<p>Lichens are symbioses between a fungus and a photosynthesizing partner such as a green alga or a cyanobacterium. Unlike mycorrhizal or rhizobial symbioses, the lichen symbiosis is not well understood either morphologically or molecularly. The lichen symbiosis has been somewhat neglected for several reasons. Lichens grow very slowly in nature (less than 1 cm a year), it is difficult to grow the fungus and the alga separately and, moreover, it remains difficult to resynthesize the mature symbiosis in the laboratory. It is not yet possible to delete genes, nor has any transformation method been established to introduce genes into the genomes of either the fungus or the alga. However, the lack of genetic tools for these organisms has been partially compensated for by the sequencing of the genomes of the lichenizing fungus <italic>Cladonia grayi</italic> and its green algal partner <italic>Asterochloris</italic> sp. This work uses the model lichen system <italic>Cladonia grayi</italic> and the associated genomes to explore one evolutionary and one developmental question concerning the lichen symbiosis.</p><p>Chapter One uses data from the genomes to assess whether there was evidence of horizontal gene transfer between the lichen symbionts in the evolution of this very intimate association; that is, whether genes of algal origin could be found in the fungal genome or vise versa. An initial homology search of the two genomes demonstrated that the fungus had, in addition to ammonium transporter/ammonia permease genes that were clearly fungal in origin, ammonium transporter/ammonia permease genes which appeared to be of plant origin. Using cultures of various lichenizing fungi, plant-like ammonium transporter/ammonia permease genes were identified by degenerate PCR in ten additional species of lichen in three classes of lichenizing fungi including the Lecanoromycetes, the Eurotiomycetes, and the Dothidiomycetes. Using the sequences of these transporter genes as well as data from publically available genome sequences of diverse organisms, I constructed a phylogy of 513 ammonium transporter/ammonia permease sequences from 191 genomes representing all main lineages of life to infer the evolutionary history of this family of proteins. In this phylogeny I detected several horizontal gene transfer events, including the aforementioned one which was demonstrated to be not a transfer from plants to fungi or vise versa, but a gene gain from a group of phylognetically unrelated hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina), which was subsequently lost in most lineages but retained in even distantly related lichenized fungi. Also demonstrated was the loss of the native fungal ammonium transporter and the subsequent replacement of this gene with a bacterial ammonium transporter during the early evolution of the fungi. Several additional recent horizontal gene transfers into lineages of eukaryotes were demonstrated as well. The phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). I show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: AMT family as defined here exhibits standard parent-to-offspring inheritance, whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs) into eukaryotes. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with ammonium transporters and ammonia permeases forming two distinct and possibly monophyletic groups.</p><p>In Chapter Two I address a follow-up question: in key lichenizing lineages for which ammonium transporter/ammonia permease (AMTP) genes were not found in Chapter One, were the genes lost? The only definitive infomation which can demonstrate absence of a gene from a genome is a full genome sequence. To this end, the genomes of eight additional lichenizing fungi in the key clades including the Caliciales (sensu Gaya 2011), the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales were sequenced using the Ilumina HiSeq technology and assembled with the short reads assembly software Velvet. These genomes were searched for ammonium transporter/ammonia permease sequences as well as 20 test genes to assess the completeness of each assembly. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacteria as a primary photobiont or living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the Acarosporomyetidae and Arthoniomycetes. In addition, the four AMTP genes from <italic>Cladonia grayi</italic> were shown to be functional by expression of the lichen genes in a strain of <italic>Saccharomyces cerevisiae</italic> in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source. </p><p>In Chapter Three I use genome data to address a developmental aspect of the lichen symbiosis. The finding that DNA in three genera of lichenizing fungi is methylated in symbiotic tissues and not methylated in aposymbiotic tissues or in the free-living fungus (Armaleo & Miao 1999a) suggested that epigenetic silencing may play a key role in the development of the symbiosis. Epigenetic silencing involves several steps that are conserved in many eukaryotes, including methylation of histone H3 at lysine 9 (H3K9) in nucleosomes within the silenced region, subsequent binding of heterochromatin-binding protein (HP1) over the region, and the recruitment of DNA methyltransferases to methylate the DNA, all of which causes the underlying chromatin to adopt a closed conformation, inhibiting the transcriptional machinery from binding. In this chapter I both identify the genes encoding the silencing machinery and determine the targets of the silencing machinery. I use degenerate PCR and genome sequencing to identify the genes encoding the H3K9 histone methyltransferase, the heterochromatin binding protein, and the DNA methyltransferases. I use whole genome bisulfite sequencing of DNA from the symbiotic structures of <italic>Cladonia grayi</italic> including podetia, squamules and soredia as well as DNA from cultures of the free-living fungus and free-living alga to determine which regions of the genome are methylated in the symbiotic and aposymbiotic states. In particular I examine regions of the genomes which appear to be differentially methylated in the symbiotic versus the aposymbiotic state. I show that DNA methylation is uncommon in the genome of the fungus in the symbiotic and aposymbiotic states, and that the genome of the alga is methylated in the symbiotic and aposymbiotic states.</p> / Dissertation

Many omics-level studies have been undertaken on Aiptasia, however, our understanding of the genes and processes associated with symbiosis regulation and maintenance is still limited. To gain deeper insights into the molecular processes underlying this association, we investigated this relationship using multipronged approaches combining next generation sequencing with metabolomics and immunohistochemistry. We identified 731 high-confident symbiosis-associated genes using meta-analysis. Coupled with metabolomic profiling, we exposed that symbiont-derived carbon enables host recycling of ammonium into nonessential amino acids, which may serve as a regulatory mechanism to control symbiont growth through a carbon-dependent negative feedback of nitrogen availability to the symbiont. We then characterized two symbiosis-associated ammonium transporters (AMTs). Both of the proteins exhibit gastrodermis-specific localization in symbiotic anemones. Their tissuespecific localization consistent with the higher ammonium assimilation rate in gastrodermis of symbiotic Aiptasia as shown by 15N labeling and nanoscale secondary ion mass spectrometry (NanoSIMS). Inspired by the tissue-specific localization of AMTs, we investigated spatial expression of genes in Aiptasia. Our results suggested that symbiosis with Symbiodiniaceae is the main driver for transcriptional changes in Aiptasia. We focused on the phagosome-associated genes and identified several key factors involved in phagocytosis and the