CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O Ãcido ascÃrbico (AA) à um dos antioxidantes mais importante na proteÃÃo das plantas contra o estresse oxidativo gerado por estresses abiÃticos. Em cÃlulas animais, diversos trabalhos tÃm demonstrado que concentraÃÃes elevadas desse Ãcido podem causar toxicidade e morte celular. O mecanismo proposto à de uma aÃÃo pro-oxidante, por meio da reduÃÃo de Fe+3para Fe+2 e aÃÃo desse Ãltimo na reaÃÃo de Fenton, com geraÃÃo de espÃcies reativas de oxigÃnio (EROs). Atà o nosso conhecimento, esse mecanismo ainda nÃo foi relatado em plantas. O objetivo deste trabalho foi elucidar mecanismos de toxicidade de concentraÃÃes elevadas de AA em plantas, utilizando como modelo mutantes de arroz com deficiÃncia (silenciamento gÃnico) nas duas APX de cloroplasto (estromal e tilacoidal). Plantas silenciadas e nÃo transformadas (NT) com 45 dias de idade foram expostas a 10 mM (concentraÃÃo moderada) e 50 mM (concentraÃÃo elevada) de AA exÃgeno (pulverizado nas folhas). Essas concentraÃÃes foram definidas a partir de experimentos dose-dependente de 0 a 50 mM utilizando segmentos foliares em placa. No sentido de avaliar o efeito protetor (antioxidante) do AA, plantas e segmentos foliares foram expostos a intensidade de luz elevada (1000 Âmol m-2 s-1) para induzir estresse fotoxidativo. ConcentraÃÃes de AA acima de 30 mM induziram estresse oxidativo (aumento no nÃvel de TBARS) em segmentos de folhas e esse efeito foi potencializado por luz elevada. AlÃm de dano oxidativo, esses nÃveis de AA induziram aumento no dano de membrana (vazamento de eletrÃlitos) e reduÃÃo na integridade do fotossistema II (Fv/Fm). à interessante notar que as concentraÃÃes de 10 e 20 mM de AA nÃo mitigaram os efeitos negativos causados pelo o excesso de luz. A concentraÃÃo elevada de AA (50 mM) induziu senescÃncia foliar na presenÃa de luz elevada, indicada por reduÃÃo nos conteÃdos de clorofilas e carotenoides. As plantas deficientes nas duas APXs de cloroplasto (APX7/8) apresentaram maior sensibilidade a toxicidade do AA, especialmente na combinaÃÃo com luz elevada. Esses efeitos foram indicados por exibirem maiores nÃveis de TBARS (peroxidaÃÃo lipÃdica), vazamento de eletrÃlitos, H2O2e radical superÃxido. Curiosamente, as plantas mutantes na presenÃa de AA elevadonÃo apresentaram diferenÃas em diversos parÃmetros da fotossÃntese: taxa de assimilaÃÃo de CO2 e indicadores de eficiÃncia doFSII (ΦPSII, ETR, NPQ e Fv/Fm) e FSI (ΦPSI, ETR e P700), alÃm da estimativa do fluxo cÃclico (CEF), quando compradas com as NT. A despeito da toxicidade de AA nÃo ter alterado os parÃmetros da fotossÃntese nas plantas deficientes em APX, a presenÃa de luz elevada, isoladamente, causou mudanÃas em alguns parÃmetros da atividade fotoquÃmica nessas plantas. O conjunto dos dados deste trabalho mostra que o excesso de AA pode causar toxicidade em plantas. A intensidade desses efeitos à fortemente potencializada pelo excesso de luz, mas eles nÃo sÃo dependentes da fotossÃntese. O papel das duas APXs de cloroplasto e da luz elevada na toxicidade de AA nÃo ficou claro neste trabalho, apesar das plantas deficientes terem mostrado maior sensibilidade. Aparentemente, o mecanismo de toxicidade de AA em plantas à semelhante ao proposto para cÃlulas animais. Esse antioxidante quando em excesso pode atuar como pro-oxidante estimulando as reaÃÃes de Fenton, induzindo a acumulaÃÃo de EROs e gerando estresse oxidativo. Novos estudos sÃo necessÃrios para elucidar o papel das APXs de cloroplasto e da luz elevada na toxicidade de Ãcido ascÃrbico em plantas. / Ascorbic acid (AA) is one of the most important antioxidants in plant protection against the oxidative stress generated by abiotic stresses. In animal cells, several works have shown that high concentrations of this acid can cause toxicity and cell death. The proposed mechanism is related to a pro-oxidant action by the reduction of Fe+3 to Fe+2 and by the action of this Fe+2 in the Fenton reaction with generation of reactive oxygen species (ROS). To the best of our knowledge, this mechanism has not been reported yet. The objective of this work was to elucidate toxicity mechanisms of high concentrations of AA in plants using rice mutants with deficiency (gene silencing) in two chloroplastic APX (stromal and thylakoidal) as model. Forty-five days old silenced plants and non-transformed (NT) were exposed to 10 mM (moderate concentration) and 50 mM (high concentration) of exogenous AA (sprayed on leaves). These concentrations were defined based on dose-dependent experiments from 0 to 50 mM using leaf segments in plates. In order to assess the protective effect (antioxidant) of the AA, plants and leaf segments were exposed to high light intensity (1000 Âmol m-2 s 1) to induce photooxidative stress. AA concentrations higher than 30 mM induced oxidative