Pheromone lures and trap crops are appealing pest management tools that use insect and/or plant volatiles to reduce pest populations on crops of interest. Generating pheromone-emitting trap plants may allow for a continuing and highly-specific attraction of insect pests without repeated and costly application of synthetic pheromones. These trap plants may also be used to develop area-wide pest management strategies. As a proof-of-principle study we tested the possibility of producing the pheromone of the crucifer flea beetle Phyllotreta cruciferae in transgenic plants. P. cruciferae is an important pest of Brassica crops. In the presence of a host plant, males emit an aggregation pheromone, which attracts both males and females. Himachaladiene, a sesquiterpene, has been identified as a key component of the aggregation pheromone of P. cruciferae. In a close relative, Phyllotreta striolata, the compound is synthesized by a two-step pathway with an isoprenyl diphosphate synthase (PsIDS3) making (Z,E)-farnesyl diphosphate (FPP), which is converted by a terpene synthase (PsTPS1) to himachaladiene. Transient transformation of N. benthamiana with PsIDS3-TPS1 co-localized to the plastid resulted in the emission of himachaladiene and other known PsTPS1 products. Daily emissions of himachaladiene were approximately 1 µg per plant, which is six-fold higher than emissions from individual male flea beetles. Stable transformation of Arabidopsis thaliana with the same vector construct resulted in transgenic plants that expressed PsTPS1 and PsIDS3 transcripts, but no himachaladiene or other PsTPS1 products were present in volatile collections or leaf extracts of these plants. Moreover, no PsTPS1 enzyme activity was observed, indicating that post-transcriptional/translational effects prevent proper expression or targeting of functional PsIDS3 and/or PsTPS1 proteins in A. thaliana. Overall, this study demonstrates that the key component of the P. cruciferae aggregation pheromone, himachaladiene, can be transiently produced and emitted in a plant system at rates that are biologically relevant for insect attraction. However, further work is required for the stable production of the pheromone in plants. In addition, preliminary results are presented for the development of simple two-choice arenas that may allow for assessment of the movement of beetles toward host plant leaf tissue. This work can inform future efforts in developing methods for the economic production of himachaladiene in a plant system or the establishment of transgenic plants for the production and deployment of himachaladiene in a field setting. / Master of Science / The crucifer flea beetle is an important pest of vegetable and oilseed Brassica crops such as broccoli, cabbage and canola. Feeding by beetles has its greatest impact on crop health and yield in the early spring, when adult beetles emerge from overwintering sites and feed on newly- emerging Brassica seedlings. Currently these insects are controlled using broad spectrum insecticides. A general awareness of the negative aspects of insecticides drives the search for alternative pest management strategies that could diversify our management strategies and reduce reliance on insecticides. Previous work has found that the crucifer flea beetle navigates to its host plants, in part, through plant-emitted volatiles. After locating the plant host, males emit a volatile aggregation pheromone that when blended with host plant volatiles increases attraction. Here work towards the development of a specialized trap crop is presented. Plants were engineered to emit a key component of the crucifer flea beetle aggregation pheromone. In an engineered non-host plant, Nicotiana benthamiana, transient production of the aggregation pheromone was established. However, in an engineered Brassica plant, Arabidopsis thaliana, no aggregation pheromone was detected despite evidence of the presence and expression of the required biosynthetic genes for its production. A discussion on alternative engineering strategies for A. thaliana is presented. In addition, preliminary results are presented for the development of a simple behavior assay to assess the attraction of beetles toward different smells. This work can inform future efforts aimed at developing methods for the economic production of the aggregation pheromone in a plant system or the establishment of plants for the production and deployment of the aggregation pheromone in a field setting.

Eukaryotic organisms require the ability to respond to their environments. They do so by utilizing signal transduction pathways that allow for signals to effect final biological responses. Many times, these final responses require new gene expression events that have been stimulated or repressed within the nucleus. Thus, much of the understanding of signal transduction pathways converges on the understanding of how signaling affects gene expression alterations (Kumar et al., 2004). The regulation of gene expression involves the modification of chromatin between condensed (closed, silent) and expanded (open, active) states. Histone modifications, such as acetylation, can determine the open versus closed status of chromatin. The PHD (Plant HomeoDomain) finger is a structural domain primarily found in nuclear proteins across eukaryotes. This domain specifically recognizes the epigenetic marks H3K4me2 and H3K4me3, which are di- and tri-methylated lysine 4 residues of Histone H3 (Loewith et al., 2000; Kuzmichev et al., 2002; Vieyra et al. 2002; Shiseki et al., 2003; Pedeux et al., 2005, Doyon et al., 2006). It is estimated that there are ~150 proteins that contain the PHD finger in humans (Solimon and Riabowol, 2007). The PHD finger is conserved in yeast and plants, however an analysis of this domain has only been performed done in Arabidopsis thaliana (Lee et al., 2009). The work presented in this report aims to extend the analysis of this domain in plants by identifying the PHD fingers of the crop species Oryza sativa (rice). In addition, a phylogenetic analysis of all PHD fingers in Arabidopsis and rice was undertaken. From these analyses, it was determined that there are 78 PHD fingers in Arabidopsis and 70 in rice. In addition, these domains can be categorized into classes and groups by defining features within the conserved motif. In a separate study, I investigated the function of two of the PHD finger proteins from Arabidopsis, ING1 (INhibitor of Growth1) and ING2. In humans, these proteins can be found in complexes associated with both open and closed chromatin. They facilitate chromatin remodeling by recruiting histone acetyltransferases and histone deacetylases to chromatin (Doyon et al., 2006, Pena et al., 2006). In addition, these proteins recognize H3K4me2/3 marks and are believed to be "interpreters" of the histone code (Pena et al., 2006, Shi et al., 2006). To understand the function of ING proteins in plants, I took a reverse genetics approach and characterized ing1 and ing2 mutants. My analysis revealed that these mutants are altered in time of flowering, as well as their response to nutrient and stress conditions. Lastly, I was able to show that ING2 protein interacts in vitro with SnRK1.1, a nutrient/stress sensor (Baena-Gonzalez et al., 2007). These results indicate a novel function for PHD proteins in plant growth, development and stress response. / Master of Science

