Les parois des cellules végétales sont des structures formées de divers polymères polysaccharidiques et de nombreuses protéines. Ces structures possèdent un fort dynamisme assuré par l'action spécifique des protéines pariétales (CWP). Parmi ces CWP, se situent les peroxydases de classe III (CIII Prx) formant une famille multigénique de 73 membres chez la plante modèle Arabidospis thaliana. Les CIII Prxs assurent de nombreux rôles dans le développement et les interactions avec l'environnement mais seuls de rares études ont pu attribuer une fonction précise à un membre individuel de la famille. Ceci est probablement du à la faible spécificité de ces enzymes in vitro et à la possible redondance fonctionnelle obligeant à considérer des études in situ-in vivo et l'utilisation de mutants multiples. Dans ce contexte, le premier objectif de cette thèse était d'établir un atlas d'expression spatiotemporelle par hybridation in situ (HIS) utilisant le développement de la graine d'A. thaliana comme modèle. Ce travail s'est appuyé sur une étude de micro-transcriptomique récemment publiée, permettant d'établir des comparaisons avec nos résultats d'HIS. Ils ont montré une complémentarité entre les deux approches et permis (1) d'établir des seuils de valeurs de transcriptomiques compatibles avec la sensibilité de l'HIS et (2) de définir des candidats d'intérêt présentant des profils d'expression spatiotemporelle suggérant des fonctions précises. Le deuxième objectif de cette thèse a été de caractériser fonctionnellement l'un de ces candidats (AtPRX36). Nous avons pu démontrer le rôle de cette protéine dans le contrôle du relargage du mucilage polysaccharidique lors de l'imbibition de la graine. Nos résultats suggèrent que l'ancrage localisé de cette protéine est assuré par un microdomaine pectique d'homogalacturonane globalement méthylestérifié formé par l'action d'un inhibiteur de une pectine méthylestérase (PMEI6), et permet un rôle d'AtPRX36 dans le relâchement polarisé de la paroi au niveau de ce microdomaine délimitant la zone de rupture des graines lors de l'imbibition. / Plant cell walls are complex structures, mainly composed of a large number of polysaccharides and proteins. The cell wall (CW) is a highly dynamic compartment which is remodeled by specific cell wall proteins (CWP). Among these CWP, the plant specific class III peroxidases (CIII Prxs) are a multigenic family of 73 members in Arabidopsis thaliana. CIII Prxs are involved in plant development but also in the interaction of the plant with its environment. However, only a few studies describing the exact function of individual members of this multigenic family have been published. This can be due to the weak in vitro substrate specificity of these enzymes and their possible functional redundancy, forcing to characterize their function in situ-in vivo and the use of multiple mutants. In this context, the first goal of my thesis was to establish the fine cellular spatio-temporal expression atlas of CIII Prxs throughout the seed development of Arabidopsis thaliana with an in situ hybridization (ISH) approach. This expression work (ISH) was partially permitted thanks to a recent published micro-transcriptomic study which allowed to compare both expression results (ISH vs micro-transcriptomic). This comparison showed that both techniques are complementary and it allowed to; (1) set a threshold for the ISH detection limit based on micro-transcriptomic expression values, and (2) to define specific spatio-temporal expression pattern that helps to select CIII Prx candidates for functional study. The second objective was to characterize the function of one of these CIII Prxs candidates (AtPRX36). We were able to determine the role of AtPRX36 during mucilage extrusion of imbibed seeds.Our results suggest that AtPRX36 is anchored within a globally methylesterified pectins CW micro-domain, putatively formed by the action of a pectin methylesterase inhibitor (PMEI6). This CW micro-domain is needed for the CW loosening action of AtPRX36 to permit CW polarized rupture during seed imbibition.