stress (increase in the TBARS level) in leaf segments and this effect was enhanced by high light. In addition to the oxidative damage, these AA concentrations induced an increase in membrane damage (electrolytes leakage) and reduction in photosystem II integrity (Fv/Fm). Interestingly, the 10 and 20 mM concentrations did not mitigate the negative effects caused by the light excess. The high AA concentration (50 mM) induced leaf senescence under high light, indicated by the decrease in chlorophyll and carotenoids contents. Plants deficient in two chloroplast APX (APX7/8) displayed higher sensibility to AA toxicity, especially in combination with high light. These effects were indicated by exhibition of higher levels of TBARS (lipid peroxidation), electrolyte leakage, H2O2 and superoxide radical. Curiously, the transgenic plants under high AA concentrations did not exhibited differences for several photosynthetic parameters: CO2 assimilation rate and PSII (ΦPSII, ETR, NPQ e Fv/Fm) and PSI efficiency indicators (ΦPSI, ETR e P700), apart from the estimation of cyclic flux (CEF), compared with NT. Despite the AA toxicity have not changed the parameters of photosynthesis in APX-deficient plants, the sole presence of high light caused changes in some parameters of photochemical activity in these plants.The data set of this work shows that the excess of AA may cause toxicity in plants. The intensity of these effects is strongly enhanced by the excess of light but they are not dependent on photosynthesis. In this work, the roles of the two chloroplastic APX and/or high light on the AA toxicity still unclear, despite the deficient plants had showed increased sensibility. Apparently, the mechanism of AA toxicity in plants is similar to the proposed for animal cells.This antioxidant, when in excess, can act as a pro-oxidant stimulating Fenton reactions, inducing the accumulation of ROS and resulting oxidative stress. Further studies are needed to elucidate the role of the chloroplast APXs and high light on the toxicity of ascorbic acid in plants.

Submitted by (edna.saturno@ufrpe.br) on 2016-07-06T11:28:13Z No. of bitstreams: 1 Maiza de Azevedo Dantas.pdf: 1426194 bytes, checksum: 248ab81985fd9f5b4bd3a17adefea341 (MD5) / Made available in DSpace on 2016-07-06T11:28:13Z (GMT). No. of bitstreams: 1 Maiza de Azevedo Dantas.pdf: 1426194 bytes, checksum: 248ab81985fd9f5b4bd3a17adefea341 (MD5) Previous issue date: 2014-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Orchids stand out among the ornamental tropical due to the exotic beauty of flowers, exuberant colors and shapes. Due to the high market demand, depredation of habitat, low rate of seed germination in natural environment and slow growth, tissue culture has been presented as an important tool for the production of seedlings of orchids on a large scale, in a reduced period of time and with greater seedling quality over conventional methods. The polysaccharide chitosan has been tested as an additive in the culture medium of some species as it stand out as a growth promoter and by acting as an antioxidant. This study aimed to evaluate the influence of dietary supplementation of chitosan to the nutrient medium in vitro cultivation of Dendrobium Phalaenopsis and Cattleya labiata in liquid and solid medium. Shoots from in vitro seedlings of both species were inoculated into vials containing 30 ml of MS medium with half the ionic strength. The experiment consisted of the combination of three chitosan concentrations (0, 15 and 20 mg.L-1) with nutrient medium in solid and liquid state. Each treatment had four replicates, each with two plants. The experimental design was completely randomized with a factorial scheme of 3x2 (chitosan concentration x physical state of the medium). For 50 days were evaluated the total fresh weight, number of shoots and roots, and were determined activities of superoxide dismutase enzyme (SOD), ascorbate peroxidase (APX) and catalase (CAT). In cultivation in liquid medium, D. phalaenopsis fresh weight showed higher treatment with 20 mg.L-1 of chitosan as the highest number of shoots was observed in both media, at the same concentration. C. labiata also exhibited higher fresh weight and number of shoots in liquid medium, but at the concentration of 15 mg.L-1 of chitosan. The solid medium favored the emission of roots in both species, but inhibited the formation of shoots of C. labiata regardless of the chitosan concentration. The best development of seedlings of both species in liquid medium coincided with increased activity of SOD directly proportional to the chitosan. This activation of SOD was not accompanied by an increase in the activity of APX or CAT, showing the action of chitosan in the dismutation of superoxide without forming hydrogen peroxide. On solid medium, the action of chitosan on