We are using the yeast two-hybrid (Y2H) system to identify novel protein-protein interactions (PPI) relevant to wood formation. Bait proteins for Y2H binary assays and screening against a xylem cDNA prey library were selected from approximately 400 Populus trichocarpa genes that are at least 8-fold more highly expressed in differentiating secondary xylem versus phloem-cambium, and designated here as poplar biomass (PB) genes. Here we report some of the interactions involving selected PB proteins and efforts to characterize their functions in Populus and Arabidopsis. Members of the ROP GTPase family, PB15 in poplar and ROP11 in Arabidopsis, interact with the domain of unknown function (DUF) 620 (DUF620) proteins (e.g., PB129 in poplar). Ectopic co-expression of PB15 and PB129 in Arabidopsis caused outgrowths at the base of flower pedicels and altered leaf morphology. Interestingly, the co-expression phenotype could not be observed in transgenic plants that are only expressing either one of the interacting partners separately. Transgenics altered in expression of PB15 and/or PB129 were prepared in Populus and characterization of transgenic trees will be performed in greenhouse and field. In addition to DUF620 family proteins, ROP11 also interacts with the COP9 subunit CSN5A in Arabidopsis. We confirmed the interaction of ROP11 and CSN5A in Y2H and employed available mutants for ROP11 and CSN5A in Arabidopsis to genetically characterize this interaction. Surprisingly, loss of ROP11 was found to rescue the csn5a-2 pleiotropic phenotype. Ectopic expression of a ROP11 dominant negative mutant in the csn5a-2 background also complemented the stunted growth phenotype. Transcript analysis and gel blot assays showed that CSN5A transcript levels remained unchanged in all rescue lines, whereas CSN5A protein levels increased relative to WT. Taken together, we concluded that ROP11 negatively regulate CSN5A protein level in plant by some as yet unknown mechanism. / Ph. D.

Computational biology enabled us to manage vast amount of experimental data and make inferences on observations that we had not made. Among the many methods, predicting metabolic functions with genome-scale models had shown promising results in the recent years. Using sophisticated algorithms, such as flux balance analysis, OptKnock, and OptForce, we can predict flux distributions and design metabolic engineering strategies at a greater efficiency. The caveat of these current methods is the accuracy of the predictions. We proposed using flux balance analysis with flux ratios as a possible solution to improving the accuracy of the conventional methods. To examine the accuracy of our approach, we implemented flux balance analyses with flux ratios in five publicly available genome-scale models of five different organisms, including Arabidopsis thaliana, yeast, cyanobacteria, Escherichia coli, and Clostridium acetobutylicum, using published metabolic engineering strategies for improving product yields in these organisms. We examined the limitations of the published strategies, searched for possible improvements, and evaluated the impact of these strategies on growth and product yields. The flux balance analysis with flux ratio method requires a prior knowledge on the critical regions of the metabolic network where altering flux ratios can have significant impact on flux redistribution. Thus, we further developed the reverse flux balance analysis with flux ratio algorithm as a possible solution to automatically identify these critical regions and suggest metabolic engineering strategies. We examined the accuracy of this algorithm using an Arabidopsis genome-scale model and found consistency in the prediction with our experimental data. / Master of Science

