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Biotic and abiotic stresses can severely limit crop productivity. In response to drought, plants close stomata to prevent water loss. Furthermore, stomata are the main entry point for several pathogens. Therefore, the development of natural products to control stomata closure can be considered a sustainable strategy to cope with stresses in agriculture. Plants respond to different stresses by releasing volatile organic compounds. Green leaf volatiles, which are commonly produced across different plant species after tissue damage, comprise an important group within volatile organic compounds. Among them, (Z)-3-hexenyl butyrate (HB) was described as a natural inducer of stomatal closure, playing an important role in stomatal immunity, although its mechanism of action is still unknown. Through different genetic, pharmacological, and biochemical approaches, we here uncover that HB perception initiates various defence signalling events, such as activation of Ca2+ permeable channels, mitogen-activated protein kinases, and production of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species. Furthermore, HB-mediated stomata closure was found to be independent of abscisic acid biosynthesis and signalling. Additionally, exogenous treatments with HB alleviate water stress and improve fruit productivity in tomato plants. The efficacy of HB was also tested under open field conditions, leading to enhanced resistance against Phytophthora spp. and Pseudomonas syringae infection in potato and tomato plants, respectively. Taken together, our results provide insights into the HB signalling transduction pathway, confirming its role in stomatal closure and plant immune system activation, and propose HB as a new phytoprotectant for the sustainable control of biotic and abiotic stresses in agriculture.

[ES] El presente trabajo final de master tiene como objetivo investigar el papel de un componente del reloj circadiano en plantas, GIGANTEA (GI), en la regulación de la respuesta de evasión de la sombra. Concretamente, se centrará en caracterizar la interacción de GI con Phytochrome interacting factor 7 (PIF7), un factor de transcripción clave en la promoción del crecimiento vegetativo en respuesta a sombra, y su relevancia fisiológica. La disminución en la cantidad y calidad de la luz es un estrés al que las plantas están sometidas en ambientes densamente poblados por otras plantas, ya que se ensombrecen unas a otras. Para enfrentarse a la sombra, aumentar su eficiencia fotosintética y en definitiva competir con sus vecinas, muchas especies vegetales han desarrollado una estrategia denominada respuesta de evasión a sombra (SAR), caracterizada por la elongación de hipocótilos o la hiponastia de las hojas entre otros fenotipos (Casal 2012). La SAR es un proceso regulado por distintos mecanismos, siendo el reloj circadiano uno de ellos (Sellaro et al. 2012). Este consiste en un oscilador endógeno, presente en múltiples organismos, que controla todo tipo de procesos metabólicos, fisiológicos y de comportamiento, siguiendo ritmos oscilatorios de aproximadamente 24 horas (Más 2005). En este trabajo se muestra una vía de la regulación circadiana de la SAR no descrita anteriormente, a través de la interacción entre el factor de transcripción Phytochrome interacting factor 7 (PIF7) y la proteína del reloj central GIGANTEA (GI). El mecanismo consiste en la represión a nivel de proteína ejercida por GI sobre PIF7, evitando que este último pueda activar la transcripción de genes implicados en la SAR, mediante lo que parece un mecanismo de secuestro independiente de la degradación de PIF7., [EN] Quantity and quality light reduction is a stress for plants that live in densely populated environments, as they shade each other. To cope with shade, increase their photosynthetic efficiency and ultimately compete with their neighbors, many plant species have developed a strategy called shade avoidance response (SAR), characterized by hypocotyl elongation or leaf hyponasty among other phenotypes (Casal 2012). SAR is a process regulated by different mechanisms and the circadian clock is one of them (Sellaro et al. 2012). The circadian clock consists of an endogenous oscillator that is present in multiple organisms, which controls all kinds of metabolic, physiological and behavioral processes, following oscillatory rhythms of approximately 24 hours (More 2005). In this work we show a previously undescribed pathway of SAR circadian regulation, through the interaction between the transcription factor Phytochrome interacting factor 7 (PIF7) and the central clock protein GIGANTEA (GI). The mechanism involves protein-level repression of PIF7 by GI, preventing the latter from activating the transcription of genes involved in SAR, by what appears to be a degradation-independent sequestration mechanism of PIF7.

