A utilização de produtos de ação fitotônica tem estimulado pesquisas em diversas áreas da agricultura, dentre elas destaca-se a utilização de fungicidas de efeito fisiológico. Um dos mais promissores é a estrobilurina Piraclostrobina, que além da ação fungicida tem ocasionado aumento de produtividade atribuída aos efeitos fisiológicos proporcionados, os quais são conhecidos nas fases vegetativa e reprodutiva. Sendo assim, foram conduzidos experimentos em campo e em casa de vegetação durante quatro anos consecutivos (2004 a 2007) com o objetivo de avaliar o efeito fisiológico em diferentes variedades cultivadas de soja à aplicação da estrobilurina Piraclostrobina. Em 2004, o crescimento das variedades cultivadas de soja CD-208, BRS-133 e Conquista foi acompanhado ao longo do ciclo de cultivo em amostragens destrutivas a cada sete dias. O fungicida Piraclostrobina + Epoxiconazol foi aplicado no momento em que as plantas se encontravam nos estádios fenológicos R5, R4 e R3, respectivamente. As parcelas pulverizadas apresentaram maior produção de massa de matéria seca, maior índice de área foliar e maior produtividade. Em 2005, foram utilizadas as variedades cultivadas Conquista, CD-208 e Monsoy- 7501. Concluiu-se que o fungicida Piraclostrobina + Epoxiconazol promove: (i) aumento da fotossíntese líquida; (ii) melhoria da eficiência de assimilação de nitrogênio devido ao aumento da atividade da enzima nitrato redutase (ANR); (iii) diminuição da síntese de etileno (SE) (redução da senescência e queda de folhas); (iv) aumento da fitomassa seca total; (v) aumento da área foliar; (vi) aumento do teor de clorofila (efeito verde); (vii) melhoria dos resultados em ambientes com mais estresse; (viii) diminuição da incidência de Phomopsis spp. e de Cercospora kikuchii na semente; e (ix) aumento (tendência) do teor de óleo na semente. Em 2006, foram confirmados os resultados referentes aos anos anteriores utilizando as variedades cultivadas Monsoy-8008-RR, Monsoy-7878-RR e BRS-245-RR. Além disso, verificou-se que a aplicação de Piraclostrobina aumentou a atividade das enzimas antioxidantes (peroxidade, superóxido dismutase e catalase) e diminuiu a atividade da enzima polifenoloxidase e aumentou os teores dos hormônios citocininas (zeatinas e isopentinil adenina), giberelinas (GA3) e ácido indolbutírico. Em 2007, foi conduzido experimento em casa de vegetação com o objetivo de avaliar o tempo de resposta de variáveis fisiológicas à aplicação da estrobilurina Piraclostrobina. Utilizou-se a variedade cultivada Conquista em vaso. Na fase vegetativa (estádios fenológicos V2-V3 e V8-V9), foram observados efeitos fisiológicos nas trocas gasosas até 7 DAA e para a ANR e SE até 16 e 18 DAA, respectivamente. Na fase reprodutiva (estádios R1-R2 e R5.1), foi verificado aumento na atividade fotossintética até 10 DAA no estádio R1-R2 e até 7 DAA no estádio R5.1. Em relação ao incremento da SE e ANR, foram evidenciados efeitos da estrobilurina Piraclostrobina somente até 7 e 6 DAA no estádio R1 e 3 e 16 DAA no estádio R5.1, respectivamente. / The use of products with tonic action in plants has stimulated research in different areas of agriculture, where the use of fungicides with physiological effect became important. One of the most promising is the strobilurin Pyraclostrobin, that in addition to the fungicidal action has caused an increase in productivity attributed to the physiological effects provided, which are known in the vegetative and reproductive phases. Therefore, experiments were carried out in greenhouse and field during four consecutive years (2004 to 2007) with the purpose of evaluating the physiological effect on different soybean cultivars to application of the strobilurin Pyraclostrobin. In 2004, the soybean growth, cultivars CD-208, BRS-133 and Conquista, was monitored during the crop cycle using destructive sampling every seven days. The Pyraclostrobin + Epoxiconazole fungicide was applied at the time the plants were in R5, R4 and R3 growth stages, respectively. The treated areas showed higher production of dry matter, greater leaf area index and greater productivity. In 2005, the cultivars Conquista, CD-208 and Monsoy-7501 were used. It was concluded that Pyraclostrobin + Epoxiconazole promotes: (i) increasing of net photosynthesis, (ii) improving the efficiency of nitrogen assimilation due to the increasing of nitrate reductase activity (NRA), (iii) decreasing the ethylene production (reduction of senescence and leaf fall), (iv) increasing the total dry mass, leaf area and chlorophyll content (green effect), (v) improving the performance in environments with more stress; (vi) reducing the incidence of Phomopsis spp. and Cercospora kikuchii in the seed, and (vii) increasing (trend) of oil content in the seed. In 2006, the results were confirmed according to the previous years using the cultivars Monsoy-8008-RR, Monsoy-7878-RR and BRS-245-RR. Besides, it was found that the application of Pyraclostrobin increased the activity of antioxidant enzymes (peroxidase, superoxide dismutase and catalase) and reduced the PPO activity and increased levels of the following hormones: cytokinins (zeatin and adenine isopentinil), gibberellins (GA3) and indole butyric acid. In 2007, a greenhouse experiment was carried out to evaluate the response time of physiological variables to the application of strobilurin Pyraclostrobin. The Conquista cultivar was used in pots. In the vegetative phase (V2-V3 and V8-V9 growth stages), physiological effects on gas exchange were observed until 7 DAA and for the NRA and ethylene synthesis (ES) until 16 and 18 DAA, respectively. In the reproductive phase (R1-R2 and R5.1 stages), an increasing of photosynthetic activity was verified until 10 DAA at R1-R2 stage and up to 7 DAA at R5.1 stage. Regarding to the increasing of the ES and NRA, the strobilurin Pyraclostrobin effects were observed only until to 7 and 6 DAA at R1 stage and 3 and 16 DAA at R5.1 stage, respectively.

