Os pigmentos naturais, além de fornecerem cor e beleza aos diferentes organismos, desempenham importantes funções e ações biológicas, como as funções vitais de fotossíntese, respiração celular e a ação antioxidante. Assim, o presente estudo investigou os potenciais citotóxicos, genotóxicos e protetores dos pigmentos naturais clorofila b (CLb) e luteína (LT), isolados e em combinação, em doses normalmente consumidas na dieta. Para isso foram utilizados o teste do micronúcleo em células da medula óssea e do sangue periférico e o ensaio cometa em células do sangue periférico, rim e fígado de camundongos. Também foram avaliados parâmetros bioquímicos de estresse oxidativo, glutationa e substâncias reativas ao ácido tiobarbitúrico no rim e no fígado, além de glutationa e catalase no sangue periférico, a fim de investigar o papel antioxidante desses pigmentos. A capacidade da LT de alterar a expressão de genes de resposta ao estresse oxidativo e defesa antioxidante foi avaliada no tecido hepático dos camundongos utilizando a técnica de PCR em tempo real (RT-qPCR) array. Para a verificação da atividade protetora dos pigmentos, a cisplatina (cDDP) foi utilizada como indutor de danos oxidativos e ao DNA. Adicionalmente, foram avaliados os potenciais citotóxicos, genotóxicos e antioxidantes da LT em cultura de células de hepatocarcinoma humano por meio do teste do MTT [3-(4,5-dimetil-2-tiazolil)-2,5-difenil-2H-tetrazólio], ensaio cometa e avaliação de parâmetros bioquímicos de estresse oxidativo, a fim de se estabelecer comparações entre resultados in vitro e in vivo, bem como propor mecanismos de ação para os efeitos antigenotóxicos da LT. Nossos resultados mostraram que os tratamentos com os pigmentos, tanto a LT quanto a CLb, isolados ou em combinação não causaram qualquer dano ao material genético nos testes empregados e ofereceram proteção frente aos danos induzidos no DNA pela cDDP tanto in vitro como in vivo. Efeitos antioxidantes para ambos pigmentos foram observados no sangue periférico e nos tecidos renais e hepáticos, e a LT também melhorou os parâmetros de estresse oxidativo avaliados in vitro. Na avaliação da expressão de genes de resposta ao estresse oxidativo em células do fígado de camundongos, a cDDP diminuiu a expressão 16 genes, entre eles, importantes genes responsáveis pela manutenção do estado redox da célula. Além disso, a LT mostrou que pode atuar como antioxidante não só agindo diretamente no seqüestro de radicais livres, mas também, induzindo a expressão de 11 dos 84 genes avaliados, e de 15 genes quando associada à cDDP. Em resumo, nossos resultados mostraram que a LT e a CLb, isoladas ou em combinação, nas doses consumidas normalmente na dieta, podem contribuir para a promoção da saúde considerando seus efeitos antigenotóxicos e antioxidantes. / The natural pigments, in addition to providing color and beauty to the different organisms, play important biological role, including the vital functions of photosynthesis, cellular respiration and antioxidant action. Thus, this study investigated the genotoxic and protective potential of natural pigments, alone and in combination, chlorophyll b (CLb) and lutein (LT), in concentrations usually consumed in the diet. For this purpose, we used the micronucleus test in bone marrow and peripheral blood cells and the comet assay in peripheral blood, kidney and liver of mice. Biochemical parameters of oxidative stress were also evaluated, such as glutathione and thiobarbituric acid reactive substances in the kidney and liver and catalase and glutathione in peripheral blood in order to investigate the antioxidant properties of these pigments. The ability of lutein to alter the expression of genes involved in oxidative stress response and antioxidant defense was evaluated in liver tissue of mice using the technique of real-time PCR (RT-qPCR) array. To verify the protective activity of the pigments, cisplatin (cDDP) was used as an inducer of oxidative stress and DNA damage. Additionally, we assessed the genotoxic and antioxidant potential of LT in cell cultures of human hepatocellular carcinoma using the test of MTT [3 - (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium], comet assay and assessment of biochemical parameters of oxidative stress, in order to make comparisons between results in vitro and in vivo, as well to propose mechanisms to antigenotoxic effects of LT. Our results showed that treatment with the pigments, both the LT and the CLb, alone or in combination, did not cause any DNA damage in the tests employed and offered protection against DNA damage induced by cDDP in both in vitro and in vivo. Antioxidant effects were observed for both pigments in peripheral blood, kidney and liver, and LT also improved the oxidative stress parameters measured in vitro. In the evaluation of the expression of genes involved in response to oxidative stress in liver cells of mice, cDDP decreased the expression of 16 genes, among them, important genes responsible for maintaining the redox status of the cell. Moreover, LT showed that it can act as an antioxidant not only acting directly in the scavenging of free radicals, but also by inducing the expression of 11 of the 84 genes evaluated and 15 when LT was associated to cDDP. In summary, our results showed that the LT and CLb, alone or in combination, at concentrations usually consumed in the diet can contribute to health promotion considering their antigenotoxic and antioxidant effects.

