1, and 68% extraction yield. Shell fibers swelling was verified by Scanning Electron Microscopy caused by the high energy of the Ultra-Turrax system and increased due to the increase in water and temperature as demonstrated by the RCCD model. Extracts obtaind under the optimized conditions were evaluated by HPLC-UV-VIS/MS to determine their phenolic compounds. + Catequin was the major component found in the extracts at 13.13 m.g-1 for X1 = 12.5 gshell.100mL-1, X2 = 30 %ethanol e X3 = 30°C. The optimization process was successfully performed and applied to other compounds.