Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence of higher auxin in both the Mtnip-1 mutant and in M. truncatula plants constitutively expressing MtNPF1.7 was observed. Previous experiments showed MtNPF1.7 expression is hormone regulated and the MtNPF1.7 promoter is active in root and nodule meristems and in the vasculature. Two potential binding sites for an auxin response factors (ARFs) were found in the MtNPF1.7 promoter. Chromatin immunoprecipitation-qRT-PCR confirmed MtARF1 binding these sites. Mutating the MtARF1 binding sites increases MtNPF1.7 expression, suggesting a mechanism for auxin repression of MtNPF1.7. Consistent with these results, constitutive expression of an ARF in wild-type plants partially phenocopies Mtnip-1 mutants’ phenotypes.

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

Chez les plantes, le nitrate est un élément essentiel mais sa disponibilité dans le sol est fluctuante. Il est donc stocké dans la vacuole grâce à un échangeur nitrate/proton appelé AtClCa. La famille de protéines ClCs comporte à la fois des échangeurs mais aussi des canaux suggérés comme issus de l’évolution des échangeurs par une conversion mécanistique. Chez Arabidopsis thaliana, seuls des ClCs échangeurs assurent la gestion du nitrate. Deux glutamates très conservés, E203 et E270 dans AtClCa, sont essentiels pour le transport des protons chez les ClCs échangeurs. La mutation du résidu E203 en une alanine, un acide aminé non protonable (E203A) a permis de produire artificiellement une telle conversion mécanistique. Afin de mieux comprendre l’importance physiologique du mécanisme d’échange, une analyse a été conduite sur des plantes exprimant la forme mutée d’AtClCa pour ce glutamate. Chez ces plantes, le stockage vacuolaire est fortement réduit au profit d’une importante assimilation accroissant la teneur en protéines. En dépit de cela, elles présentent un défaut de production de biomasse résultant en grande partie d’une perturbation de l’homéostasie hydrique. Elles sont également plus sensibles aux stress hydrique et probablement azoté. La conservation d’un échangeur est donc requise pour croitre en dépit des fluctuations environnementales. En parallèle, la mutation E270A a été introduite en plante afin d’étudier son importance sur la physiologie d’Arabidopsis. Une analyse préliminaire de la biomasse et des contenus en nitrate et eau de plantes exprimant la forme mutée de ce glutamate est donc présentée dans la seconde partie de cette thèse. / Nitrate is a major element for plant but its availability is very fluctuant in soils. Then, it is stored in vacuoles thanks to a nitrate/proton exchanger named AtClCa. In ClCs, exchangers but also channels were identified, the latest were suggested to be evolved from exchanger in which a mechanistic switch happened. In Arabidopsis thaliana, only exchangers are involved in nitrate management. Two conserved glutamate, E203 and E270 in AtClCa, are essential for protons transport in ClCs exchangers. The mutation of E203 into an alanine, a non-protonable amino acid (E203A) artificially produces such a mechanistic switch. To better understand the physiological importance of this exchange mechanism, a study was conducted in plants expressing the mutated form of AtClCa for this glutamate. In those plants, the vacuolar storage is highly restricted whereas the assimilation is favoured and the protein content increased. Despite that, the biomass production is decreased mostly because of a hydric homeostasis disruption. Those plants are also more sensitive to hydric and probably nitrogenous stress. The exchanger conservation is then required for plant growth whatever the environmental fluctuations. In parallel, the mutation E270A was introduced in planta to study its physiological importance. A preliminary analysis of plant biomass and nitrate and water contents was then performed in plants expressing the E270A mutated form of AtClCa and the results are presented in the second part of the manuscript.

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