Flavonoid biosynthesis is an important secondary metabolic pathway in higher plants with a range of vital functions in plants and animals. This pathway has been developed as a model system for the study of multi-enzyme complexes. The goal of the work presented here was to structurally characterize a series of loss-of-function chalcone synthase (CHS) alleles and to define the molecular basis of the interaction between CHS and the second enzyme of flavonoid biosynthesis, chalcone isomerase (CHI). CHS proteins encoded by five previously characterized alleles were characterized by homology modeling in an effort to explain the alterations in function, stability, and dimerization exhibited by these variants. Four of the encoded proteins have a single amino acid substitution and the fifth is a truncated protein resulting from a frameshift. Models for each of these proteins were generated in silico and analyzed after molecular dynamics simulations. This analysis suggested reasons for changes in catalytic ability and stability for three of the five CHS variants. To characterize the molecular basis of the CHS-CHI interaction, a model was developed using X-ray crystallography, small-angle neutron scattering (SANS), in silico docking, molecular dynamics simulations, and yeast 2-hybrid analyses. These enzymes appear to be interacting in a manner that could facilitate the flow of intermediates from one active site to another. These experiments also identified a series of amino acids that appear to be involved in the interaction, which are currently undergoing alteration and analysis using a yeast 2-hybrid assay to verify the authenticity of the model. The data presented herein could be used in future engineering experiments to alter pathway flux to control the levels or types of flavonoid endproducts, resulting in more nutritious plants or flowers with novel pigments. These experiments advance the study of the structure of multi-enzyme complexes, an area that currently contains little information. As well, this is the first known use of SANS for the investigation of the architecture of metabolons. The techniques described herein could easily be applied to other systems in an effort to better understand the organization of multi-enzyme complexes and the implications of these assemblies on metabolic regulation. / Ph. D.

Plants have evolved multiple surveillance mechanisms to detect the presence of disease-causing organisms. One mode of surveillance is based on dozens of constitutively expressed resistance (R) genes. R genes recognize pathogen gene products as signals of invasion. We are interested in how plants evolve R genes to keep pace with rapidly evolving pathogen populations. The mechanisms that drive the evolution of new R genes are poorly understood. There is data that supports the relevance of recombination in the evolution of resistance gene clusters in plants. However, a more comprehensive understanding of the molecular biology of recombination and the impact recombination has on R gene evolution is necessary. The objectives of this dissertation were to develop a genetic screen that models meiotic unequal crossing over at a synthetic RPP8 (synthRPP8) resistance gene cluster and to assess the effect of abiotic stress on recombination with the synthetic RBCSB gene cluster (synthRBCSB) in Arabidopsis. The genetic screen utilized in these studies specifically identifies a novel recombinant gene and a concomitant gene duplication that results from meiotic unequal crossing-over by coupling chimeric gene formation to the activation of the firefly luciferase gene. Two synthRPP8 clusters were constructed and extensive optimization of screening conditions were performed. An initial screen of ~1 million synthRPP8 transgenic plants was performed and plants that expressed the luc+ phenotype were isolated and analyzed. Unexpectedly, background bioluminescence was found to interfere with the identification of bona fide luc+ synthRPP8 recombinants. An abiotic stress response assay was performed and the data suggests activation of a putative stress response element in the promoter of RPP8 is responsible for background levels of in vivo luciferase activity. The background bioluminescence could not be sufficiently reduced. Therefore, two additional synthRPP8 constructs, synthRPP8-3 and synthRPP8-4, were constructed and are currently being examined for their utility to model meiotic unequal crossing-over. UV-C treatment was shown to stimulate somatic unequal crossing over, as well as upregulate defense/stress response genes and transcription factors. Meiotic recombination may also be affected by stress. Therefore, the effect of UV-C irradiation on the frequency of unequal meiotic recombination between paralogous RBCSB genes and on the expression of genes associated with the defense/stress response was examined. We observed a ~2-fold increase in the frequency of meiotic recombination after UV-C irradiation but this increase was not statistically significant. We did not detect a significant alteration in the steady-state MYB10, PR-1 and HSF-3 mRNA levels by semi-quantitative RT-PCR. The expression data we gathered provided minimal support for whether the UV-C treatment was an effective DNA damaging agent. / Ph. D.

