A produção vegetal é dependente do processo fotossintético. As técnicas de biologia molecular e transformação genética de plantas trouxeram boas perspectivas para a alteração do metabolismo fotossintético. Plantas transgênicas de tabaco (Nicotiana tabacum, L.) que superexpressam o gene quimérico Lhcb1*2 de ervilha têm sido estudadas por apresentarem uma série de alterações no desenvolvimento e no metabolismo fotossintético, em relação à linhagem selvagem. Vários autores observaram mudanças morfológicas, fisiológicas, bioquímicas e adaptativas que favorecem essas plantas em diversas condições de cultivo. O objetivo desse trabalho foi o de avaliar o impacto da superexpressão desse gene na formação plastidial de plântulas de tabaco germinadas e mantidas no escuro por sete dias e depois transferidas à luz, com coletas periódicas de 0, 6, 18 e 120 horas pós-iluminação. O desenvolvimento plastidial, avaliado por microscopia de luz, mostra um provável adiantamento na formação dos cloroplastos dos materiais vegetais transgênicos (TR1 e TR2) em relação à selvagem (WT). A análise de ultraestrutura dos plastídios por microscopia eletrônica de transmissão, demostrou um real adiantamento na formação dos cloroplastos maduros nas duas linhagens transgênicas A análise de Western blot confimou a presença de proteínas específicas do fotossistema II (Lhcb 1-2 e D1). Este fato implica que a montagem do aparato fotossintético é antecipada nos transgênicos, assim como o desenvolvimento morfológico e estrutural observado nos plastídios. / The vegetal production is strictly dependent on the photosynthetic process. Techniques of molecular biology and genetic transformation of plants brought good perspectives for the alteration of the photosynthetic metabolism. Transgenic tobacco plants (Nicotiana tabacum, L.) which express the chimeric pea Lhcb1*2 gene were pbtained and presenta series of alterations on development and photosynthetic metabolism in relation to the wild type. Previous analysis have demonstrated morphological, physiological, biochemical and adaptative changes that favour these transgenic lines in various conditions of culture. The aim of this work was to evaluate the impact of the expression of this gene in the plastid formation, of tobacco seedlings. Seeds were germinated and kept in darknes for seven days, and transferred to light. The seedlings were then collected after 0, 6, 18 and 120 hours of exposure to continuous ilumination. The plastidial development evaluated by light microscopy, showed an advanced chloroplast formation of the transgenic lines (TR1 and TR2) in relation to the wild type (WTSR1). The ultrastructural analysis of the plastids by electronic microscopy showed, indeed on advanced formation of mature chloroplasts in the transgenic lines. The Western blot analysis confirmed the presence of two specific proteins (CAB and D1), of the photosystem II. This fact implies that the assembly of the photosynthetic apparatus might occurs earlier in the transgenic lines, as well as the morphological and structural development of the plastids.

Made available in DSpace on 2016-08-12T19:18:28Z (GMT). No. of bitstreams: 1 CristhyanAGC_TESE.pdf: 2158176 bytes, checksum: f755e84c9ed7d1a46f3c72723b738900 (MD5) Previous issue date: 2015-01-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The survey was conducted in Embrapa tropical agribusiness, in Fortaleza, Ceará, and divided into three separate experiments carried out to evaluate the effects caused by heat stress (Experiments I and II) and by combining temperature and high CO2 concentration (Experiment III) on the growth, physiology, biochemistry and flowering hybrid yellow melon "Goldex" grown in the greenhouse. In all three experiments, the design was completely randomized, the two first were made up of 4 treatments and 8 repetitions and the third was in a 2x2 factorial design with 8 replications. In the experiments I and II, plants were placed in germination chambers with different temperatures (30, 34, 38 and 42 °C), simulating a heat stress. However, in the second experiment, the plants were removed from the chambers at 18 DAT and forwarded to a greenhouse up reaching the flowering phase. In the