ABSTRACT: Formation of organ shape is an intriguing yet largely unanswered question in developmental biology. Shapes arise as a result of tightly controlled spatial variation in the rates and directions of tissue expansion over the course of development; therefore, quantifying these growth patterns could provide information about the underlying mechanisms of morphogenesis. Here we present a novel technique and computational tools for quantifying growth and shape changes in developing leaves, with a few unique capabilities. This includes the ability to compute growth from three-dimensional (3D) coordinates, which makes this the first method suitable for studying leaf growth in species or mutants with non-flat leaves, as well as small leaves at early stages of development, and allows us to simultaneously capture 3D shape changes. In the following, we apply these methods to study growth and shape changes in the first rosette leaf of Arabidopsis thaliana. Results reveal clear spatiotemporal patterns in growth rates and directionality, and tissue deformation maps illustrate an intricate balance involved in maintaining a relatively flat leaf surface in wild type leaves. Semi-automated tools presented make a high throughput of data possible with this method, and algorithms for generating mean maps of growth will make it possible to perform standardized comparative analyses of growth patterns between wild type and mutants and/or between species. The methods presented in this thesis will therefore be useful for studying leaf growth and shape, to further investigate the mechanisms of morphogenesis.   RÉSUMÉ: Comment un organe acquiert sa forme particulière au cours du développement est une question intéressante mais largement non résolue. La forme d’un organe résulte de la façon dont les taux et directions de croissance de ses tissues varient dans l’espace et dans le temps. Quantifier les motifs de croissance est donc nécessaire pout élucider les mécanismes sous-jacents de la morphogenèse. Nous présentons ici une nouvelle méthodologie pour quantifier la croissance et les changements de forme dans les feuilles en développement. Cette méthodologie s’appuie sur le développement de nouvelles techniques expérimentales et de programmes informatiques, et présente des avantages uniques : la croissance de la surface des feuilles et le changement de forme peuvent être analysés en trois dimensions (3D), pour une longue période et de large déformations. De plus l’analyse de multiples échantillons permet de générer une cartographie moyenne des motifs de croissance à la surface des feuilles au cours de leur développement, ainsi qu’une description quantitative de la déformation des tissus sous l’effet de leur croissance. Dans cette thèse, nous présentons les résultats de croissance et de changements de forme de la première feuille de rosette d'Arabidopsis thaliana au cours de son développement. Les cartes moyennes de croissance révèlent des motifs spatio-temporels évidents tant pour les taux que pour les directions de croissance. De plus, la description de la déformation des tissus démontre l'équilibre complexe impliqué dans le maintien d'une surface relativement plane dans les feuilles. La méthode proposée et les logiciels associés permettra d’effectuer des analyses comparative de la croissance entre feuilles de type sauvage et feuilles de mutants aux formes altérées, afin d’élucider les mécanismes de la morphogenèse foliaire.

Plants respond to pathogen attack by inducing a coordinated resistance strategy, which results in the expression of defense gene products. When a plant-pathogen interaction results in disease establishment, parasite colonization is caused by a delayed plant defense response, not due the absence of any response. Thus, the speed and intensity of the plant response and intracellular signalling determines the outcome of a plant-pathogen interaction. The acceleration of plant responses by the application of resistance inducers could provide a commercially, biologically and environmentally feasible alternative to existing pathogen control methods. Lipopolysaccharides are amphipathic lipoglycans that are attached to the outer bacterial membrane by a lipidic entity inserted into the bacterial phospholipid monolayer, with the saccharidic part oriented towards the exterior. The general structure of this compound is comprised of an anchor named lipid A associated with a core polysaccharide, which bears an O-antigen domain. LPS has been described as one of the pathogen-associated molecular patterns (PAMPs) capable of eliciting the activation of the plant innate immune system. LPS present in the outer membranes of plant growth-promoting rhizobacteria (PBPR) are major determinants