Proteomics is defined as a tool to explore how proteins control and regulate important molecular and physiological processes. Further, peptide-centric approaches, or bottom-up methods, provide more comprehensive coverage of a proteome compared to whole-protein approaches. This body of work assesses the technical feasibility of several bottom-up proteomics technologies applied to Arabidopsis thaliana, including gel-based methods, those that require peptide derivitization, and those that do not. Selected-reaction monitoring (SRM) for targeted proteomics, and data-independent acquisition (MSE) was also evaluated. In addition to assessing the capabilities of these technologies, we then applied them to the context of uncovering new insights into the flavonoid biosynthetic pathway and the auxin and ethylene signaling pathways. Chapter one provides background information related to secondary metabolism, phytohormone signaling, and the status of proteomics in plants. In Chapter 2 and Appendix A, we establish the methodology to apply traditional and DiGE-based 2D-GE strategies to global proteomics in Arabidopsis. Our results suggest that while 2D-GE is applicable to Arabidopsis, there are practical and conceptual limitations that must be understood. Further, our results suggest that pertubations in the flavonoid pathway do not affect the abundance of proteins in Arabidopsis seedlings, roots, or flowers that can be studied using 2D-GE and DiGE. Additionally, we demonstrated the first parallel comparison of the effects of auxin and ethylene on the Arabidopsis root proteome and observed no overlap among the proteins regulated by the two phytohormones, at least for the most abundant proteins observed by 2D-GE. Chapter 3 explores the efficacy of selected reaction monitoring for relative peptide quantification in Arabidopsis roots. Our results suggest that while the technology parallels application in yeast and humans, there are substantial analytical challenges that much be addressed. In Chapter 4 we explore the MSE data acquisition scheme for global proteomics in Arabidopsis. We observe that treatment with exogenous auxin affects the abundance of many proteins representing diverse biological processes. Interestingly, we observe minimal overlap among genes and proteins regulated by exogenous auxin. Appendix B explores the efficacy of iTRAQ labeling for relative peptide quantification in Arabidopsis roots. / Ph. D.

Inositol pyrophosphates (PP-InsPs) are a family of molecules recently discovered to be implicated in a number of plant pathways such as auxin regulation, phosphate (Pi) sensing, and jasmonate-(JA)-regulated plant defense. Transgenic plants that overexpress inositol tetrakisphosphate 1-kinase (ITPK1) and the kinase domain of the dual domain diphosphoinositol pentakisphosphate kinase 2 (VIP2KD) have been previously studied to display uniquely elevated PP-InsPs. Here it is reported that the JA defense pathway is constitutively upregulated in VIP2KD OX plants, resulting in a lower rate of herbivory on the transgenic plants. ITPK1 OX, although also having elevated PP-InsPs, was fed upon by insect larvae comparably to Wild-Type Arabidopsis (WT). The data implicate VIP2, InsP8, and possibly the PP-InsP biosynthesis as a whole. / Master of Science in Life Sciences / Plants and insects have been evolving defenses against each other since they first emerged together post-Cambrian explosion. They each have evolved targeted metabolic pathways to produce chemicals with which to repel, harm, or even trick one another. In Arabidopsis thaliana, one of the most widely studied defense mechanisms is the jasmonic acid defense pathway, which responds to the herbivory of insects like caterpillars by setting off an array of genetic switches. The plant enters a stressed state wherein it represses the genes focused on growth and development and encourages the expression of genes focused on protecting vital resources and thwarting the attacker. This work examines a connection between the phosphate-sensing pathways and the jasmonic acid defense pathways in plants, and the following data show that plants with elevated inositol pyrophosphates (a phosphate storage molecule) are resistant to the herbivory of common pest caterpillars.