Made available in DSpace on 2017-07-10T19:23:35Z (GMT). No. of bitstreams: 1 Davi.pdf: 4681290 bytes, checksum: 2364ed0a7d9e06ef6976e7af50609d99 (MD5) Previous issue date: 2013-02-15 / The Brazilian agriculture, propelled by the globalization of world s economy, needs to develop new technologies and studies that will increase yield and decrease the costs of production. This increase can be achieved through the management of adequate productive areas and must be cautious about its environmental impact, attempting to cause less impact and more production. Since nitrogen is one of the most useful agricultural inputs, it is important the development of researches aiming local application and evaluating the influence of this and other nutrients on the agricultural production. Determining the amount of chlorophyll is a method that has been being utilized to predict the necessity of nitrogen fertilization in various cultures. This research had the objective of evaluating spatial dependence and creating thematic maps referring to the index of chlorophyll in comparison with the growing of soybean and corn culture for correlation analysis with chemical and physical attributes and yield. It was used a portable device to measure the index of chlorophyll (non-destructive, inexpensive and fast, when compared to laboratory process). It were applied techniques of spatial dependence analysis on data constructing thematic maps through interpolation by kriging. All chemical and physical soil properties showed spatial dependence, allowing site-specific management of these attributes. The yield presented spatial correlation with chemical and physical soil properties and spatial correlation between chlorophyll and productivity was proved significant, mostly in vegetative stages. / A agricultura brasileira, impulsionada pela globalização da economia mundial, necessita desenvolver novas tecnologias e estudos que aumentem a produtividade e diminuam os custos de produção. Este aumento pode ser obtido através do manejo adequado das áreas produtivas e deve observar o impacto ambiental gerado, buscando-se máxima produção. Sendo o nitrogênio um dos insumos agrícolas mais consumidos e este estar relacionado diretamente com o teor de clorofila presente nas plantas, é importante que sejam desenvolvidas pesquisas visando a aplicação localizada e a avaliação da influência deste e de outros nutrientes para a produção agrícola. A determinação do teor de clorofila por meio de clorofilômetro vem sendo utilizado para predizer a necessidade de adubação nitrogenada em várias culturas. O presente projeto teve por objetivo avaliar a dependência espacial e confeccionar mapas temáticos referentes ao índice de clorofila em função do crescimento das culturas de soja e milho para análise da correlação com atributos químicos, físicos e da produtividade. Para isto, foi utilizado um aparelho portátil de medição de índice de clorofila (não destrutivo, de baixo custo e de rápida realização, se comparado com processos laboratoriais). Foram aplicadas técnicas de análise da dependência espacial nos dados, construindo mapas temáticos por meio do interpolador krigagem. Todos os atributos químicos e físicos do solo estudados apresentaram estrutura de dependência espacial, permitindo o manejo localizado destes atributos. A produtividade apresentou correlação espacial com atributos químicos e físicos do solo, e a correlação espacial entre índice de clorofila e produtividade se mostrou significativa em sua maior parte, nos estádios vegetativos.

Made available in DSpace on 2016-08-17T17:11:02Z (GMT). No. of bitstreams: 1 DISSERTACAO_ANTONIA DE LIMA DA SILVA.pdf: 596277 bytes, checksum: 87785a3c189c598f2fcb892036144118 (MD5) Previous issue date: 2015-05-07 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / In the humid tropics, construction and soil fertility maintenance represent a challenge for farmers and also for the researchers who are dedicated to the implementation of sustainable farming systems. The objective was to evaluate the influence of ground cover use of inorganic nitrogen and increase efficiency of water use on the soil mechanical resistance to penetration, chlorophyll content and productive corn parameters in the agri-environmental conditions of the humid tropics. The treatments were: soil cover with corn + inorganic nitrogen (Ninorg) + shift watering every 4 days (4CN); Corn with ground cover + shift watering every 4 days (4C); Without mulching corn + shift + Ninorg watering every 4 days (4SN); Corn without soil cover + shift watering every 4 days (4); Corn with ground cover + shift + Ninorg watering every 8 days (8CN); Corn with ground cover + shift watering every 8 days (8C); Without mulching corn + Ninorg + shift watering every 8 days (8SN); Corn without cover soil + shift watering every 8 days (8S). In reading I to relative chlorophyll content (CRT I) 4CN treatment differed from all the other treatments being similar only with the 4SN treatment. Soil moisture levels differ between treatments in the evaluations performed at 4 and 8 days after irrigation, the evaluation performed at 4 days after irrigation, the 4CN treatments, 4C and 8C with higher percentages differ from 4SN treatments, 4S and 8SN . Regarding the soil penetration resistance (RP), in assessing four days after irrigation was found that there were no differences between treatments in the superficial layers of 0-5 cm, as in the layer 6-10 cm, the 8S treatment differed from other treatments, with higher PR of 1.53 MPa. With respect to the evaluation performed on the 8th day after the irrigation, it was found differences between treatments within each depth and maximum penetration resistance (PMAX). Most productivity components 4CN demonstrates that treatment promoted significant differences when