Es erfolgte eine Analyse der Regulation der chloroplastidären NADP-MDH und deren Rolle als poising-Mechanismus in Arabidopsis thaliana. Es wurden WT-Pflanzen im Kurztag (KT) oder im Langtag (LT) angezogen und dann einer Überreduktion durch Erhöhung der bereitgestellten Energie unterzogen. In KT- und LT-Pflanzen konnten unterschiedliche Strategien ermittelt werden, um überschüssige Elektronen zu entsorgen. Während KT-Pflanzen redox-vermittelte Akklimatisierungssignale benutzen, um eine bessere Lichtnutzung und ein optimiertes Redox-poising zu erreichen, herrscht in LT-Pflanzen der Schutz vor oxidativem Schaden vor. Es erfolgte eine Charakterisierung von im KT angezogenen NADP-MDH-knock-out-Pflanzen. Da schon unter Kontrollbedingungen erhöhte Aktivitäten von antioxidativen Enzymen vorlagen, reagieren NADP-MDH-KO-Pflanzen wie Pflanzen, die Starklicht-Intensitäten ausgesetzt sind. Die Kapazität der NADP-MDH wird transkriptional reguliert. Um die Signale und Interaktionspartner zu identifizieren, die die verstärkte Transkription der NADP-MDH vermitteln, wurde der Promotorbereich des Enzyms analysiert. Es konnten insgesamt 39 Proteine identifiziert werden, die möglicherweise die verstärkte Transkription der NADP-MDH vermitteln. Unter anderem wurde die Bindung von cytosolischer GAPDH innerhalb der kodierenden Sequenz des NADP-MDH-Gens wahrscheinlich gemacht und in vitro verifiziert. Die Bindestelle der cytosolischen GAPDH wurde auf einen 257 bp umfassenden Bereich eingegrenzt. Es wird postuliert dass es sich um einen regulierten Promotor mit upstream- und downstream-Elementen, sowie mit cis-Elementen innerhalb der CDS handelt. Daher handelt es sich bei dem Gen der NADP-MDH um einen null-core- oder einen distinct-INR-Promotor.

Drought is a major environmental factor limiting crop productivity. Considering a significant area of crop production under water-limited rained conditions, there is a great need to develop production systems to sustain yield potentials under drought stress. Silicon has recently been recognized as an important element in plant nutrition. In this study, it was shown that supplying soybean with soluble silicon in the soil could improve vegetative growth and drought tolerance under water limiting conditions. In order to understand the molecular mechanism how silicon alleviates drought stress, the effects of silicon application on protein expression and antioxidant enzymes were examined. Soybean plants were grown in sand-containing pots supplied with 4 millimolar solutions of sodium silicate. To cancel the effect of sodium, the same amount of sodium chloride was used along with control plants. Soluble proteins were isolated from the leaves and roots of silicon-treated and control plants subjected to water deficit stress. Two-dimensional gel electrophoresis and mass spectrometry approaches were used to identify differentially expressed leaf and root proteins in response to silicon application under water deficit stress. Proteins that showed differential expression in response to silicon application included metabolic enzymes and proteins involved in the proteasome-dependent degradation pathway. These results indicate that silicon application could affect enzymes important for carbohydrate metabolism and stabilize aldehyde dehydrogenases and malic enzyme under water deficit stress, which may be attributable to drought tolerance.