El gineceo tiene un papel fundamental en la continuidad de la especie ya que está implicado en la recepción y germinación del polen, asegurando así la fertilización de los óvulos. Tras dicho proceso el gineceo pasa a denominarse fruto, y es el encargado de proteger las semillas durante su desarrollo y dispersarlas cuando estén maduras. En Arabidopsis se han identificado muchos de los genes necesarios para la morfogénesis del fruto y la formación de los tejidos necesarios para la dispersión de las semillas, aunque en la mayoría de los casos no se conocen con precisión las rutas genéticas implicadas en estos procesos. Por ello es importante ampliar los estudios genéticos y moleculares encaminados a desvelar las interacciones funcionales entre los genes implicados, así como identificar nuevos factores con funciones clave en la morfogénesis del gineceo. En la presente tesis se ha llevado a cabo la caracterización funcional de la pequeña subfamilia de factores de transcripción NGATHA (NGA) de Arabidopsis, y se ha estudiado el papel que desempeñan estos factores en las rutas genéticas responsables de la morfogénesis del gineceo. Para la caracterización funcional de los genes NGA se han identificado y caracterizado mutantes de pérdida de función para cada uno de estos loci, y se han generado y analizado combinaciones múltiples de estas mutaciones. Estos estudios han puesto de manifiesto el papel esencial que los genes NGA desempeñan en la morfogénesis de los tejidos apicales del gineceo en Arabidopsis. El análisis de los patrones de expresión temporal y espacial de los genes NGA muestra que su expresión tiene lugar en dominios que experimentan una proliferación activa y coincide, en general, con regiones que acumulan niveles altos de auxinas. Para profundizar en la relación entre los genes NGA y las rutas de señalización de auxinas se han realizado diferentes análisis, como el estudio de las respuestas clásicas a auxinas en los mutantes nga, la caracterización del efecto fenotípico de las auxinas y de inhibidores del transporte de auxinas en estos mutantes, y el análisis de expresión de genes implicados en la biosíntesis de auxinas, como los genes YUCCA (YUC), en los mutantes nga. Los resultados obtenidos indican que los fenotipos observados en el gineceo de los mutantes nga probablemente son debidos a una menor síntesis de auxinas en la región apical del gineceo como consecuencia de la falta de inducción de los genes YUC en este dominio. Por tanto, los factores NGA parecen estar implicados en la biosíntesis de auxinas a través de la regulación de los genes YUC. Además de las auxinas, los genes NGA podrían regular respuestas a brasinosteroides en la raíz, ya que se ha demostrado la interacción física entre las proteínas NGA y BRX. Así mismo, se ha llevado a cabo un exhaustivo análisis genético para determinar el papel de los genes NGA en las rutas que dirigen la morfogénesis del gineceo. Los estudios realizados con combinaciones múltiples de mutantes, tanto de pérdida como de ganancia de función, junto con análisis de expresión han puesto de manifiesto la existencia de una relación funcional entre los factores de transcripción de la familia STY/SHI y los factores NGA, pese a no estar relacionados estructuralmente. En este trabajo se muestra que estas dos familias probablemente no se regulan mutuamente, sino que actúan de manera cooperativa sobre dianas comunes. Los resultados obtenidos indican que un posible trímero NGA/STY1/CRC sería el responsable de especificar la formación del estilo. Se muestra también que los genes NGA participan en la regulación de genes que dirigen la formación de la zona de dehiscencia y la apertura del fruto y que también parecen tener un papel en la formación de las regiones apicales del gineceo. Los análisis genéticos realizados indican que los factores NGA podrían estar implicados en la inducción de los genes SHATTERPROOF (SHP) e interferir con la activación del gen FRUITFULL (FUL). / Navarrete Gomez, ML. (2011). Análisis funcional de genes reguladores del desarrollo del fruto y su relación con las vías reguladas por auxinas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/10940

The plant hormones gibberellins (GAs) regulate multiple processes of plant development. Most of this regulation occurs at the transcriptional level, through the activity of the DELLAs, which are nuclear-localized proteins subjected to GA-mediated proteolitic degradation. DELLAs are encoded by five genes, and genetic studies show that each DELLA displays specific, but also partially overlapping roles with respect to their paralogs. In this Thesis, we have addressed two issues: (1) the contribution of DELLA multiplication to the diversification of functions controlled by GAs; and (2) the identification of direct targets regulated by DELLAs in etiolated seedlings with special attention to those involved in differential growth processes. Using combinations of mutants and transgenic lines expressing two phylogenetically distant DELLA genes (RGA and RGL2), we have found that these two DELLA proteins can perform each other's role as long as they are expressed under the reciprocal promoters, indicating that DELLA subfunctionalization relies mainly on their differential expression patterns. To identify direct DELLA targets, we have performed transcriptomic analyses of dark-grown seedlings expressing an inducible version of gai-1, a stable, dominant allele of a DELLA gene. This approach rendered a list of over 150 genes differentially expressed after induction of gai-1. The presence of several auxin-related genes among the primary targets of DELLA proteins has allowed us to establish a new role for GAs in the modulation of hypocotyl gravitropism through the repression of IAA19/MASSUGU2 expression by DELLAs. Moreover, the repression of HOOKLESS1 and the auxin efflux carriers PIN3 and PIN7 by DELLAs, is proposed as the molecular mechanism to explain the already known physiological regulation of apical hook development by GAs. / Gallego Bartolomé, J. (2011). Della protein function during differential growth processes in arabidopsis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11403