compared to treatments with irrigation every 8 days. Regarding the efficiency of water use, treatment with 8-day irrigation schedule, proved to be more efficient, with the maximum value of 17.28 kg / m3. In view of the foregoing, it is concluded that the efficiency of water usage was higher than in treatments with high turn independently of the presence or non-watering of the soil cover. / No trópico úmido, a construção e manutenção da fertilidade dos solos representam grande desafio para os agricultores e também para os pesquisadores que se dedicam à implantação de sistemas agrícolas sustentáveis. Objetivou-se avaliar a influência do uso de cobertura do solo, do incremento de nitrogênio inorgânico e eficiência do uso da água sobre a resistência mecânica do solo à penetração, conteúdo de clorofila e os parâmetros produtivos do milho nas condições agroambientais do trópico úmido. Os tratamentos avaliados foram: Milho com cobertura do solo + nitrogênio inorgânico (Ninorg) + turno de rega a cada 4 dias (4CN); Milho com cobertura do solo + turno de rega a cada 4 dias (4C); Milho sem cobertura do solo + Ninorg + turno de rega a cada 4 dias (4SN); Milho sem cobertura do solo + turno de rega a cada 4 dias (4S); Milho com cobertura do solo + Ninorg + turno de rega a cada 8 dias (8CN); Milho com cobertura do solo + turno de rega a cada 8 dias (8C); Milho sem cobertura do solo + Ninorg + turno de rega a cada 8 dias (8SN); Milho sem cobertura o solo + turno de rega a cada 8 dias (8S). Na leitura I do Teor Relativo de Clorofila (TRC I), o tratamento 4CN diferiu de todos os outros tratamentos, sendo semelhante apenas com o tratamento 4SN. Os teores de umidade do solo diferiram entre os tratamentos nas avaliações realizadas aos 4 e 8 dias após a irrigação, na avaliação realizada aos 4 dias após a irrigação, os tratamentos 4CN, 4C e 8C com maiores porcentagens diferiram dos tratamentos 4SN, 4S e 8SN. Com relação a resistência do solo à penetração (RP), na avaliação 4 dias após a irrigação foi constatado que não houve diferenças entre os tratamentos nas camadas superficiais de 0-5 cm, já na camada 6-10 cm, o tratamento 8S diferiu dos demais tratamentos, apresentando maior RP de 1,53 MPa. Com relação à avaliação realizada aos 8 dias após a irrigação, verificou-se diferenças entre os tratamentos dentro de cada profundidade e resistência máxima à penetração (PMAX). A maioria dos componentes de produtividade demonstra que o tratamento 4CN promoveu diferenças significativas quando comparado aos tratamentos que receberam turno de rega de 8 dias. Em relação a eficiência do uso da água, os tratamentos com turno de rega de 8 dias, mostraram-se mais eficientes, apresentando o valor máximo de 17,28 kg/m3. Diante o exposto, conclui-se que a eficiência do uso da água foi superior nos tratamentos com maior turno de rega independente da presença ou não de cobertura do solo.

O déficit hídrico é o principal fator limitante na produtividade das culturas agrícolas. Com a crescente expansão da cultura canavieira rumo a regiões de marcantes déficits hídricos, torna-se essencial o desenvolvimento de variedades tolerantes a este tipo de estresse. O objetivo deste trabalho foi estudar os efeitos do déficit hídrico, em 20 variedades de cana-de-açúcar, visando identificar características para auxiliar o programa de melhoramento da cultura. O conteúdo relativo de clorofila, a eficiência quântica potencial do fotossistema II (Fv/Fm), o teor relativo de água (TRA), o incremento em altura dos colmos (IAC) foram utilizados como parâmetros fisiológicos além das características tecnológicas da matéria prima Brix e Pol. O déficit hídrico foi imposto pela suspensão da irrigação durante 3, 10 e 20 dias. O déficit hídrico reduziu o conteúdo relativo de clorofila, TRA, Fv/Fm, IAC e Pol para a maioria das variedades analisadas enquanto o teor de sólidos solúveis, mensurada através do Brix, não foi afetado. O período de suspensão de rega de 03 dias não foi suficiente para induzir déficit hídrico e, as melhores respostas para identificação de diferenças fisiológicas dentro de variedades foi obtida aos 20 dias. Não foi observado padrão de resposta que possa ser relacionado com o nível de tolerância das variedades para o resultado dos parâmetros IAC, TRA, Brix e Pol. Contudo constatou-se que os parâmetros Fv/Fm e conteúdo relativo de clorofila mostraram-se eficientes para diferenciar variedades de cana-de-açúcar tolerantes e sensíveis à deficiência hídrica. / Drought is a major limitation to crop productivity worldwide. The sugarcane is a crop in expansion towards areas of marked water deficit and it is essential the development of drought tolerant varieties. The aim of this work was to study the effects of drought in 20 sugarcane genotypes to identify parameters to improve the genotype screening in the breeding program. The relative chlorophyll content, potential quantum efficiency of photosystem II (Fv/Fm), the relative water content (TRA), the increase in height of stalks (IAC) were evaluated as physiological parameters in addition to the technological parameters Brix and Pol. The water deficit was imposed by withholding irrigation for 3, 10 and 20 days. The water deficit reduced the relative chlorophyll content, TRA, Fv/Fm, Pol and IAC for most varieties evaluated while the soluble solids content, measured by ° Brix, was unaffected. The withhold treatment of 03 days was not sufficient to induce water deficit and the best results to identify physiological differences within genotypes were obtained with 20 days. The IAC, TRA, Brix and Pol results were not related to the level of tolerance of genotypes. However, Fv/Fm and relative chlorophyll content traits were effective to distinguish between drought tolerant and susceptible sugarcane genotypes subjected to water stress.