experiment III, the plants were placed in germination chambers and subjected to treatment 34 °C; 34 °C+900 ppm CO2; 42 °C and 42 °C+900 ppm CO2. In the first experiment, it was found in most of the variables that at least one assessment showed significant differences between treatments, which is not observed for stem diameter and fructose. Plant height, leaf number (NF), leaf area (LA), sucrose and fresh and dry shoot and root (MFA, MSA, MFR and MSR) were negatively impacted by temperature 42 °C. For chlorophyll and gas exchange, despite significant differences between treatments of positive or negative trends, were observed variables. Glucose was positively influenced by the temperatures of 38 and 42°C. In the second trial, there was no significant difference between treatments in the variables except the stem diameter. Plant height, NC, AF and fresh and dry shoot and root mass were negatively impacted by a temperature of 42 °C. Chlorophyll and gas exchange showed significant differences between treatments, however, there was no trend, positive or negative, of the variables in the time periods. The flowering was influenced by the treatments, because of the higher temperature, the later the beginning of this phase is observed. In the experiment III, plant height and stem diameter at 7; 14 e 23 DAT significant differences between the temperatures, concentrations of CO2 and interaction entres them, however, the same result was observed on analysis at 0 DAT fact also checked for the trunk diameter at 23 different concentrations in the DAT CO2. NF to 14 e 23 DAT and the AF and fresh and dry shoot were significantly influenced by factors and interaction entres them. Chlorophyll revealed significant differences only between the temperatures at 7 DAT. Already 14 DAT, there were no significant differences of the factors and interaction between them, at 23 DAT significant differences existed in temperatures and interaction of temperature and CO2. Gas exchange were not affected by temperature, and no interaction between factors at 14 DAT, still the same resource, the CO2 enrichment decreased gs and Ci/Cref not influencing the A and E. Have at 21 DAT, the responses were quite different, and the gas and suffered the effects of temperature, CO2 and the interaction between them. While for Ci/Cref and A were not observed significant effects of the factors nor its interaction with the exception of the increase in the in plants under elevated temperature with or without enrichment of CO2, an effect that was also observed in the gas and the leaves and subjected to 42 ºC + 900 ppm de CO2. The Fv/Fm and F0 were influenced only by temperature. Already Fm and qP suffered no effects of the factors nor the interaction between them. The qN showed significant differences for both factors, with no significant effect only the interaction between them. The ETR was affected by concentrations of CO2 and interaction of temperature and CO2. It follows that the melon subjected to high temperatures (38 and 42 °C) up to 24 days after transplanting showed lower growth. This fact was also observed when plants were subjected to initial thermal stress with 42 °C temperature, with a negative effect also on flowering, slowing within 10 days compared to the temperature of 30 °C. The melon showed further growth in plants under the combination of 34 °C+900 ppm CO2 and lower under high temperature, enriched or not CO2. Most physiological analysis of gas exchange and chlorophyll fluorescence were not affected by treatments / A pesquisa foi realizada na Embrapa Agroindústria Tropical, situada em Fortaleza, Ceará, e dividida em três experimentos distintos que tiveram como objetivos avaliar os efeitos causados pelo estresse térmico (Experimentos I e II) e pela combinação temperatura e elevada concentração de CO2 (Experimento III) no crescimento inicial, fisiologia, bioquímica e florescimento do meloeiro amarelo híbrido Goldex cultivados em casa de vegetação. Em todos os três experimentos, o delineamento utilizado foi o inteiramente casualizado; os dois primeiros foram constituídos de 4 tratamentos e 8 repetições e o terceiro foi em esquema fatorial 2x2 com 8 repetições. Nos experimentos I e II, as plantas