the antioxidative mechanism is also provided by the activation of the APX and CAT. Verified the prevalence of the liquid medium for micropropagation of the species studied and confirmed the combination of chitosan in controlling levels of reactive oxygen species in joint action with the antioxidant enzyme system action. / As orquídeas destacam-se entre as ornamentais tropicais, devido à beleza exótica das flores, exuberância de cores e formas. Em virtude da alta demanda do mercado, depredação do habitat, baixa taxa de germinação das sementes em ambiente natural e o lento crescimento, a cultura de tecidos vem se apresentando como uma importante ferramenta para a produção de mudas de orquídeas em larga escala, em período reduzido de tempo e com maior qualidade das mudas em relação aos métodos convencionais. O polissacarídeo quitosana tem sido testado como aditivo no meio de cultura de algumas espécies por se destacar como promotor de crescimento e por sua ação como antioxidante. O presente trabalho teve por objetivo avaliar a influência da suplementação de quitosana ao meio nutritivo no cultivo in vitro de Dendrobium phalaenopsis e Cattleya labiata, em meio líquido e sólido. Brotações provenientes de mudas in vitro de ambas as espécies foram inoculadas em frascos com 30 ml de meio MS com metade da força iônica. O experimento constou da combinação de três concentrações de quitosana (0, 15 e 20 mg L-1 de quitosana) em meio nutritivo em estado sólido e líquido. Cada tratamento contou com quatro repetições, cada uma com duas plantas. O delineamento experimental foi inteiramente casualizado em esquema fatorial de 3x2 (concentrações de quitosana x estado físico do meio). Aos 50 dias avaliaram-se o peso fresco total, número de brotos e de raízes, e foram determinadas as atividades das enzimas superóxido dismutase (SOD), ascorbato peroxidase (APX) e catalase (CAT). No cultivo em meio líquido, D. phalaenopsis apresentou maior peso fresco no tratamento com 20 mg.L-1 de quitosana, enquanto o maior número de brotos foi observado em ambos os meios, nessa mesma concentração. C. labiata também exibiu maior peso fresco e número de brotos em meio líquido, porém na concentração de 15 mg.L-1 de quitosana. O meio sólido favoreceu a emissão de raízes das duas espécies, mas inibiu a formação de brotos de C. labiata, independente das doses de quitosana. O melhor desenvolvimento das mudas das duas espécies em meio líquido coincidiu com aumento da atividade de SOD diretamente proporcional ao da concentração de quitosana. Essa ativação da SOD não foi acompanhada pelo aumento na atividade da APX nem da CAT, evidenciando a ação da quitosana na dismutação do superóxido sem formação de peróxido de hidrogênio. Em meio sólido, a ação da quitosana no mecanismo antioxidativo se deu pela ativação da APX e CAT. Constatou-se a prevalência do meu líquido para a micropropagação das espécies estudadas e confirmou-se a ação combinada da quitosana no controle dos níveis de espécies reativas de oxigênio, em ação conjunta com o sistema enzimático antioxidativo.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24670号 / 農博第2553号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5451(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 梅澤 俊明, 教授 矢﨑 一史, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

A cana-de-açúcar é uma das principais culturas agrícolas no cenário econômico e social brasileiro. Na cultura de cana-de-açúcar o estresse hídrico é o principal fator limitante para o aumento de produtividade, sendo responsável por alterações fisiológicas, bioquímicas e moleculares nas plantas, que podem deflagrar perturbações metabólicas que ativam a morte celular programada (MCP). Sabendo-se que o gene BI-1 apresenta o potencial de reduzir os efeitos da MCP desencadeado por estresses bióticos e abióticos em plantas, este trabalho teve como objetivo analisar plantas transgênicas de cana-de-açúcar que expressam o gene BI-1 de Arabidopsis thaliana (AtBI-1) em condições de estresse hídrico. Também, plantas transgênicas e controle foram inoculadas com o fungo Puccinia melanocephala demonstrando que o processo de transformação genética com o gene AtBI-1 alterou as características pré existentes de resistência a ferrugem marrom nas plantas transgênicas. Os estudos de tolerância ao défict hídrico foram realizados em dois experimentos, o experimento 1 com plantas transgênicas e controles de 90 dias e o experimento 2 com plantas de 60 dias. Plantas do experimento 1 foram analisadas quanto características morfológicas como número de estômatos e tricomas, altura e circunferência do colmo e após ficarem 24 dias sem água foram analisadas quanto a taxa fotossintética, comportamento estomático e conteúdo relativo de água nas folhas, enquanto no experimento 2 as plantas foram analisadas quanto aos teores de prolina, atividades