O conhecimento do comportamento vegetal quanto ao uso de nutrientes permite manusear ou modificar o sistema de cultivo para melhorar a eficiência na utilização de nitrogênio. O fornecimento de potássio para as plantas estimula o aproveitamento do nitrogênio, possibilitando que sua absorção, assimilação e, conseqüentemente, a produtividade sejam aumentadas. Com base nestes fatos, o objetivo deste estudo foi avaliar o efeito de combinações de doses de nitrogênio e de potássio na nutrição, no teor de clorofila e na atividade da enzima redutase do nitrato em plantas de trigo (Triticum aestivum L.). O experimento foi conduzido em casa de vegetação do Departamento de Ciência do Solo da ESALQ/USP, localizada no município de Piracicaba-SP, utilizando-se vasos de três litros onde foram acondicionadas amostras da camada 0-20 cm de um Argissolo Vermelho Amarelo Abrúptico. Utilizou-se esquema fatorial 52 fracionado, com combinações de cinco doses de nitrogênio (0; 70; 140; 210 e 280 mg dm -3) e de cinco doses de potássio (0; 50; 100; 150 e 200 mg dm-3), as quais foram distribuídas segundo delineamento experimental de blocos aleatorizados, com quatro repetições. As leituras SPAD das folhas do terço-médio das plantas de trigo foram realizadas 30 dias após a semeadura. Aos setenta e nove dias após a semeadura foi realizada determinação da atividade da enzima redutase do nitrato. Aos 80 dias após a semeadura, as plantas foram colhidas, separada em parte aérea, espiga e raízes, secadas, pesadas e submetidas à determinação da concentração de nutrientes e acúmulo de nitrogênio e potássio. Os resultados foram analisados por meio do programa estatístico SAS e mostraram que o equilíbrio entre o fornecimento de nitrogênio e potássio foi essencial para o crescimento adequado das plantas de trigo. Ficou evidente que a combinação entre as doses de nitrogênio e de potássio interagiu positivamente na produção de fitomassa e na concentração de nitrogênio, fósforo, potássio, cálcio e enxofre na parte aérea das plantas; o nitrogênio foi determinante para a concentração de magnésio no tecido vegetal. A interação entre as doses de nitrogênio e de potássio influenciou positivamente na produção de fitomassa e na concentração de nitrogênio na espiga. A concentração de potássio na espiga dependeu apenas do fornecimento das doses de nitrogênio. A produção de fitomassa das raízes foi incrementada com o fornecimento das doses de nitrogênio e de potássio, sendo suas concentrações de nitrogênio, magnésio e de enxofre influenciadas pelo suprimento de nitrogênio. A concentração de potássio nas raízes diminuiu e a de cálcio aumentou com as doses de nitrogênio, conforme equação de segundo grau. O fornecimento de potássio promoveu decréscimos lineares na concentração de magnésio na parte aérea e nas raízes das plantas de trigo. O nitrogênio aumentou a atividade da redutase do nitrato até as doses intermediárias, diminuindo nas doses elevadas. O teor de clorofila das folhas do terço-médio da planta foi influenciado pela interação entre as doses de nitrogênio e potássio e refletiu diretamente na produtividade de fitomassa da parte aérea e de espigas, assim como no acúmulo de nitrogênio pelas plantas de trigo. / The knowledge about plant nutrition allows managing crop systems in order to improve the nitrogen use efficiency. Supplying potassium to plants stimulates the efficiency use of nitrogen through plant uptake and assimilation, consequently increasing crop yields. Based on these facts, the aim of this study was to evaluate the effect of combined rates of nitrogen and potassium on the nutrition, chlorophyll content, and nitrate reductase activity in wheat (Triticum aestivumaestivum L.) plants. The experiment was conducted in a greenhouse of the Soil Science Department of the ESALQ/USP in Piracicaba State of São Paulo, Brazil. Three-liter pots were filled with 0-20 cm depth samples of Arenic Hapludult. A fractionated 52 factorial was used, with combinations of five nitrogen rates (0; 70; 140; 210 e 280 mg dm-3) and five potassium rates (0; 50; 100; 150 e 200 mg dm-3), which were set in a randomized block design with four replications. Thirty days after seeding SPAD values readings were taken on the leaves. Nitrate reductase activity was determined seventy-nine days after seeding and one day later plants were harvested and separated in shoot, roots, and ears. These plant materials were weighted and submitted to nutrient content and accumulation of nitrogen and potassium. Statistical analyses were performed by means of SAS software. Results presented that the balance between nitrogen and potassium supplies was essential to adequate wheat plant growth. It was clear that the combination of nitrogen and potassium rates had a positive influence on wheat dry mass yield as well as on nitrogen, phosphorus, potassium, calcium, and sulphur contents in plant shoot. Nitrogen was essential to maintain adequate magnesium content in plant tissues. The interaction between nitrogen and potassium rates was important for plant yield and ear nitrogen content. Potassium content in the ears was affected only by nitrogen rates. Root dry matter yield increased with rates of nitrogen and potassium. Nitrogen, magnesium and sulphur contents in the roots were influenced only by nitrogen application. Potassium concentration in the roots reduced and calcium concentration increased with nitrogen rates according to a quadratic equation. Potassium supply caused linear decreases in magnesium content of shoots and roots. Nitrogen increased nitrate reductase activity up to the medium rates of application and reduced for the highest rates. Chlorophyll content in the leaves was influenced by the interaction between nitrogen and potassium rates, reflecting on the dry matter yield of plant shoot and ears, as well as on nitrogen accumulation in the wheat plants.

