Made available in DSpace on 2015-03-26T13:36:37Z (GMT). No. of bitstreams: 1 texto completo.pdf: 407532 bytes, checksum: b633523fe7dd877278a161e6da72a14a (MD5) Previous issue date: 2008-10-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Physiological changes and oxidative stress were studied in soybean leaves, Glycine max, variety MG / BR 46 (Conquista) during their development. In a group of plants the reproductive organs were removed, in an attempt to extend their life cycle, retarding their leaf senescence. In the control plants the floweres were not removed and the plants followed their normal cycle until natural senescence. Control plants developed normally, exhibiting progressive degradation of chlorophylls and carotenoids (in low proportion), with the characteristic yellow color of senescent leaves. On the other hand, plants with no flower kept their leaves green until the end of the experiment. The degradation of the pigments led to a sharp decline in carbon assimilation rates (A) in control plants, in which also occurred a decrease in stomatal conductance (gs) and increase in the ratio between the internal and ambient CO2 concentration (Ci/Ca). In contrast, plants without flowers showed a minor drop in A, gs and Ci/Ca. The proportion of electrolyte leakage increased in the control plants throughout their development. Leaves of control plants showed lower levels of hydrogen peroxide (H2O2), in contrast with a great increase observed in plants with no flowers. The low activity of superoxide dismutase (SOD) in control plants in addition to the high activity of peroxidases (POX) and ascorbate peroxidase (APX) are likely responsible for the low levels of H2O2 in those plants. Conversely, the intense activity of SOD and the low performance of POX and APX in plants without flowers might have contributed to the high levels of hydrogen peroxide. Catalase (CAT) activity dropped continuously in the leaves of the two kind of plants, indicating that this enzyme does not play a fundamental role in the removal of reactive oxygen species during this senescence. In both kind of plants, the activity of glutathione reductase had initially increased, followed by a sharp decrease until the end of the experiment. The results of this experiment suggest that oxidative stress is not the determinant factor associated with leaf senescence process in soybean plants. / Foram avaliadas diversas alterações fisiológicas e o estresse oxidativo ao longo do desenvolvimento de folhas de soja, Glycine max, variedade MG/BR 46 (Conquista), em dois diferentes grupos de plantas: com órgãos reprodutores intactos, cujas plantas seguiram o ciclo normal até a senescência e com órgão reprodutores removidos, a fim de prolongar o ciclo de vida, retardando a senescência foliar. Foram avaliados: teores de clorofilas e carotenóides, parâmetros de trocas gasosas, atividades de algumas enzimas do sistema antioxidativo, teores de peróxido de hidrogênio, além de danos celulares. As folhas analisadas das plantas desenvolvendo normalmente apresentaram progressiva degradação de clorofilas e carotenóides (em menor proporção), fato que resultou na coloração amarela característica de folhas senescentes, enquanto as plantas desfloradas mantiveram suas folhas verdes. A degradação dos pigmentos resultou em queda acentuada das taxas de assimilação líquida de carbono (A) nas plantas com órgãos reprodutores intactos, além de ter ocorrido queda na condutância estomática (gs) e aumento na razão entre a concentração interna e ambiente de CO2 (Ci/Ca). Ao contrário, as plantas com órgãos reprodutores removidos apresentaram menor queda em A, gs e Ci/Ca, que se mantiveram constantes após a ligeira queda inicial. O percentual de extravasamento de eletrólitos em plantas em senescência natural aumentou ao longo do desenvolvimento, mas não foi acompanhado de aumento de aldeído malônico (MDA). Em plantas desfloradas o extravasamento foi inicialmente constante, seguido de queda com o início da senescência, mas, por outro lado, os níveis finais de MDA foram duas vezes maiores que os iniciais. As folhas das plantas intactas apresentaram baixos teores de peróxido de hidrogênio (H2O2), ao contrário das plantas desfloradas. A baixa atividade da dismutase do superóxido (SOD) em plantas senescendo normalmente, além das atividades elevadas de peroxidase (POX) e peroxidase do ascorbato (APX), devem ter sido as responsáveis pelos baixos níveis de H2O2 nessas plantas. Inversamente, a atividade intensa de SOD e a baixa atuação de POX e APX em plantas desfloradas contribuíram para os altos teores de peróxido de hidrogênio. A catalase (CAT) teve sua atividade em queda ao longo do experimento, nas folhas de ambos os tratamentos, indicando que a enzima não teve participação importante na remoção de H2O2. Nos trifólios das plantas dos dois tratamentos, a redutase da glutationa (GR) teve sua atividade inicialmente elevada, seguida de queda drástica até o final do experimento. Tais resultados indicam que o estresse oxidativo não foi o fator determinante da senescência foliar natural das plantas de soja utilizadas no presente experimento.