das enzimas guaiacol peroxidase (GPOX), ascorbato peroxidase (APX) e catalase (CAT) após as plantas ficarem 17 dias sob déficit hídrico. Estas enzimas estão envolvidas em processos de desativação de elementos ativos de oxigênio. Os resultados demonstraram que as plantas transgênicas expressando o gene AtBI-1 possuem fenótipo de menor altura, e maior taxa fotossintética, maior comportamento estomático e maior conteúdo relativo de água nas folhas, e assim apresentam maior tolerância ao déficit hídrico que plantas controle. Contudo, houve baixo acúmulo de prolina, baixa atividade da GPOX, APX e CAT nas plantas transgênicas durante o estresse hídrico comparada com as plantas controle do mesmo tratamento. Porém foi observado alta atividade constitutiva da catalase nas plantas transgênicas. A atividade da catalase nestas plantas transgênicas sugere a possibilidade da interação entre AtBI-1 e calmudolinas. Futuros estudos podem contribuir para elucidar se a proteína BI-1 é essencial para a ativação das catalases por calmudolinas. / Sugarcane is one of the main agricultural crops in the Brazilian social and economic scenario. Water stress in the culture of sugarcane is the main limiting factor for increasing productivity accounting for physiological, biochemical and molecular plants that can trigger metabolic disturbances activating programmed cell death (MCP). Knowing that the BI-1 gene has the potential to reduce the effects of MCP triggered by biotic and abiotic stresses in plants, this study aimed to analyze transgenic sugarcane that express the BI-1 gene of Arabidopsis thaliana (AtBI-1) under water stress. Also, transgenic and control plants were inoculated with Puccinia melanocephala fungus demonstrating that the genetic transformation process with the AtBI-1 gene altered the pre-existing characteristics of brown rust resistance in transgenic plants. Studies of tolerance to water deficit were performed in two experiments, the experiment 1 was prepared with transgenic and control plants with 90 days and the experiment 2 used plants with 60 days. Plants from experiment 1 were analyzed as for morphological characteristics such as number of stomata and trichomes, height and diameter of stem after plants being under water for 24 days as were analyzed photosynthetic rate, stomatal behavior, relative water content in leaves while in the experiment 2, plants were analyzed for the levels of proline, enzyme activities of guaiacol peroxidase (GPOX), ascorbate peroxidase (APX) and catalase (CAT) under water deficit for 17 days. These enzymes are involved in deactivation of active elements oxygen. The results demonstrated that the transgenic plants expressing the AtBI-1 gene presented the phenotype of lower height, higher index of leaf area, higher photosynthetic rate, higher stomatal behavior and higher relative water content in leaves than control plants increasing tolerance to drought stress. However, there were low levels of proline, low activity of GPOX activity, APX and CAT in transgenic plants during drought stress compared to control plants of the same treatment, but the observed high constitutive activity of catalase in transgenic plants. Catalase activity in these transgenic plants suggests the possibility of interaction between AtBI-1 and calmudolinas. Future studies may contribute to understand whether the BI-1protein is essential for the activation of catalase by calmudolinas.

There is an influx in heavy metals into soils and ground water due to activities such as increased mineral mining, improper watering and the use of heavy metal contaminated fertilizers. These heavy metals are able to increase the ROS species within plants which may result in plant metabolism deterioration and tissue damage. Heavy metals may also directly damage plants by rendering important enzymes non-functional through binding in metal binding sites of enzymes. The heavy metal focused on in this study was vanadium due to South Africa being one of the primary produces of this metal. Two related Brassica napus L cultivars namely Agamax and Garnet which are economically and environmentally important to South Africa were exposed to vanadium. Physiological experiments such as cell death, chlorophyll and biomass determination were conducted to understand how these cultivars were affected by vanadium toxicity. A low cost, sensitive and robust vanadium assay was developed to estimate the amount of vanadium in samples such as water, soils and plant material. The oxidative state as well as the antioxidant profile of the two cultivars were also observed under vanadium stress. A chlorophyll assay which was conducted on the two cultivars xiv exposed to vanadium showed a marked decrease in chlorophyll A in the suspected sensitive cultivar which was Garnet. However, the suspected tolerant cultivar