Advances in bioinformatics and computational biology have enabled integration of an enormous amount of known biological interactions. This has enabled researchers to use models and data to design experiments and guide new discovery as well as test for consistency. One such computational method is constraint-based metabolic flux modeling. This is performed using genome-scale metabolic models (GEMs) that are a collection of biochemical reactions, derived from a genome's annotation. This type of flux modeling enables prediction of net metabolite conversion rates (metabolic fluxes) to help understand metabolic activities under specific environmental conditions. It can also be used to derive metabolic engineering strategies that involve genetic manipulations. Over the past decade, GEMs have been constructed for several different microbes, plants, and animal species. Researchers have also developed advanced algorithms to use GEMs to predict genetic modifications for the overproduction of biofuel and valuable commodity chemicals. Many of the predictive algorithms for microbes were validated with experimental results and some have been applied industrially. However, there is much room for improvement. For example, many algorithms lack straight-forward predictions that truly help non-computationally oriented researchers understand the predicted necessary metabolic modifications. Other algorithms are limited to simple genetic manipulations due to computational demands. Utilization of GEMs and flux-based modeling to predict in vivo characteristics of multicellular organisms has also proven to be challenging. Many researchers have created unique frameworks to use plant GEMs to hypothesize complex cellular interactions, such as metabolic adjustments in rice under variable light intensity and in developing tomato fruit. However, few quantitative predictions have been validated experimentally in plants. This research demonstrates the utility of GEMs and flux-based modeling in both metabolic engineering and analysis by tackling the challenges addressed previously with alternative approaches. Here, a novel predictive algorithm, Node-Reward Optimization (NR-Opt) toolbox, was developed. It delivers concise and accurate metabolic engineering designs (i.e. genetic modifications) that can truly improve the efficiency of strain development. As a proof-of-concept, the algorithm was deployed on GEMs of E. coli and Arabidopsis thaliana, and the predicted metabolic engineering strategies were compared with results of well-accepted algorithms and validated with published experimental data. To demonstrate the utility of GEMs and flux-based modeling in analyzing plant metabolism, specifically its response to changes in the signaling pathway, a novel modeling framework and analytical pipeline were developed to simulate changes of growth and starch metabolism in Arabidopsis over multiple stages of development. This novel framework was validated through simulation of growth and starch metabolism of Arabidopsis plants overexpressing sucrose non-fermenting related kinase 1.1 (SnRK1.1). Previous studies suggest that SnRK1.1 may play a critical signaling role in plant development and starch level (a critical carbon source for plant night growth). It has been shown that overexpressing of SnRK1.1 in Arabidopsis can delay vegetative-to-reproductive transition. Many studies on plant development have correlated the delay in developmental transition to reduction in starch turnover at night. To determine whether starch played a role in the delayed developmental transition in SnRK1.1 overexpressor plants, starch turnover was simulated at multiple developmental stages. Simulations predicted no reduction in starch turnover prior to developmental transition. Predicted results were experimentally validated, and the predictions were in close agreement with experimental data. This result further supports previous data that SnRK1.1 may regulate developmental transition in Arabidopsis. This study further validates the utility of GEMs and flux-based modeling in guiding future metabolic research. / Ph. D.

Secondary xylem, commonly known as wood, is a valuable commercial commodity. Among the major components of wood are the elongated, thick-walled water-conducting cells known as tracheary elements. Understanding tracheary element differentiation and maturation is of scientific and commercial importance as it may lead to broad understanding of cellular differentiation processes as well as ways to increase both the quality and quantity of wood produced by economically important tree species. One way to begin to understand the regulation of tracheary element differentiation is to identify elements that control expression of genes associated with tracheary elements. In Arabidopsis thaliana, Xylem Cysteine Protease 1 (XCP1) is specifically expressed in tracheary elements where it catalyzes microautolysis. Thus XCP1 can serve as a useful model for identifying factors that regulate tracheary element-specific gene expression. A deletion analysis of the XCP1 promoter was conducted to identify promoter elements that are necessary and sufficient for tracheary element-restricted gene expression. Two regions required for tracheary element-specific gene expression were identified. One of these was assembled as a multimeric bait construct and used in yeast one-hybrid assays to identify candidate transcription factors that bind to the XCP1 promoter region. Subsequently, a southwestern blot analysis was used to identify transcription factors displaying specific binding to a previously reported cis-element, CTTCAAAGCCA, found in the XCP1 promoter and other tracheary element-associated genes from multiple species. / Master of Science

Cysteine proteases are associated with xylem tracheary element differentiation. XCP1 was recently identified as a xylem-specific cysteine protease in Arabidopsis (Zhao, et al., 2000). For this study a recombinant polyhistidine-tagged XCP1 (XCP1H6) was expressed and purified from an E. coli expression system. A polyclonal anti-XCP1 antibody was produced using purified XCP1H6. Immunoblot analysis of a developmental time course of xylem and bark protein extracted from root-hypocotyl segments demonstrated that XCP1 was expressed in xylem only. Further analysis under optimized immunoblot conditions, however, revealed that anti-XCP1 antibody reacted with protein present in both xylem and bark. The vast majority of immunoreactivity, however, was restricted to xylem. Cell-type localization of GUS expression under the control of a putative XCP1 promoter indicated that the XCP1 promoter specifies expression of XCP1 in tracheary elements in leaves, stems, roots and flowers. XCP1 promoter-driven GUS activity was not associated with senescing tissues. / Master of Science

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]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11301

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]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/38108

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]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153809 / Compendio