L'autophagie est importante pour le recyclage et la mobilisation des éléments nutritifs dans les plantes. Plusieurs lignées sur-exprimant les gènes AtATG8a-i d’Arabidopsis ont été sélectionnées afin de déterminer l'effet de l'augmentation de l'activité autophagique sur l'efficacité de la remobilisation de l'azote. Les lignées sur-expresseur ont présenté une meilleure remobilisation de l'azote des feuilles de la rosette jusqu'aux graines qque le contrôle, mais uniquement dans des conditions de culture en nitrate pléthorique. Les lignées sur-exprimant ATG8a et ATG8g ont été les plus performantes. Une vaste collection de mutants atg, comprenant les mutants atg8a-i, a ensuite été utilisée pour mesurer la remobilisation de l’azote des rosettes, aux tiges et aux semences, afin de déterminer quels gènes ATG sont essentiels pour la remobilisation. Une attention particulière a été accordée à la famille de gènes ATG8a-i afin de déterminer si un membre de la famille ATG8 pourrait être plus spécifiquement dédié au recyclage de l'azote lors de la sénescence des feuilles pour la remobilisation. Bien que les divers mutants atg8 n’aient pas présenté de différence majeure par rapport au sauvage, l'un d'entre eux a montré un léger phénotype de sénescence précoce, suggérant que cette isoforme pourrait être plus spécialisée dans la remobilisation de l'azote. Afin d'améliorer l'activité de l'autophagie chez l'orge, nous avons surexprimé HvATG5 chez l'orge et testé la sensibilité des sur-expresseurs à plusieurs conditions de stress. Nous avons constaté que les sur-expresseurs de HvATG5 étaient plus tolérants à une faible alimentation en nitrate, à une faible teneur en soufre, et surtout moins sensibles aux effets de l'obscurité prolongée. Afin d'estimer les rôles respectifs des protéases induites par la sénescence et de l'autophagie, plusieurs mutants de protéase (sag12, rd21A, cathB3) ont été croisés avec des mutants d'autophagie (atg5, atg7. Leur contribution à la remobilisation de l'azote a été mesurée par marquage 15N. / Autophagy is important for nutrient recycling and mobilization in plants. Several Arabidopsis AtATG8a-i overexpressing lines were selected in order to determine the effect of increasing autophagy on nitrogen remobilization efficiency. The overexpressing lines remobilized more nitrogen from the rosette leaves to the seeds but only when cultivated under high nitrate conditions. The lines overexpressing ATG8a and ATG8g were the most performant. A large collection of atg mutants including the atg8a-i mutants was then used to monitor N-remobilization from the rosettes to the stems and seeds in order to determine which ATG genes are essential for N remobilization. A special focus was dedicated to the ATG8a-i gene family in order to determine whether a member of the ATG8 family could be more specifically dedicated to N-recycling during leaf senescence for remobilization. Although the various atg8 mutants were not different from wild-type, one of them presented slight early senescence phenotype, suggesting this isoform could be more specialized in N remobilization. In order to enhance autophagy activity in barley, we overexpressed HvATG5 in barley and tested sensitivity of over-expressors to several stress conditions. We found that barley HvATG5 over-expressors were more tolerant to low nitrate supply, to low sulfur, and especially less sensitive to dark-stress effects. In order to estimate the respective roles of autophagy and senescence induced proteases, several protease mutants (sag12, rd21A, cathB3) were crossed with autophagy mutants (atg5, atg7) in order to monitor their respective contributions to nitrogen remobilization.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23744号 / 理博第4834号 / 新制||理||1691(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 工藤 洋, 准教授 本庄 三恵, 教授 松下 智直 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Handroanthus chrysotrichus is a Brazilian native forest species which presents several important aspects both under the economic and ecological points of view, because its ability to provide timber products as well as for being indicated for degraded and permanent preservation areas recovery. Its seeds present problems concerning loss of viability when subjected to storage. This way, micropropagation emerges as an alternative for the species propagation. However, some characteristics inherent in in vitro grown plants may promote physiological and anatomic changes in tissues under that condition. Thus, the aim of this study was to evaluate aspects related to micropropagation and leaf anatomy