Agamax fared better and the decrease in chlorophyll A was much less. A similar trend was observed for the two cultivars when the cell death assay was conducted. The vanadium assay showed that Garnet had higher concentrations of vanadium within its leaves and lower concentrations in its roots when compared to Agamax. This observation displayed that Agamax had inherent mechanisms which it used to localize vanadium in its roots and which assisted in its tolerance to the vanadium stress. The oxidative state was determined by doing assays for the specific reactive oxygen species namely hydrogen peroxide and superoxide. It was observed that vanadium treated Garnet leaves had higher reactive oxygen species (ROS) production when compared to the Agamax treated leaves. In-gel native PAGE activity gels were conducted to determine the antioxidant profile for the two cultivars which were exposed to vanadium. The antioxidant enzymes which were under investigation were ascorbate peroxide (APX), superoxide dismutase (SOD) and glutathione-dependent peroxidases (GPX-like) as these enzymes are known to be responsible for controlling the ROS produced in the plants. The GPX-like profile consisted of three isoforms. No isoforms were inhibited by vanadium treatments but one isoform had increased activity in both the Garnet and Agamax treated samples. The SOD profile for Garnet consisted of six isoforms xv and Agamax had seven isoforms. One isoform which was visualized in both Agamax as well as Garnet was inhibited by vanadium treatments. Agamax also had two isoforms which were up-regulated however the corresponding isoforms in Garnet showed no change. The Ascorbate peroxidase profile consisted of seven isoforms for both Garnet and Agamax. No isoforms were inhibited by vanadium treatment. Three isoforms were up-regulated in Garnet and Agamax under vanadium treatments. Here, it is illustrated that Garnet lacked certain mechanisms found in Agamax (and thus experienced more cell death, yield and chlorophyll loss) and performed worst under high vanadium concentrations. Although Garnet increased the activity of some of its antioxidant isoforms in response to increasing ROS levels it was not adequate to maintain a normal oxidative homeostasis. This disruption in oxidative homeostasis lead to plant damage. Agamax was observed to produce less ROS than Garnet and was able to control the ROS produced more effectively than Garnet and thus less damage was observed in Agamax. / Magister Scientiae - MSc

Magister Scientiae - MSc (Biotechnology) / Soybean have been listed as a priority commodity crop in South Africa (SA) and provide a good source of protein to the population. Therefore, soybean has been earmarked as an important food security crop and strategies are currently being discussed at governmental level to increase and sustain soybean production. However, the SA landscape poses many challenges to the agricultural sector such as prolong drought periods, flooding, nutrient poor soils, saline soils and heavy metal contaminated soils. Heavy metal (HM) contamination is becoming a serious concern and is aggravated by historical mining in SA. Indeed, SA has established itself as the number one ranked mining country in the world and is frequently mining metals such as chromium, vanadium, gold, zirconium, platinum, and antimony. Prolong rainfall near mining areas leads to acid mine drainage which lowers the soil pH to approximately two. These highly acidic soils will solubilize the metals and cause the metals to leach into river systems as well as the water table leading to increase heavy metal contamination in nearby soil sites. This increase metal content negatively affects seed germination and overall plant development. Nonetheless, plants have evolved numerous internal mechanisms that help them to survive HM toxicity; by either avoiding or tolerating the stress. Two stress-activated pathways that help the plant tolerate stress have attracted much interest i.e. the glyoxalase system and reactive oxygen species (ROS) - antioxidant system as they detoxify methylglyoxal (MG) and ROS. / 2021-08-31