[ES] La duración de las horas de luz y oscuridad es una señal importante en la determinación de distintos procesos fisiológicos y del desarrollo. El reloj circadiano es un mecanismo molecular que permite a los organismos medir el tiempo y anticiparse a los ciclos de luz-oscuridad que ocurren a lo largo del día. Desde un punto de vista ecológico, se entiende que esta capacidad de anticipación conferida por el reloj circadiano permite a los organismos optimizar el aprovechamiento de los recursos disponibles, siendo capaces de destinar dichos recursos a aquellos procesos más relevantes en cada momento del día. El reloj circadiano de las plantas regula el funcionamiento de una gran cantidad de procesos fisiológicos, pero en muchos casos se desconocen los mecanismos moleculares subyacentes. Así, el presente trabajo se centra en el estudio de la regulación circadiana de la defensa frente a patógenos a nivel molecular a través del estudio de conexiones específicas entre un componente clave del reloj circadiano en plantas y elementos centrales de la ruta de señalización del ácido jasmónico (JA). Se hipotetiza que la modulación de la actividad de dichos elementos señalizadores por parte del reloj podría contribuir a la temporalización de la respuesta a patógenos mediada por JA a lo largo del día y, por tanto, a la adaptación de las plantas al ambiente., [EN] The Earth's rotation on its own axis has promoted the evolution of the circadian clock as a molecular mechanism that allows organisms to measure time and anticipate the light-dark cycles occurring throughout the day. On an ecological level, it is understood that this capacity for anticipation conferred by the circadian clock allows organisms to optimize the use of available resources, being able to allocate those resources to the most relevant processes at each moment of the day. The circadian clock in plants regulates many physiological processes, including many of agronomic interest, but in many instances the underlying molecular mechanisms are not known. Thus, the present work focuses on the study of the circadian regulation of the defense response against pathogens at the molecular level through the study of specific connections between a key component of the circadian clock in plants and central elements of the jasmonic acid (JA) signaling pathway. It is hypothesized that the modulation of the activity of these signaling elements by the clock could contribute to the gating of JAmediatedresponses to pathogens across the day and, therefore, to plant adaptation to the environment.

[ES] Debido a su naturaleza sésil, las plantas han de enfrentarse "in situ" a variaciones ambientales que pueden comprometer su supervivencia y éxito reproductivo. Para ello las plantas han desarrollado mecanismos con los que monitorizar constantemente el ambiente (intensidad y calidad de la luz, temperatura, disponibilidad de agua y nutrientes, etc.) y reajustar su desarrollo en consecuencia. Por ejemplo, cuando las condiciones ambientales son adversas (estrés abiótico), las plantas frenan el crecimiento y simultáneamente activan los mecanismos de defensa. Las proteínas DELLA juegan un papel central en esta respuesta coordinada gracias a su capacidad de interaccionar con más de 300 proteínas, principalmente factores de transcripción (TFs), que regulan procesos tan diversos como la división celular, la elongación celular o la biosíntesis de compuestos antioxidantes. En concreto, las DELLA reprimen el crecimiento mediante la interacción con TFs de las familias TCP, PIF y BES1/BZR principalmente mientras que desencadenan las respuestas defensivas mediante la interacción con proteínas de la familia JAZ entre otras. Aunque se ha argumentado que el freno del crecimiento es imprescindible para la redistribución de recursos energéticos en la planta, estudios más recientes indican que esto no es así, sino que la percepción del estrés provoca la acumulación de DELLAs por su papel positivo en defensa y esta acumulación es la que causa la represión colateral del crecimiento. En este trabajo se propone la generación y caracterización de alelos arísticos DELLA (del inglés `edgetic alleles"), es decir, versiones de las proteínas DELLA que pierdan específicamente la capacidad de interaccionar con factores promotores del crecimiento pero que mantengan la capacidad de interaccionar con proteínas con efecto positivo en defensa. Por tanto, nuestro