formation of symbiosome. Our study provided the first insights into the tissue specific complexity of gene expression in Aiptasia. To investigate symbiosis-induced response in symbiont and to find further evidence for the hypotheses generated from our host-focused analyses, we explored the growth and gene expression changes of Symbiodiniaceae in response to the limitations of three essential nutrients: nitrogen, phosphate, and iron, respectively. Comparisons of the expression patterns of in hospite Symbiodiniaceae to these nutrient limiting conditions showed a strong and significant correlation of gene expression profiles to the nitrogen-limited culture condition. This confirmed the nitrogen-limited growing condition of Symbiodiniaceae in hospite, and further supported our hypothesis that the host limits the availability of nitrogen, possibly to regulate symbiont cell density. In summary, we investigated different molecular aspects of symbiosis from both the host’s and symbiont’s perspective. This dissertation provides novel insights into the function of nitrogen, and the potential underlying molecular mechanisms, in the metabolic interactions between Aiptasia and Symbiodiniaceae.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-05-26T20:35:35Z No. of bitstreams: 1 2012 - Renata Aparecida Costa Silva.pdf: 1687897 bytes, checksum: 4d1af8e117daa1842ae1766319ddf8d6 (MD5) / Made available in DSpace on 2017-05-26T20:35:35Z (GMT). No. of bitstreams: 1 2012 - Renata Aparecida Costa Silva.pdf: 1687897 bytes, checksum: 4d1af8e117daa1842ae1766319ddf8d6 (MD5) Previous issue date: 2012-02-15 / CAPES / Nitrogen is one of the nutrient elements most limiting for plant growth. Thus, increasing plant nitrogen usage efficiency (NUE) is an essential factor for sustainable agriculture, leading to an increased food production with less fertilizer input and less environment impact. The aim of this study was to evaluate the effect of OsDof25 overexpression on N-NO3- and N-NH4+ uptake. In transgenic rice plants, OsDof25 was expressed under control of maize ubiquitin promoter (UBIL:OsDof25:3xHA). Two experiments were conducted: one to evaluate the kinetic parameters Vm?x and KM, and another to analyze the expression level of nitrate (NRT2.1~2.2 and NAR) and ammonium transporters (AMT1.1~1.3), both under high and low NO3- and NH4+ supply. The untransformed plants showed higher growth that transformed lineages. The L1 and L2 showed a lower value of the KM in the resupply treatment of 0.2 mM N-NO3-. In the resupply with 0.2 mM N-NH4 + the L4 showed higher Vmax and L1 lower KM. There were no large variations in uptake kinetics between the transformed and untransformed plants. At the root the NRT2 showed low expression in lineages L1 and L4, when under constant supply of N-NO3-, in contrast, in the treatment under resupply with 0.2 mM N-NO3-was increased expression of OsNTR2.1 ~ 2.2, and NAR in both transformed lineages, but in the root the concentration of NO3- was opposed to the expression of NRT2 and NAR, in both conditions. In the leaves, the line L4 showed high expression of the transporter OsNRT2.1 with the resupply of 0.2 and 2.0 mM N-NO3-. In plants grown under constant supply of N-NH4+, L1 showed lower expression of AMT1 in the root compared to L4 and untransformed plants. When subjected to nitrogen deficiency, there was an increased expression of the OsAMT1.2. However, there was no correlation between N transporter expression levels and NO3- and NH4+ content in the transformed plants, indicating a possible change in enzyme activity and reduction or assimilation of N in these plants. The transformed plants when subjected to resupply with low levels of nitrate and ammonium showed better response parameters Vmax and KM compared to the untransformed. In the plants transformed the resupply with nitrate at low concentration resulted in increasing the gene expression of the transporters (OsNTR2.1 ~ 2.2 and protein