foram acondicionadas em câmaras de germinação com diferentes temperaturas (30; 34; 38 e 42 ºC), simulando um estresse térmico. Porém, no experimento II as plantas foram retiradas das câmaras aos 18 DAT e encaminhadas para casa de vegetação até tingirem a fase de florescimento. No experimento III, as plantas foram acondicionadas em câmaras de germinação e submetidas aos tratamentos 34 ºC; 34 ºC + 900 ppm de CO2;42 ºC e 42 ºC + 900 ppm de CO2. No experimento I, verificou-se na maioria das variáveis que pelo menos uma avaliação apresentava diferenças significativas entre os tratamentos, fato este não observado para o diâmetro do caule e frutose.Altura da planta, número de folhas (NF), área foliar (AF), sacarose e massa fresca e seca de parte aérea e raiz (MFA, MSA, MFR e MSR) foram influenciados negativamente pela temperatura 42 ºC. A clorofila e as trocas gasosas, apesar das diferenças significativas entre os tratamentos, não foram observadas tendências positiva ou negativa das variáveis. A glicose foi influenciada positivamente pelas temperaturas de 38 e 42 ºC. No experimento II, houve diferença significativa entre os tratamentos nas variáveis, exceto o diâmetro do caule. Altura da planta, NF, AF e massa fresca e seca de parte aérea e raiz foram influenciados negativamente pela temperatura de 42 ºC. A clorofila e as trocas gasosas apresentaram diferenças significativas entre os tratamentos, no entanto, não se observou tendência, positiva ou negativa, das variáveis nos tempos avaliados. O florescimento foi influenciado pelos tratamentos, pois quanto maior a temperatura, mais tardiamente é observado o início desta fase. No experimento III, a altura das plantas e o diâmetro do caule aos 7; 14 e 23 DAT apresentaram diferenças significativas entre as temperaturas, concentrações de CO2 e interação entres eles, no entanto, o mesmo resultado não foi observado na análise aos 0 DAT, fato também verificado para o diâmetro do caule aos 23 DAT nas diferentes concentrações de CO2. O NF aos 14 e 23 DAT e a AF e massas fresca e seca de parte aérea foram influenciadas significativamente pelos fatores e interação entres eles. A clorofila revelou diferenças significativas apenas entre as temperaturasaos 7 DAT. Já aos 14 DAT houve diferenças significativas dos fatores e interação entre eles, aos 23 DAT as diferenças significativas existiram nas temperaturas e interação temperatura e CO2. As trocas gasosas não foram influenciadas pelas temperaturas e nem pela interação entre os fatores aos 14 DAT, ainda na mesma avaliação, o enriquecimento de CO2 diminuiu a gs e a razão Ci/Cref não influenciando a A e E. Já aos 21 DAT, as respostas foram bem divergentes, a gs e a E sofreram efeitos da temperatura, do CO2 e da interação entre eles, ao passo que para a razão Ci/Cref e para A, não foram observados efeitos significativos dos fatores e nem de sua interação, com exceção do aumento da A nas plantas submetidas a elevada temperatura com ou sem enriquecimento de CO2, efeito que também foi observado na gs e E das folhas submetidas a 42 ºC + 900 ppm de CO2. A Fv/Fme a F0 foram influenciadas apenas pelas temperaturas. Já a Fm e a qP não sofreram efeitos dos fatores e nem da interação entre eles. A qN apresentou diferenças significativas para ambos os fatores, não apresentando efeito apenas da interação entre eles. A ETR foi influenciada significativamente apenas pelas concentrações de CO2 e interação temperatura e CO2. Conclui-se que o meloeiro submetido a elevadas temperaturas (38 e 42 ºC) até os 24 dias após o transplantio apresentaram menor crescimento, fato que também foi verificado quando as plantas foram submetidas a estresse térmico inicial com temperatura de 42 ºC, com efeito negativo também no florescimento, retardando em até 10 dias na comparação com a temperatura de 30 ºC. O meloeiro apresentou ainda maior crescimento nas plantas submetidas à combinação de 34 ºC + 900 ppm de CO2 e menor sob elevada temperatura, enriquecida ou não de CO2. Já as análises fisiológicas de trocas gasosas e de fluorescência da clorofila, em sua maioria, não foram influenciadas pelos tratamentos