objetivo último es utilizar las DELLA como herramienta biotecnológica para obtener plantas cuyo crecimiento no se vea penalizado en condici, [EN] In a stressful situation, plants slow down their growth and simultaneously activate their defense mechanisms. defense mechanisms. The DELLA proteins play a central role in this coordinated coordinated response through their interaction with more than 300 proteins, mainly transcription factors (TFs). transcription factors (TFs). Specifically, DELLAs repress growth by interacting with TFs of the through interaction with TFs from the TCP, PIF and BES1/BZR and ARR families mainly, while they promote defensive responses by interacting with proteins of the JAZR and interaction with JAZ family proteins. The aim of this master thesis has been to functionally characterize the activity of potential alleles of GAI, variants of this DELLA that have selectively lost certain functions. functions. Previous work in the group identified several GAI clones that did not interact with PIF but did interact with PIF but did interact with ARR1 and JAZ in yeast assays. The phenotypic phenotypic analysis performed in this TFM shows that the GAI aristatic alleles complemented root root development (DELLA-ARR1 interaction-dependent) and defense gene activation (DELLA-ARR1-dependent) and (DELLA-JAZ interaction-dependent) in loss-of-function dellaKO mutants. dellaKO loss-of-function mutants. However, these alleles did not complement the development of the development (DELLA-PIF interaction-dependent). Thus, it is concluded that a strategy has been the development of a strategy to uncouple different DELLA activities in plants. DELLA activities.

Over time, plants have evolved flexible self-organizing patterning mechanisms to adapt tissue functionality for continuous organ growth. An example of this process is the multicellular organization of cells into a vascular network in foliar organs. An important, yet poorly understood component of this process is secondary vein branching, a mechanism employed to extend vascular tissues throughout the cotyledon surface. Here, we uncover two distinct branching mechanisms during embryogenesis by analyzing the discontinuous vein network of the double mutant cotyledon vascular pattern 2 (cvp2) cvp2-like 1 (cvl1). Similar to wild-type embryos, distal veins in cvp2 cvl1 embryos arise from the bifurcation of cell files contained in the midvein, whereas proximal branching is absent in this mutant. Restoration of this process can be achieved by increasing OCTOPUS dosage as well as by silencing RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) expression. Although RPK2-dependent rescue of cvp2 cvl1 is auxin- and CLE peptide-independent, distal branching involves polar auxin transport and follows a distinct regulatory mechanism. Our work defines a genetic network that confers plasticity to Arabidopsis embryos to spatially adapt vascular tissues to organ growth.

[ES] Las proteínas DELLA son reguladores de procesos del desarrollo y el crecimiento de las plantas, involucradas en la vía de señalización de las giberelinas (GAs). Las proteínas DELLA no pueden unirse al DNA por sí solas y su mecanismo de acción es la interacción mediante su dominio GRAS con factores de transcripción (FT) y con reguladores de la transcripción (RT), a través de los cuales modula la expresión de sus genes diana. Los modos de acción de las proteínas DELLA son el secuestro de FTs y RTs, impidiendo su actividad normal, y la coactivación, promoviéndola. Al dominio N-terminal de las DELLA solamente se le ha atribuido la función del control de la estabilidad dependiente de GAs, a través de la interacción con el receptor de GAs codificado por GID1. Sin embargo, aunque sólo hay genes GID1 en plantas vasculares, las DELLA de las plantas no vasculares (briófitas) presentan una sorprendente conservación de su domino N-terminal, que se ha atribuido a su papel adicional como activador de la transcripción por contener motivos denominados ¿dominios de transactivación de nueve aminoácidos¿ (9aaTAD). Estos dominios frecuentemente interaccionan con dominios de interacción inducibles por quinasas (KIX), presentes en complejos coactivadores de la transcripción. Trabajo previo del laboratorio indica que la capacidad transactivadora del dominio N-terminal de las DELLA de Arabidopsis depende de su interacción con MED15, una subunidad de la cola del