of H. chrysotrichus. Firstly, health and germination tests were applied to seeds from different lots of that species. As for in vitro multiplication of H. chrysotrichus explants, different sources and concentrations of cytokines combined or not with auxin were tested, as well as the influence of successive subculture execution on multiplication rates. Aspects concerning leaf senescence of sprouts maintained in vitro in Woody Plant Medium (WPM) were also assessed. Concerning rooting in vitro, different concentrations of 3-Indolebutyric acid (IBA) combined with the presence or absence of vermiculite in ½ WPM were tested and, later, the ex vitro acclimatization of plants micropropagated was assessed. Finally, the test was conducted in order to compare the leaf anatomy of H. chrysotrichus plants grown in vivo, in vitro and acclimatized. The seed lots of H. chrysotrichus showed differences in health quality and geminal capacity of the seeds. As for in vitro multiplication of H. chrysotrichus, the use of cytokinins was not considered necessary. High rates of in vitro multiplication were obtained, when considered in vitro formation of buds. As for leaf senescence, the species showed great potential, remaining long periods in the same nutrient medium. There was rooting in vitro in more than 50% of the shoots cultivated in the presence of vermiculite, regardless IBA concentration. It was not possible to satisfactorily promote the acclimatization of micropropagated plants, as only 7.12% of the seedlings survived. Differences in leaf anatomy of plants grown in vivo, in vitro or acclimatized were verified. The results can contribute with information about H. chrysotrichus, concerning aspects related to its in vitro culture and leaf anatomy of the tissues grown in different environments. / Handroanthus chrysotrichus é uma espécie florestal nativa do Brasil, que apresenta diversos aspectos importantes tanto sob o ponto de vista econômico, pela produção de produtos madeiráveis, como, também, ecologicamente, uma vez que é indicada para a recuperação de áreas degradadas e de preservação permanente. As sementes desta espécie apresentam problemas referentes à perda da viabilidade quando submetidas ao armazenamento. Sendo assim, a micropropagação emerge como uma alternativa de propagação para a espécie. No entanto, algumas características inerentes às plantas cultivadas in vitro podem promover alterações fisiológicas e anatômicas nos tecidos mantidos nessas condições. Desse modo, o objetivo deste estudo foi avaliar aspectos relacionados à micropropagação e à anatomia foliar de H. chrysotrichus. Primeiramente, foram realizados testes de sanidade e germinação nas sementes provenientes de diferentes lotes desta espécie. Já para a multiplicação in vitro de explantes de H. chrysotrichus foram testadas diferentes fontes e concentrações de citocininas combinadas ou não à auxina, bem como a influência da execução de sucessivos subcultivos nas taxas de multiplicação. Também foram avaliados aspectos relacionados à senescência foliar de brotações desta espécie conservadas in vitro em meio nutritivo WPM. Em relação ao enraizamento in vitro, testaram-se diferentes concentrações de Ácido 3-Indolbutírico - AIB combinadas à presença ou ausência de vermiculita no meio nutritivo ½WPM e, posteriormente, avaliou-se a aclimatização ex vitro das plantas micropropagadas. Por fim, foi realizado o ensaio visando comparar a anatomia foliar de plantas de H. chrysotrichus cultivadas in vivo, in vitro ou aclimatizadas. Os lotes de sementes de H. chrysotrichus apresentaram diferenças na qualidade sanitária e capacidade geminativa das sementes. Na multiplicação in vitro de H. chrysotrichus, o uso de citocininas foi considerado dispensável. Foram obtidas elevadas taxas de multiplicação in vitro em H. chrysotrichus, quando considerada a formação in vitro de gemas. Quanto à senescência foliar, a espécie demonstrou grande potencial, podendo permanecer longos períodos no mesmo meio nutritivo. Houve rizogênese in vitro em mais de 50% das brotações cultivadas na presença de vermiculita, independentemente das concentrações de AIB. Não foi possível promover a aclimatização das plantas micropropagadas de maneira satisfatória, sendo que apenas 7,12% das mudas sobreviveram. Foram verificadas diferenças na anatomia foliar de plantas desenvolvidas in vivo, in vitro ou aclimatizadas. Os resultados obtidos poderão contribuir com informações a respeito de H. chrysotrichus quanto a aspectos relacionados ao seu cultivo in vitro e anatomia foliar dos tecidos cultivados em diferentes ambientes.