The CO dehydrogenase~linked ferredoxin from acetate-grown <i>Methanosarcina thermophiIa</i> was characterized to determine the structure and biochemical properties of the iron-sulfur clusters. Chemical and spectroscopic analyses indicated that the ferredoxin contained two [4Fe-4S] clusters per monomer of 6,790 Da, although a [3Fe-4S] species was also detected in the oxidized protein. The midpoint potentials of the [4Fe-4S] and [3Fe~4S] clusters at pH 7 were -407 m V and + 103 m V, respectively. Evidence from biochemical and spectroscopic studies indicated that the [3Fe-4S] species may have been formed from [4Fe-4S] clusters when ferredoxin was oxidized. The gene encoding the CO dehydrogenase-linked ferredoxin (<i>fdxA</i>) in <i>Ms. thermophila</i> had the coding capacity for a 6,230-Da protein which contained eight cysteines with spacings typical of 2[4Fe-4S] ferredoxins. A second open reading frame (ORF1) was also identified which had the potential to encode a 2[4Fe-4S] bacterial-like ferredoxin (5,850 Da). The deduced proteins from <i>fdxA</i> and ORF1 were 62% identical. <i>fdxA</i> and ORFI were present as single copies in the genome and each was transcribed on a monocistronic mRNA. Both <i>fdxA</i> and ORF1 were transcribed in cells grown on methanol and trimethylamine, but only the <i>fdxA</i> -specific transcript was detected in acetate-grown cells. The apparent transcriptional start sites of <i>fdxA</i> and ORFI were downstream of sequences which had high identity with the consensus methanogen promoter. The heterodisulfide of two cofactors unique to the methanogenic microorganisms, HS-HTP and HS-CoM, was enzymatically reduced in cell extracts of <i>Ms. thermophila</i> using electrons from the oxidation of either H₂ or CO. The homodisulfides of either HS-HTP or HS-CoM were not reduced under the same conditions. The results indicated that methane is formed by reductive demethylation of CH₃-S-CoM using HS-HTP as a reductant in <i>Ms. thermophila</i>. Coupling of CO oxidation with reduction of the heterodisulfide suggested that the CO dehydrogenase-linked ferredoxin may be involved, although the details of electron flow are not known. / Ph. D.

Orientadores: Anibal Eugênio Vercesi, Helena Coutinho Franco de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-24T17:38:21Z (GMT). No. of bitstreams: 1 Moraes_Audreyde_M.pdf: 2287376 bytes, checksum: 667ac52246453deec2cd72f05541e70c (MD5) Previous issue date: 2014 / Resumo: Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital quando liberada / Abstract: Note: The complete abstract is available with the full electronic document / Mestrado / Fisiopatologia Médica / Mestra em Ciências

As células trofoblásticas provenientes de cones ectoplacentários na fase de pós-implantação expressam as subunidades do complexo NAD(P)H-oxidase de membrana p22-phox, gp91-phox, p47-phox, p40-phox, p67-phox e Rac1 previamente descritas em fagócitos e envolvidas com a atividade fagocitária destas células. Ocorre modulação da expressão das subunidades do complexo enzimático NAD(P)H-oxidase sob o estímulo com PMA, agente capaz de fosforilar a subunidade p47-phox e ativar esta enzima em fagócitos profissionais. Nossos ensaios experimentais indicam que as células trofoblásticas são capazes de gerar espécies reativas de oxigênio dependente da atividade do complexo NAD(P)H-oxidase de membrana de baixa intensidade quando não estimuladas e de forma muito mais intensa quando estimuladas pelo PMA. As características de expressão e atividade do complexo enzimático NAD(P)H-oxidase de membrana encontradas neste estudo sugerem que esta enzima é semelhante a encontrada em outros fagócitos e, desta forma possa talvez estar também envolvida com processos de defesa da interface materno-fetal. / In macrophages and neutrophils, the activation of the phagocytosis is defined by the acquisition of competence for microbicide, tumoricide and cytolytic functions resultant of generation and release of oxygen reactive species, besides the phagocytic process per se. Like these phagocytes, trophoblast cells in many species are also phagocytic. This cellular population involves completely the embryo and exhibits different and specific characteristics along the gestation. In rodents and primates, these cells are strategically positioned between maternal and fetal circulation and exhibit invasive and phagocytic activity, respectively responsible for anchorage of the embryo into the endometrium and uptake of an adequate nutritional supply for the embryo development. In rodents, these activities present a maximum degree during the implantation period, gradually declining, as the placenta develops. In the presence of strange particles at the maternal-placental interface, however, this process can be reactivated and, in this case, may be related to defense\'s mechanisms. Previous studies performed in our laboratory showed the potential of the trophoblast in producing and releasing reactive species of oxygen/nitrogen, in a very similar manner to that observed in macrophages and neutrophils. The production of such molecules is associated to different enzymes, but the localization of hydrogen peroxide on trophoblast cell surface has suggested a NAD(P)H-oxidase activity. NAD(P)H-oxidase is formed by the cytochrome b558 associated to the cellular membrane (subunits p22-phox + gp91-phox), the cytosolic subunits p47-phox, p67-phox and p40phox and, the GTPases Rac1 and Rac2 in an electron generator system that uses NADH or NADPH as substratum. Once activated, the enzymatic complex is responsible for the electron inflow to the molecular oxygen, yields superoxide anion. Thus, based on the literature and results previously obtained by our group, this study analyzed the protein and gene expression of the NAD(P)H-oxidase complex subunits respectively by immunolocalization and Westernblotting and rt-PCR, in the mouse trophoblast stimulated with PMA. Rt-PCR semi-quantitative analyses showed increase expression of the subunits p22-phox, gp91-phox, p47-phox, p67-phox, p40phox and Rac1 in PMA-treated in comparison with non treated ectoplacental cones. The expression of the subunits gp91-phox, p47-phox and p67-phox were confirmed by Western blotting and, like gene expression also increased in the presence of PMA. These subunits were mostly located in the trophoblast giant cell population, associated to the phagocytic process at the maternal-placental interface. Increased expression of such subunits may be related to an increase in the NAD(P)H-oxidase activity. To analyze this possibility and to determine the role played by NAD(P)H-oxidase activity in the reactive oxygen species produced by trophoblast cells, cellular assays were performed using the oxyethidium fluorescence, a product of dihydroethidium oxidation by superoxide anion. Thus, under PMA stimulus and antimycin A that blocks the mitochondrial NAD(P)H-oxidase activity and, apocynin and allopurinol, respectively blocking the membrane NAD(P)H-oxidase and xhantine oxidase and, still, using specific superoxide and hydrogen peroxide scavengers (superoxide dismutase enzyme and catalase) we showed the generation of reactive species of oxygen-NAD(P)H-oxidase dependent by trophoblast cells, mostly when stimulated. These results come to add important information about the potential of the trophoblast in producing reactive species at the maternal-fetal interface and, open a new investigation interest on the NADPH-oxidase regulatory processes and its involvement in defense functions of the embryo in both healthy and pathological processes that can determine the failure of the gestation.