Foram levantados dados referentes a Rede de Monitoramento da Qualidade das Águas Superficiais do Estado de São Paulo, da CETESB (Companhia de Tecnologia de Saneamento Ambiental da Secretaria de Estado de Meio Ambiente do Estado de São Paulo, Brasil ), do período de 1996 a 2001, totalizando 69 pontos de amostragem, (35 em rios e 34 em reservatórios). As variáveis examinadas foram clorofila a, nutrientes e variáveis complementares utilizadas na avaliação do grau de trofia desses ambientes através do Índice de Estado Trófico de Carlson, modificado por Toledo (1983). Os pontos de amostragem em ambientes lóticos apresentaram maiores valores de fósforo total, e os em ambientes lênticos apresentaram maiores valores de clorofila a, DBO e nitrogênio amoniacal. Não houve diferença significativa entre os valores de fósforo total ou de nitrogênio total entre a estação seca e chuvosa.. Em ambos os tipos de ambientes o nutriente limitante foi o fósforo. A clorofila a, corrigida para feofitina a, foi adotada como indicadora da biomassa fitoplanctonica, tanto para reservatórios como para rios, sendo que a relação clorofila a:feofitina a foi de 2:1 em reservatórios e de 1:1 em rios. Os valores médios de clorofila a, calculados através de média geométrica, podem ser utilizados para indicar o grau de eutrofização, bem como a probabilidade de ocorrência de valores máximos. As regressões obtidas entre clorofila a e fósforo total, para rios e reservatórios, foram consideradas distintas, sendo que ambas apresentaram alta correlação. O modelo proposto para a previsão da clorofila a, através das concentrações de fósforo total, em reservatórios, integra os dados do estudo do CEPIS (Centro Panamericano de Ingenería Sanitária y Ciencias del Ambiente), sendo que a introdução do tempo de residência melhora, significativamente, o referido modelo. Foram estimados os valores basais de fósforo total, ortofosfato solúvel, nitrogênio total e de clorofila a, além de transparência para reservatórios e de turbidez para rios, os quais foram considerados valores limites entre a classe oligotrófica e a mesotrófica. Foram propostas ainda novas classificações de trofia, bem como novos índices de estado trófico, distintos para ambientes lóticos e lênticos, nos quais foi introduzida uma nova classe, entre as classes eutrófica e a hipereutrófica, denominada supereutrófica. Estes novos índices foram testados com os dados de 2001, 2002 e 2003 e apresentaram maior sensibilidade e maior coerência entre os índices calculados através das concentrações de clorofila a e de fósforo total do que a metodologia anterior. A eutrofização também afeta o Índice de Proteção da Vida Aquática (IVA), não só por meio do índice de estado trófico, mas também da toxicidade, a qual é detectada em florações de cianobactérias ou quando da aplicação de sulfato de cobre. / Data from 1996 to 2001, from CETESB’s Surface Water Monitoring Program for the State of São Paulo, amounting to 69 sampling sites, (35 located in rivers and 34 in reservoirs)was gathered, and analyzed for chlorophyll a, nutrients and other variables used on the evaluation of the trophic status, through a modified Carlson’s Trophic State Index (TSI), (Toledo, 1983). Higher concentrations of total phosphorus were found in lotic environments, and higher values of chlorophyll a, BOD and nitrogen were registered in lentic ones. There was no significant difference between total phosphorus and total nitrogen between the dry and wet seasons. Phosphorus was the limiting nutrient in both environments. Chlorophyll a, corrected for phaeophytin a, was adopted as an indicator of phytoplanktonic biomass for reservoirs, as well as for rivers. The average ratio between chlorophyll a and phaeophytin a, was of 2:1 in reservoirs and of 1:1 in rivers. Average concentrations of chlorophyll a, calculated as geometric mean, can be used to predict trophic status as well as the probabilities of maximum values in a year period. Chlorophyll a versus total phosphorus regressions were significantly different for rivers and reservoirs, both with high correlation. The prediction of chlorophyll a through phosphorus, in reservoirs, was found to be statistically similar to the one proposed by CEPIS study, and was improved by considering the residence time information. Background values of total phosphorus, soluble orthophosphate, nitrogen, and chlorophyll a, were estimated, as well as transparency for reservoirs and turbidity for rivers, and were considered the boundaries between the oligotrophic and mesotrophic categories. New classifications of trophic status were proposed, as well as new trophic state indexes, specific for rivers and reservoirs, introducing a new trophic category, supereutrophic, which divides the eutrophic and hypereutrophic classes. These new indexes were tested with the data of 2001, 2002 and 2003, and showed higher sensibility and better agreement between chlorophyll a and phosphorus idexes, than the methodology previously used. Eutrophication, not only influences the Aquatic Biota Protection Index (IVA) through the TSI, but through toxicity, detected in cyanobacteria blooms, as well as resulting of the use of copper sulfate to control algae populations.

A doença cancro cítrico é considerada uma das mais importantes doenças da citricultura devido ao seu poder de proliferação nas fazendas, e aos danos causados às plantas e frutos. Os prejuízos causados pela presença da doença são consideravelmente preocupantes, pois as principais medidas de controle pelos órgãos responsáveis envolvem a erradicação de plantas infectadas e demais plantas vizinhas, inviabilizando economicamente grandes áreas produtivas. A legislação brasileira exige um extenso protocolo de atividades que necessita ser realizado antes da confirmação do diagnóstico. Atrasos na confirmação do diagnóstico favorecem a proliferação da doença. Assim, qualquer esforço em acelerar esta detecção deve com certeza ter um grande impacto nesta área. Esta é a motivação de nosso trabalho, onde aplicamos a técnica de espectroscopia por imagens de fluorescência em folhas de culturas cítricas com a intenção de avaliar a capacidade de diagnóstico desta técnica em plantas assintomáticas contaminadas no laboratório com cancro cítrico. O objetivo é determinar o instante de tempo mínimo necessário entre a infecção e o diagnóstico preciso da doença. Este estudo foi aplicado para experimentos envolvendo amostras destrutivas e não-destrutivas. Os resultados mostram a possibilidade de aplicar tal técnica na detecção de cancro cítrico. / The citrus canker disease is considered one of the most important citrus diseases due to its ability to spread on farms, and to damage plants and fruits. The damage, caused by the citrus canker, can be devastating, because the main control actions involve the eradication of infected plants and other plants nearby, causing large economic losses. Brazilian law requires an extensive testing protocol to confirm the diagnosis. Delays in diagnosis tests allows the spread of the disease. Therefore, any effort to accelerate this procedure will have a major impact in this area. This is the motivation of our work, where we apply the fluorescence imaging spectroscopy technique on citrus leaves with the goal to evaluate the diagnostic capability of this technique in asymptomatic plants infected with citrus canker in the laboratory. The goal is to determine the minimum time delay between infection and accurate diagnosis of the disease. This study was applied to experiments involving non-destructive and destructive samples. The results show the possibility of applying this technique in the detection of citrus canker.