of induced systemic resistance (ISR). In addition, LPS may function as an activator of systemic acquired resistance (SAR), providing non-specific immunization against later infection. Evidence suggests that LPS may advance plant disease resistance using the mechanism of ISR or SAR through its application to plants as a sensitizing agent, priming them to respond more effectively to subsequent pathogen attack. Phosphorylation plays a major role during the plant defense response, exemplified by its phosphorylation of transcription factors, required for the expression of defense-related genes. One of the most extensively documented phosphorylation responses is that of MAP kinase activation by phosphorylation in response to elicitation by race-specific and non-racespecific elicitors in various plant species.Proteins that undergo differential phosphorylation as a result of elicitation could be components of signal transduction pathways which connect pathogen perception with defense responses. Thus the identification of protein kinases, protein phosphatases and their substrates is essential in the elucidation of plant defense responses. The hypothesis behind this dissertation is that LPS elicitation results in alterations in the phosphorylation profile of Arabidopsis thaliana proteins. In this study, LPS was extracted from the cell walls of Burkholderia cepacia, a bacterial endophyte, and characterized by SDS-PAGE. The exposure of Arabidopsis callus culture cells to LPS resulted in distinctive changes in the phosphoprotein profile of the cells. Radioactive phosphorous labelling of proteins provided evidence that phosphorylation occurs in Arabidopsis following LPS perception, as part of a defense response related to LPS elicitation. Further investigation of differential protein phosphorylation via immunoblotting with antiphosphotyrosine antibodies revealed that tyrosine phosphorylation of Arabidopsis proteins occurs in response to LPS. One of the tyrosine-phosphorylated proteins was found to be a 42 kDa kinase, activated in response to LPS elicitation. The identity of the kinase as a mitogen-activated protein (MAP) kinase was confirmed by immunoblotting with anti-active MAP kinase antibodies. In addition, an assay of MAP kinase activity demonstrated the ability of the LPS-responsive MAP kinase to phosphorylate the ERK-MAP kinase substrate Elk1. In terms of the global phosphoproteome of Arabidopsis in response to LPS, phosphopeptides were purified from a crude protein digest by immobilized metal affinity chromatography and analyzed by liquid chromatography-tandem mass spectrometry (LCMS/ MS). While LC indicated both quantitative and qualitative differences resulting from LPS elicitation, no peptides could be positively identified as phosphopeptides by MS analysis. This work can however be repeated with further precautions to prevent the loss of phosphate groups prior to analysis. The results obtained in this study indicate that LPS causes specific alterations in Arabidopsis protein phosphorylation as a post-translational modification in response to the perception of LPS during a plant-pathogen interaction, proving the original hypothesis. / Prof. I.A. Dubery

La presente tesis doctoral se enmarca dentro del tema de la homeostasis de cationes en células vegetales. El conocimiento de los transportadores involucrados en la homeostasis de cationes y sus mecanismos de regulación, puede tener aplicaciones biotecnológicas para el desarrollo de plantas tolerantes a estreses abióticos como la salinidad o el pH ácido. El objetivo general del presente trabajo es determinar y estudiar los mecanismos implicados en la homeostasis de pH en Arabidopsis thaliana. La metodología empleada ha consistido, por un lado, en la realización de micromatrices para estudiar la respuesta transcripcional al estrés por ácido acético. Por otro lado, se ha llevado a cabo un rastreo genético para encontrar genes determinantes de la tolerancia a este estrés. Los resultados del análisis transcripcional han servido para identificar posibles genes cuya expresión es relevante para la homeostasis de pH. De hecho, estos resultados han servido de base para otra tesis doctoral realizada en el departamento. Los resultados del rastreo genético han permitido aislar y caracterizar un mutante dominante negativo (wat1-1D: "weak acid tolerant") que expresa una versión truncada de la adaptina ß3 de A. thaliana y que posee una alteración en la homeostasis de pH y de otros cationes monovalentes, como el sodio o el potasio. / Niñoles Rodenes, R. (2011). Biología molecular de la regulación de la homeostasis de pH en Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11301 / Palancia