A produção vegetal é dependente do processo fotossintético. As técnicas de biologia molecular e transformação genética de plantas trouxeram boas perspectivas para a alteração do metabolismo fotossintético. Plantas transgênicas de tabaco (Nicotiana tabacum, L.) que superexpressam o gene quimérico Lhcb1*2 de ervilha têm sido estudadas por apresentarem uma série de alterações no desenvolvimento e no metabolismo fotossintético, em relação à linhagem selvagem. Vários autores observaram mudanças morfológicas, fisiológicas, bioquímicas e adaptativas que favorecem essas plantas em diversas condições de cultivo. O objetivo desse trabalho foi o de avaliar o impacto da superexpressão desse gene na formação plastidial de plântulas de tabaco germinadas e mantidas no escuro por sete dias e depois transferidas à luz, com coletas periódicas de 0, 6, 18 e 120 horas pós-iluminação. O desenvolvimento plastidial, avaliado por microscopia de luz, mostra um provável adiantamento na formação dos cloroplastos dos materiais vegetais transgênicos (TR1 e TR2) em relação à selvagem (WT). A análise de ultraestrutura dos plastídios por microscopia eletrônica de transmissão, demostrou um real adiantamento na formação dos cloroplastos maduros nas duas linhagens transgênicas A análise de Western blot confimou a presença de proteínas específicas do fotossistema II (Lhcb 1-2 e D1). Este fato implica que a montagem do aparato fotossintético é antecipada nos transgênicos, assim como o desenvolvimento morfológico e estrutural observado nos plastídios. / The vegetal production is strictly dependent on the photosynthetic process. Techniques of molecular biology and genetic transformation of plants brought good perspectives for the alteration of the photosynthetic metabolism. Transgenic tobacco plants (Nicotiana tabacum, L.) which express the chimeric pea Lhcb1*2 gene were pbtained and presenta series of alterations on development and photosynthetic metabolism in relation to the wild type. Previous analysis have demonstrated morphological, physiological, biochemical and adaptative changes that favour these transgenic lines in various conditions of culture. The aim of this work was to evaluate the impact of the expression of this gene in the plastid formation, of tobacco seedlings. Seeds were germinated and kept in darknes for seven days, and transferred to light. The seedlings were then collected after 0, 6, 18 and 120 hours of exposure to continuous ilumination. The plastidial development evaluated by light microscopy, showed an advanced chloroplast formation of the transgenic lines (TR1 and TR2) in relation to the wild type (WTSR1). The ultrastructural analysis of the plastids by electronic microscopy showed, indeed on advanced formation of mature chloroplasts in the transgenic lines. The Western blot analysis confirmed the presence of two specific proteins (CAB and D1), of the photosystem II. This fact implies that the assembly of the photosynthetic apparatus might occurs earlier in the transgenic lines, as well as the morphological and structural development of the plastids.

Magister Scientiae (Biodiversity and Conservation Biology) / Wheat is a key global commodity in terms of acreage and tradeable value and as a staple in household diets. Many factors including biotic stress conditions have detrimental effects on global wheat production and yield. The increasing prevalence of biotic stress inflicted by fungal species such as Fusarium has significantly reduced yield and quality of cereal crops thus, threatening sustainable agriculture and food security. Interactions between wheat and Fusarium spp. such as Fusarium proliferatum triggers the accumulation of reactive oxygen species (ROS) to levels toxic to the plant thus leading to oxidative damage and ultimate cellular death. In order to maintain redox homeostasis, plants rely on ROS-scavenging antioxidants (enzymatic and non-enzymatic) to control ROS molecules to levels less toxic to plants. This study investigated the impact of F. proliferatum on the physio-biochemical responses of two wheat cultivars (SST 015 and SST 088). Changes in seed germination, growth, biomass, chlorophyll and mineral contents were monitored. Furthermore, changes in ROS accumulation and antioxidant enzyme activity was measured in the shoots of both wheat cultivars.

