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Metabolismo de nitrog?nio em plantas de arroz (Oryza sativa L.) em associa??o com bact?rias diazotr?ficas endof?ticas / Nitrogen metabolism in rice plants (Oryza sativa, L.) inoculated with endophytic diazotrophic bacteriaSabino, Daniele Cristina Costa 26 March 2003 (has links)
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Previous issue date: 2003-03-26 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Rice is colonized by diazotrophic bacteria that can act as root growth promoting, as well as to increase plant nutrition throw the biological nitrogen fixation process. These associations can modify the plant physiology since they compete for carbohydrate and other compounds necessary to host plants. The aim of this work was to evaluate the influence of the endophytic diazotrophic bacteria, Burkholderia brasilensis and Herbaspirillum seropedicae on nitrogen metabolism of two rice cultivars (IR42 and IAC4440). The experiment was carried out in pots containing 3 kg of soil, fertilized with 3 nitrogen levels (0, 40, and 80 kg N ) as (NH4)2SO4, and three inoculation treatments (H. seropedicae (ZAE94), B. brasilensis (M130) and the mixture of both). The plants were harvested at vegetative (45 days of inoculation) and flowering stages (according to the cycle of each cultivar). Bacterial counting, dry matter, N content and the concentration of nitrogen compounds were measured at each harvest. The results showed no statistical differences on bacterial counting. The number of root bacteria was higher than in shoots. A population reduction occurred on the flowering stage as compared to vegetative one. Cultivar IR42 responded to inoculation more noticiable than IAC4440. The dry matter accumulation of the IR42 cultivar at the vegetative stage varied with inoculation and nitrogen dose applied. This cultivar accumulated higher amounts of nitrogen when inoculated with ZAE94 strain, independent of the nitrogen applied. Concentrations of nitrate, free amino acid and soluble sugar in the aerial parts were higher in plants fertilized with 40kgN/ha and inoculated with the mixture of bacteria. These results suggested that the diazotrophic bacteria inoculation could modify the plants nitrogen metabolism and theses effects were variable according to the rice cultivars and nitrogen fertilization level applied. / O arroz (Oryza sativa L.) ? colonizado por bact?rias diazotr?ficas que, podem atuar como promotoras de crescimento das ra?zes, assim como melhorar a nutri??o da planta atrav?s do N2 fixado biologicamente. No entanto, estas associa??es podem modificar a fisiologia das plantas, uma vez que as bact?rias diazotr?ficas para fixarem o nitrog?nio necessitam de carboidratos e outros componentes, competindo, deste modo, com as plantas hospedeiras. Assim sendo, este trabalho teve como objetivo avaliar a influ?ncia da inocula??o de bact?rias diazotr?ficas endof?ticas Burkholderia brasilensis e Herbaspirillum seropedicae no metabolismo de nitrog?nio de duas cultivares de arroz adubadas ou n?o com nitrog?nio mineral. Um experimento em condi??es de vasos foi realizado na ?rea experimental da Embrapa Agrobiologia com as cultivares de arroz IR42 e IAC4440. A inocula??o consistiu no uso das estirpes ZAE94 de Herbaspirillum seropedicae, e M130 de Burkholderia brasilensis , a mistura destas duas estirpes e da testemumha (controle n?o inoculado). A aduba??o na forma de (NH4)2SO4, foi aplicada nas concentra??es de 0, 40 e 80 kgN/ha. As coletas foram realizadas no per?odo vegetativo (45 dias) e no florescimento (de acordo com o ciclo de cada cultivar). Os par?metros avaliados foram a presen?a das bact?rias nos tecidos das plantas, o ac?mulo de mat?ria seca da parte a?rea, o ac?mulo de N, e a concentra??o dos compostos nitrogenados na planta. Os resultados mostraram que n?o houve diferen?as nas popula??es das bact?rias em fun??o da inocula??o, por?m, o n?mero de bact?rias na raiz foi ligeiramente maior do que na parte a?rea. Foi tamb?m observado uma redu??o na popula??o no per?odo de florescimento, em rela??o ao per?odo vegetativo. De um modo geral, a cultivar IR42 apresentou maiores respostas ? inocula??o, quando comparada com a cultivar IAC4440. O ac?mulo de mat?ria seca da cultivar IR42 no per?odo vegetativo, variou com a inocula??o e a dose de nitrog?nio aplicada. Por?m, a percentagem de nitrog?nio (%N) acumulada na parte a?rea destas plantas, foi maior nos tratamentos inoculados com a estirpe ZAE94, independente da dose de nitrog?nio aplicada. As plantas que receberam 40 kgN/ha e foram inoculadas com a mistura, apresentaram as maiores concentra??es de nitrato, amino?cidos livres e a??cares sol?veis na parte a?rea. Os resultados obtidos sugerem que a inocula??o das bact?rias diazotr?ficas modifica o metabolismo de nitrog?nio e que este efeito foi diferenciado em fun??o da variedade e da dose de nitrog?nio aplicada.