Las poliaminas son pequeñas moléculas de naturaleza policatiónica que se encuentran de manera natural todos los organismos estudiados. Estas sustancias son esenciales para el crecimiento de los organismos, no obstante, a día de hoy se desconoce cual es exactamente su función. Por otro lado, altas concentraciones de estas sustancias son tóxicas y, de igual modo, los mecanismos que median su toxicidad también son desconocidos. La Norespermidina (NE), una poliamina infrecuente que no se encuentra de manera natural en la mayoría de organismos, ha sido usada en este laboratorio por su naturaleza policatiónica como agente de selección de mutantes que presenten tolerancia a cationes tóxicos (Alejandro et al., 2007). Debido a ello, se han querido esclarecer las bases moleculares que median su toxicidad. El tratamiento con NE reduce los niveles de proteínas poliubiquitiladas, sugiriendo que esta sustancia puede interferir con las reacciones de poliubiquitilación. Efectivamente, ensayos in vitro han demostrado que el tratamiento con NE inhibe la reacción de poliubiquitilación, posiblemente a nivel de las enzimas E3 ubiquitin-ligasas. Esto a su vez provoca una inhibición del sistema de degradación de proteínas ubiquitín-26S-proteasoma, que altera los niveles de aminoácidos disponibles para la síntesis de proteínas (Suraweera et al., 2012). Esta disminución de los niveles de aminoácidos disponibles inhibe la síntesis de proteínas en general y provoca una consecuente inhibición del crecimiento que puede desencadenar muerte celular. / Sayas Montañana, EM. (2014). Mecanismos de toxicidad de poliaminas: inhibición del recambio de proteínas [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/38108 / TESIS

The lipoxygenase (LOX) pathway plays an important role in the oxidative metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). The LOX pathway functions in conjugation with hydrolysis by fatty acid amide hydrolase (FAAH) and to produce oxidized NAEs during seed germination and early seedling development. When Arabidopsis seedlings were grown in low micromolar concentrations of lauroylethanolamide (NAE 12:0), growth retardation and elevated endogenous PU-NAE levels were observed due to the competitive inhibition of LOX by NAE 12:0. The elevated levels of endogenous PU-NAEs were more pronounced in genotypes with reduced NAE hydrolase capacity (faah knockouts), and less evident with overexpression of FAAH. Alterations in PU-NAE metabolism were studied in seedlings of various lox and FAAH mutants. The partitioning of PU-NAEs into oxylipin metabolites was exaggerated in the presence of exogenous linolenoylethanolamide (NAE18:3) and resulted in bleaching of cotyledons. The bleaching phenotype was restricted to a narrow developmental window (3-to-5 days after sowing), and was attributed to a reversible disruption of thylakoid membranes in chloroplasts. Biochemical and genetic evidence suggested that 9-hydro(pero)xy and 13-hydro(pero)xy octadecatrienoylethanolamides (9- and 13-NAE-H(P)OT), but not their corresponding hydro(pero)xy free fatty acids, induced cotyledon bleaching. The LOX-mediated metabolites of NAE18:3 shared some overlapping effects on seedling development with those of linoleoylethanolamide (NAE18:2) such as a reduction in seedling root growth. On the other hand, NAE18:3 oxylipin metabolites also exhibited distinct effects during seedling development such as the inhibition of photomorphogenesis. Biochemical and genetic evidence indicated that a LOX-mediated metabolite of NAE18:2, 9-hydro(pero)xy octadecadienoylethanolamide (9-NAE-H(P)OD), acted as a potent negative regulator of seedling root development, and this depended on an intact abscisic acid (ABA) signaling pathway. Synergistic inhibition of root elongation between 9-NAE-H(P)OD and ABA was restricted to a narrow developmental window (3-to-5 d after sowing) of seedling development. Genetic evidence with Arabidopsis mutants in ABA synthesis (aba1, aba2), perception (pyr1, pyl2, pyl4, pyl5, pyl8) and transcriptional regulation (abi3-1) suggested that negative regulation of growth by 9-NAE-H(P)OD likely was mediated through an increase in ABA synthesis, and this was confirmed biochemically. Induction of a secondary dormancy program in Arabidopsis seedlings by environmental stresses also requires an intact ABA signaling cascade, and our study has shown that this regulatory seedling program is dependent, in large part, on NAE oxylipin formation. Together, results presented here indicated that LOX-mediated metabolites of NAE18:3 and NAE18:2 in Arabidopsis represent a newly-discovered group of bioactive metabolites, and their accumulation during the embryo-to-seedling transition of plant development may act to synchronize seedling establishment with environmental cues.

