Amino acids are essential molecules in plant metabolism. Amino acids carry reduced nitrogen while serving as precursors for protein synthesis and secondary metabolites. Translocation of amino acids in the cell is mediated by amino acid transporters. While about 100 transporters have been identified, only a dozen have been fully characterized. The regulation of amino acid transporters is not fully understood and stands as the basis of this study. Previous toxicity-based screenings of Arabidopsis thaliana mutants led to the isolation of a loss-of-function line and the phenylalanine insensitive growth (pig1) mutant capable of growth on toxic concentrations of phenylalanine (1). The pig1-1 mutants also displayed a deregulated metabolism (1). We followed this work with a similar forward genetic screening of Arabidopsis thaliana that led to the identification of 18 mutants capable of growth in the presence of amino acids at toxic concentrations. From this screen, seven mutations were confirmed to affect the amino acid transporter AAP1. Here I demonstrate that, when expressed in yeast deficient for endogenous amino acid transporters, three variant aap1 proteins restored growth similar to yeast complemented by wild type AAP1. Transport of radiolabeled Pro was abolished by variant aap1 proteins while deletion of an intracellular loop spanning the 8th and 9th transmembrane domains reduced Pro transport in yeast. Site directed mutagenesis of this loop conferred a variant aap1 protein which augmented Pro transport in yeast. Amino acid transport in loss-of-function aap1 plants display decreased uptake and increased efflux. In addition, aap1 mutant plants accumulated between 2 and 8 times more free amino acids in the leaves than the wild type. These observations are not fully compatible with the accepted role of AAP1 in transport by the root. The present work describes how the amino acid transporter AAP1 could play a role in regulating amino acid metabolism. We hypothesize that the amino acid transporter AAP1 functions as a senor that is involved in amino acid homeostasis in addition to its established role as a transporter. Is true, this would make AAP1 the first identified amino acid sensor in plants. Knowledge of the mechanism of amino acid sensing would enable us to engineer crops for improved nutrition in a more efficient way than affecting metabolic enzymes. / MSLFS

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.

Les plantes sont capables de percevoir dans leur environnement la disponibilité en nitrate (NO3-), un macro-nutriment essentiel. Chez Arabidopsis thaliana, le transporteur de NO3- NRT1.1 constitue un système de perception qui active de nombreuses réponses au NO3-, notamment la régulation de l'expression de gènes et le développement des racines latérales. Dans ce dernier cas, un mécanisme de transduction du signal a été proposé. Celui-ci met en jeu une activité de transport d'auxine par NRT1.1 qui est inhibée par le NO3-. Cependant, le(s) mécanisme(s) moléculaire(s) permettant à NRT1.1 de contrôler un large panel de réponses au NO3- reste(nt) largement inconnu(s). L'objectif de ce travail était donc d'approfondir nos connaissances sur les voies de signalisation du NO3- dépendantes de NRT1.1. Grâce à l'analyse de mutants et de lignées transgéniques exprimant des versions de NRT1.1 présentant des mutations ponctuelles, nous avons pu découpler certaines des réponses NRT1.1-dépendantes et montré que cette protéine peut percevoir/transduire le signal NO3- au travers d'au moins trois mécanismes distincts, possédant des bases structurales différentes au sein de la protéine. D'autre part, ce travail a permis de valider l'hypothèse selon laquelle NRT1.1, en intervenant comme transporteur d'auxine, contrôle directement le développement des racines latérales, et ce indépendamment des autres transporteurs d'auxine qui y sont exprimés. Enfin, nous avons montré qu'en plus de sa régulation transcriptionnelle déjà connue, NRT1.1 est soumis à une puissante et complexe régulation post-transcriptionnelle. En effet, le transcrit NRT1.1 est stabilisé en présence de NO3- dans la racine alors que l'accumulation de la protéine NRT1.1 est réprimée par le NO3- spécifiquement au niveau des primordia de racines latérales. Les résultats obtenus au cours de ce travail ont permis d'élaborer un modèle cohérent du rôle de signalisation joué par NRT1.1, et ouvrent de nombreuses perspectives pour comprendre comment, chez les plantes, un « transcepteur » (transporteur/senseur) peut contrôler une vaste gamme de réponses adaptatives aux facteurs de l'environnement. / Plants are able to sense the external availability of nitrate (NO3-), a major macro-nutrient. In Arabidopsis thaliana, the NO3- transporter NRT1.1 acts as a sensor that triggers many different adaptive responses, including the regulation of gene expression and lateral