Made available in DSpace on 2014-12-17T14:03:41Z (GMT). No. of bitstreams: 1 HelaineCS_DISSERT.pdf: 3181905 bytes, checksum: 5c020989070824dc8493d5f191c83a23 (MD5) Previous issue date: 2013-01-29 / Ag?ncia Brasileira da Inova??o / Sugarcane (Saccharum spp.) is a plant from Poaceae family that has an impressive ability to accumulate sucrose in the stalk, making it a significant component of the economy of many countries. About 100 countries produce sugarcane in an area of 22 million hectares worldwide. For this reason, many studies have been done using sugarcane as a plant model in order to improve production. A change in gravity may be one kind of abiotic stress, since it generates rapid responses after stimulation. In this work we decided to investigate the possible morphophysiological, biochemical and molecular changes resulting from microgravity. Here, we present the contributions of an experiment where sugarcane plants were submitted to microgravity flight using a vehicle VSB-30, a sounding rocket developed by Aeronautics and Space Institute teams, in cooperation with the German Space Agency. Sugarcane plants with 10 days older were submitted to a period of six minutes of microgravity using the VSB-30 rocket. The morphophysiological analyses of roots and leaves showed that plants submitted to the flight showed changes in the conduction tissues, irregular pattern of arrangement of vascular bundles and thickening of the cell walls, among other anatomical changes that indicate that the morphology of the plants was substantially influenced by gravitational stimulation, besides the accumulation of hydrogen peroxide, an important signaling molecule in stress conditions. We carried out RNA extraction and sequencing using Illumina platform. Plants subjected to microgravity also showed changes in enzyme activity. It was observed an increased in superoxide dismutase activity in leaves and a decreased in its activity in roots as well as for ascorbate peroxidase activity. Thus, it was concluded that the changes in gravity were perceived by plants, and that microgravity environment triggered changes associated with a reactive oxygen specie signaling process. This work has helped the understanding of how the gravity affects the structural organization of the plants, by comparing the anatomy of plants subjected to microgravity and plants grown in 1g gravity / A cana-de-a??car (Saccharum spp.) ? um planta da fam?lia Poaceae que possui uma impressionante capacidade de armazenar sacarose no colmo, o que a torna um significante componente da economia de muitos pa?ses. Aproximadamente 100 pa?ses produzem cana-de-a??car em uma ?rea de 22 milh?es de hectares no mundo. Por essas raz?es, diversos estudos sobre a resposta de culturas a estresse ambiental contemplam a cana-de-a??car. Uma mudan?a na gravidade pode ser um tipo de estresse abi?tico, uma vez que ? capaz de gerar respostas r?pidas ap?s a estimula??o gravitacional. No presente trabalho procurou-se investigar as poss?veis altera??es morfofisiol?gicas, bioqu?micas e moleculares decorrentes da microgravidade. Aqui s?o apresentadas as contribui??es do experimento de submiss?o de plantas de cana-de-a??car ? microgravidade atrav?s de voo em um ve?culo VSB-30, Plantas de cana-de-a??car cultivadas em condi??es controladas, com 10 dias de desenvolvimento, foram assim submetidas a um per?odo de seis minutos de microgravidade real. As an?lises morfofisiol?gicas de ra?zes e folhas mostraram que as plantas sofreram altera??o nos tecidos de condu??o da seiva e ?gua, padr?o de disposi??o irregular de feixes vasculares, espessamento de paredes celulares, entre outras modifica??es anat?micas que indicam que a morfologia das plantas foi substancialmente influenciada pela aus?ncia de est?mulo gravitacional, al?m do ac?mulo de per?xido de hidrog?nio, importante mol?cula de sinaliza??o em condi??es de estresse. Foi realizada a extra??o do RNA e o sequenciamento do RNA atrav?s da plataforma Illumina, e estas sequencias est?o sendo analisadas. Foram tamb?m observadas altera??es nas atividades enzim?ticas, com aumento na atividade de super?xido dismutase em folhas e redu??o da atividade de super?xido dismutase e ascorbato peroxidase em ra?zes. Assim, estes resultados permitem concluir que a altera??o da gravidade foi percebida pelas plantas de cana-de-a??car e o ambiente de microgravidade desencadeou altera??es associadas a um processo de sinaliza??o por meio de esp?cies reativas de oxig?nio em condi??es de estresse. O presente trabalho auxiliou, portanto, a compreender como a gravidade interfere na organiza??o estrutural das plantas, atrav?s da compara??o da anatomia de plantas submetidas ? microgravidade e plantas crescidas em gravidade 1g

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O objetivo do presente trabalho foi caracterizar aspectos fisiolÃgicos e bioquÃmicos que mostrem se plantas de arroz (Oryza sativa) deficientes da enzima citosÃlica peroxidase do ascorbato (cAPX) sÃo mais suscetÃveis ao estresse oxidativo do que as plantas com cAPX. A APX Ã uma importante enzima do metabolismo oxidativo de plantas, atuando na regulaÃÃo dos nÃveis endÃgenos do perÃxido de hidrogÃnio (H2O2). Para isso, plantas deficientes em cAPX (Apx1/2s) e plantas nÃo transformadas (WT) foram utilizadas para a experimentaÃÃo. As plantas foram silenciadas pela tÃcnica do RNA de interferÃncia (iRNA) e cultivadas por 35 dias em vasos de 1.5 L contendo soluÃÃo nutritiva sob condiÃÃes de casa de vegetaÃÃo. O experimento I foi realizado com segmentos de folhas imersos em