N-Acylethanolamines (NAEs) are fatty acid derivatives in plants that negatively influence seedling growth. N-Lauroylethanolamine (NAE 12:0), one type of NAE, inhibits root length, increases radial swelling of root tips and reduces root hair numbers in a dose dependent manner in Arabidopis thaliana L. (ecotype Columbia). A forward genetics approach was employed by screening a population of T-DNA “activation-tagged” developed by the Salk Institute lines for NAE resistance to identify potential genes involved in NAE signaling events in Arabidopsis thaliana L. (ecotype Columbia). Seeds of the activation tagged lines were grown at 0, 25, 30, 50, 75 and 100 µM N-lauroylethanolamime (NAE 12:0). Ten plants which displayed NAE tolerance (NRA) seedling phenotypes, compared with wildtype (Columbia, Col-0) seedlings were identified. I focused on one mutant line, identified as NRA 25, where the tolerance to NAE 12:0 appears to be mediated by a single dominant, nuclear gene. Thermal asymmetric interlaced (TAIL) PCR identified the location of the T-DNA insert as 3.86 kbp upstream of the locus At1g68510. Quantitative PCR indicated that the transcript level corresponding to At1g68510 is upregulated approximately 20 fold in the mutant relative to wildtype. To determine whether the NAE tolerance in NRA 25 is associated with overexpression of At1g68510 I created overexpressing lines of At1g68510 with and without GFP fusions behind the 2X35S CaMV promoter. As predicted, results with overexpressing lines of At1g68510 also exhibited enhanced resistance to NAE when compared with the wildtype. Confocal images of the fusion proteins suggest that GFP-At1g68510 is concentrated in the nucleus and this was confirmed by counterstaining with 4', 6-Diamidino-2-phenylindol (DAPI). Futhermore, At1g68510 overexpressing lines and NRA 25 line also exhibited tolerance to abscisic acid (ABA) during seedling germination. The findings suggests that At1g68510 overexpression mediates seedling tolerance to both ABA and NAE, a mechanism independent of fatty acid amide hydrolase in which its overexpression leads to NAE resistance but hypersensitivity to ABA. The next steps are to identify the association of At1g68510 with specific genomic regions or interacting proteins that may be additional components of NAE signaling in plants.

Plant natriuretic peptides (PNPs) comprise a novel class of hormones that share some sequence similarity in the active site with their animal analogues that function as regulators of salt and water balance. A PNP present in Arabidopsis thaliana (AtPNP-A) has been assigned a role in abiotic and biotic stress responses, and the recombinant protein has been demonstrated to elicit cyclic guanosine monophosphate (cGMP)-dependent stomatal guard cell opening, regulate ion movements, and induce osmoticum-dependent water uptake. Although the importance of the hormone in maintaining ion and fluid homeostasis has been established, key components of the AtPNP-A-dependent signal transduction pathway remain unknown. Since identification of the binding partners of AtPNP-A, including its receptor(s), is fundamental to understanding the mode of its action at the molecular level, comprehensive protein-protein interaction studies, involving yeast two-hybrid screening, affinity-based assays, protein cross-linking and co-immunoprecipitation followed by mass spectrometric (MS) analyses have been performed. Several candidate binding partners of AtPNP-A identified with at least two independent methods were subsequently expressed as recombinant proteins, purified, and the specificity of their interactions with the recombinant AtPNP-A was verified using surface plasmon resonance. Several specific binary interactants of AtPNP-A were subjected to functional assays aimed at unraveling the consequences of the interactions in planta. These experiments have revealed that reactive oxygen species (ROS) are novel secondary messengers involved in the transduction of AtPNP-A signal in suspension-cultured cells of A. thaliana (Col-0). Further insight into the AtPNP-A dependent signalling events occurring in suspension-cultured cells in ROS-dependent or ROS-independent manner have been obtained from the large-scale proteomics study employing tandem mass tag (TMT) labelling followed by MS analysis to identify and relatively quantify proteins that are differentially expressed upon the treatment with nano- and picomolar concentrations of the biologically active AtPNP-A peptide at different time-points post-treatment. Characterization of both the AtPNP-A interactome and AtPNP-A dependent proteome afforded novel insights into the signal transduction pathways altered by PNPs and shed new light on the mechanisms by which these candidate interactants operate. Taken together, indications are that PNP dependent mechanisms can be harnessed for possible biotechnological applications.