Las Giberelinas (GAs) intervienen en la regulación de numerosos procesos en el desarrollo a lo largo de la vida de la planta, y lo hacen promoviendo la degradación de las proteínas DELLA (DELLAs) [1], reguladoras negativas de las respuestas a GAs. Las DELLAs son nucleares y carecen de sitios de unión a DNA, sin embargo son capaces de interaccionar con otras proteínas y de esta manera regular la expresión génica [2-6]. Una de las preguntas que se abordaran en este proyecto es el de entender como las GAs son capaces de regular diversos procesos del crecimiento y del desarrollo, nuestra hipótesis se basa en que las proteínas DELLA se encuentran reprimiendo o potenciando la actividad de otras proteínas involucradas en diferentes procesos del desarrollo. / Marín De La Rosa, NA. (2014). DELLA proteins as hubs in signaling networks in plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37193

Chemical Genetics is a powerful approach that uses small molecules to probe gene function. In this Theses, we have used it to identify compounds that help us to i) dissect a plant signaling pathway, and ii) to solve a problem in agriculture. In both cases, we have screened a library of 10.000 compounds. Gibberellins are plant hormones that regulate multiple transitions in plant development and that allow them to tune their growth and development according to the environmental conditions. Gibberellin signaling proceeds through the degradation of DELLA proteins, the negative regulators of the pathway that regulate gene expression through the interaction with multiple transcription factors. To further dissect this pathway, we have sought to identify compounds with gibberellin-like activity affecting only a subset of processes. For that purpose, we have performed two screenings, in one of them we have searched for compounds affecting cell expansion in Arabidopsis seedlings, and in the other for compounds that prevent the interaction of a DELLA protein with a particular transcription factor in yeast two-hybrid assays. We have identified compounds with the expected behavior: compounds that activate the pathways downstream of the receptor, and others that prevent interaction with ARR1, BZR1, PIF4 or KAN1. Phosphorus is an essential element for plants and it is only absorbed in phosphate form, which is obtained from limited, not renewable sources and for which there is no alternative. As consequence, the cost of phosphate application in the field is steadily increasing. We have established collaboration with Dadelos Agrosolutions S.L. to identify compounds that improve phosphate assimilation. We have screened a chemical library and selected compounds based in their ability to down regulate the Arabidopsis IPS1::GUS reporter under suboptimal phosphate concentrations, under which it is normally on. Importantly, these compounds enhance the growth capacity of the plants under suboptimal phosphate concentrations. / La Genética química es una potente herramienta basada en el empleo de moléculas para estudiar funciones génicas conocidas o descubrir nuevas funciones, mediante la identificación de compuestos químicos que interfieran o promuevan ciertos procesos biológicos de interés. En esta tesis se ha explorado el uso de la genética química con el fin de diseccionar rutas de señalización en plantas, tanto con un objetivo de generar conocimiento fundamental, como con un objetivo más aplicado. En ambos casos, el abordaje ha consistido en el rastreo de una quimioteca de 10 mil compuestos, para encontrar aquéllos con las propiedades deseadas. Las giberelinas son hormonas vegetales que regulan distintas transiciones del desarrollo de las plantas, y permiten optimizar el patrón de crecimiento en función de las condiciones ambientales. La señalización por giberelinas supone la inducción de la degradación de las proteínas DELLA, proteínas de localización nuclear que regulan la expresión génica mediante la interacción física con más de 100 factores de transcripción. El hecho de que estas hormonas generen efectos tan amplios nos ha impulsado a la búsqueda de compuestos equivalentes a las giberelinas pero que afecten sólo a algunos procesos en concreto, sin alterar el resto. Para ello hemos realizado dos rastreos: el primero buscando compuestos agonistas de giberelinas en la expansión celular en Arabidopsis, y el segundo buscando compuestos que impidan de forma selectiva la interacción entre DELLAs y sólo un subgrupo reducido de factores de transcripción mediante el ensayo de doble híbrido en levadura. En ambos casos, los rastreos han permitido identificar moléculas con el comportamiento deseado: moléculas que activan la ruta de giberelinas de forma independiente de su receptor (es decir, en etapas posteriores de la señalización), y moléculas que inhiben la interacción de DELLAs preferentemente con ARR1, BZR1, PIF4 y KAN1. El fósforo es un componente esencial para las plantas, y éstas lo toman en forma de fosfato inorgánico, cuya fuente, la roca fosfórica, es un recurso limitado, no renovable y del que no se dispone alternativa. Debido a ello la fertilización fosfatada sufre un encarecimiento sostenido. En colaboración con Dadelos Agrosolutions S.L. se han buscado compuestos que mejoren la capacidad de asimilación de fosfato, mediante el rastreo de la quimioteca