O conhecimento do comportamento vegetal quanto ao uso de nutrientes permite manusear ou modificar o sistema de cultivo para melhorar a eficiência na utilização de nitrogênio. O fornecimento de potássio para as plantas estimula o aproveitamento do nitrogênio, possibilitando que sua absorção, assimilação e, conseqüentemente, a produtividade sejam aumentadas. Com base nestes fatos, o objetivo deste estudo foi avaliar o efeito de combinações de doses de nitrogênio e de potássio na nutrição, no teor de clorofila e na atividade da enzima redutase do nitrato em plantas de trigo (Triticum aestivum L.). O experimento foi conduzido em casa de vegetação do Departamento de Ciência do Solo da ESALQ/USP, localizada no município de Piracicaba-SP, utilizando-se vasos de três litros onde foram acondicionadas amostras da camada 0-20 cm de um Argissolo Vermelho Amarelo Abrúptico. Utilizou-se esquema fatorial 52 fracionado, com combinações de cinco doses de nitrogênio (0; 70; 140; 210 e 280 mg dm -3) e de cinco doses de potássio (0; 50; 100; 150 e 200 mg dm-3), as quais foram distribuídas segundo delineamento experimental de blocos aleatorizados, com quatro repetições. As leituras SPAD das folhas do terço-médio das plantas de trigo foram realizadas 30 dias após a semeadura. Aos setenta e nove dias após a semeadura foi realizada determinação da atividade da enzima redutase do nitrato. Aos 80 dias após a semeadura, as plantas foram colhidas, separada em parte aérea, espiga e raízes, secadas, pesadas e submetidas à determinação da concentração de nutrientes e acúmulo de nitrogênio e potássio. Os resultados foram analisados por meio do programa estatístico SAS e mostraram que o equilíbrio entre o fornecimento de nitrogênio e potássio foi essencial para o crescimento adequado das plantas de trigo. Ficou evidente que a combinação entre as doses de nitrogênio e de potássio interagiu positivamente na produção de fitomassa e na concentração de nitrogênio, fósforo, potássio, cálcio e enxofre na parte aérea das plantas; o nitrogênio foi determinante para a concentração de magnésio no tecido vegetal. A interação entre as doses de nitrogênio e de potássio influenciou positivamente na produção de fitomassa e na concentração de nitrogênio na espiga. A concentração de potássio na espiga dependeu apenas do fornecimento das doses de nitrogênio. A produção de fitomassa das raízes foi incrementada com o fornecimento das doses de nitrogênio e de potássio, sendo suas concentrações de nitrogênio, magnésio e de enxofre influenciadas pelo suprimento de nitrogênio. A concentração de potássio nas raízes diminuiu e a de cálcio aumentou com as doses de nitrogênio, conforme equação de segundo grau. O fornecimento de potássio promoveu decréscimos lineares na concentração de magnésio na parte aérea e nas raízes das plantas de trigo. O nitrogênio aumentou a atividade da redutase do nitrato até as doses intermediárias, diminuindo nas doses elevadas. O teor de clorofila das folhas do terço-médio da planta foi influenciado pela interação entre as doses de nitrogênio e potássio e refletiu diretamente na produtividade de fitomassa da parte aérea e de espigas, assim como no acúmulo de nitrogênio pelas plantas de trigo. / The knowledge about plant nutrition allows managing crop systems in order to improve the nitrogen use efficiency. Supplying potassium to plants stimulates the efficiency use of nitrogen through plant uptake and assimilation, consequently increasing crop yields. Based on these facts, the aim of this study was to evaluate the effect of combined rates of nitrogen and potassium on the nutrition, chlorophyll content, and nitrate reductase activity in wheat (Triticum aestivumaestivum L.) plants. The experiment was conducted in a greenhouse of the Soil Science Department of the ESALQ/USP in Piracicaba State of São Paulo, Brazil. Three-liter pots were filled with 0-20 cm depth samples of Arenic Hapludult. A fractionated 52 factorial was used, with combinations of five nitrogen rates (0; 70; 140; 210 e 280 mg dm-3) and five potassium rates (0; 50; 100; 150 e 200 mg dm-3), which were set in a randomized block design with four replications. Thirty days after seeding SPAD values readings were taken on the leaves. Nitrate reductase activity was determined seventy-nine days after seeding and one day later plants were harvested and separated in shoot, roots, and ears. These plant materials were weighted and submitted to nutrient content and accumulation of nitrogen and potassium. Statistical analyses were performed by means of SAS software. Results presented that the balance between nitrogen and potassium supplies was essential to adequate wheat plant growth. It was clear that the combination of nitrogen and potassium rates had a positive influence on wheat dry mass yield as well as on nitrogen, phosphorus, potassium, calcium, and sulphur contents in plant shoot. Nitrogen was essential to maintain adequate magnesium content in plant tissues. The interaction between nitrogen and potassium rates was important for plant yield and ear nitrogen content. Potassium content in the ears was affected only by nitrogen rates. Root dry matter yield increased with rates of nitrogen and potassium. Nitrogen, magnesium and sulphur contents in the roots were influenced only by nitrogen application. Potassium concentration in the roots reduced and calcium concentration increased with nitrogen rates according to a quadratic equation. Potassium supply caused linear decreases in magnesium content of shoots and roots. Nitrogen increased nitrate reductase activity up to the medium rates of application and reduced for the highest rates. Chlorophyll content in the leaves was influenced by the interaction between nitrogen and potassium rates, reflecting on the dry matter yield of plant shoot and ears, as well as on nitrogen accumulation in the wheat plants.