metil-viologÃnio (MV) 50 M durante 24h, e o experimento II foi realizado pela aplicaÃÃo dos seguintes estresses abiÃticos: salinidade, alta luminosidade e MV. Os resultados do experimento I mostraram que as plantas Apx1/2s possuem um nÃvel basal de H2O2 maior do que os nÃveis encontrados nas plantas WT, sugerindo que as plantas Apx1/2s apresentam um nÃvel de H2O2 prÃximo de um nÃvel de sinalizaÃÃo celular. O fato das plantas WT terem acumulado H2O2 apÃs 1h de tratamento sugere a necessidade de um nÃvel sinalizador de H2O2 para ativaÃÃo dos sistemas de defesa. As plantas Apx1/2s ao contrario das WT apresentaram uma queda constante no conteÃdo de H2O2, indicando uma provÃvel remoÃÃo do excesso de H2O2. ApÃs 3h de tratamento as enzimas SOD, APX e PHGPx de cloroplasto apresentaram atividade superior nas plantas Apx1/2s, tanto no controle quanto no estresse, comparadas com as plantas WT. Esses resultados sugerem existÃncia de um sistema antioxidante bastante ativado nas plantas Apx1/2s. No experimento II as plantas Apx1/2s nÃo apresentaram diferenÃas nos parÃmetros fotoquÃmicos quando comparadas com as plantas WT, mesmo possuindo uma menor fotossÃntese em condiÃÃes controle. A dissipaÃÃo do excesso de energia (NPQ) nas plantas Apx1/2s tratadas com luz foi, em mÃdia, maior que das plantas WT, indicando uma possÃvel maior eficiÃncia na dissipaÃÃo de energia. Mesmo com eficiente dissipaÃÃo de energia ambas as plantas nÃo conseguiram evitar energia excessiva no fotossistema e acabaram sofrendo fotoinibiÃÃo e danos no aparato fotossintÃtico (Fv/Fm). Em relaÃÃo Ãs plantas WT, as Apx1/2s apresentaram maior atividade das enzimas antioxidativas SOD, CAT e PHGPx nas condiÃÃes controle, na provÃvel tentativa de compensar a ausÃncia da cAPX. No tratamento com MV a isoforma cloroplÃstica da PHGPx foi estimulada em mais de 100% nas plantas Apx1/2s, indicando que essas plantas podem reparar danos oxidativos com mais rapidez que as WT. Os dados sugerem que as plantas Apx1/2s, apesar da ausÃncia da cAPX, ativam sistemas adicionais de proteÃÃo antioxidativa para compensar essa ausÃncia e responder mais rÃpida e eficientemente a situaÃÃes de estresse. / The aim of this study was to characterize physiological and biochemical aspects that show if rice plants (Oryza sativa) knockdown on cytosolic ascorbate peroxidase enzyme (cAPX) are more susceptible to oxidative stress than the wild type plants. APX is an important enzyme from oxidative metabolism of plants, acting on regulation of the endogenous levels of hydrogen peroxide (H2O2). For this, rice plants knockdown on cAPX (Apx1/2s) and wild type (WT) were used for the experimentation. The plants were silenced by interference RNA technical (iRNA) and were grown for 35 days into 1.5 L pots containing nutritive solution under greenhouse conditions. The experiment I was performed with leaves segments immersed in methyl viologen (MV) 50 μM for 24h and the experiment II was performed by application of the following treatments: salt stress, high light and MV. The results from experiment I shown Apx1/2s plants have a higher level of H2O2 high than the levels found on WT rice plants, suggesting that Apx1/2s plants present a level of H2O2 near a level of cell signaling. The fact of WT plants had accumulated H2O2 1h after the treatment suggest the necessity of a signaling H2O2 level for stimulate defense systems. Apx1/2s plants unlike of WT plants presented a constant decline on H2O2 content, indicating a likely H2O2 scavenging excess. After 3h of treatment the chloroplastic enzymes SOD, APX and PHGPx presented upper active in Apx1/2s plants, in control and stress, compared with WT plants. These results suggest the existence of an antioxidant system quite active in the Apx1/2s plants. In experiment II the Apx1/2s plants presented no differences in the photochemical parameters when compared with WT plants, even possessing a smaller photosynthesis under controlled conditions. The energy dissipation (NPQ) in the Apx1/2s plants under high light was, in average, higher than WT plants, suggesting better energy dissipation. Even with efficient energy dissipation, the plants could not avoid the excess of energy in the photosystem and they suffered photoinhibition and damage in photosynthetic apparatus (Fv/Fm). In relation the WT plants, Apx1/2s plants presented a higher activity of antioxidant enzymes SOD, CAT and PHGPx under controlled conditions, probably intending compensate the lack of cAPX. In the MV treatment the chloroplastic PHGPx was stimulated above 100% in the Apx1/2s plants, indicating that these plants can repair oxidative damage faster than the WT plants. The results suggest that Apx1/2s plants, despite the absence of cAPX, activated additional security systems to compensate the lack of cAPX and respond quickly and efficiently to stress situations.