OsNAR2.1), and the treatment with constant supply provided greatest nitrate accumulation in these plants. The results of both kinetic parameters and accumulation of fresh matter suggest that plants transformed for the expression of the OsDof25 presented highest tolerance to nutritional stress. / O nitrog?nio ? um dos elementos minerais que mais limita o desenvolvimento das plantas. Assim, aumentar a efici?ncia de uso de nitrog?nio (EUN) ? um fator ? essencial para uma agricultura sustent?vel, levando a um aumento da produ??o de alimentos com menor uso de insumos e menos impactos ao ambiente. Este trabalho teve por objetivo avaliar o efeito da superexpress?o do fator de transcri??o OsDof25 sobre a absor??o de nitrog?nio em duas linhagens transformadas de arroz (L1 e L4) da variedade Nipponbare comparando-as com plantas n?o transformadas (WT). Nas plantas transformadas, o OsDof25 foi expresso sob o controle do promotor da ubiquitina 1 de milho (UBIL:OsDof25:3xHA). For feitos dois experimentos: um para avaliar os par?metros cin?ticos Vm?x e KM, sob condi??es de alto e baixo suprimento de N-NO3- e N-NH4+, e outro para analisar a express?o dos transportadores de NO3- (NRT2.1~2.2 e NAR) e NH4+ (AMT1.1~1.3) tamb?m sob alto e baixo suprimento desses ?ons. As plantas n?o transformadas apresentaram maior crescimento do que as linhagens transformadas. As L1 e a L2 mostraram menor valor de KM no tratamento com ressuprimento de 0,2 mM de N-NO3-. No ressuprimento com 0,2 mM de N-NH4+ a L4 apresentou maior Vm?x e L1 menor KM, mas, n?o houve grandes varia??es nos par?metros cin?ticos de absor??o entre as plantas transformadas e n?o transformadas. Na raiz os NRT2 mostraram baixa express?o nas linhagens L1 e L4 quando submetidas ao suprimento constante de N-NO3-, em contrapartida, no tratamento sob ressuprimento com 0,2 mM de N-NO3- ocorreu aumento de express?o dos OsNTR2.1~2.2 e NAR nas duas linhagens transformadas, por?m na raiz a concentra??o de N-NO3- foi oposta a express?o dos NRT2 e NAR, em ambas as situa??es. Nas folhas, a linhagem L4 apresentou alta express?o do transportador OsNRT2.1 com o ressuprimento de 0,2 e 2,0 mM de N-NO3-. Nas plantas submetidas ao suprimento constante de N-NH4+, a L1 apresentou menor express?o dos AMT1 na raiz quando comparada a L4 e a planta n?o transformada. Quando submetida a defici?ncia de N-NH4+, a express?o do OsAMT1.2 aumentou nas ra?zes de todas as plantas. Entretanto, n?o houve correla??o positiva entre a express?o dos transportadores de N e os teores de NO3- e NH4+ nas linhagens transformadas, indicando uma poss?vel altera??o na atividade das enzimas de redu??o e ou assimila??o de N. As plantas transformadas quando submetidas ao ressuprimento com baixos teores de nitrato e am?nio apresentaram melhor resposta dos par?metros Vm?x e KM em rela??o a n?o transformadas. Nas plantas transformadas o ressuprimento com nitrato em baixa concentra??o resultou em maior express?o dos genes dos transportadores OsNTR2.1~2.2 e da prote?na OsNAR2.1 e o tratamento com suprimento constante proporcionou maior ac?mulo de nitrato nestas plantas. Os resultados tanto dos par?metros cin?ticos quanto do ac?mulo de mat?ria fresca sugerem que as plantas transformadas para express?o do OsDof25 apresentaram maior toler?ncia ao estresse nutricional.

Black Cottonwood (Populus trichocarpa) is a fast-growing, economically important tree species. P. trichocarpa was the first tree to have its genome fully sequenced and is considered the model organism for genomic research in trees. Of the macronutrients in plants, Nitrogen (N) is required in the greatest amounts and is generally the limiting nutrient in terrestrial ecosystems. Inorganic N-transport is performed by four families of transporter proteins, AMT1 and AMT2 for ammonium (NH4+) and NRT1 and NRT2 for nitrate (NO3-). I have created phylogenetic reconstructions of each of these transporter families in 22 members of Viridiplantae whose genomes have been fully