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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.

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

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

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.

Chez les plantes supérieures, l’organogénèse est principalement post-embryonnaire et assurée par les méristèmes. Les familles de gènes CLE (CLAVATA3/ENDOSPERMSURROUNDING REGION related) et WOX (WUSCHEL-LIKE HOMEOBOX) sont des régulateurs majeurs de l’activité des méristèmes. Des analyses phylogénétiques des gènes WOX de différentes espèces ont identifié trois groupes d’orthologie, dont le groupe WOX13OG. Ce dernier contient le seul gène WOX d’Ostreococcus tauri, les trois gènes WOX de Physcomitrella patens, ainsi que trois des gènes WOX parmi les quinze que compte Arabidopsis thaliana. L’objectif de ma thèse a été de caractériser la fonction des gènes du groupe WOX13 OG chez A. thaliana.Parmi les mutants nuls identifiés pour ces gènes, seul le mutant wox14 présente des phénotypes forts, à savoir un retard de transition florale et des défauts de développement vasculaire. Ces résultats sont en accord avec l’induction de l’expression de WOX14 à la jonction des faisceaux vasculaires lors de la transition florale. Les résultats indiquent que WOX14 est impliqué dans le contrôle du nombre de faisceaux vasculaires initiés lors de cette dernière. La restauration conjointe de la transition florale et du nombre de faisceaux vasculaires, par complémentation du mutant wox14 ou application de gibbérellines (GA),suggère un lien direct entre ces deux processus. Cette étude nous a permis d’identifier le gène CLE46 comme étant dérégulé dans le mutant wox14. Son expression dans les cellules du xylème, associée à l’augmentation du nombre de faisceaux vasculaires chez wox14, suggèrent que CLE46 est un élément d’une voie de signalisation de contrôle du nombre de faisceaux vasculaires. L’importance des interactions WOX-CLE dans le développement vasculaire est soulignée par l’induction de WOX4 et WOX14 par le peptide CLE41. Les analyses du transcriptome du mutant wox14 révèlent que la signalisation des GA est déficiente. Ce qui confirme les résultats de la complémentation du phénotype wox14 par les GA. De plus, le gène GA3ox1 a été identifié comme une cible potentielle de régulation de la biosynthèse de GA par le gène WOX14. / Whilst primary meristems are initiated during embryogenesis, in higher plants additionalsecondary meristems initiate post-embryonically and contribute to the plant architecture andthe vascular strand development. Differentiation of the plant vascular cambium into xylemand phloem was shown to be regulated by cell to cell communication. The large CLE(CLAVATA3/ENDOSPERM SURROUNDING REGION related) signaling peptide familyand the WOX (WUSCHEL-LIKE HOMEOBOX) transcription factor family are thought to beconserved regulators of stem cell fate. In this thesis we report the presence of supernumeraryvascular bundles in the young inflorescence stem of the wox14 mutant. Our data indicate thatWOX14 prevents additional cambium cell to differentiate into vascular bundles during floraltransition. Moreover, the data suggest that vascular differentiation and floral transition arelinked. Consistently, WOX14 expression is induced within the connecting vascular strandduring floral transition. Furthermore, the application of gibberellins (GA) fully rescued boththe floral transition and the vascular bundle phenotype of the wox14 mutants. A detail analysisof GA biosynthesis and target genes showed that WOX14 controls the amount of bioactiveGA within the vasculature. However, WOX14 is also specifically expressed in the phloem ofthe inflorescence stem indicating a function in late vascular bundle development. We alsoshowed that not only WOX4 but also WOX14 are the target of the CLE41 peptide duringvascular development. Furthermore, the data indicate that another CLE gene, namely CLE46,is misregulated in the wox14 mutant. These results suggest that CLE46 might be the firstidentified CLE signal from the xylem that impacts vascular differentiation.