As células trofoblásticas provenientes de cones ectoplacentários na fase de pós-implantação expressam as subunidades do complexo NAD(P)H-oxidase de membrana p22-phox, gp91-phox, p47-phox, p40-phox, p67-phox e Rac1 previamente descritas em fagócitos e envolvidas com a atividade fagocitária destas células. Ocorre modulação da expressão das subunidades do complexo enzimático NAD(P)H-oxidase sob o estímulo com PMA, agente capaz de fosforilar a subunidade p47-phox e ativar esta enzima em fagócitos profissionais. Nossos ensaios experimentais indicam que as células trofoblásticas são capazes de gerar espécies reativas de oxigênio dependente da atividade do complexo NAD(P)H-oxidase de membrana de baixa intensidade quando não estimuladas e de forma muito mais intensa quando estimuladas pelo PMA. As características de expressão e atividade do complexo enzimático NAD(P)H-oxidase de membrana encontradas neste estudo sugerem que esta enzima é semelhante a encontrada em outros fagócitos e, desta forma possa talvez estar também envolvida com processos de defesa da interface materno-fetal. / In macrophages and neutrophils, the activation of the phagocytosis is defined by the acquisition of competence for microbicide, tumoricide and cytolytic functions resultant of generation and release of oxygen reactive species, besides the phagocytic process per se. Like these phagocytes, trophoblast cells in many species are also phagocytic. This cellular population involves completely the embryo and exhibits different and specific characteristics along the gestation. In rodents and primates, these cells are strategically positioned between maternal and fetal circulation and exhibit invasive and phagocytic activity, respectively responsible for anchorage of the embryo into the endometrium and uptake of an adequate nutritional supply for the embryo development. In rodents, these activities present a maximum degree during the implantation period, gradually declining, as the placenta develops. In the presence of strange particles at the maternal-placental interface, however, this process can be reactivated and, in this case, may be related to defense\'s mechanisms. Previous studies performed in our laboratory showed the potential of the trophoblast in producing and releasing reactive species of oxygen/nitrogen, in a very similar manner to that observed in macrophages and neutrophils. The production of such molecules is associated to different enzymes, but the localization of hydrogen peroxide on trophoblast cell surface has suggested a NAD(P)H-oxidase activity. NAD(P)H-oxidase is formed by the cytochrome b558 associated to the cellular membrane (subunits p22-phox + gp91-phox), the cytosolic subunits p47-phox, p67-phox and p40phox and, the GTPases Rac1 and Rac2 in an electron generator system that uses NADH or NADPH as substratum. Once activated, the enzymatic complex is responsible for the electron inflow to the molecular oxygen, yields superoxide anion. Thus, based on the literature and results previously obtained by our group, this study analyzed the protein and gene expression of the NAD(P)H-oxidase complex subunits respectively by immunolocalization and Westernblotting and rt-PCR, in the mouse trophoblast stimulated with PMA. Rt-PCR semi-quantitative analyses showed increase expression of the subunits p22-phox, gp91-phox, p47-phox, p67-phox, p40phox and Rac1 in PMA-treated in comparison with non treated ectoplacental cones. The expression of the subunits gp91-phox, p47-phox and p67-phox were confirmed by Western blotting and, like gene expression also increased in the presence of PMA. These subunits were mostly located in the trophoblast giant cell population, associated to the phagocytic process at the maternal-placental interface. Increased expression of such subunits may be related to an increase in the NAD(P)H-oxidase activity. To analyze this possibility and to determine the role played by NAD(P)H-oxidase activity in the reactive oxygen species produced by trophoblast cells, cellular assays were performed using the oxyethidium fluorescence, a product of dihydroethidium oxidation by superoxide anion. Thus, under PMA stimulus and antimycin A that blocks the mitochondrial NAD(P)H-oxidase activity and, apocynin and allopurinol, respectively blocking the membrane NAD(P)H-oxidase and xhantine oxidase and, still, using specific superoxide and hydrogen peroxide scavengers (superoxide dismutase enzyme and catalase) we showed the generation of reactive species of oxygen-NAD(P)H-oxidase dependent by trophoblast cells, mostly when stimulated. These results come to add important information about the potential of the trophoblast in producing reactive species at the maternal-fetal interface and, open a new investigation interest on the NADPH-oxidase regulatory processes and its involvement in defense functions of the embryo in both healthy and pathological processes that can determine the failure of the gestation.