>Magister Scientiae - MSc / Biotic stress is one of the main causes for agricultural loss of economically important cereal crops. The increasing prevalence of biotic stress inflicted by fungal species such as Fusarium has significantly reduced yields and quality of cereals, threatening sustainable agriculture and food security worldwide. Interactions between wheat and Fusarium spp. such as Fusarium oxysporum promotes the accumulation of reactive oxygen species (ROS). Overproduction of ROS can become toxic to plants depending on the scavenging ability of antioxidant systems to maintain redox homeostasis. This study investigated the effects of F. oxysporum on the physiological and biochemical response of three wheat cultivars namely, SST 056, SST 088 and SST 015. Physiological responses were monitored by measuring changes observed in plant growth parameters including shoot and root growth and biomass, relative water content as well as photosynthetic metabolism and osmolyte content in all three wheat cultivars. Downstream biochemical analysis involved monitoring the accumulation of ROS biomarkers (superoxide and hydrogen peroxide) as well as the detection of enzymatic activity of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD). These biochemical responses were only monitored on the two wheat cultivars which presented contrasting responses to F. oxysporum infection. Results showed that F. oxysporum significantly reduced plant growth, biomass, chlorophyll pigments and relative water content of all three cultivars, with the highest reduction observed for SST 088 relative to SST 015 and SST056. On the other hand, proline content was significantly enhanced in all three wheat cultivars, with the highest increase observed for SST 015 relative to SST 056 and SST 088. Based on the contrasting physiological results observed for these three cultivars, downstream biochemical analysis was focused on SST 015 and SST 088. F. oxysporum trigged an increased in superoxide and hydrogen peroxide contents in both cultivars, with the highest increase observed for SST 088. A similar trend was observed for the extent of lipid peroxidation, manifested as enhanced MDA levels. Furthermore, F. oxysporum differentially altered antioxidant enzyme activity relative to the control of both wheat cultivars. A Significant increase in SOD activity was observed for both cultivars in response to F. oxysporum. However, contrasting responses in APX and POD activity (as seen for the band intensities of individual isoforms) was observed in these wheat cultivars in response to F. oxysporum. Based on the results obtained in this study we suggest that F. oxysporum infection has varying degrees of severity in different wheat cultivars. In light of the significant reduction of plant development coupled with enhanced ROS accumulation and differential antioxidant capacity for SST 015 relative SST 088, we suggest that SST 015 is more resilient to F. oxysporum. We thus conclude that a direct relationship exists between ROS accumulation and antioxidant scavenging in regulating plant tolerance against F. oxysporum pathogens.

Made available in DSpace on 2017-07-10T17:37:00Z (GMT). No. of bitstreams: 1 Thaisa_Muriel_Mioranza.pdf: 2175568 bytes, checksum: 5bdfcf6dcf5708e0c3a818711f1c959a (MD5) Previous issue date: 2015-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this work was to study the influence of homeopathic or high diluted solution of Thuya occidentalis in the control of root-knot nematode Meloidogyne incognita in tomato plants and physiological responses promoted in the plant. The experiment consisted of in vitro and in vivo assay. The treatments were different dynamization of T. occidentalis 6, 12, 24, 50, 100, 200 and 400CH (centesimal hahnemaniana dilution) and the water (control). In the in vitro experiment nematodes were treated with 0.1% homeopathic solution of the treatments for 24 hours for the motility test and 15 days for hatching test. For the in vivo test, the experiment was carried out in two years, the first year in completely randomized design and the second year in a randomized block design, both with eight treatments and with four replications. At the moment that the tomato seedlings were transplanted, they were submerged in a 0.1% solution of treatments. Subsequently were inoculated 4188 eggs and 661 second-stage juveniles (J2) in the first experiment and 4350 eggs and 700 J2 in the second experiment. Were performed weekly sprays of 0.1% solution of the treatments in the shoots of plants. The plants were cultivated until the nematodes complete a development cycle. We evaluated the number of galls, egg masses, number of eggs and J2 in the roots, number of eggs and J2 in the soil, plant height, shoot fresh and dry weight, root volume, number of clusters, number of fruits per bunch, fresh and dry fruit weight and relative chlorophyll content. In the 2014 experiment were chosen the dynamizations 6, 24 and 50CH, and the control with nematode and treated with water and