sequenced. Based on these phylogenies, I have introduced a new classification system for the transporter families that better represents the evolutionary and functional relatedness of the proteins. These phylogenies were supplemented with topology predictions, subcellular localization predictions, and in silico expression profiling in order to suggest functional characterization of the groups. This facilitated candidate gene selection for NH4+ and NO3- uptake transporters from P. trichocarpa. Expression profiling was performed on two of these candidates. Results suggest that PtAMT1-1 may be a high-affinity, root-localized NH4+ transporter. In contrast, PtNRT2-6 is a high-affinity NO3- transporter localized to the dormant bud, but its physiological functions remain largely enigmatic. Flux profiles of NH4+, NO3-, and H+ in the first 1.4 cm of root tips of three-week-old P. trichocarpa seedlings and cuttings were measured using the Microelectrode Ion Flux mEasurement (MIFE) system to demonstrate the activity of AMTs and NRTs under nutrient-abundant and nutrient-deficient conditions. I found mainly N-efflux from roots of cuttings while seedling roots exhibited N-uptake. This is the first report of such a difference. This highlights an unexpected but clear physiological difference between seedling and cutting roots, which are frequently used in experimental setups. / Graduate / 0817 / 0369 / 0715 / neilvonw@gmail.com

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-07-26T18:47:21Z No. of bitstreams: 1 2014 - Marcela Jacques de Lemos Neves.pdf: 434472 bytes, checksum: c9817e97c5d136299acb936ad4b87b6f (MD5) / Made available in DSpace on 2017-07-26T18:47:21Z (GMT). No. of bitstreams: 1 2014 - Marcela Jacques de Lemos Neves.pdf: 434472 bytes, checksum: c9817e97c5d136299acb936ad4b87b6f (MD5) Previous issue date: 2014-05-30 / FAPERJ / The main goal of this study was evaluate the effect of downregulation of the ammonium transporter OsAMT1.3 in the ammonium uptake through the high affinity system, as well as the effects on nitrogen metabolism. To perform the OsAMT1.3 gene silencing, it was used the artificial micro RNA (amiRNA) technology. In this system, the OsAMT1.3 coding region sequence (cds) is inserted in W marcelaMD3 website and putatives amiRNAs to silencing OsAMT1.3 were made. The amiRNA was inserted by PCR using the pNW55 vector as template. The amiRNA was inserted in the IRS154 vector using the T4 DNA ligase. The IRS154 plus amiRNA was cloned in the E. coli by electroporation. After rice transformation of Nipponbare variety through Agrobacterium and Hygromycin selection, 14 lineages were obtained. Six lineages showed high seed production and only one lineage with abnormal growth. After seed production in a greenhouse, the lineages L1, L2 and L6 were selected to further experiments evaluating the effects of OsAMT1.3 downregulation. First, the lineages selected were evaluated about the levels of OsAMT1.3 downregulation. The rice lineages transformed with amiRNA showed lower level of OsAMT1.3 expression compared to control plants, however, different levels of OsAMT1.3 downregulation was observed. The lineage L1 showed lower levels of OsAMT1.3 downregulation, L2 and L6 showed higher levels of OsAMT1.3 downregulation. The lineages L1, L2 and L6 as well as IRS control plant (transformed with empty vector) were grown in growth chamber at 30 days after germination, and the treatments used were: N starvation for three days, resupply with 0.15 mM of NH4+-N (low level) and 2.0 mM of NH4+-N (high level). The lineages L1, L2 and L6 showed lower NH4+ uptake with 0.15 mM of NH4+-N compared to control plants (IRS), on the other hand, the plants grown with 2.0 mM of of NH4+-N did not show NH4+ uptake differences, except L1. The expression of OsAMT1.1, OsAMT1.2 and OsAMT1.3 ammonium transporters were upregulated with 0.15 mM of NH4+-N in the control plants (IRS); in the lineages L1, L2 and L6 showed downregulation