Here, we embarked a bioprospecting project that focuses on the isolation and characterization of plant root endophytes, collected from the Thar Desert. A total of 381 endophytes were isolated and based on their 16S rRNA gene sequences, genus Bacillus (58 strains) was identified as the major taxon and only endophytes from this genus were isolated from all plant types. Of the 58 Bacillus strains, only 16 strains were selected for screening of plant growth promotion traits such as P and Zn solubilization, indole-3-acetic acid and siderophore production, and antimicrobial activity. Based on the presence of specific plant growth promotion traits 10 strains were shortlisted for further in vitro screening with A. thaliana; to confirm that these bacteria can confer resilience to plants under salt stress conditions. B. circulans (PK3-15 and PK3-109), B. cereus (PK6-15) B. subtilis (PK3-9) and B. licheniformis (PK5-26) displayed the ability to increased the fresh weight of A. thaliana under salt stress conditions by more than 50 % compared to the uninoculated control. An interesting observation was that B. circulans (PK3-109) (shown to produce IAA exopolysaccharide) and B. circulans (PK3-138) (shown to produce IAA) in vitro results were substantially different as B. circulans (PK3-138) decreased the total fresh weight of A. thaliana by 47 %, whilst B. circulans (PK3-109) was one of the best performing strains. Thus, the genomes of these two strains were sequences to unravel the molecular versatility of B. circulans strains, specifically with respect to their interaction with plants. Most of the genome of these strains is identical but the most interesting feature was the presence of 1/ the DegS–DegU two-component system that is known to mediate the salt stress response and DegU also represses toxin wapA similar to antitoxin wapI, and 2/ YxiG, a gene in the unique orthogroup of PK3-109 was found to be linked to WapI. Thus, PK3-138 substantially decreasing the total fresh weight of A. thaliana under salt stress conditions suggests that the toxic activity of a toxin such as WapA is not effectively ameliorated by the antitoxin such as WapI in the absence of a functional YxiG gene.

Tesis por compendio / [ES] La proteína quinasa GCN2 es una proteína conservada en todos los eucariotas implicada en el control de la traducción en condiciones de estrés. Está considerada un punto clave en el control de la homeostasis celular y un sensor de distintas condiciones de estrés. El estrés que inició su caracterización en levaduras y células animales es el ayuno de aminoácidos, pero recientemente se ha observado activación de este sistema ante multitud de estreses tanto bióticos como abióticos. El sistema GCN se ha descrito ampliamente en Saccharomyces cerevisiae: GCN2 se une a las proteínas GCN1 y GCN20, permitiendo la activación de la quinasa en situaciones de ayuno de aminoácidos. GCN2 se activa por tRNA no cargados, y posteriormente fosforila al factor de traducción eIF2¿, lo que conlleva una reducción de la síntesis global de proteínas, pero también una mayor traducción de mRNA específicos, como los que codifican a GCN4. Este factor de transcripción regulará la expresión de nuevos genes, lo que permite que la célula pueda iniciar una respuesta de adaptación al estrés. En plantas se desconoce con detalle como el sistema GCN contribuye a mitigar el estrés y controlar la homeostasis. Las tres proteínas conocidas de este sistema tienen homólogos en Arabidopsis. Diversos estudios indican que el mecanismo de actuación de GCN2 en plantas presenta muchas incógnitas. Mientras que la quinasa GCN2 de plantas se activa bajo diferentes situaciones de estrés, la participación de los homólogos de GCN1 y GCN20 en estos procesos es controvertida, y recientemente se ha propuesto un nuevo papel para GCN1 en la traducción, independiente de GCN2. El homólogo de GCN1 en plantas está implicado en la inmunidad innata y adquirida y sus líneas mutantes presentan fenotipos muy diferentes a los de las líneas mutantes en GCN2. La relación funcional entre estos dos genes sigue siendo difícil de definir en plantas. En esta tesis, demostramos que, aunque los genes GCN1 y GCN2 de Arabidopsis son necesarios para mediar la fosforilación de eIF2¿ tras tratamientos con glifosato, inhibidor de la biosíntesis de aminoácidos aromáticos, los mutantes de pérdida de función de ambas líneas desarrollan distintos fenotipos de raíz y cloroplasto. Los experimentos de microscopía electrónica revelan que los mutantes en GCN1, pero no en GCN2, se ven afectados en la biogénesis de cloroplastos, lo que explica el fenotipo macroscópico observado previamente para estos mutantes. Los