Os ritmos circadianos coordenam grande parte dos mecanismos fisiológicos, principalmente no metabolismo do nitrogênio, inclusive a assimilação do nitrato, que é a forma de nitrogênio mais incorporada pelos organismos fotossintéticos marinhos. Sua assimilação é feita pela enzima nitrato redutase (NR) que é regulada de forma sistêmica e em vários níveis: transcricional, pós-transcricional e pós-traducional. Tal modulação é exercida por diversos fatores como nitrato, luz, glutamato, quinases e por fatores de transcrição centrais do ritmo circadiano. A fotossíntese fornece os esqueletos de carbono e poder redutor para a incorporação do nitrogênio na síntese de glutamato. Gracilaria birdiae é uma macroalga endêmica do litoral brasileiro e ainda precisa de mais estudos em fisiologia, apesar da sua grande importância econômica. Este estudo desenvolveu um novo protocolo para o ensaio em larga escala da atividade enzimática da NR e analisou a oscilação do desempenho fotossintético ao longo do dia em G. birdiae. A alga, proveniente do Rio Grande do Norte, foi isolada a nível unialgal e apresentou uma taxa de crescimento relativo diário de 6,5% em laboratório. O novo protocolo descrito propõe as seguintes adaptações: amostragem de 20 mg de biomassa algal em vez de 100 mg, extrato bruto com 100 µL em vez de 1 mL, ensaio enzimático de 50 µL e parada da reação com sulfanilamida em vez de EtOH/ZnSO4. Estas modificações permitem o ensaio enzimático da NR semi-purificada de 8 testes com réplicas experimentais e biológicas e controle negativo (96 ensaios no total) em 3 horas. A caracterização enzimática demonstrou os seguintes parâmetros ótimos para atividade máxima: tampão fosfato pH 8, temperatura 25°C, 60 minutos de incubação, 1,25 mMol de nitrato e 50 µMol de NADH. A atividade máxima de 5,4 ± 0,7 nMol.min-1.mg-1 de proteínas e o KM 6 ± 2,2 µMol.L-1 para NADH e de 109 ± 11 µMol.L-1 para o nitrato. A enzima não apresentou atividade na presença de NADPH e demonstrou atividade 48 vezes maior que no protocolo inicial. Descrevemos um método preciso e reprodutível para análise em larga escala da atividade da NR que pode ser utilizado em estudos comparativos de cinética enzimática da NR de diferentes espécies ou em estudos de ritmos biológicos. A análise do desempenho fotossintético, feita em coletas de 15 mg de alga, foi caracterizada pela fluorescência da clorofila, demonstrando os seguintes parâmetros máximos: taxa de transporte de elétrons relativa (rETR) de 15,29 µMol elétrons.m-2.s-1, eficiência fotossintética (alfa) de 0,37 fótons/elétrons, irradiância de saturação (IK) foi de 41,32 µE.m-2.s-1 e houve fotoinibição a partir de 300 µE.m-2.s-1. Apenas o parâmetro alfa apresentou oscilação significativa em claro constante e manteve o período de 24 horas, apesar de ter diminuído sua amplitude em 50%. Isso pode ser devido a um controle pelo mecanismo central dos ritmos circadianos sobre proteínas ou pigmentos ligados ao fotossistema II, influenciando na capacidade de transformação de fótons em elétrons. Paralelamente, a aclimatação da alga à luz constante pode ter levado a uma queda na produção de pigmentos e fotoxidação, justificando a queda nos valores médios de alfa. G. birdiae é uma alga com grande importância econômica e ecológica e este estudo, ligado a outros trabalhos de fisiologia, confirma seu grande potencial para estabelecimento como organismo modelo para estudos fisiologia e fotossíntese / Circadian rhythms control most of physiological processes, including nitrate assimilation. This nutrient is the most incorporated form of nitrogen by marine plants and its assimilation is made by the nitrate reductase enzyme (NR) that is highly regulated in all levels expression, since mRNA transcription until protein degradation. Many factors coordinate this regulation, including the nitrate itself, light glutamate, kinases and by transcription factors of the central oscillator of the circadian rhythms. Photosynthesis also plays an especial role in NR regulation through carbon skeleton and NADPH synthesis that allows the last step of nitrate assimilation: glutamate synthesis. Gracilaria birdiae is an endemic marine rhodophyte from Brazil that still lacks physiological and molecular studies despite its extensively exploitation for agar-agar extraction. This study aimed the development of a large scale enzymatic assay of NR (NRA) and the analysis of daily oscillation of photosynthesis performance for G. birdiae. The algae, collected ant Rio Grande do Norte state, was isolated to a unialgal level and presented an average of relative daily growth rate of 6.5% in laboratory. The new protocol here described proposes the following adaptations for NRA: sampling of 20 mg instead of 100 mg, 100 µL of crude extract rather than 1 mL, NRA in 50 µL in place of 1 mL and reaction stop with sulphanilamide instead of an EtOH/ZnSO4 system. These changes allow the assay of the semi-purified NR of 24 samples all with its experimental and biological triplicates and negative control in 3 hours. Phosphate buffer at pH 8, 25°C, 60 minutes of incubation, 1.25 mMol of nitrate and 50 µMol of NADH. The maximum activity was 5.4 ± 0.7 nMol.min-1.mg-1 of protein and a KM of 6 ± 2.2 µMol.L-1 for NADH and of 109 ± 11 µMol.L-1 for nitrate. NR didn\'t show any activity in the presence of only NADPH as the electron donor and showed and 48 times greater activity in the new protocol, compared to the old one. We describe here an accurate and reproducible method for large scale