Dans les expériences modélisant la photosynthèse, une bonne dispersion des molécules de chlorophylle est requise. Afin d'obtenir cette bonne dispersion, une solution consiste à les ancrer à un hôte. Dans cette étude, des nouveaux matériaux hybrides sont synthétisés en utilisant des hydrotalcites et un dérivé de la chlorophylle : la chlorophylline. La chlorophylline est incorporée et dispersée dans les hydrotalcites par co-précipitation ou pendant la reconstruction des hydrotalcites. Dans cette thèse est également discuté l'effet d'une irradiation par micro-onde. Dans tous les cas sont obtenus des matériaux stables, où les molécules de chlorophylline sont isolées et liées aux hydrotaltices par des liaisons avec les groupes hydroxy OH. Dans ce travail, de nouveaux matériaux sont synthétisés en utilisant les hydrotalcites et la chlorophylle a. Dans ce cas là, il est mis en valeur que la stabilisation de la chlorophylle a peut dépendre du support, à la fois par la nature et le rapport des métaux constituants les hydrotalcites. Ces matériaux hybrides obtenus (chlorophylle a/hydrotalcites) sont également stables. Ces matériaux ont été testés par lixiviation à l'acétone. Dans tous les cas, les composés lixiviés sont de la chlorophylle a, montrant ainsi que la nature et la composition des molécules sont préservées même après trente jours, bien qu'il soit à noter que dans le cas des hydrotalcites Ni/Al une faible fraction de la chlorophylle est décomposée en phéophytine. Finalement, bien que les hydrotalcites soient des composés relativement simples, ils s'avèrent tout à fait adaptés pour empêcher la dégradation des molécules de chlorophylle. / In experiments modeling photosynthesis, well dispersed chlorophyll molecules are required. A solution could be to anchor them on some host. In this work, new hybrid materials are synthesized using hydrotalcite and a chlorophyll derivative : chlorophyllin. Chlorophyllin is incorporated and dispersed in hydrotalcite through simultaneous precipitation or during hydrotalcite reconstruction. The effect of microwave irradiation on the crystallization step is discussed. In all cases stable materials are obtained, chlorophyllin molecules are isolated and bonded through hydrotalcite OH groups. Also new hybrid materials are synthesized using hydrotalcites and chlorophyll a. The effect of the support composition on chlorophyll a stabilization is discussed. The stability of the adsorbed chlorophyll a is affected by the support nature and the metal ratio of the hydrotalcite. The obtained hybrid compounds (chlorophyll a on hydrotalcite) are stable. Those hybrids were tested in lixiviation with acetone. In all cases, the lixiviated compound was chlorophyll a showing that the composition and nature of the molecule was preserved even after thirty days, although Ni/Al hydrotalcites decomposed a small fraction of the chlorophyll to pheophytin. Hydrotalcites being basic compounds turn out to be an adequate material to avoid degradation of chlorophyll molecules.

Rose-scented geranium (Pelargonium capitatum x P. radens) belongs to the family Geraniaceae and it is a multi-harvest, high value, commercially important essential oil yielding aromatic plant. The essential oil extracted from the herbage of the plant is widely used in the fragrance and cosmetics industry and scenting of soaps. The essential oil is extracted by steam distillation. South Africa is now producing significant quantities of geranium oil. However, previous experience by this research group showed that seedlings often take long to establish, resulting in high death rates and sometimes poor growth after establishment. Stunted growth and yellowing of leaves was also observed in some cases. Poor vegetative growth causes low herbage yield and, consequently, low total essential oil production per hectare. Poor growth is believed to be due to a combination of factors, including nutrient deficiencies and acidic soil conditions and has not been studied under South Africa condition. Production of the crop is also reported to respond differently to rate and source of nutrition in various agro-ecological regions of the world. Therefore, field trials were conducted at the Hatfield Experimental Farm, University of Pretoria, in order to investigate how the plant responds to agronomic practices, such as source and amount of nitrogen, time of N fertilizer application, season of N fertilization and liming. Response of rose-scented geranium to source and amount of N showed that, at the first harvest (summer/autumn), there was no significant effect of conventional N on fresh herbage and oil yield, probably due to leaching of N by rainfall. However, organic N at 100 kg•ha-1 increased fresh herbage and oil yields by 58% and 48% over the control, respectively. In the second harvest (spring/summer), fresh herbage yield increased by 46% (conventional N) and 60% (organic N) at 100 kg•ha-1 compared to the control. Compared to the control, 100 kg•ha-1 conventional and organic N also increased essential oil yields by 94% and 129%, respectively. For both N sources nitrogen use efficiency (NUE) and LAI decreased with an increase in N level, and organic N gave highest essential oil production efficiency and LAI. Essential oil content (% fresh mass basis) also varied between the harvests, being greater in the second harvest (September to December 2005; spring/summer) than the first harvest (February to May 2005; summer/autumn). This was due to environmental variations that occurred between the harvesting periods. N level and source were found to have no noticeable effect on essential oil composition. This study revealed that rose-scented geranium produced higher fresh herbage and essential oil yield when organic fertilizer was used as a source of N. Nitrogen management in terms of rate and time of application is important in rose-scented geranium production. Delaying nitrogen topdressing (conventional N in the form of LAN; N 28%) after harvest to between the 7th and 9th week after cut back, was found to have a significant positive effect on biomass and essential oil production. Essential oil content of the plant did not show any response to a delay in nitrogen topdressing. A delay in nitrogen topdressing, in the first re-growth resulted into a lower citronellol to geraniol (C:G) ratio, which favour essential oil quality of the crop. Generally, the characteristics of the essential