plants without nematodes and without treatment, with four repetitions each for the measurement of photosynthesis rates (A), stomatal conductance (gs), transpiration (E), internal CO2 concentration on leaf (Ci) and leaf temperature (Tleaf) varying the photosynthetic photon flux density (PPFD). It was also calculated the efficiency of water use (USA) (A/E), intrinsic water use efficiency (EUIA) (A/gs) and the instantaneous efficiency of carboxylation (A/C). The treatments did not show nematostatic and nematicide effect even did not influence in hatches eggs for in vitro assay. To the in vivo assay, in 2013, T. occidentalis 100CH caused a reduction in the population of J2 in the roots, as well as 200CH promoted an increase in the volume root and fruit fresh weight of the first bunch. In 2014, 100CH caused a decrease in the number of egg masses and J2 in the soil, and influenced the development of the root, while 50CH increased the fresh weight of shoots of tomato plants. The specific measures of gas exchange did not differ between treatments. Plants infected with M. incognita and without treatment caused an increase in the net photosynthesis and the carboxylation capacity for response curve to light. Already T. occidentalis 24CH inhibited the effect of increased CO2 fixation in tomato plants inoculated with M. incognita, providing similar behavior to healthy plants in different densities of photons. Plants of homeopathic treatments showed higher leaf temperature. T. occidentalis showed control capability of root-knot nematode and influenced in tomato growth and physiology / O objetivo desse trabalho foi estudar a influência de soluções ultradiluídas de Thuya occidentalis no controle do nematoide de galhas Meloidogyne incognita em plantas de tomateiro e as respostas fisiológicas promovidas na planta. O experimento foi constituído de ensaios in vitro e in vivo. Os tratamentos utilizados foram diferentes dinamizações de T. occidentalis 6, 12, 24, 50, 100, 200 e 400CH (centesimal hahnemanniana) e a testemunha água (controle). No experimento in vitro os nematoides ficaram em contato direto com a solução de 0,1% dos tratamentos, por 24 horas para o teste de motilidade e 15 dias para o teste de eclosão. Para o teste in vivo, o experimento foi realizado em dois anos, sendo o primeiro ano em delineamento inteiramente casualizado e o segundo ano em delineamento de blocos ao acaso, ambos com oito tratamentos e quatro repetições. No momento em que as mudas de tomateiro foram transplantadas, estas foram submersas em uma solução de 0,1% dos tratamentos. Posteriormente foram inoculados 4188 ovos e 661 juvenis de segundo estágio (J2) no primeiro experimento e 4350 ovos e 700 J2 no segundo experimento. Foram realizados pulverizações semanais da solução de 0,1% dos tratamentos na parte aérea das plantas. As plantas foram conduzidas até que os nematoides completassem um ciclo de desenvolvimento. Foram avaliados o número de galhas, massas de ovos, número de ovos e J2 nas raízes, número de ovos e J2 no solo, altura de plantas, massa fresca e seca da parte aérea, volume de raiz, número de cachos, número de frutos por cacho, massa fresca e seca dos frutos e teor relativo de clorofila. No experimento de 2014, foram escolhidas as dinamizações de 6, 24 e 50CH, além da testemunha com nematoide e tratada com água e testemunha sem nematoide e sem tratamento, com quatro repetições cada, para a mensuração da taxa de assimilação líquida de CO2 (A), transpiração foliar (E), condutância estomática (gs), concentração interna de CO2 (Ci) e temperatura foliar (°C), variando-se a densidade de fluxo de fótons fotossinteticamente ativos (DFFFA). Também foi calculada a eficiência no uso da água (EUA) (relação A/E), eficiência intrínseca no uso da água (EUIA) (relação A/gs) e a eficiência instantânea da carboxilação (A/Ci) (relação A/Ci). Os tratamentos não mostraram efeito nematostático e nematicida assim como não influenciaram na eclosão de ovos para o ensaio in vitro. Já para o in vivo, em 2013, T. occidentalis 100CH causou redução na população de J2 nas raízes, assim como 200CH promoveu aumento no volume de raiz e massa fresca de frutos do primeiro cacho. Em 2014, 100CH promoveu diminuição no número de massas de ovos e J2 no solo, e influenciou no desenvolvimento da raiz, enquanto 50CH incrementou a massa fresca da parte aérea de tomateiro. As medidas pontuais de trocas gasosas não apresentaram diferença entre os tratamentos. Plantas infectadas com M. incognita e sem tratamento promoveram aumento na fotossíntese líquida e capacidade de carboxilação pela curva de resposta à luz. Já T. occidentalis 24CH inibiu o efeito de maior fixação de CO2 em tomateiros inoculados por M. incognita, proporcionando comportamento semelhante às plantas sadias em diferentes densidades de fótons. Plantas submetidas aos tratamentos homeopáticos apresentaram maior temperatura foliar. T. occidentalis apresentou capacidade de controle de nematoide de galhas e influenciou no crescimento e fisiologia do tomateiro