NRA that can be used in comparative studies of NR kinetics or in biological rhythms of NR. The analysis of photosynthetic performance, made in 15 mg sampling of algal biomass, was characterized through chlorophyll fluorescence, revealing the maximum values of these parameters: relative electrons transfer rate (rETR) of 15,29 µMol elétrons.m-2.s-1, photosynthetic efficiency (the alpha parameter) of 0,37 photons/electrons, the saturating irradiance (IK) was 41.32 µE.m-2.s-1 and there was photoinhibition below the irradiance of 300 µE.m-2.s-1. The only variable that oscillated during the continuous light experiment and maintained the 24-hour period was the Alfa parameter, besides its 50% lower amplitude. This oscillation can be due to an endogenous control of the central circadian oscillator into proteins or pigments bound to the photosystem II, influencing the capacity of photon-electron transformation. At the same time, the acclimation to constant light could have driven the algae to a damping of pigment production and photoxidation, what explains the fall of alpha average values. G. birdiae is an alga with great economic and ecological relevance and this study confirms the potential for its establishment as a model organism.

As nitrogen (N) is a critical nutrient for plant growth, the development of synthetic N fertilisers dramatically changed agricultural production in the twentieth century. Improvement in N use efficiency (NUE) has been a focus of breeding for grain crop species, since protein is an important component of the harvested product. The study of NUE in sugarcane has lagged behind grain crops, mainly because N is not a component of sucrose, the primary product of the traditional sugarcane industry. Recently, improvement in NUE has become a focus of sugarcane breeding, due largely to environmental concerns regarding pollution from high N fertilisation, and the increasing cost of N fertilisers. This thesis aimed to gain an initial understanding of the genetic basis for variability in NUE in sugarcane. This was achieved through: (i) the screening of 168 sugarcane genotypes under limiting and non-limiting N supply in two glasshouse experiments; (ii) the mapping of marker-trait associations (MTA) for biomass and physiological traits under limiting and non-limiting N supply in a sugarcane mapping population; (iii) the analysis of expression of candidate genes encoding enzymes involved in the central processes of N assimilation and remobilisation in plants; and (iv) the mapping of candidate genes in a sugarcane genetic map. Genetic variation was identified for growth traits as well as physiological traits including %N, internal NUE (iNUE, g dry weight g-1 N) and leaf glutamine synthetase (GS) activity in a sugarcane mapping population. These traits were also analysed for linkage with genetic markers. Genetic variation in the screened genotypes was higher under limiting N supply, a finding that was reflected by the fact that marker-trait associations (MTA) for increases in iNUE were not identified under non-limiting N supply in the commercial parent of the mapping population. Contrary to findings in grain crop species, there was no link between GS activity and other traits, either through phenotypic correlations or co-location of MTA. The expression of candidate genes encoding GS, nitrate reductase (NR) and alanine amintotransferase (AlaAT) was quantified with Sequenom™ MassARRAY technology. Plants were grown under growth-limiting N supply, non-limiting N supply, or a N-pulse treatment, which consisted of growth-limiting N supply followed by non-limiting N supply 24 hours prior to sampling. Two genes, scAlaAT.d and scGS1.a, encoding AlaAT and GS respectively, were identified as non-responsive to changes in N supply, whereas scAlaAT.a, scGS1.b and scGS1.c had significantly (p<0.05) increased expression under a N-pulse, indicating an important role for these genes in the response of sugarcane to a sudden increase in N availability. The location of candidate genes associated with variation in NUE in a sugarcane genetic map were sought through restriction fragment length polymorphism (RFLP) markers. Twenty-two probes were screened, of which two generated single-dose markers, allowing the mapping of a single allele of scAspAT, encoding aspartate aminotransferase, and two alleles of scGS2, encoding plastidic GS. Because of the economic and environmental consequences of inefficient N fertiliser application, the development of sugarcane cultivars with improved NUE is essential. Since variation for NUE exists, especially in unimproved sugarcane varieties, this may be achieved through traditional breeding methods by screening existing breeding populations under limiting N supply. Additionally, an improved understanding of the genetic basis of variation for NUE in sugarcane should be pursued by further analysis of candidate gene response to changing N availability by screening widely varying cane species for differences in gene expression, enzyme activity and metabolite profiles. The further addition of candidate gene locations to sugarcane genetic maps will aid both future marker-assisted selection in breeding, and a fundamental understanding of genetic control of NUE variation. Through the development of sugarcane cultivars with improved NUE and an enhanced knowledge of the genetic control underpinning sugarcane N physiology, concerns regarding high N fertiliser applications may be mitigated and sustainability ensured.