oil were within the internationally acceptable range for rose-scented geranium essential oil. In addition, production of rose-scented geranium during cooler periods is not advisable due to limited biomass production which might encourage leaching of nitrogen. The net benefits from N application is dependent on the growing period and in the present study spring and summer were more beneficial than winter and autumn. Organic N at 100 kg•ha-1•year-1 increased herbage and essential oil yield of the crop in spring and summer but further increases in organic or conventional N levels had no significant effect. N application either in winter or autumn did not improve production of the crop. Application of more N than what is required for optimum growth of the plant had no positive effect on essential oil production. Application of organic N also resulted in higher N use efficiency than conventional N. The essential oil contents (% fresh mass basis) achieved in the present study generally fell within the range of 0.04 to 0.2%. Citronellol and geraniol concentration (%), were at peak in spring season followed by summer and autumn and lowest in winter. The ratio between these two components (C:G ratio) is also used as an indicator of rose-scented geranium essential oil quality and most desirable (low C:G ratio) essential oil was attained in spring, summer and autumn harvesting seasons and least desirable oil was attained in winter. The relationship between SPAD-502 chlorophyll meter readings (SPAD units) and leaf N content (% dry weight basis) was a quadratic function. SPAD-502 chlorophyll meter readings (SPAD units) matched well with that of leaf N concentration data of rose-scented geranium. Regardless of the factors that affect the readings, this instrument can be used as an indicator of leaf N status of rose-scented geranium. Soil pH above 5.5 and soil base saturation above 55% increased fresh herbage and essential oil yield (per ha), which corresponded in this case with 2 to 6 t•ha-1 of lime application. Oil content (%) was not significantly affected by application of lime. Therefore, optimum growth of rose-scented geranium can be achieved by application of lime when plants are grown on acidic soils, but without any effect on oil content and essential oil composition. It can be concluded that N rate, source and season of production and soil pH should be considered to ensure optimal rose-scented geranium production. / Thesis (PhD)--University of Pretoria, 2012. / Plant Production and Soil Science / PhD / Unrestricted

River flow is important in controlling phytoplankton distribution in estuaries. Data on the effect of river inflow on phytoplankton distribution patterns in temporarily open/closed estuaries is lacking. This study investigated the influence of river inflow on size-fractionated phytoplankton biomass (Chl a), community composition and environmental parameters measured monthly over three years in two temporarily open/closed estuaries in the Eastern Cape, South Africa. A once-off primary production study over an annual cycle was completed in the Van Stadens and Maitland estuaries. The study monitored physical, chemical and biological characteristics in both estuaries to examine the effects of changes in environmental factors and river inflow. Daily sampling of physico-chemical and biological variables from river to sea was carried out in the Van Stadens to investigate short-time scale effects of changes in environmental factors and river inflow on the phytoplankton biomass. Five and three stations in the main channel of the Van Stadens and Maitland estuaries respectively were sampled at 0.5 m below the water surface and 0.5 m above the sediment surface for biological and chemical variables and at the surface, 0.25 m and every 0.5 m thereafter for physical parameters. Five stations adjacent to the main channel along the estuary were monitored for groundwater macronutrient concentrations and five additional sites located within the upper catchment of the Van Stadens River were sampled on a quarterly basis over two years. Both estuaries were characterised by distinct hydrological conditions, an overwash, an open, a closed and a semi-closed mouth phase. Flooding in the Maitland and Van Stadens estuaries in 2001 and 2002 caused sediment scour, altered channel morphology and brought about breaching of the mouth. Flood driven mouth-breaching events occurred three and four times in each of the estuaries during the study. The mouth stayed open 20 – 25 percent and was closed 60 – 65 percent of the time. In the Van Stadens the closed overwash mouth condition occurred approximately 10 – 20 percent of the time while in the Maitland it occurred less with the semi-closed mouth condition occurring 10 – 20 percent of the time. Incidents related to mouth opening not associated with strong river floods occurred approximately 10 – 15 percent of the time, although in the Maitland a semi-closed mouth state persisted more frequently than in the Van Stadens Estuary. During flooding events salinity dropped to low levels (< 5 psu) but soon recovered to brackish conditions when river flow was reduced and marine water penetrated deep upstream. Reduction in river flow combined with marine sediment deposition resulted in the closure of the mouth. During closed mouth conditions strong onshore storm surges and spring high tides introduced marine water through overwash that kept salinity high. In both estuaries salinity showed a negative correlation with rainfall (R2 = 0.12), indicative of the strong influence of marine overwash that kept salinity high thus masking the influence of freshwater. High rainfall in the Van Stadens Estuary caused high levels of turbidity that reduced light penetration at depth. Light attenuation was positively correlated with the high rainfall (R 2 = 0.26) suggesting that increased turbidity was linked to rainfall induced discharge. In contrast, in the Maitland Estuary light attenuation did not show any correlation with increased rainfall possibly because of the reduced water depth and increased euphotic zone following the floods in 2002. High river inflow introduced macronutrients in both estuaries such that dissolved inorganic phosphates (DIP) and dissolved inorganic nitrogen (DIN) concentrations in the Van Stadens Estuary were strongly correlated with rainfall (R2 = 0.78 and 0.57 respectively). In the Maitland Estuary