y la selección de moléculas que permitan reprimir la expresión del marcador IPS1::GUS en Arabidopsis en condiciones subóptimas de fosfato en las que este testigo está normalmente encendido. Se han podido encontrar tres compuestos que aumentan la capacidad del fosfato de reprimir la expresión génica, a la vez que aumentan la capacidad de crecimiento de las plantas en presencia de concentraciones subóptimas de fosfato. / La Genètica química és una potent eina basada en l'ús de molècules per estudiar funcions gèniques conegudes o descobrir noves funcions, mitjançant la identificació de compostos químics que interfereixin o promoguin certs processos biològics d'interès. En aquesta tesi s'ha explorat l'ús de la genètica química per tal de disseccionar rutes de senyalització en plantes, tant amb un objectiu de generar coneixement fonamental, com amb un objectiu més aplicat. En ambdós casos, l'abordatge ha consistit en el rastreig d'una quimioteca de 10 mil compostos, per trobar aquells amb les propietats desitjades. Les gibberel·lines són hormones vegetals que regulen diferents transicions del desenvolupament de les plantes, i permeten optimitzar el patró de creixement en funció de les condicions ambientals. La senyalització per gibberel·lines suposa la inducció de la degradació de les proteïnes DELLA, proteïnes de localització nuclear que regulen l'expressió gènica mitjançant la interacció física amb més de 100 factors de transcripció. El fet que aquestes hormones generin efectes tan amplis ens ha impulsat a la recerca de compostos equivalents a les gibberel·lines però que afectin només a alguns processos en concret, sense alterar la resta. Per això hem realitzat dos rastrejos: el primer buscant compostos agonistes de gibberel·lines en l'expansió cel·lular en Arabidopsis, i el segon buscant compostos que impedeixin de manera selectiva la interacció entre DELLAs i només un subgrup reduït de factors de transcripció mitjançant l'assaig de doble híbrid en llevat. En tots dos casos, els rastrejos han permès identificar molècules amb el comportament desitjat: molècules que activen la ruta de gibberel·lines de forma independent del seu receptor (és a dir, en etapes posteriors de la senyalització), i molècules que inhibeixen la interacció de DELLAs preferentment amb ARR1, BZR1, PIF4 i KAN1. El fòsfor és un component essencial per a les plantes, i aquestes ho prenen en forma de fosfat inorgànic, del qual la font, la roca fosfòrica, és un recurs limitat, no renovable i del qual no es disposa alternativa. A causa d'això la fertilització fosfatada pateix un encariment sostingut. En col·laboració amb Dadelos Agrosolutions S.L. s'han buscat compostos que millorin la capacitat d'assimilació de fosfat, mitjançant el rastreig de la quimioteca i la selecció de molècules que permetin reprimir l'expressió del marcador IPS::GUS en Arabidopsis en condicions subòptimes de fosfat en les que aquest està normalment encès. S'han pogut trobar tres compostos que augmenten la capacitat del fosfat per a reprimir l'expressió gènica, alhora que augmenten la capacitat de creixement de les plantes en presència de concentracions subòptimes de fosfat. / Grau Enguix, F. (2017). Genética Química aplicada a la señalización por giberelinas y fosfato en Arabidopsis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90566

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 / Amino acids play essential role in crop metabolism. Amino acids are nitrogen containing molecules that are used to make protein and many other molecules. They are located through-out the plant and move from organ to organ by amino acid transporters. A dozen of approximately 100 known amino acid transporters have been studied in depth and are well understood. Interestingly, not much is known about these transporters and what controls their activity. A mutant weed, Arabidopsis thaliana mutant phenylalanine insensitive growth (pig1), was identified by its ability to survive in toxic environments with high amounts of the amino acid phenylalanine and also showed an irregular metabolism of amino acids (1). The Pilot Lab and I were able to identify 18 more mutants with similar abilities to survive in toxic amino acid conditions by performing similar experiments. Seven of the new mutants were found to have mutations that effected the amino acid transporters AAP1. Using yeast incapable of growing in nitrogen restricted conditions where amino acids are the only source of nitrogen, I found that three of the variants app1 proteins we identified were able to restore growth like wild type AAP1 yeast. These variant aap1 yeast did not show the ability to transport the acid proline, while other alter versions of the aap1 protein made to alter its structure and proposed significant parts were able to increase proline transport. Plants with no or mutant aap1 proteins showed a decreased ability to uptake amino acids in addition to increased efflux of amino acids. These plants also had a higher level of amino acids in their leaves than normal wild type plants. These results obtained in both plants and yeast with altered amino acid transporter aap1 do not agree with what we understand to be the accepted function of AAP1 transporting amino acids in plant roots. The work presented in this thesis discusses how AAP1 could be involved with controlling plant amino acid metabolism. It is my hypothesis that the transporter is serving two functions by both transporting and sensing amino acids. As a sensor, AAP1 serves to maintain a proper balance of amino acids for plant metabolism. If AAP1 does this, it would be the first of its kind to be identified in plants and help enhance crop engineering for better nutrition to better feed growing populations.