<p>Sulfite oxidase, a metabolically important enzyme, catalyzes the physiologically critical conversion of sulfite to sulfate in the terminal step of the degradation of sulfur containing compounds. The enzyme has been the focus for much research since its discovery in the 1950's. A central question to understanding the mechanism of molybdoenzymes such as sulfite oxidase and nitrate reductase concerns the roles of active site residues and the coordination chemistry of the Mo atom in the structure and function of the enzyme. The goal of this work was directed towards the characterization and determination of the structures of active site variants of sulfite oxidase using a spectroscopic, kinetic, and protein crystallographic approach.</p><p>Earlier studies have identified a single, highly conserved cysteine residue as the donor of a covalent bond from the protein to molybdenum in sulfite oxidase and nitrate reductase. The C185S and C185A variants of chicken sulfite oxidase exhibited severely attenuated activity. Crystallographic and spectroscopic analysis of both variants revealed a change in the metal coordination, from a dioxo to a trioxo form of Mo. </p><p>Assimilatory nitrate reductase is a member of the sulfite oxidase family of molybdopterin enzymes. The crystal structure of the Mo domain of the enzyme from Pichia angusta revealed high structural homology in the active sites of nitrate reductase and sulfite oxidase. Both enzymes utilize the same form of the molybdenum cofactor and have three out of five residues conserved at the active site. Substitution of two active site residues in sulfite oxidase alters the substrate affinity of chicken SO from sulfite to nitrate, resulting in an increase of nitrate reductase activity over wild-type sulfite oxidase. Additionally we identified an additional amino acid position in sulfite oxidase that corresponds to a non-conserved position in NR that further increased NR activity. Finally, these nitrate reductase variants of sulfite oxidase were crystallized and the structures solved. This represents the first example of the transmutation of a molybdenum enzyme to change activity and substrate affinity to those of a homologous enzyme.</p> / Dissertation

The main goal of this study was to assess the impact that nitrate and light have on the relationship between the gene expression of the enzyme nitrate reductase and the incorporation of nitrate in the cosmopolitan diatom Thalassiosira pseudonana, both in laboratory experiments and in natural environments. Continuous cultures were grown at different nitrate (NO3-) concentrations (i.e., 60, 120, and 400 µM) to evaluate their effects on the expression levels of different genes of the nitrogen metabolic pathway (i.e., nitrate and ammonium transporters, nitrate and nitrite reductases, glutamine synthetases II and III). Semi-continuous cultures were grown under different irradiances (i.e., 50, 110, 200, and 320 µmol photon cm-2 s-1) to assess the influence of light intensity (irradiance) on the relationship between the expression of those genes, uptake, and assimilation of nitrate. The expression of all of the genes that were tested decreased significantly (p < 0.05) at the highest concentration of NO3- (i.e., 400 µM), with nitrate transporters showing the most pronounced change from 27.97 to 0.59 fold change cell-1 x 10-6, at 60 and 400 µM NO3- concentrations respectively. Ammonium transporters were detected at all concentrations of NO3-, suggesting that cells are always ready to metabolize ammonium. Growth was limited (µ = 0.99 d-1) by the low irradiance treatment, was maximum (µ = 2.04 d-1) at 200 µmol photon cm-2 s-1 and was inhibited (µ = 1.54 d-1) at the highest irradiance. These trends were reflected in gene expression and uptake rates, with minimum values at the lowest and highest irradiance levels. However, results from the enzymatic assay did not show any significant differences between treatments (p > 0.05). The trends observed in the enzymatic rates could be explained by the gene expression of NO3- reductase and the uptake and growth rates in a multiple regression analysis (R2 = 0.66, p < 0.05). The results of this study show that uptake is independent of gene expression, probably because of a decoupling between transcription and protein synthesis. Not all of the newly synthesized transcripts will inevitably be translated into proteins. And even if they were, there could be post-translational mechanisms preventing the enzymes to become active. This indicates that uptake can be independent of the expression. It was attempted to measure the expression of T. pseudonana genes involved in the metabolism of NO3- in natural diatom assemblages. The use of gene expression as a proxy for metabolic processes carried out by a phytoplankton assemblage in the field is limited and depends on environmental factors, since the current methods of assessing expression rely on genomic sequences that are particularly variable in phytoplankton. The assessment of gene expression provides a useful insight into physiological studies of phytoplankton, and it should be complemented with other measurements, such as the biomass and taxonomic composition of the assemblage for a more complete picture of marine ecosystem nutrient dynamics. / Graduate