root development. In the latter case, a transduction mechanism that involves a NO3--inhibited auxin transport activity dependent of NRT1 has been proposed. However, the molecular mechanism(s) allowing NRT1.1 to control such a large palette of NO3- responses is still largely unknown. Thus the aim of this work was to better understand and characterize the NRT1.1-dependent NO3- signaling pathway(s). Using mutants and transgenic lines expressing point mutated forms of NRT1.1, we uncoupled several of the NRT1.1-dependent responses and thus demonstrated that NR1.1 can sense/transduce NO3- signal through at least three distinct mechanisms at the protein level. This work also largely confirmed the hypothesis that NRT1.1 directly controls lateral root development through its auxin transport activity regardless of the other auxin transporters expressed in lateral root primordia. Finally, we showed that, besides the already well characterized transcriptional NO3--dependent regulation of NRT1.1, this gene is also subjected to complex post-transcriptional regulations. Indeed, on the one hand, NRT1.1 mRNA is stabilized by NO3- in roots whereas, on the other hand, protein accumulation is specifically repressed by NO3- in lateral root primordia. Altogether, these results allowed us to build a comprehensive model of the complex NRT1.1 signaling and open many perspectives to understand how plant “transceptors” (transporter/sensor) can monitor a large variety of adaptive responses to environmental factors.

The dimorphic transition from the yeast to the filamentous form of growth allows cells to explore their environment for more suitable niches and is often crucial for the virulence of pathogenic fungi. In contrast to their Mep1/3 paralogues, fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family have been assigned an additional receptor role required to trigger the filamentation signal in response to ammonium scarcity. Here, genetic, kinetic, expression and structure-function analyses were used to shed light on the poorly characterized signaling role of Saccharomyces cerevisiae Mep2. We show that Mep2 variants lacking the C-terminal tail conserve the ability to induce filamentation, revealing that signaling can proceed in the absence of exclusive binding of putative partners to the largest cytosolic domain of the protein. Our data support that filamentation signaling requires the conformational changes accompanying substrate translocation through the pore crossing the hydrophobic core of Mep2. pHluorin reporter assays show that the transport activity of Mep2 and of non-signaling Mep1 differently affect yeast cytosolic pH in vivo, and that the unique pore variant Mep2H194E, with apparent uncoupling of transport and signaling functions, acquires increased ability of acidification. Functional characterization in Xenopus oocytes reveals that Mep2 mediates electroneutral substrate translocation while Mep1 performs electrogenic transport. Our findings highlight that the Mep2-dependent filamentation induction is connected to its specific transport mechanism, suggesting a role of pH in signal mediation. We also show that the signaling process is conserved for the Mep2 protein from the human pathogen Candida albicans. Our results allow to propose a model for the sensing function of Mep2 where pH and calcium are key players. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Amino acids are necessary for protein synthesis and specialized metabolism in plants. Yet very little is known about how plants sense and regulate when and where to allocate amino acids to meet the demand for nitrogen in growing tissues. In particular, while characterized in yeast and mammals, no amino acid sensor has been identified in plants. Amino Acid Permease 1 (AAP1) has been previously characterized and was shown to mediate amino acid uptake from the soil. aap1 knockout plants and several EMS mutants affected in AAP1 sequence display enhanced tolerance to toxic concentrations of amino acids. Yet, two of the corresponding variant proteins appear to be functional transporters, effectively dissociating amino acid transport and phenotype. To understand this apparent discrepancy, I precisely studied AAP1 localization of expression at the plant and cellular level, and in specific tissue types of the root where AAP1 function is required for the tolerance phenotype and the amino acid uptake activity. I showed that AAP1 protein is present in the endoplasmic reticulum of the cortex in wild type plants Yet, its ectopic expression in root tip and phloem increased amino acid uptake, while expression in cortex could not. This and other of my results do not support the current model of AAP1 functioning in amino acid uptake by the root. I propose that the main effect of mutations in AAP1 is a disturbance in amino acid metabolism, possibly triggered by altered