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Orientador: Marlene Aparecida Schiavinato / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T08:53:28Z (GMT). No. of bitstreams: 1 Longatti_ClaudiaAparecida_M.pdf: 1449126 bytes, checksum: be3aefaf910ddbe46531e367581164f3 (MD5) Previous issue date: 2012 / Resumo: O metabolismo geral de uma planta pode ser afetado pelo excesso de sais no solo, visto que a salinidade pode causar reduções no crescimento e acelerar a senescência. Os brassinosteróides são hormônios vegetais envolvidos no aumento da tolerância a vários tipos de estresses, inclusive o estresse salino. Com o objetivo de avaliar a influência da aplicação de brassinosteróide no crescimento e metabolismo do nitrogênio em plantas de Cajanus cajan sob diferentes condições de estresse salino, plantas noduladas de C. cajan foram cultivadas em casa de vegetação na ausência ou presença de brassinosteróide (24-epibrassinolídeo), sob diferentes concentrações de NaCl (0 mM, 100 mM e 200 mM). A salinidade afetou o crescimento, reduzindo a área, o comprimento do caule, a longevidade foliar, o teor de umidade de folhas, caules e raízes, bem como a atividade da nitrogenase, os teores de pigmentos fotossintetizantes, de malondialdeído e de nitrogênio total em folhas verde maduras, e a proficiência e eficiência do uso de nitrogênio. A aplicação de brassinosteróide amenizou o efeito do NaCl nas reduções da área foliar e teores de clorofila a e total. Também atenuou o efeito do estresse salino na atividade da nitrogenase. Esse efeito positivo do brassinosteróide sobre a atividade da enzima refletiu-se no aumento da concentração de compostos nitrogenados nas folhas verde-maduras e reduziu, ainda, a perda de N nas folhas senescentes. A maior concentração de N total nas folhas verde-maduras de plantas tratadas com o 24-epibrassinolídeo levou à redução da eficiência de utilização desse elemento / Abstract: The general metabolism of a plant can be affected by salinity excess in soil, due to the fact that salinity can cause growth reduction and accelerate senescence. The brassinosteroids are plant hormones involved in increasing tolerance in several types of stress, including salt stress. In order to verify the influence of the application brassinosteroid on development and nitrogen metabolism in Cajanus cajan plants under different levels of salt stress, nodulated plants of C. cajan were cultivated in the greenhouse in the absence or presence of 24-epibrassinolide, under different concentrations of NaCl (0 mM, 100 mM and 200 mM). Salinity affected growth reducing leaf area, branch length, leaf longevity, moisture content of leaves, stems and roots, as well as activity of nitrogenase, amounts of photosynthetic pigments, malondialdehyde and total nitrogen in mature green leaves; nitrogen use proficiency and efficiency. The application of brassinosteroid attenuated the effect of NaCl on the leaf area and on the concentrations of chlorophyll a and total chlorophyll. The hormone also attenuated the effect of salt stress on the activity of nitrogenase. The positive effect of the hormone on the activity of the enzyme led to an increase in the concentration of nitrogenous compounds in the mature green leaves and reduced the lost of nitrogen in senescent leaves. The higher concentration of total nitrogen in mature green leaves of the plants treated with 24-epibrassinolide led to a reduction in nitrogen use efficiency / Mestrado / Biologia Vegetal / Mestre em Biologia Vegetal

The ability of plants to sense and respond to environmental and endogenous signals is essential to their growth and development. As part of these diverse cellular functions, ubiquitin-mediated proteolysis has emerged to be an important process involved in how plant signalling pathways can be regulated in response to such cues. Of the three enzymes involved in linking ubiquitin to protein targets, E3 ubiquitin ligases are of interest as they confer substrate specificity during this ubiquitination process. The overall focal point of this research is on plant U-box (PUB) E3 ubiquitin ligases, a family that has undergone a large gene expansion possibly attributable to the regulation of biological processes unique to the plant life cycle. In Arabidopsis there are 64 predicted PUBs, many for which biological roles have yet to be determined. And as research continues to uncover PUB functions, the functional diversity in the gene family will likely expand. Specifically the focus of this research is on characterizing two ARM repeat-containing PUBs – AtPUB18 and AtPUB19. General analysis of pub18 and pub19 T-DNA insertion lines for growth defects did not yield distinct altered phenotypes. Closer inspection of selected lines showed independent gene assortment phenotypes that, with further inordinately convoluted pursuit, proved to have an AtPUB18/19-unrelated outcome. The availability of Arabidopsis microarray databases provided exploratory expression profiling as a starting point to elucidate PUB function. AtPUB19 and closely related AtPUB18 are notable for their increased expression during abiotic stresses. While condition-directed germination assays showed a decreased sensitivity to salt and ABA for pub18 pub19 double insertion lines, no related change in susceptibility to these or other abiotic stress treatments were seen with condition-directed root growth assays. Thus, this preliminary work has begun to reveal insight into the complex abiotic stress-related roles AtPUB18 and AtPUB19 have during mediation of environmental stress acclimation in Arabidopsis.