A cana-de-açúcar é uma das principais culturas agrícolas no cenário econômico e social brasileiro. Na cultura de cana-de-açúcar o estresse hídrico é o principal fator limitante para o aumento de produtividade, sendo responsável por alterações fisiológicas, bioquímicas e moleculares nas plantas, que podem deflagrar perturbações metabólicas que ativam a morte celular programada (MCP). Sabendo-se que o gene BI-1 apresenta o potencial de reduzir os efeitos da MCP desencadeado por estresses bióticos e abióticos em plantas, este trabalho teve como objetivo analisar plantas transgênicas de cana-de-açúcar que expressam o gene BI-1 de Arabidopsis thaliana (AtBI-1) em condições de estresse hídrico. Também, plantas transgênicas e controle foram inoculadas com o fungo Puccinia melanocephala demonstrando que o processo de transformação genética com o gene AtBI-1 alterou as características pré existentes de resistência a ferrugem marrom nas plantas transgênicas. Os estudos de tolerância ao défict hídrico foram realizados em dois experimentos, o experimento 1 com plantas transgênicas e controles de 90 dias e o experimento 2 com plantas de 60 dias. Plantas do experimento 1 foram analisadas quanto características morfológicas como número de estômatos e tricomas, altura e circunferência do colmo e após ficarem 24 dias sem água foram analisadas quanto a taxa fotossintética, comportamento estomático e conteúdo relativo de água nas folhas, enquanto no experimento 2 as plantas foram analisadas quanto aos teores de prolina, atividades das enzimas guaiacol peroxidase (GPOX), ascorbato peroxidase (APX) e catalase (CAT) após as plantas ficarem 17 dias sob déficit hídrico. Estas enzimas estão envolvidas em processos de desativação de elementos ativos de oxigênio. Os resultados demonstraram que as plantas transgênicas expressando o gene AtBI-1 possuem fenótipo de menor altura, e maior taxa fotossintética, maior comportamento estomático e maior conteúdo relativo de água nas folhas, e assim apresentam maior tolerância ao déficit hídrico que plantas controle. Contudo, houve baixo acúmulo de prolina, baixa atividade da GPOX, APX e CAT nas plantas transgênicas durante o estresse hídrico comparada com as plantas controle do mesmo tratamento. Porém foi observado alta atividade constitutiva da catalase nas plantas transgênicas. A atividade da catalase nestas plantas transgênicas sugere a possibilidade da interação entre AtBI-1 e calmudolinas. Futuros estudos podem contribuir para elucidar se a proteína BI-1 é essencial para a ativação das catalases por calmudolinas. / Sugarcane is one of the main agricultural crops in the Brazilian social and economic scenario. Water stress in the culture of sugarcane is the main limiting factor for increasing productivity accounting for physiological, biochemical and molecular plants that can trigger metabolic disturbances activating programmed cell death (MCP). Knowing that the BI-1 gene has the potential to reduce the effects of MCP triggered by biotic and abiotic stresses in plants, this study aimed to analyze transgenic sugarcane that express the BI-1 gene of Arabidopsis thaliana (AtBI-1) under water stress. Also, transgenic and control plants were inoculated with Puccinia melanocephala fungus demonstrating that the genetic transformation process with the AtBI-1 gene altered the pre-existing characteristics of brown rust resistance in transgenic plants. Studies of tolerance to water deficit were performed in two experiments, the experiment 1 was prepared with transgenic and control plants with 90 days and the experiment 2 used plants with 60 days. Plants from experiment 1 were analyzed as for morphological characteristics such as number of stomata and trichomes, height and diameter of stem after plants being under water for 24 days as were analyzed photosynthetic rate, stomatal behavior, relative water content in leaves while in the experiment 2, plants were analyzed for the levels of proline, enzyme activities of guaiacol peroxidase (GPOX), ascorbate peroxidase (APX) and catalase (CAT) under water deficit for 17 days. These enzymes are involved in deactivation of active elements oxygen. The results demonstrated that the transgenic plants expressing the AtBI-1 gene presented the phenotype of lower height, higher index of leaf area, higher photosynthetic rate, higher stomatal behavior and higher relative water content in leaves than control plants increasing tolerance to drought stress. However, there were low levels of proline, low activity of GPOX activity, APX and CAT in transgenic plants during drought stress compared to control plants of the same treatment, but the observed high constitutive activity of catalase in transgenic plants. Catalase activity in these transgenic plants suggests the possibility of interaction between AtBI-1 and calmudolinas. Future studies may contribute to understand whether the BI-1protein is essential for the activation of catalase by calmudolinas.

The aim of this study was to verify the possibility of ibuprofen degradation by selected plant cultures and determination of activities of antioxidant enzymes (peroxidase, catalase, ascorbate peroxidase and glutathione-S-transferase) as markers of oxidative stress caused by ibuprofen. Nicotiana tabaccum (cv. La Burley 21, cv. SR 1 and their GMOs) and Nicotiana glauca were used as experimental plants. The rate of removal of ibuprofen tested by tobacco was decreasing in the following order: N. tabaccum SR1 > N. tabaccum Zm-P60-1-T4 > N. tabaccum TRI 2T2 > N. glauca > N. tabaccum TRI 2T1 > N. tabaccum cv. La Burley > N. tabaccum Zm-P60-1-T5. As the most suitable tobacco for the removal of ibuprofen seemed untransformed N. tabaccum SR1. The long-term experiment showed that plant stress is being manifested even after longtime. N. tabaccum cv. La Burley 21 seemed to be the most tolerant to ibuprofen in compare with the total enzyme activities in cultures with the presence of ibuprofen and controls. N.glauca was the least tolerant cultivar. Keywords: phytoremediation, ibuprofen, Nicotiana tabaccum, Nicotiana glauca, HPLC, peroxidase, catalase, ascorbate peroxidase, glutathion-S-transferase