O gene thi1 foi isolado a partir de uma biblioteca de cDNA de A. thaliana devido à sua capacidade de complementar mutantes de E. coli para rotas de reparo de DNA. O posterior seqüenciamento desse gene permitiu a identificação de similaridade com genes de fungos ativados em condições de estresse ou ativados na ausência de tiamina (vitamina B1). A síntese de tiamina é de grande importância já que na forma pirofosfatada é coenzima essencial para vários processos vitais das células. No presente estudo, foi realizada a caracterização funcional do gene thi1 utilizando-se, para tanto, linhagens de A. thaliana mutante e/ou com expressão diferencial desse gene. Foram analisados parâmetros biológicos como viabilidade de sementes, antioxidantes, danos no DNA, e atividade transcricional e traducional do gene thi1. Foi iniciado, também, o processo de padronização da quantificação de tiamina em plantas. Foi observado, para a linhagem mutante, menor viabilidade de sementes, maior produção de antioxidantes e maior quantidade de danos no DNA de cloroplastos. Quanto à atividade transcricional e traducional, foi observado que o gene thi1 apresenta um pico de expressão no período da tarde com um ritmo circadiano em potencial. Além disso, o acúmulo da proteína nos tecidos acompanha o perfil de expressão de RNAm thi1, o que sugere que o modo de regulação primária do gene é em nível transcricional. A análise comparativa de proteína por gel bi-dimensional entre as linhagens selvagem e mutante permitiu a identificação de quatro proteínas em maior quantidade na mutante, sendo duas identificadas por seqüenciamento: enolase e fosfoglicerato desidrogenase. Na análise de tiamina foi observado que a linhagem mutante acumula um composto, não identificado, que emite fluorescência no mesmo comprimento de onda que as tiaminas, sendo, provavelmente, um precursor do tiazol. Os resultados obtidos nesse trabalho indicam que THI1 defectivo acarretaria em desbalanço metabólico e, não necessariamente, que o gene thi1 estaria envolvido em dupla função. Em bactérias, entre os precursores de tiazol, estão a cisteína e o gliceraldeído 3-fosfato (G3P). O G3P em excesso seria deslocado para o ciclo de Calvin a fim de regenerar a ribulose 1,5-bisfosfato. Maior quantidade de G3P, maior taxa fotossintética, maior produção de ROS, maior produção de antioxidantes na mutante. A maior disponibilidade de G3P aumentaria o fluxo de glicólise e, consequentemente, de respiração mitocondrial, aumentando a taxa de ROS. A fosfoglicerato desidrogenase, em maior quantidade na mutante, está envolvida na síntese de cisteina que acarreta na produção de glutationa. A glutationa e a cisteína, por sua vez, atuariam induzindo o promotor da SOD, acarretando, então, na produção de mais antioxidantes. Todos esses antioxidantes estariam envolvidos na detoxificação de ROS presente em excesso na linhagem mutante, que levaria à menor viabilidade de sementes e maior quantidade de danos no DNA. Devido à grande quantidade de ROS, haveria superexpressão de enolase, envolvida no bloqueio da proliferação celular e, subsequentemente, em morte celular programada, o que explicaria o retardo no desenvolvimento da linhagem mutante / thi1 gene was previously isolated from A. thaliana cDNA library due to its capacity to complement mutant Escherichia coli defective in DNA repair. The late analyse of this gene showed its similarity with yeast genes activated under stress conditions or activated in the absence of thiamine. It means that THI1 has bifunctional activity, being involved in thiamine biosynthesis and repair/tolerance to DNA damage. The thiamine biosynthesis is important because its phosphorilated form is a coenzyme essential to several vital process at the cell. The repair/tolerance to DNA damage shows its importance because it is necessary to maintenance the genetic stability of the individual. In the present study, we report the functional characterization of thi1 gene using A. thaliana lines with differential expression of this gene. We analyzed the seed viability, the fresh weight of different lines, thi1 mRNA expression, the amount of protein produced and the expression in situ using the thi1-GUS construction in different conditions. Besides that we quantified free radicals in the wild-type (WT) and mutant lines and analysed the response of the mutant line, with defective THI1, to the production of antioxidant enzymes and non-enzimatics antioxidants. We also quantified DNA damage in chloroplast of WT and mutant lines and we did comparative proteomic analysis between and began the standardization of the thiamine quantification in plants. All these results together lead us up to a better understanding about THI1 activity at the cellular metabolism. Previous results suggested that besides thiamine synthesis, THI1 would be involved in repair/tolerance to DNA damage. Results obtained in this study strengthen the hypothesis of this another function of the thi1 gene. Considering that the mutant line does not produce a higher quantity of ROS, as indicated by hydrogen peroxide quantification, but shows more antioxidants and more DNA damage, probably the genetic material of this line is more susceptible to damage, showing that the defective THI1 protein could not protect it efficiently.