Thesis (M.S.)--University of Michigan, 1949.

L’azote est un des macronutriments essentiels pour les plantes. Dans les sols, l’azote est présent sous différentes formes organiques et inorganiques. Les plantes utilisent préférentiellement le nitrate, qui n’est pas toujours disponible en quantités suffisantes dans les sols. Récemment, une étude a permis de montrer que le facteur de transcription NLP7 est un régulateur majeur de la réponse primaire au nitrate. La localisation subcellulaire de cette protéine est régulée par le nitrate : en son absence, elle est localisée dans le cytoplasme alors qu’après ajout de nitrate, une rétention nucléaire est activée. Les mécanismes moléculaires de cette rétention restent encore à comprendre ainsi que la transmission du signal nitrate, de l’extérieur de la plante à la protéine NLP7. Le transporteur de nitrate NPF6.3 a été montré comme jouant un rôle dans la perception du nitrate, c’est donc un transcepteur. Notre hypothèse était que NPF6.3 est le récepteur de nitrate en amont de NLP7. Pour tester cette hypothèse, nous avons étudié par des approches génétiques les liens d’épistasie entre les deux gènes. L’étude de la biomasse et de l’expression des gènes sentinelles en réponse au nitrate chez les simples et double mutants a permis d’observer des phénotypes additifs. Nous avons pu montrer que le mécanisme de relocalisation rapide de la protéine NLP7 dans le noyau est toujours actif dans le fond mutant npf6.3. Ces résultats ont donc permis de montrer que NLP7 et NPF6.3 n’appartiennent pas à la même voie de signalisation mais que ces deux voies pourraient être dépendantes selon les conditions. D’autre part, peu de régulateurs de la réponse au nitrate sont connus. De manière intéressante, les gènes cibles de NLP7 sont enrichis en protéines régulatrices comme par exemple d’autres facteurs de transcription ou encore des protéines kinases, ce qui place NLP7 à un haut niveau hiérarchique de régulation dans la voie de signalisation en réponse au nitrate. En effet, ces cibles directes de NLP7 pourraient elles-mêmes être impliquées dans des voies de signalisation en aval de NLP7. Dans le but de disséquer la voie de signalisation en aval de NLP7, nous avons étudié deux cibles directes de NLP7, des Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs), MAPKKK13 et MAPKKK14. Les MAPKs sont connues pour leur mode d’action en cascades de phosphorylations. Par des approches biochimiques en protoplastes, nous avons montré que MAPKKK13/14 sont capables d’activer des MAPKs du groupe C via MKK3. De plus, nous avons obtenu de premières indications montrant que certaines réponses développementales au nitrate ainsi que la réponse primaire au nitrate seraient partiellement modifiées dans les simples mutants mapkkk13 et mapkk14, et dans le double mutant mapkkk13/14. / Nitrogen is one of the most important macronutrients for plants. In the soils, nitrogen can be found under different organic and inorganic forms. Plants preferentially use nitrate, which is not always available for plant uptake and assimilation in soils. Recently, it was shown that the NLP7 transcription factor is a master regulator of the primary nitrate response. Its subcellular protein localization is regulated by nitrate: without nitrate, the protein is localised in the cytoplasm whereas after nitrate resupply, a nuclear retention is observed. Molecular mechanisms of this nuclear-cytosolic shuttling and of the nitrate signal transduction from the external medium to the NLP7 protein are still unknown. It has been shown that the NPF6.3 nitrate transporter plays a role in the nitrate signal sensing, which made this protein a transceptor. Our hypothesis was that NPF6.3 is the nitrate sensor upstream of NLP7. To test this hypothesis, we studied by genetic approaches the epistasis link between the two genes. By studying the simple and double mutant’s biomass and sentinel gene regulation in response to nitrate, we observed additive phenotypes. We showed that the nuclear relocation mechanism of NLP7 is still active in the npf6.3 mutant background. All together, these results showed that NLP7 and NPF6.3 are not in the same signalling pathway but there would be an interplay depending on the conditions. On the other hand, only a few regulators of the nitrate response are known. Interestingly the direct target genes of NLP7 are highly enriched for regulatory proteins such as other transcription factors or protein kinases, which places NLP7 at a high hierarchical place in the nitrate signalling pathway. Indeed these direct targets of NLP7 may themselves be involved in signalling cascades downstream of NLP7. In order to identify molecular events downstream of NLP7, we studies two NLP7 direct targets, Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs), MAPKKK13 and MAPKKK14. MAPKs are known to act as phosphorylation cascades. Using biochemical approaches in protoplasts, we have shown that MAPKKKK13/14 are able to activate Group C MAPKs via MKK3. In addition we showed in planta that nitrate addition indeed triggered the activation of group C MAPKs and that this activation is dependent on NLP7 and MKK3. Furthermore we obtained first indications that nitrate-dependent developmental traits and the primary nitrate response are partially impaired in the single mutants mapkkk13 and mapkkk14, and the double mutant mpkkk13mpkkk14.