of the OsAMT1.1, OsAMT1.2 and OsAMT1.3 genes. The lower NH4+ uptake with 0.15 mM of NH4+-N resulted in lower levels of NH4+-N and Amino-N in the roots in the lineages, while the NH4+-N and Amino-N in the plants grown with 2.0 mM of NH4+-N was minimally changed. The results indicate that OsAMT1.3 downregulation leads to OsAMT1.1 and OsAMT1.2 downregulation as well, decreasing the NH4+ uptake. Despite the OsAMT1.3 lower expression compared to OsAMT1.1 and OsAMT1.2, the OsAMT1.3 transporter may be involved in the nitrogen uptake efficiency in low levels of NH4+. / O objetivo deste trabalho foi estudar o efeito do silenciamento do gene do transportador OsAMT1.3 em arroz sobre a habilidade das plantas em absorver o N-NH4+ atrav?s do sistema de alta afinidade, bem como os reflexos sobre o metabolismo de nitrog?nio. Para o silenciamento do gene OsAMT1.3 foi usada a tecnologia do miRNA artificial (amiRNA). Para tanto, a sequ?ncia codante do gene (cds) OsAMT1.3 ? inserida no sistema WMD3 que indica sequ?ncias de amiRNA?s para silenciar o pr?prio OsAMT1.3. A inser??o do amiRNA foi feita por PCR usando o vetor pNW55 como molde. O miRNA foi inserido no vetor IRS154 por corte e liga??o usando a enzima T4 DNA ligase. O produto da liga??o foi introduzido em E. coli por eletropora??o. Ap?s a transforma??o de arroz da variedade Nipponbare mediada por Agrobacterium. A sele??o das plantas transformadas foi feita com o antibi?tico Higromicina. No total foram obtidas 14 linhagens transformadas. Para confirmar que as plantas mutantes possuiam a constru??o, foi realizado o teste com a folha bandeira em solu??o de higromicina. Seis linhagens apresentaram boa produ??o de sementes e houve uma linhagem com crescimento anormal. Ap?s a multiplica??o das linhagens em casa de vegeta??o, foram selecionadas as linhagens L1, L2 e L6 para os experimentos de an?lise dos efeitos do silenciamento do gene OsAMT1.3. Primeiramente, as linhagens selecionadas foram avaliadas quanto ao n?vel de silenciamento do gene OsAMT1.3. As linhagens de arroz transformadas apresentaram maior n?vel de silenciamento do gene OsAMT1.3 quando comparadas ?s plantas controle, no entanto, diferentes n?veis de silenciamento foram observados. A linhagem L1 apresentou menor n?vel de silenciamento do gene OsAMT1.3, enquanto L2 e L6 apresentaram maior silenciamento. As linhagens L1, L2 e L6 e a planta controle IRS (transformada com o vetor vazio) foram cultivadas em c?mara de crescimento at? os 30 dias ap?s a germina??o, com a aplica??o dos seguintes tratamentos: sem N por tr?s dias, ressuprimento com 0,15 mM de N-NH4+ ap?s tr?s dias de priva??o de N (baixa dose) e 2,0 mM de N-NH4+ constante (alta dose). As linhagens L1, L2 e L6 apresentaram menor absor??o de NH4+ com 0,15 mM de N-NH4+ quando comparadas com as plantas controle (IRS), enquanto as plantas cultivadas com 2,0 mM de N-NH4+ n?o apresentaram diferen?as na absor??o de NH4+. A express?o dos genes dos transportadores de NH4+ OsAMT1.1, OsAMT1.2 e OsAMT1.3 foi induzido pelo tratamento com 0,15 mM de N-NH4+ nas plantas controle (IRS), enquanto as linhagens transformadas apresentaram repress?o dos genes OsAMT1.1, OsAMT1.2 e OsAMT1.3. A menor absor??o de NH4+ com 0,15 mM de N-NH4+ causou menor n?vel de N-NH4+ e N-amino nas ra?zes das linhagens transformadas, enquanto nas plantas com 2,0 mM de N-NH4+ houve pouca altera??o nos conte?dos de N-NH4+ e N-amino. Os resultados indicam que o silenciamento do gene OsAMT1.3 provoca regula??o negativa dos transportadores OsAMT1.1 e OsAMT1.2, alterando a absor??o de NH4+. Apesar do gene OsAMT1.3 ser menos expresso que os genes OsAMT1.1 e OsAMT1.2, o transportador OsAMT1.3 pode estar envolvido na efici?ncia de absor??o em baixas doses de NH4+.

Made available in DSpace on 2016-04-26T19:39:32Z (GMT). No. of bitstreams: 1 2009 - Leandro Azevedo Santos.pdf: 3069700 bytes, checksum: dbd1726a5b683e7f290e1829143eacac (MD5) 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.