DIP and DIN concentrations remained significantly higher (p < 0.05) compared to that in the Van Stadens suggesting that the Maitland catchment contributed greater nutrient input into the estuary and may be associated with farming activities. Phytoplankton chlorophyll a (Chl a) ranged from 0.8 – 13.9 μg L-1 in the Van Stadens and in the Maitland Estuary from 5.3 – 138 μg L-1 during the 3-year study. During the open mouth condition Chl a biomass and primary production ranged from 5.4 – 52.9 μg Chl a L-1 and 1.2 – 11.7 mg C m-2 d-1 in the Maitland and in the Van Stadens from 1.6 – 9.8 μg Chl a L-1 and 1.2 - 14 mg C m-2 d-1 respectively. Maximum annual primary production in the Maitland and Van Stadens estuaries was 8.8 and 5.1 g C m-2 y-1 respectively. When the mouth was open in the Van Stadens Estuary the microphytoplankton (> 20 μm) accounted for > 65 percent of the Chl a, whereas during closed mouth conditions they accounted for about 55 percent of the Chl a biomass. Chlorophytes became the dominant taxon in the dry summer months but were replaced by cryptophytes and dinoflagellates during the wet season. When nutrient concentrations were low during low flow conditions in the Van Stadens Estuary mixotrophic microphytoplankton became an important fraction of the water column together with phototrophic dinoflagellates and cryptophytes. In the Maitland large sized chlorophytes were the dominant taxa in late spring and summer seasons and made up more than 80 percent of the cell numbers. In the Maitland before the floods in 2002 cyanophytes were the dominant group in late spring contributing more than 75 percent in cell abundance. Data from the short-term study in the Van Stadens Estuary showed similarities and differences in the Chl a response to increased river inflow. High river inflow initially reduced Chl a biomass followed by a recovery period of a couple of days compared to a 8 – 10 week recovery period in studies monitored over seasonal and annual temporal scales. The responses may be dissimilar but help to illustrate that there are similar response patterns to environmental forcing necessary to support phytoplankton biomass at different temporal scales. This study has demonstrated that flooding events caused by strong river flow cause breaching of the mouth, a reduction in salinity and marked nutrient input. Although the causes of flooding can be similar in both estuaries the resultant effects are varied and can alter the ability of the estuary to retain water. This study was able to demonstrate that the supply of macronutrients from the catchment was strongly correlated with rainfall (R2 = 0.67) and that phytoplankton growth mainly depended on an allochthonous source of macronutrients although internal supplies could be critical at times in controlling microalgal biomass.

Leaves are responsible for the majority of the fixed carbon in most plant species. Along leaf development, the photosynthetic capacity increases until the organ reaches maturity. Consequently, at the onset of senescence the leaves have the highest photosynthetic activity, then, as the chloroplasts are dismantled and the photosynthetic machinery is degraded, leaves gradually lose the rate of carbon assimilation. Although the capacity to fix carbon declines as senescence progresses, nutrient remobilization from macromolecule degradation nourishes the developing sink organs. In this regard, delaying leaf senescence stands out as a promising strategy to increase plant yield as extends the window of time with maximum carbon fixation rate. Another approach that is receiving much attention is the manipulation of chlorophyll degradation once it potentially regulates photosynthetic capacity and affects the nutritional quality of harvestable organs. As chlorophyll is degraded, the released phytol is recycled and can be either stored (i.e. as fatty acid phytyl esters), used for chlorophyll synthesis or be incorporated in tocopherol biosynthesis. Tocopherols have high nutraceutical value due to their antioxidant properties. However, the majority of the studies regarding senescence and chlorophyll degradation were carried out in the model plant Arabidopsis thaliana or grasses, creating a knowledge gap about these processes in fleshy fruit-bearing plants of human diet interest. In this regard, the tomato, Solanum lycopersicum, is an excellent model not only for the genetic and genomic resources, but also for its agronomic and nutritional importance. Thus, this project aims to extend what is known about the effects of chlorophyll degradation and senescence manipulation over the metabolism and yield of tomato plants, as well as fruit nutritional quality. In order to evaluate the consequences of alteration in chlorophyll degradation, first the enzymes chlorophyllase and pheophytinase, both capable of dephytylating the chlorophyll molecule, were identified and characterised. An extensive phylogenetic, evolutive and transcriptional analysis allowed the identification of two groups of chlorophyllases, one putatively involved in the response to different stimuli, while the other may act in chlorophyll homeostasis. As for pheophytinase, only one group was identified, being related to physiologically programmed processes that trigger chlorophyll degradation (i.e. leaf senescence and fruit ripening). Given this scenario, pheophytinase was chosen to be constitutively knocked-down in order to evaluate the effects over the metabolism of leaves and fruits. As consequence of this manipulation, transgenic plants were impaired in the leaf senescence-associated chlorophyll breakdown, but, although with an initial delay, fruit ripening-associated degreening was not compromised. Several photosynthetic and biochemical parameters were signs of photoinhibition, possibly due to a deficiency in chlorophyll recycling in leaves. This led to an increase in sugar exportation towards fruits, ultimately increasing soluble sugar content of ripe fruits. However, as a consequence, carotenoid levels were reduced, what, at least partially, it was compensated by an increase in tocopherol content. The results indicated that pheophytinase plays a role beyond senescence-associated chlorophyll degradation and its manipulation led to the development of fruit with increased soluble sugars and tocopherols at the cost of lowering carotenoid levels. Thus, these evidences support the manipulation of chlorophyll