The ability of plants to sense and respond to environmental and endogenous signals is essential to their growth and development. As part of these diverse cellular functions, ubiquitin-mediated proteolysis has emerged to be an important process involved in how plant signalling pathways can be regulated in response to such cues. Of the three enzymes involved in linking ubiquitin to protein targets, E3 ubiquitin ligases are of interest as they confer substrate specificity during this ubiquitination process. The overall focal point of this research is on plant U-box (PUB) E3 ubiquitin ligases, a family that has undergone a large gene expansion possibly attributable to the regulation of biological processes unique to the plant life cycle. In Arabidopsis there are 64 predicted PUBs, many for which biological roles have yet to be determined. And as research continues to uncover PUB functions, the functional diversity in the gene family will likely expand. Specifically the focus of this research is on characterizing two ARM repeat-containing PUBs – AtPUB18 and AtPUB19. General analysis of pub18 and pub19 T-DNA insertion lines for growth defects did not yield distinct altered phenotypes. Closer inspection of selected lines showed independent gene assortment phenotypes that, with further inordinately convoluted pursuit, proved to have an AtPUB18/19-unrelated outcome. The availability of Arabidopsis microarray databases provided exploratory expression profiling as a starting point to elucidate PUB function. AtPUB19 and closely related AtPUB18 are notable for their increased expression during abiotic stresses. While condition-directed germination assays showed a decreased sensitivity to salt and ABA for pub18 pub19 double insertion lines, no related change in susceptibility to these or other abiotic stress treatments were seen with condition-directed root growth assays. Thus, this preliminary work has begun to reveal insight into the complex abiotic stress-related roles AtPUB18 and AtPUB19 have during mediation of environmental stress acclimation in Arabidopsis.

Red alder (Alnus rubra Bong.) is the only tree in British Columbia and the Northwest US to engage in actinorhizal symbiosis to fix atmospheric nitrogen. This study was conducted to explore the plasticity in growth and physiology among 58 17-year-old red alder families in response to variation in climate in two common garden plots, one at Bowser, BC and one at Terrace, BC. Physiological assessments included height and diameter growth, bud flush, water use efficiency as measured by δ13C, cold hardiness as measured by controlled freezing and electrolyte leakage, autumn leaf senescence, and instantaneous and seasonally integrated rates of nitrogen fixation as measured by acetylene reduction and natural abundance δ15N isotope analysis, respectively. Significant differences were identified among families for growth (height and diameter), bud burst stage, leaf senescence, cold hardiness, and bud nitrogen content. No significant differences among families were identified for water use efficiency as measured by δ13C, or for rates of nitrogen fixation as measured by either acetylene reduction or natural abundance δ15N. This study identified possible adaptive differences among red alder genotypes, especially in traits such as bud flush timing, cold hardiness, or nitrogen fixation and their respective contributions to growth. These differences often reflected a tradeoff between growth and the ability to tolerate an extreme environment. Cold hardiness results indicate that red alder families are well adapted to their climate of origin, and may not be able to acclimate sufficiently to a northward assisted migration of genotypes. Nitrogen fixation results demonstrated gaps in our current knowledge of Frankia distribution and impact on the actinorhizal symbiosis in British Columbia. / Graduate