La fixation de l’azote diatomique est un processus très important à la vie, vu sa nécessité dans la biosynthèse de plusieurs molécules de base; acides aminés, acides nucléiques, etc. La réduction de l’azote en ammoniaque est catalysée par la nitrogénase, une enzyme consommatrice de beaucoup d’énergie étant donné qu’elle nécessite 20 à 30 moles d’ATP pour la réduction d’une mole d’azote. De ce fait une régulation rigoureuse est exigée afin de minimiser le gaspillage d’énergie. Plusieurs systèmes de contrôle sont connus, aussi bien au niveau post-traductionnel que traductionnel. Chez la bactérie photosynthétique pourpre non-sulfureuse R. capsulatus, la régulation de l’activité de la nitrogénase nécessite une panoplie de protéines dont la protéine membranaire AmtB, qui est impliquée dans le transport et la perception d’ammonium, et les protéines PII qui jouent plusieurs rôles clés dans la régulation de l’assimilation d’azote. Suite à l’ajout de l’ammonium dans le milieu, une inhibition réversible de l’activité de la nitrogénase est déclenchée via un mécanisme d’ADP-ribosylation de la nitrogénase. La séquestration de GlnK (une protéine PII) par l’AmtB permet à DraT, une ADP-ribosyltransférase, d’ajouter un groupement ADP-ribose sur la protéine-Fe de la nitrogénase l’empêchant ainsi de former un complexe avec la protéine-MoFe. Donc, le transfert d’électrons est bloqué, engendrant ainsi l’inhibition de l’activité de la nitrogénase qui dure aussi long que la concentration d’azote fixé reste élevé, phénomène appelé le « Switch-off/Switch-on » de la nitrogénase. Dans ce mémoire, pour mieux comprendre ce phénomène de régulation, des mutations ponctuelles au niveau de certains résidus conservés de la protéine AmtB, dont D338, G367, H193 et W237, étaient générées par mutagénèse dirigée, afin d’examiner d’avantage leur rôle dans le transport d’ammonium, la formation du complexe AmtB-GlnK, ainsi que dans le « Switch-off » et l’ADP-ribosylation. Les résultats permettent de conclure l’importance et la nécessité de certains résidus telle que le G367 dans la régulation de la nitrogénase et le transport d’ammonium, contrairement au résidu D338 qui ne semble pas être impliqué directement dans la régulation de l’activité de la nitrogénase. Ces résultats suggèrent d’autres hypothèses sur les rôles des acides aminés spécifiques d’AmtB dans ses fonctions comme transporteur et senseur d’ammonium. / The reduction of diatomic nitrogen is a very important biological process given the need of all organisms for fixed nitrogen for the biosynthesis of basic key molecules such as, amino acids, nucleic acids, etc.. The reduction of nitrogen to ammonia is catalyzed by nitrogenase, an enzyme with high energy demands since it requires 20 to 30 moles of ATP for the reduction of one mole of nitrogen. Therefore a strict control is required to minimize energy waste. Several systems of regulation are known, both at the translational and post-translational level. In the purple non-sulfur photosynthetic bacterium R. capsulatus, the post-translational regulation of nitrogenase activity requires an array of proteins, including; the membrane protein AmtB, implicated in the perception and transport of ammonium, and PII proteins, which play key roles in the regulation of nitrogen assimilation. Following the addition of ammonium to the medium nitrogenase activity is reversibly inhibited (nitrogenase switch-off) via a mechanism of ADP-ribosylation of nitrogenase. Sequestration of GlnK (PII protein) by AmtB allows DraT, an ADP-ribosyltransferase, to add an ADP-ribose group to the Fe protein preventing it from forming a complex with the MoFe protein and nitrogenase activity is consequently inhibited. To better understand this phenomenon, in this Master’s thesis point mutations were created by site-directed mutagenesis at specific conserved residues of the AmtB protein, namely, D338, G367, H193 and W237, in order to examine their role in ammonium transport, formation of an AmtB-GlnK complex, and the regulation of nitrogenase (Switch-off/ADP-ribosylation). Plasmid-borne mutant alleles were transferred to a ∆AmtB strain of R. capsulatus, and the resultant strains were subjected to a series of tests. These demonstrated the importance and necessity of certain residues, such as G367, in the regulation of nitrogenase and ammonium transport, in contrast to residue D338, which seems to have no direct role in the regulation of nitrogenase activity. These results suggest further hypotheses about the roles of specific amino acids of AmtB in its functions as a sensor and transporter for ammonium.