breakdown as a strategy for improvement of fleshy fruit plants. In order to address the effects of senescence over yield and fruit quality, the transcription factor ORESARA1, which has been widely characterised in A. thaliana and is considered a key regulator of senescence initiation, was targeted. After a comprehensive phylogenetic analysis and the characterization of the regulatory mechanisms, one putative ortholog was selected to be silenced. As consequence of this manipulation, leaves displayed increased chlorophyll content. Moreover, as senescence was delayed, the extent of photosynthetic activity of the leaves was also expanded. As the number of fruits was increased in the knockdown lines, this reflected in an increment in the harvest index. Ripe fruits accumulated more soluble sugars and tocopherols. Collectively, the results support the manipulation of leaf senescence as a strategy for tomato yield improvement. Altogether, data obtained enhance the knowledge about the impacts of chlorophyll degradation and leaf senescence over the metabolism of fleshy-fruit plants, providing strategies to increase yield and nutritional quality of fruits / As folhas, para a maioria das espécies vegetais, são o principal órgão responsável pela fixação de carbono. Durante o desenvolvimento foliar, o potencial fotossintético aumenta até a folha atingir a sua maturidade. Consequentemente, no momento que o programa de senescência se inicia, a folha apresenta a maior taxa de fotossíntese, a qual passa então a declinar conforme o cloroplasto se desorganiza e a maquinaria fotossintética é degradada. Apesar da redução na fixação de carbono, o catabolismo de macromoléculas possibilita a remobilização de nutrientes para os órgãos dreno em desenvolvimento. Neste contexto, atrasar a senescência destaca-se como uma promissora estratégia para aumento da produtividade, uma vez que estende o período de máxima fixação de carbono das folhas. Outra estratégia que tem recebido atenção por, potencialmente, regular a capacidade fotossintética e afetar a qualidade nutricional dos órgãos coletáveis é a manipulação da degradação da clorofila. Durante o catabolismo deste pigmento, o fitol liberado é reciclado podendo ser armazenado (i.e. na forma de ésteres de fitil com ácidos graxos), ser utilizado na síntese de novas moléculas de clorofila ou ser incorporado na rota biossintética de tocoferóis. Estes últimos compostos, por seu potencial antioxidante, possuem alto valor nutracêutico. No entanto, a maior parte dos estudos sobre senescência e degradação de clorofila foi realizada na planta modelo Arabidopsis thaliana ou em gramíneas, tornando escassas as informações relativas a plantas com frutos carnosos de interesse para a dieta humana. Nesse âmbito, o tomateiro, Solanum lycopersicum, é um excelente modelo de estudo não apenas pela disponibilidade de recursos genético e genômicos, mas também pela importância agronômica e nutricional desta espécie. Assim, este trabalho pretende expandir o conhecimento acerca dos efeitos da manipulação da degradação de clorofila e da senescência sobre o metabolismo e produtividade do tomateiro, bem como sobre a qualidade nutricional dos frutos. De modo a se avaliar as consequências de alterações na degradação de clorofila, iniciou-se por identificar e caracterizar em tomateiro as enzimas clorofilase e feofitinase, as quais catalisam a defitilação da molécula de clorofila. Uma vasta análise filogenética, evolutiva e transcricional permitiu a identificação de dois grupos de clorofilases, um dos quais estaria envolvido na plasticidade de respostas a estímulos e o outro na homeostase dos níveis de clorofila. Já para feofitinase, somente um grupo foi identificado, o qual está relacionado a processos fisiologicamente programados que levam à degradação de clorofila (i.e. senescência foliar e amadurecimento de frutos). Dado o panorama obtido, a feofitinase foi escolhida para ser constitutivamente silenciada de modo a se avaliar as consequências para o metabolismo de folhas e frutos. Como consequência do silenciamento, as linhagens transgênicas mostraram-se incapazes de degradar clorofila durante a senescência, mas, embora com um atraso nas etapas iniciais, a degradação ao longo do amadurecimento de frutos não foi comprometida. Diversos parâmetros fotossintéticos e bioquímicos foram indicativos de fotoinibição, possivelmente em virtude de uma deficiência na reciclagem da clorofila em folhas. Isto acarretou em um aumento na exportação de açúcares para frutos, incrementando a concentração de açúcares solúveis nos frutos maduros, que, em contrapartida, resultou na queda nos teores de carotenoides. A queda nestes compostos antioxidantes foi, ao menos parcialmente, compensada por um aumento nos níveis de tocoferóis. Os resultados indicaram que a feofitinase possui um papel além da degradação de clorofila associada à senescência, e que sua manipulação leva ao desenvolvimento de frutos com maior teor de açúcares solúveis e de tocoferóis ao custo da redução no de carotenoides. Desta forma, estas evidências suportam a manipulação da clorofila como estratégia para o melhoramento de frutos carnosos. Para investigar o efeito da senescência sobre a produtividade e qualidade de frutos foi escolhido o fator de transcrição ORESARA1, o qual está amplamente caracterizado em A. thaliana e é considerado um regulador chave no desencadeamento deste processo. A partir de uma extensa análise filogenética e da caracterização de sua regulação, um putativo ortólogo foi selecionado como alvo para silenciamento. Como consequência desta manipulação, folhas apresentaram os níveis de clorofila incrementados. Além disto, taxas fotossintéticas maiores que as do genótipo controle foram mantidas por maior tempo indicando que a iniciação da senescência foi retardada. Assim, estas plantas produziram um maior número de frutos, consequentemente, aumentando o índice de colheita dessas linhagens. Os frutos maduros apresentaram maiores teores de açúcares solúveis e de tocoferóis. Os resultados demostraram que o retardo do início da senescência é uma estratégia efetiva para aumento da produtividade de tomateiro. Coletivamente, os resultados obtidos aprofundam o conhecimento acerta dos impactos da degradação de clorofila e senescência sobre o metabolismo de plantas com frutos carnoso, além de prover estratégias para se incrementar a produtividade e a qualidade nutricional de frutos