The metabolism of plants grown in the cultivation medium is influenced by its composition and availability of CO2. In this project the effect of cultivation medium on the activity phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), NADP-malic enzyme (NADP-ME, EC 1.1.1.40), pyruvate, phosphate dikinase (PPDK, EC 2.7.9.1) and enzymes of nitrogen metabolism: nitrate reductase (NR, EC 1.7.1.1), glutamine synthetase (GS, EC 6.3.1.2), glutamate synthase (GOGAT, EC 1.4.1.13) and glutamate dehydrogenase (GDH, EC 1.4.1.2) was studied. The tobacco plant Nicotiana tabacum L., cv. Petit Havana SR1 were grown in vitro in containers fitted with a filter, which caused limited access of CO2. The cultivation medium was modified Murashige-Skoog agar with decreased amounts of phosphate, nitrate, ammonium, or with casein as source of nitrogen and with or without of 1.5% sucrose as additional carbon source Activity of PEPC was higher in plants grown in medium in the presence of sucrose. Reduced concentrations of phosphates, nitrates or NH4 + ions or when casein was the only source of N in medium caused decreased activity of PEPC. Under these conditions, also activity of NADP-ME and of enzymes of nitrogen metabolism: NR, GS decreased. Activity of all enzymes was also negatively affected by limited CO2. On the other...

Biotic stress in the form of viral infection, as well as abiotic salt stress, cause leaves injuries, stomata closure and decreased rate of photosynthesis. These factors lead to the limitation of plant growth and to reduced amount of coenzyme NADPH. However NADPH is an important coenzyme for many metabolic pathways such as synthesis of fatty acids, amino acids and secondary metabolites involved in stress responses. NADPH is also a coenzyme for key enzymes of antioxidant system and for many regulatory enzymes. NADP-dependent enzymes are alternative source of NADPH in plants under stress conditions. In this work, activities of four NADP-dependent enzymes: Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), NADP-malic enzyme (decarboxylating) (NADP-ME, EC 1.1.1.40) and Shikimate dehydrogenase (SDH, EC 1.1.1.25) were studied. Activities of all these enzymes but SDH increased in leaves of tobacco plants (Nicotiana tabacum L.) infected by PVYNTN , The most sensitive enzymes to viral infection were NADP-ICDH and NADP-ME, whose activity was increased in comparison with control plants 3-fold and 2,4-fold, respectively. Changes in activity of studied enzymes were also determined in plants exposed to viral infection in combination with heat-shock...