mutantes en GCN1 presentan una compleja reprogramación transcripcional que afecta, entre otros, a las respuestas relacionadas con los mecanismos de defensa, fotosíntesis y al correcto plegamiento de las proteínas. Por otro lado, mostramos que ninguno de los cinco genes homólogos a GCN20 en Arabidopsis en necesario para la fosforilación de eIF2¿. Además, los fenotipos bajo estrés abiótico de plantas mutantes en los mismos, y el desarrollo de sus cloroplastos, sugiere que GCN20 está funcionalmente relacionado con GCN1, pero no con GCN2, algo que se confirma ya que los mutantes gcn1 y gcn20 comparten una reprogramación transcripcional similar, afectando a la fotosíntesis y a las respuestas frente al estrés. Identificamos la proteína quinasa GCN2 como un componente celular que fomenta la acción del glifosato en Arabidopsis. Los estudios comparativos que utilizan plántulas mutantes de pérdida de función de GCN2 muestran que el programa molecular que la planta despliega después del tratamiento con el herbicida no está teniendo lugar. Además, las plantas adultas gcn2 muestran una menor inhibición de la fotosíntesis, y acumulan menos ácido siquímico que las de tipo silvestre después del tratamiento con glifosato. Algo similar ocurre tras el tratamiento con luz ultravioleta UV-B, donde mutantes de pérdida de función son más resistentes. La activación de GCN2 ante este estrés es independiente del fotorreceptor UV-B (UVR8) y de sus componentes de señalización aguas abajo y de la vía de señalización de estrés de las MAP quinasas. / [EN] The GCN2 protein kinase is a conserved protein in all eukaryotes involved in translation control under stress conditions. It is considered a key point in the control of cellular homeostasis and a sensor for a wide variety of stress conditions. Aminoacid fasting was the stress that started its characterization in yeast and animal, but recently activation of this system has been observed in both biotic and abiotic stresses. The GCN system has been extensively described in Saccharomyces cerevisiae: GCN2 binds to GCN1 and GCN20 proteins, allowing kinase activation in aminoacid fasting situations. GCN2 is activated by uncharged tRNAs, and subsequently phosphorylates the translation factor eIF2¿, leading to a reduction in overall protein synthesis, but also a greater translation of specific mRNAs, such as those encoding GCN4. This transcription factor will regulate the expression of new genes, allowing the cell to initiate an adaptive response to stress. In plants, it is not deeply known how the GCN system helps to alleviate stress and control homeostasis. All three known proteins in this system have homologs in Arabidopsis. Some studies indicate that the mechanism of action of GCN2 in plants presents many gaps. While plant GCN2 kinase is activated under different stress situations, the involvement of GCN1 and GCN20 homologs in these processes is controversial, and recently it has been proposed a new role for GCN1 in translation, independent from GCN2. The GCN1 homolog in plants is involved in innate and acquired immunity and its mutant lines present very different phenotypes from those of the GCN2 mutant lines. The functional relationship between these two genes is difficult to define in plants. In this thesis, we prove that, although the Arabidopsis GCN1 and GCN2 genes are necessary to mediate in the phosphorylation of eIF2¿ after treatments with glyphosate, an inhibitor of aromatic aminoacid biosynthesis, the loss of function mutants of both lines develop different phenotypes of root and chloroplast. Electron microscopy experiments reveal that the mutants in GCN1, but not in GCN2, are affected in chloroplast biogenesis, which explains the macroscopic phenotype previously observed for these mutants. The mutants in GCN1 present a complex transcriptional reprogramming that affects, among others, the responses related to defense mechanisms, photosynthesis and the correct folding of proteins. On the other hand, we show that none of the five GCN20 homologous genes in Arabidopsis is necessary for the phosphorylation of eIF2¿. Furthermore, the phenotypes under abiotic stress of mutant plants in them, and the development of their chloroplasts, suggest that GCN20 is functionally related to GCN1, but not to GCN2, which is confirmed because the gcn1 and gcn20 mutants share a similar transcriptional reprogramming and affects photosynthesis and stress responses. We identify the GCN2 protein kinase as a cellular component that promotes the action of glyphosate in Arabidopsis. Comparative studies using GCN2 loss-of-function mutant seedlings show that the molecular program that the plant develops