Le fer est un élément essentiel pour les plantes, mais toxique lorsqu'il est accumulé en excès. Chez Arabidopsis thaliana, le transporteur IRT1 joue un rôle essentiel dans l'acquisition du Fe depuis la solution du sol, en conditions limitantes en cet élément. Le gène IRT1 est régulé transcriptionnellement par le fer conduisant à une accumulation des transcrits IRT1 dans l'épiderme des racines carencées en fer. Par homologie avec les mécanismes décrits pour le transporteur de zinc ZRT1 de levure, une régulation post-traductionnelle d'IRT1, contrôlant la stabilité de celui-ci en présence de fer a été envisagée. IRT1 a donc été utilisé comme modèle pour caractériser le système endocytique des plantes. Nos travaux révèlent que la protéine IRT1 est localisée au niveau des endosomes précoces (TGN/EE) des cellules de poils racinaires. Des approches pharmacologiques ont permis de révéler un cyclage d'IRT1 entre la membrane plasmique et le TGN/EE ainsi qu'une dégradation vacuolaire. Nous avons également pu montrer que l'internalisation et la dégradation d'IRT1 ne sont pas affectées par la disponibilité en fer et sont sous le contrôle de la monoubiquitination de résidus lysines présents dans les parties cytosoliques de la protéine IRT1. Nos travaux suggèrent un modèle où l'internalisation d'IRT1 depuis la membrane plasmique, contrôlée par monoubiquitination, permet aux plantes de se prémunir contre la toxicité des métaux transportés par IRT1. Enfin, nous avons réalisé un crible double hybride en utilisant la boucle cytosolique d'IRT1 afin d'identifier des protéines contrôlant son trafic et/ou sa dégradation. Ce crible a permis notamment l'identification d'une protéine à domaine FYVE, localisée aux endosomes et dont la caractérisation fonctionnelle a été initiée / Iron is an essential element for plants but toxic when present in excess. IRT1 is the major root iron transporter responsible for iron uptake from the soil under iron limitation in Arabidopsis thaliana. IRT1 is transcriptionally regulated by iron, resulting in a high IRT1 expression in iron-starved root epidermal cells. In addition, IRT1 was suggested to be controlled at the post-translational level, with iron affecting IRT1 protein stability, in a similar fashion with the yeast ZRT1 zinc transporter. To shed light on two poorly-understood phenomena in plants, endocytosis and degradation of plasma membrane proteins, we studied the proposed post-translational regulation of IRT1 in Arabidopsis thaliana. Interestingly IRT1 protein is found in early endosomes of root hair cells. Pharmacological approaches reveal that IRT1 cycles back and forth with the plasma membrane to perform iron uptake, and is sent to the vacuole for proper turnover. We also demonstrate that iron nutrition have no effect on the levels and the subcellular localization of IRT1 protein. The internalization of IRT1 is dependent on the monoubiquitination of several cytosol-exposed lysine residues. Together, these data suggest a model where monoubiquitin-dependent endocytosis/recycling of IRT1 keeps the plasma membrane pool of IRT1 low, to better deal with metal uptake. Finally, in order to indentify genes involves in IRT1 endocytosis/recycling and turnover, we perform a yeast two-hybrid screen with IRT1 cytosolic loop. This screen allows the identification of a FYVE domain-containing protein localized in endocytic compartment which functional characterization was initiated.