Submitted by DENISE APARECIDA CHICONATO (denise.ac@hotmail.com) on 2016-02-04T14:10:02Z No. of bitstreams: 1 Tese_Denise_Aparecida_Chiconato.pdf: 1322746 bytes, checksum: 5a673f4bca29c82501e188fb0ce8d28d (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-02-05T12:07:51Z (GMT) No. of bitstreams: 1 chiconato_da_dr_jabo.pdf: 1322746 bytes, checksum: 5a673f4bca29c82501e188fb0ce8d28d (MD5) / Made available in DSpace on 2016-02-05T12:07:51Z (GMT). No. of bitstreams: 1 chiconato_da_dr_jabo.pdf: 1322746 bytes, checksum: 5a673f4bca29c82501e188fb0ce8d28d (MD5) Previous issue date: 2016-01-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A cana-de-açúcar é uma cultura mundialmente importante. O Brasil é o maior produtor mundial de cana-de-açúcar e se destaca na comercialização e exportação de açúcar e etanol originados a partir desta cultura. Os diferentes estresses abióticos alteram negativamente o desenvolvimento das plantas e, entre os estresses, a salinidade de solos se apresenta pela grave redução de produtividade, sendo o principal fator estressante em regiões de clima árido e semiárido. Este trabalho objetivou verificar os efeitos do NaCl sobre o desenvolvimento de plantas jovens de cana-de-açúcar, cultivares SP 81-3250 e IAC 87-3396, mediante o estudo de variáveis fisiológicas-bioquímicas: (I) os osmoprotetores prolina, glicina betaína e sacarose, (II) a ocorrência de alterações nutricionais pela relação Na+/K+, (III) a peroxidação lipídica (IV) a influência do NaCl na atividade da enzima redutase do nitrato; (V) o efeito do estresse salino nas trocas gasosas; (VI) o crescimento de parte aérea e raízes das diferentes cultivares de cana-de-açúcar sob estresse salino. Para tanto, foram utilizadas quatro concentrações de NaCl (0, 40, 80 e 160 mM) e realizadas as seguintes avaliações: teores de Na+ e K+ nas folhas e raízes, os osmólitos compatíveis prolina, glicina betaína e sacarose, potencial hídrico, a peroxidação lipídica, atividade da enzima redutase do nitrato, trocas gasosas, clorofilas totais, rendimento quântico do FSII e crescimento de parte aérea e raiz aos 15 e 30 dias de desenvolvimento após aplicação do estresse. A partir dos resultados, observou-se maior suscetibilidade das plantas da cv. IAC 87-3396 pelo maior acúmulo de Na+ e relação Na+/K+, maior peroxidação lipídica pelo acúmulo de MDA e maior efeito negativo sobre as trocas gasosas. Ambas as cultivares apresentaram acúmulo de prolina sob estresse, porém, plantas da cv. SP 81-3250 apresentaram, além de mais rápido acúmulo deste aminoácido, menor peroxidação lipídica e crescimento superior. Sob estresse salino severo a cv. IAC 87-3396 apresenta maior suscetibilidade. / Sugarcane is a globally important crop. Brazil is the largest producer of sugarcane and stands out in the marketing and exportation of sugar and ethanol derived from this culture. The various abiotic stresses adversely change plant growth and between the stresses, soil salinity is presented by the severe reduction in productivity, it is the main stressor in arid and semi-arid climates. This study aimed to verify the effects of NaCl on the development of sugarcane young plants, SP 81- 3250 and IAC 87-3396 cultivars, through the study of physiological-biochemical variables: (I) the osmoprotectors proline, glycine betaine and sucrose, (II) the occurrence of nutritional changes the ratio Na+/K+ (III) lipid peroxidation (IV) effect of NaCl in the activity of nitrate reductase enzyme; (V) the effect of salt stress on gas exchange; (VI) the growth of shoots and roots of the sugarcane cultivars under salt stress. Thus, it was used four concentrations of NaCl (0, 40, 80 and 160 mM) and made the following assessments: Na+ and K+ accumulation in the leaves and roots, compatible osmolytes: proline, glycine betaine and sucrose, water potential, peroxidation lipid, activity of nitrate reductase enzyme, gas exchange, total chlorophyll, quantum yield of PSII and shoot and root growth at 15 and 30 days of development after stress application. The results showed higher susceptibility of the cv. IAC 87-3396 by higher accumulation of Na+ and Na+/K+ ratio, increase in lipid peroxidation by MDA accumulation and greater negative effect on gas exchange. Both cultivars showed proline accumulation under stress, however, the cv. SP 81- 3250 showed in addition faster accumulation of this amino acid, reduced lipid peroxidation and higher growth. Under severe salt stress cv. IAC 87-3396 is more susceptible.

Orientador: Silvio José Bicudo / Banca: Dirceu Maximino Fernandes / Banca: Helio Grassi Filho / Banca: José Roberto Santos / Banca: Hideaki Wilson Takahashi / Resumo: Considerando a hipótese de que a eficácia da utilização do molibdênio é dependente do ajuste em etapas importantes da determinação de sua concentração no solo, bem como da dose e forma de aplicação, realizou-se a presente pesquisa com o objetivo de aprimorar a etapa de retirada do molibdênio da resina trocadora de íons, durante o procedimento analítico de determinação de molibdênio na solução do solo; e avaliar a eficácia de doses e formas de aplicação de molibdênio na cultura do milho. Para o aprimoramento da etapa de retirada do molibdênio da resina trocadora de íons, durante o procedimento analítico de determinação de molibdênio, foram realizados estudos no laboratório para avaliar qual a solução que retira o molibdênio retido na resina melhor representava o molibdênio adicionado ao solo, sendo aplicados nesses estudos o delineamento inteiramente casualizado, com quatro repetições. Para avaliar a eficácia de doses e formas de aplicação de molibdênio na cultura do milho, foram instalados três experimentos em casa de vegetação pertencente à FCA/UNESP, Campus de Botucatu, em dois anos. No primeiro ano foram realizados dois experimentos, um com calagem: Experimento I; e outro sem calagem: Experimento II; no segundo ano foi realizado mais um experimento com calagem: Experimento III. O delineamento experimental, para os três experimentos foi o de blocos casualizados, com quatro repetições. Os experimentos I e II obedeceram a um arranjo fatorial de 2x3x4+1, sendo seus tratamentos compostos por dois tipos solos com texturas diferentes: Neossolo Litólico (RL) e Latossolo Roxo álico (LRa); três formas de aplicação: via folha, via semente e via solo; e quatro doses de molibdênio: 25, 100, 400 e 1600 g ha-1 (para a aplicação via folha e via solo) e 5,625; 11,25; 22,5 e 45,0 g ha-1 (para a aplicação via semente), mais uma testemunha absoluta... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Assuming that the effective use of molybdenum is dependent on accurate determination of its concentration in soil as well as the dose and method of application, put into practice this research with the objective of improving the determination of the concentration of molybdenum in soil and evaluate the effectiveness of application methods and doses in maize. To improve the determination of molybdenum in soil, studies were carried in the laboratory to evaluate which solution mixed extraction of molybdenum contained in the resin that best represented the molybdenum added to soil, being applied in these studies the entirely randomized design, with four replications. To evaluate the effectiveness of doses and methods of application of molybdenum in maize, essays were carried in a greenhouse belonging to the FCA/UNESP, Botucatu, in two years. In the first year two experiments had been carried through, one with lime: Experiment I; another without lime: Experiment II; in the second year it was carried through an experiment with lime: Experiment III. The experimental design, in the three experiments, was a randomized block with four replications. In the experiments I and II used a factorial arrangement of 2x3x4+1, being their treatments made of two soils with different textures: soil Neossolo Litólico (LR) and soil Latassolo Roxo álico (LRa), three application forms (leaf, seeds, and soil aplications) and four levels of molybdenum: 25, 100, 400 and 1600 g ha-1 (for leaf and soil applications) and 5.625, 11.25, 22.5 and 45.0 g ha-1 (for seeds application) and a control without molybdenum fertilization. The Experiment III was installed in only kind of soil, the Nitossolo Litólico (RL), in factorial arrangement of 3x4+1, being their treatments made of by same application forms and levels of molybdenum used in Experiments I and II. The essays were conducted on experimental plots... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:30:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-17Bitstream added on 2014-06-13T19:19:11Z : No. of bitstreams: 1 pereira_frs_dr_botfca.pdf: 1161993 bytes, checksum: 12e776fae4e5259fe0e366a10986b5ed (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Considerando a hipótese de que a eficácia da utilização do molibdênio é dependente do ajuste em etapas importantes da determinação de sua concentração no solo, bem como da dose e forma de aplicação, realizou-se a presente pesquisa com o objetivo de aprimorar a etapa de retirada do molibdênio da resina trocadora de íons, durante o procedimento analítico de determinação de molibdênio na solução do solo; e avaliar a eficácia de doses e formas de aplicação de molibdênio na cultura do milho. Para o aprimoramento da etapa de retirada do molibdênio da resina trocadora de íons, durante o procedimento analítico de determinação de molibdênio, foram realizados estudos no laboratório para avaliar qual a solução que retira o molibdênio retido na resina melhor representava o molibdênio adicionado ao solo, sendo aplicados nesses estudos o delineamento inteiramente casualizado, com quatro repetições. Para avaliar a eficácia de doses e formas de aplicação de molibdênio na cultura do milho, foram instalados três experimentos em casa de vegetação pertencente à FCA/UNESP, Campus de Botucatu, em dois anos. No primeiro ano foram realizados dois experimentos, um com calagem: Experimento I; e outro sem calagem: Experimento II; no segundo ano foi realizado mais um experimento com calagem: Experimento III. O delineamento experimental, para os três experimentos foi o de blocos casualizados, com quatro repetições. Os experimentos I e II obedeceram a um arranjo fatorial de 2x3x4+1, sendo seus tratamentos compostos por dois tipos solos com texturas diferentes: Neossolo Litólico (RL) e Latossolo Roxo álico (LRa); três formas de aplicação: via folha, via semente e via solo; e quatro doses de molibdênio: 25, 100, 400 e 1600 g ha-1 (para a aplicação via folha e via solo) e 5,625; 11,25; 22,5 e 45,0 g ha-1 (para a aplicação via semente), mais uma testemunha absoluta... / Assuming that the effective use of molybdenum is dependent on accurate determination of its concentration in soil as well as the dose and method of application, put into practice this research with the objective of improving the determination of the concentration of molybdenum in soil and evaluate the effectiveness of application methods and doses in maize. To improve the determination of molybdenum in soil, studies were carried in the laboratory to evaluate which solution mixed extraction of molybdenum contained in the resin that best represented the molybdenum added to soil, being applied in these studies the entirely randomized design, with four replications. To evaluate the effectiveness of doses and methods of application of molybdenum in maize, essays were carried in a greenhouse belonging to the FCA/UNESP, Botucatu, in two years. In the first year two experiments had been carried through, one with lime: Experiment I; another without lime: Experiment II; in the second year it was carried through an experiment with lime: Experiment III. The experimental design, in the three experiments, was a randomized block with four replications. In the experiments I and II used a factorial arrangement of 2x3x4+1, being their treatments made of two soils with different textures: soil Neossolo Litólico (LR) and soil Latassolo Roxo álico (LRa), three application forms (leaf, seeds, and soil aplications) and four levels of molybdenum: 25, 100, 400 and 1600 g ha-1 (for leaf and soil applications) and 5.625, 11.25, 22.5 and 45.0 g ha-1 (for seeds application) and a control without molybdenum fertilization. The Experiment III was installed in only kind of soil, the Nitossolo Litólico (RL), in factorial arrangement of 3x4+1, being their treatments made of by same application forms and levels of molybdenum used in Experiments I and II. The essays were conducted on experimental plots... (Complete abstract click electronic access below)