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Efeito da superexpress?o dos fatores de transcri??o ZmDof1 e OsDof25 sobre a efici?ncia de uso de nitrog?nio em Arabidopsis. / Effects of ZmDof1 and OsDof25 transcriptional factors superexpression on nitrogen usage efficiency in Arabidopsis.Santos, Leandro Azevedo 03 June 2009 (has links)
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Previous issue date: 2009-06-03 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / To improve nitrogen usage efficiency in plants the rice transcriptional factor OsDof25 was
identified and cloned, whose probably orthologe is the maize ZmDof1, already identified and
partially characterized. The ZmDof1 was also cloned for comparative analysis with OsDof25,
in order to confirm this last one as ZmDof1 orthologe in rice. The constructions for
Arabidopsis superexpression of these transcriptional factors were made using the cloning
system of gateway technology (Invitrogen), to obtain the expression vectors 35S:ZmDof1:HA
and 35S:OsDof25:HA. Lineages with different expression levels of these genes were
obtained, but with only one inserted copy. These transgenic lineages when grown in a half
strength of MS medium (10mM of NH4
+ and 20mM of NO3
-) showed phenotypes with
chloroses and growth difficulty; although when they were cultured in soil they showed great
vegetative development and delay in the inflorescence emission. When analyzed the gene
expression changes induced by the superexpression of these transcriptional factors, it was
observed that both genes produced an increase in the expression levels of high and low
affinity ammonium transporters (AMT1.1 and AMT2.1, respectively), indicating that these
phenotypes may be due to the toxic effect of an excess of ammonium uptake. We also verified
an increase of expression for pyruvate kinase (PK1 and PK2), and phosphoenolpyruvate
carboxylase (PEPC1 and PEPC2). Pyruvate kinase converts phophoenolpyruvate (PEP) to
pyruvate, and phosphoenolpyruvate carboxylase converts PEP to oxalacetate, which is
substrate for malate dehydrogenase to form malate. Both pyruvate and malate may feed the
Krebs cycle. In addition, there was an increase in the expression of isocitrate dehydrogenase,
which is present in the citosol and mitochondria, needed for converting isocitrate to 2-
oxoglutarate. Thus, it was hypothesized that the increase of expression levels of these carbon
metabolism enzymes was necessary to increase the production of 2-oxoglutarate and,
consequently, to reduce the toxic effect of ammonium uptaked. Besides, it was observed an
increase of expression levels and activity of glutamate dehydrogenase (GDH). This enzime
may work as much in the direction of glutamate amination as in deamination, when the plants
were submitted to ammonium excess or carbon limitation conditions, respectively. / Com o objetivo de aumentar a efici?ncia de uso de nitrog?nio (EUN) em plantas, foi
identificado e clonado o fator de transcri??o OsDof25 de arroz, cujo prov?vel ort?logo ? o
ZmDof1 de milho, j? identificado e parcialmente caracterizado. Tamb?m foi clonado o
ZmDof1 para an?lises comparativas com o OsDof25, a fim de comprovar que este ?ltimo ?
realmente ort?logo do ZmDof1. As constru??es para superexpress?o destes fatores de
transcri??o em Arabidopis foram feitas utilizando o sistema gateway de clonagem para
obten??o dos vetores de express?o 35S:ZmDof1:HA e 35S:OsDof25:HA. Foram obtidas
linhagens com diferentes n?veis de express?o destes genes, mas com apenas uma inser??o. As
linhagens transg?nicas obtidas quando crescidas em meio MS ? for?a i?nica (10mM de NH4
+
e 20mM de NO3
-) apresentaram fen?tipos como clorose e dificuldade de desenvolvimento, ao
passo que quando cultivadas em solo mostraram desenvolvimento vegetativo mais intenso e
atraso para emiss?o da infloresc?ncia. Quando analisadas as modifica??es de express?o
g?nica causadas pela superexpress?o destes fatores de transcri??o, observou-se que ambos os
fatores de transcri??o provocaram aumento de express?o dos transportadores de am?nio de
alta e baixa afinidades (AMT1.1 e AMT2.1 respectivamente), indicando que o fen?tipo
observado pode ser devido ao efeito t?xico do excesso de am?nio absorvido. Verificou-se
tamb?m aumento de express?o das enzimas piruvato quinase (PK1 e PK2) e fosfoenolpiruvato
carboxilase (PEPC1 e PEPC2). A piruvato quinase converte o fosfoenolpurato (PEP) a
piruvato, enquanto a fosfoenolpiruvato carboxilase converte o PEP a oxalacetato (OAA) que
pode sofrer a??o da malato desidrogenase originando o malato. Ambos os metab?litos,
piruvato e malato, alimentam o ciclo de Krebs. Houve tamb?m aumento de express?o da
isocitrato desidrogenase, enzima presente na mitoc?ndria (ciclo de Krebs) e no citosol que
converte isocitrato a 2-oxoglutarato (2-OG). Assim, ? prov?vel que o aumento da express?o
destas enzimas do metabolismo de carbono foi necess?rio para aumentar a produ??o de 2-OG
e, por conseguinte, diminuir o efeito t?xico do excesso de am?nio absorvido. Al?m disso,
observou-se aumento de express?o e atividade da glutamato desidrogenase (GDH). Essa
enzima pode atuar tanto na dire??o da amina??o, quanto na dire??o da desamina??o, em
condi??es de excesso de am?nio e/ou sob condi??es de limita??o de carbono nas plantas,
respectivamente.
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