after the treatment with the herbicide is not taking place. Furthermore, adult gcn2 plants show less inhibition of photosynthesis, and accumulate less shikimic acid than wild-type ones after glyphosate treatment. Something similar happens after treatment with UV-B ultraviolet light, where loss-of-function mutants are more resistant. Activation of GCN2 in the face of this stress is independent of the UV-B photoreceptor and its downstream signaling components and the stress signaling pathway of MAP kinases. / [CA] La proteína quinasa GCN2 és una proteïna conservada en tots els organismes eucariotes implicada en el control de la traducció en condicions d'estrés. Està considerada un punt clau en el control de l'homeòstasis cel.lular i es un sensor de diferents i variades condicions d'estrés. L' estrés que va iniciar la seua caracterització en llevats i cèl.lules animals és el dejuni d' aminoàcids, però recentment s'ha observat l'activació d'aquest sistema davant de multitud d'estressos tant biòtics com abiòtics. El sistema GCN ha segut descrit ampliament en Saccharomyces cerevisiae: GCN2 s'uneix a les proteïnes GCN1 y GCN20, permitint l'activació de la quinasa en situacions de dejuni d'aminoàcids. GCN2 s'activa per tRNA no carregats, i posteriorment fosforila el factor de traducció eIF2¿, donant lloc a una reducció de la síntesi global de proteïnes, però també una major traducció de mRNA específics, com els que codifiquen a GCN4. Aquest factor de transcripció regularà l'expressió de nous gens, el que permet que la cèl·lula puga iniciar una resposta d'adaptació a l'estrés. En plantes es desconeix amb detall com el sistema GCN contribueix a mitigar l'estrés i controlar l'homeòstasi. Les tres proteïnes conegudes d'aquest sistema tenen homòlegs en Arabidopsis. Diversos estudis indiquen que el mecanismo d'actuació de GCN2 en plantes presenta moltes incògnites. Mentres que la quinasa GCN2 de plantes es activada en diferents situacions d'estrés, la participació dels homòlegs de GCN1 i GCN20 en aquests processos és controvertida, i recentment s'ha proposat un nou paper per a GCN1 en la traducció, independent de GCN2. L'homòleg de GCN1 en plantes està implicat en la inmunidad innata i adquirida i les seues línies mutants presenten fenotips molt diferents als de les línies mutants en GCN2. La relació funcional entre estos dos gens continua sent difícil de definir en plantes. En esta tesi, demostrem que, encara que els gens GCN1 i GCN2 d' Arabidopsis són necessaris per a donar lloc a la fosforilació d'eIF2¿ després de ser tractada amb glifosato, inhibidor de la biosíntesi d' aminoàcids aromàtics, els mutants de pèrdua de funció d'ambes línies desenvolupen distints fenotips d'arrel i cloroplast. Els experiments de microscòpia electrònica revelen que els mutants en GCN1, però no en GCN2, es veuen afectats en la biogènesis de cloroplastos, el que explica el fenotip macroscòpic observat prèviament per a estos mutants. Els mutants en GCN1 presenten una complexa reprogramació transcripcional que afecta, entre d'altres, a les respostes relacionadaes amb els mecanismes de defensa, fotosíntesi i al correcte plegament de les proteïnes. D'altra banda, demostrem que ningun dels cinc gens homòlegs a GCN20 en Arabidopsis és necessari per a la fosforilació d' eIF2¿. Ademés, els fenotips baix estrés abiòtic de plantes mutants en ells mateix, i el desenvolupament dels seus cloroplasts, sugereixen que GCN20 està funcionalment relacionat amb GCN1, però no amb GCN2, cosa que es confirma ja que els mutants gcn1 i gcn20 compartixen una reprogramació transcripcional similar, afectant a la fotosíntesi i les respostes davant l'estrés. Identifiquem la proteïna quinasa GCN2 com un component cel.lular que fomenta l'acció del glifosato en Arabidopsis. Els estudis comparatius que utilitzen plántules mutants de pèrdua de funció de GCN2 mostren que el programa mol.lecular que la planta desplega després del tractament amb herbicida no está ocorreguent. Ademés, les plantes adultes gcn2 presenten una menor inhibició de la fotosíntesi, i acumulen menys àcid siquímic que les de tipus silvestre després de ser tractades amb glifosato. S'obté un resultat semblant després del tractament amb llum ultravioleta UV-B, on els mutants de pèrdua funció són més resistents. La activación de GCN2 ante este estrés es independiente del fotorreceptor UV-B (UVR8) y de sus componentes de señalización aguas abajo y de la vía de señalización de estrés de las MAP quinasas. / Faus Ferrer, MI. (2020). Caracterización del sistema GCN en plantas mediante la utilización de mutantes de pérdida de función [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153809 / TESIS / Compendio