amino acid sensing. In this new model, AAP1 would be necessary for sensing amino acid status of cortex cells, possibly in the endoplasmic reticulum, and adjust amino acid metabolic activity and uptake to current availability. In effect, disruption of the sensing function, either by complete loss of AAP1 function (knockout) or by uncoupling the transport and sensing function (EMS mutants), would lead to the various characteristics of the phenotype of the aap1 mutants I observed. My main hypothesis is that AAP1 is a transporter endowed with sensing function, i.e., an amino acid transceptor. / Doctor of Philosophy / Changing environments create challenges for plants to grow under harsher, nutrient limiting conditions. Nitrogen is an essential nutrient for plant growth, used for the synthesis of amino acids and other nitrogen-containing metabolites. Amino acids are necessary for protein synthesis and other specialized metabolism – being targets for manipulation for improving agronomic traits. Protein content is a complex trait that involves many genes, possibly including amino acid transporters. In addition, the amount of nitrogen needed by and available to the plant increases or decreases depending on the environment conditions. How plants control nitrogen need and use at the molecular level is not well understood. The data presented here challenge a current model and I report how a protein (AAP1) involved in the acquisition of amino acids from the soil provides regulatory control over these processes. . This valuable information is useful for better understanding how plants use nitrogen and more precise breeding methods can be used to improve traits, such as protein content in agronomically important crops.

Submitted by Maria Suzana Diniz (msuzanad@hotmail.com) on 2015-11-05T14:36:51Z No. of bitstreams: 1 arquivototal.pdf: 4812802 bytes, checksum: e5276e62e6294058de196c6252bedc95 (MD5) / Made available in DSpace on 2015-11-05T14:36:51Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 4812802 bytes, checksum: e5276e62e6294058de196c6252bedc95 (MD5) Previous issue date: 2015-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This work deals with the evaluation of the communication system by radio frequency using the NRF-24L01+® transceiver to be used in monitoring of electrical systems on the concept of smart grid. The complete system consists of one or more Units Remote Data Acquisition - URDAs; multiple Smart Sensing Units - SSUs; and Supervisory Control Subsystem - SCS. The connection between URAD and SSUs may occur via wired connection (Ethernet, RS232, USB, CAN or PLC) and wireless (RF). URADs fit to the acquisition, processing and communication of variables with low time constant while the USIs are primarily responsible for the acquisition of magnitudes with larger time constants (temperature, pressure, humidity, etc.). In this work, we focus on development and communication of SSUs. For these tests the units are of two types: a master unit, responsible for requesting data (wireless) and sending the SCS (Communication RS232, USB, CAN or RF); and a slave unit, which may account for the measured variables of interest to send to the master unit when requested. For wireless communication (RF), the transceiver nRF - 24L01+® from NORDICTM was used, because its processing characteristics and communication satisfactorily meet the needs and requirements of the project, which will be addressed in the course of this work. / O presente trabalho trata da avaliação do sistema de comunicação por meio de rádio-frequência utilizando o transceiver nRF-24L01+® para ser utilizado no monitoramento de sistemas elétricos no conceito de smart grid. O sistema completo é composto de uma ou mais Unidades Remotas de Aquisição de Dados – URADs; de várias Unidades de Sensoriamento Inteligente – USIs; e um Subsistema de Controle Supervisório – SCS. A conexão entre a URAD e as USIs pode ocorrer através de conexão cabeada (Ethernet, RS232, USB, CAN ou PLC) e sem fio (RF). Cabem às URADs a aquisição, processamento e comunicação das grandezas com pequena constante de tempo, enquanto que as USIs encarregam-se da aquisição de grandezas com constantes de tempo maiores (temperatura, pressão, umidade, etc.). Neste trabalho, tratamos do desenvolvimento e de testes de comunicação da USI. Para estes testes as unidades são de 2 tipos: uma unidade mestre, responsável pela requisição dos dados (sem fio) e pelo envio ao SCS (comunicação RS232, USB, CAN ou RF); e uma unidade escravo, que pode ser responsável pela medição de grandezas de interesse para envio à unidade mestre quando requisitada. Para a comunicação sem fio (RF), utilizou-se o transceptor nRF-24L01+® da NORDICTM, pois suas características de processamento e comunicação atendem satisfatoriamente às necessidades e exigências do projeto, que serão abordadas no transcurso deste trabalho.