La fixation de l’azote diatomique est un processus très important à la vie, vu sa nécessité dans la biosynthèse de plusieurs molécules de base; acides aminés, acides nucléiques, etc. La réduction de l’azote en ammoniaque est catalysée par la nitrogénase, une enzyme consommatrice de beaucoup d’énergie étant donné qu’elle nécessite 20 à 30 moles d’ATP pour la réduction d’une mole d’azote. De ce fait une régulation rigoureuse est exigée afin de minimiser le gaspillage d’énergie. Plusieurs systèmes de contrôle sont connus, aussi bien au niveau post-traductionnel que traductionnel. Chez la bactérie photosynthétique pourpre non-sulfureuse R. capsulatus, la régulation de l’activité de la nitrogénase nécessite une panoplie de protéines dont la protéine membranaire AmtB, qui est impliquée dans le transport et la perception d’ammonium, et les protéines PII qui jouent plusieurs rôles clés dans la régulation de l’assimilation d’azote. Suite à l’ajout de l’ammonium dans le milieu, une inhibition réversible de l’activité de la nitrogénase est déclenchée via un mécanisme d’ADP-ribosylation de la nitrogénase. La séquestration de GlnK (une protéine PII) par l’AmtB permet à DraT, une ADP-ribosyltransférase, d’ajouter un groupement ADP-ribose sur la protéine-Fe de la nitrogénase l’empêchant ainsi de former un complexe avec la protéine-MoFe. Donc, le transfert d’électrons est bloqué, engendrant ainsi l’inhibition de l’activité de la nitrogénase qui dure aussi long que la concentration d’azote fixé reste élevé, phénomène appelé le « Switch-off/Switch-on » de la nitrogénase. Dans ce mémoire, pour mieux comprendre ce phénomène de régulation, des mutations ponctuelles au niveau de certains résidus conservés de la protéine AmtB, dont D338, G367, H193 et W237, étaient générées par mutagénèse dirigée, afin d’examiner d’avantage leur rôle dans le transport d’ammonium, la formation du complexe AmtB-GlnK, ainsi que dans le « Switch-off » et l’ADP-ribosylation. Les résultats permettent de conclure l’importance et la nécessité de certains résidus telle que le G367 dans la régulation de la nitrogénase et le transport d’ammonium, contrairement au résidu D338 qui ne semble pas être impliqué directement dans la régulation de l’activité de la nitrogénase. Ces résultats suggèrent d’autres hypothèses sur les rôles des acides aminés spécifiques d’AmtB dans ses fonctions comme transporteur et senseur d’ammonium. / The reduction of diatomic nitrogen is a very important biological process given the need of all organisms for fixed nitrogen for the biosynthesis of basic key molecules such as, amino acids, nucleic acids, etc.. The reduction of nitrogen to ammonia is catalyzed by nitrogenase, an enzyme with high energy demands since it requires 20 to 30 moles of ATP for the reduction of one mole of nitrogen. Therefore a strict control is required to minimize energy waste. Several systems of regulation are known, both at the translational and post-translational level. In the purple non-sulfur photosynthetic bacterium R. capsulatus, the post-translational regulation of nitrogenase activity requires an array of proteins, including; the membrane protein AmtB, implicated in the perception and transport of ammonium, and PII proteins, which play key roles in the regulation of nitrogen assimilation. Following the addition of ammonium to the medium nitrogenase activity is reversibly inhibited (nitrogenase switch-off) via a mechanism of ADP-ribosylation of nitrogenase. Sequestration of GlnK (PII protein) by AmtB allows DraT, an ADP-ribosyltransferase, to add an ADP-ribose group to the Fe protein preventing it from forming a complex with the MoFe protein and nitrogenase activity is consequently inhibited. To better understand this phenomenon, in this Master’s thesis point mutations were created by site-directed mutagenesis at specific conserved residues of the AmtB protein, namely, D338, G367, H193 and W237, in order to examine their role in ammonium transport, formation of an AmtB-GlnK complex, and the regulation of nitrogenase (Switch-off/ADP-ribosylation). Plasmid-borne mutant alleles were transferred to a ∆AmtB strain of R. capsulatus, and the resultant strains were subjected to a series of tests. These demonstrated the importance and necessity of certain residues, such as G367, in the regulation of nitrogenase and ammonium transport, in contrast to residue D338, which seems to have no direct role in the regulation of nitrogenase activity. These results suggest further hypotheses about the roles of specific amino acids of AmtB in its functions as a sensor and transporter for ammonium.