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Mycobacterium avium is human and animal opportunistic pathogen responsible for disseminated disease in immunocompromised patients. Mycobacteria have a capacity to adapt to the environmental conditions by inducing enzyme activities and altering their metabolism. M. avium A5 cells were grown in a defined minimal medium (Nitrogen Test Medium) with glutamine, nitrite, nitrate, or ammonia as sole nitrogen source at a concentration of 2 mM at 370C aerobically. The strain grew well on all the nitrogen sources except nitrite. It grew slowly on nitrite with a generation time of 6 days and cultures were not viable after 4 weeks of storage. These data confirm that M. avium can utilize a single nitrogen source in a defined minimal medium as documented by McCarthy (1987). M. avium genome has been sequenced and contains genes sharing sequence similarities to respiratory nitrate reductase and dissimilatory nitrite reductases. Because, M. avium can use nitrate or nitrite as sole nitrogen source for growth (McCarthy, 1987), it must have assimilatory nitrate- and nitrite-reductases. Nitrate- and nitrite-reductase activities of M. avium cells growing aerobically or undergoing anaerobic shift in the presence of ammonia, nitrate or ammonia and nitrate in combination were measured. M. avium produced nitrate- as well as nitrite-reductase activity. Nitrite- and nitrate-reductases used either NADH or NADPH as an electron donor. Nitrite reductase activity was greater than nitrate reductase activity. This observation supports the rapid reduction of nitrite and slow reduction of nitrate in M. avium as documented by McCarthy (1987) and explained why M. avium gives a negative result by the standard nitrate reductase test. In addition to assimilatory enzyme activity, M. avium A5 also produced dissimilatory nitrate- and nitrite-reductase activities. / Master of Science

Excess nitrate-N concentration (>10 mg L-1) in drinking water can cause significant risk to human health. Also, at very low concentration (0.035-0.1 mg P L-1), phosphorus is considered as a pollutant due to its effects of promoting algal growth and eutrophication of surface waters. This thesis’ research was conducted at two different sites. The first study was conducted at Carman on a sandy loam soil with cropping system, perennial versus annual, as the main plot and manure nutrient management system, as the subplot to measure nitrate and phosphorus leaching from hog manures. The second field experiment, located northwest of the town of Carberry, Manitoba, was conducted on a loamy sand soil. A two year rotation was employed for the annual cropping systems with a randomized complete block design. Treatments included two rates of liquid hog manure (LH-5000, LH-2500), two rates of fertilizers (F5000, F2500) corresponding to the amount of available nitrogen in the two rates of hog manure a compost treatment (Com-2500) and a control for a total of six treatments. The results from Carman site showed that while a substantial amount of nitrate-nitrogen was lost from the annual plots (40 to 60 kg ha-1 in 2010 and 23 to 60 kg ha-1 in 2011), a negligible amounts of nitrate was lost from the perennial (< 1 kg ha-1). There was no evidence of significant downward movement of phosphorus below the top 15 cm soil layer in this study. However, repeated, annual application of manure at an N-based rate resulted in increased soil test P. In Carberry, total N leaching of fertilizer amended plots was greater than in plots that received manure. Based on the results, application of liquid hog manure at the rate of 2500 gallon ac-1 was economically and environmentally more desirable and is recommended. We applied the multi-layer water balance model, VSMB, to the data that we generated in the field to gain an understanding of how well the model will simulate the loss of water that we measured from the lysimeters. The simulation study showed that the VSMB model grossly underestimated the amount of leached water, possibly due to an overestimation of evapotranspiration. / October 2015

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