L'ozone (O3), polluant atmosphérique et gaz à effet de serre, est responsable des pertes de production pour plusieurs espèces végétales. Actuellement, les seuils de risques ne prennent pas en considération la capacité de détoxication intrinsèque des cellules, qui dépend en partie de la régénération du NADPH. Dans cette dernière perspective, l'étude des différentes NADP-déshydrogénases cytosoliques dans les feuilles de deux génotypes de peuplier euraméricain (Populus deltoides x Populus nigra), montre que les activités de la cG6PDH (glucose-6-phosphate déshydrogénase) et de la ME (enzyme malique) sont plus élevées chez le génotype tolérant Carpaccio en réponse à un traitement O3 (120 ppb pendant 17 jours). Dans ces conditions, le maintien des teneurs en NADPH chez Carpaccio, nous a permis de conclure que la capacité des cellules à régénérer ce nucléotide, contribue à améliorer la tolérance face à l'O3. Par une approche de génétique inverse chez Arabidospis, nous avons étudié l'importance de l'activité isocitrate déshydrogénase (ICDH) et de la glutathion réductase 1 (GR1) sous O3, dans un contexte de modulation de la durée du jour. Nous montrons ainsi pour les différents génotypes une plus grande sensibilité à l'O3 lorsque les plantes croissent sous un régime de jours longs, nous soulignons l'interconnexion entre déshydrogénases NADPdépendantes chez le mutant icdh et nous mettons en évidence un possible rôle du glutathion dans le contrôle des interactions entre les fonctions de signalisation oxydante et antioxydante chez le mutant gr1. Enfin, nous avons montré que le monoxyde d'azote (NO) pouvait être en partie impliqué dans la régulation post-traductionnelle de la PEPC sous O3. En effet, nous avons montré que le SNP (donneur de NO) mime les effets de l'O3 alors que le prétraitement de rcd1, mutant surproducteur de NO sous O3, avec du cPTIO + LNNA prévient partiellement ces effets. De plus, l'incubation in vitro d'extraits protéiques foliaires avec du GSNO stimule fortement l'activité PEPC. Tous ces résultats corroborent l'hypothèse que sous O3, l'activité PEPC pourrait être modulée via S-nitrosylation. Cette hypothèse est confortée par la prédiction dans la séquence d'acides aminés de la PEPC de trois cystéines S-nitrosylées, très conservées chez les plantes / Ozone (O3), both an air pollutant and a greenhouse gas, is responsible for yield production losses. Current flux based threshold indices do not take in consideration intrinsic cell capacity for detoxification, which partly depends on NADPH regeneration. In this optic, the study of the NADP-dehydrogenases within leaves of two hybrid poplar (Populus deltoides x Populus nigra) genotypes showed that the activation of cG6PDH (glucose-6-phosphate dehydrogenase) and of ME (malic enzyme) was higher in the tolerant genotype (Carpaccio) in response to O3 treatment (120 ppb for 17 days). The maintenance of NADPH levels in Carpaccio, supported the hypothesis that the capacity for cells to regenerate this nucleotide, contributes to improve tolerance to O3.To investigate the importance of a specific cytosolic NADP?dehydrogenase, we used a reverse genetic approach to test isocitrate dehydrogenase (icdh) and glutathione reductase 1 (gr1) mutants to O3 in a context of day?length modulation. Our study reveals an important role for day length conditions in influencing O3 responses with extended damage for all the genotypes in long days, we pointed to overlapping functions of NADP-dependent dehydrogenases in icdh mutants, and highlights novel roles for the glutathione system in controlling the interactions between antioxidative and oxidative signaling functions in gr1 mutant. Finally, we showed that nitric oxide (NO) could be partly implied in the posttranslational regulation of PEPC under ozone. Treatment of the wild ecotype Col?0 with the NO donor SNP mimicked the O3 effects concerning development of visible injuries and the stimulation of PEPC activity. The pre-treatment of the NO-overproducer mutant rcd1, with cPTIO+L-NNA partially prevented both of these O3 effects. Additionally, in vitro incubation of a protein extract with GSNO strongly enhanced PEPC activity. All together, these results corroborated the hypothesis of the modulation of PEPC activity via Snitrosylation under O3, which is reinforced by the presence of three predicted and highly preserved Snitrosylated cysteines in the protein sequence

El presente trabajo ha sido subencionado en parte por los proyectos: BI093-0660-CO4-03 CICYT y GV-1170793 Generalitat Valenciana.