complejo Mediator que facilita la actividad de la RNA Polimerasa II. En este trabajo nos planteamos la pregunta de si éste es el mecanismo que utilizan también las DELLA de briófitas para ejercer como coactivadoras. Para resolverlo, decidimos trabajar con la briófita Marchantia polymorpha, cuyo linaje se separó de las plantas vasculares en un punto muy temprano tras la colonización de la superficie terrestre. Mediante análisis filogenéticos hemos identificado una secuencia MpMED15 con su dominio KIX conservado, a través del cua, [EN] DELLA proteins are regulators of plant growth and development processes, involved in the gibberellin signalling pathway (GAs). DELLA proteins can¿t bind to DNA by themselves. Their mechanism of action is the interaction through their GRAS domain with transcription factors (FT) and transcription regulators (RT), through which they modulate the expression of their target genes. The modes of action of DELLA proteins are the sequestration of FTs and RTs, preventing their normal activity, and coactivation, promoting it. Only the GA-dependent stability control function has been attributed to the N-terminal domain of DELLA, through interaction with the GAs receptor encoded by GID1. However, although there are only GID1 genes in vascular plants, the DELLAs of non-vascular plants (bryophytes) show a surprising conservation of their N-terminal domain, which has been attributed to their additional role as an activator of transcription because they contain motifs called "Nine amino acid transactivation domains" (9aaTAD). These domains frequently interact with kinase-inducible interaction domains (KIX), present in transcriptional coactivator complexes. Previous laboratory work indicates that the transactivating capacity of the N-terminal domain of Arabidopsis DELLAs depends on its interaction with MED15, a subunit of the tail of the Mediator complex that facilitates RNA polymerase II activity. In this work we are wondering whether this is the mechanism that the DELLA of bryophytes also use to act as co-activators. To solve this, we decided to work with the bryophyte Marchantia polymorpha, whose lineage separated from vascular plants at a very early point after colonization of the earth's surface. Through phylogenetic analysis we have identified an MpMED15 sequence with its conserved KIX domain, by which it interacts with the N-terminal domain of MpDELLA, according to yeast two hybrid (Y2H) and Bimolecular Fluorescence Complementation (BiFC) assays. We have also verified tha

[CA] El desenvolupament de la vasculatura en plantes ha suposat un gran avantatge per a l’adaptació de les espècies vegetals a situacions climatològiques canviants i nombrosos factors que condicionen la seua supervivència. La formació de xilema, floema o de cèl·lules especialitzades en el transport de nutrients son alguns dels canvis a nivell fenotípic que experimenten les plantes vasculars. La regulació d’estes variacions fenotípiques s’integra dins d’una extensa xarxa de regulació genètica on podríem establir-ne 3 nivells: un primer nivell de gens reguladors upstream, un segon nivell de gens reguladors perse i un 3r nivell de gens efectors. Esta cascada de regulació de gens que codifiquen proteïnes (ex: factors de transcripció, enzims etc. ) atribueix una àmplia plasticitat al procés global de diferenciació en xilema. A més, gràcies al coneixement actual de la seqüència d’estos gen, és possible cercar dintre d’aquestes xarxes reguladores les diferències existents entre diferents ecotips d’un mateix organisme i entendre millor la seva adaptació genètic a l’entorn per selecció natural. Mitjançant eines estadístiques hem establert relacions significatives entre polimorfismes d’un sol nucleòtid (SNPs) d’una població de la Península Ibérica de l’organisme Arabidopsis thaliana i variables climatològiques dels ecosistemes d’on són originals. Algunes d’estes modificacions reflecteixen en variants de gens codificants i d’altra banda poden servir per esclarir quina ha sigut l’evolució que ha portat les accessions d’una zona geogràfica concreta a tenir el seu genotip actual, íntimament correlacionat amb l’històric climatològic o la geografia d’una zona. La crisi climàtica a la qual ens enfrontem i la recerca de noves fonts d’energia alternatives als combustibles fòssils impulsen nous plantejaments biotecnològics entorn a l’aprofitament de biomassa. Comprendre la regulació de la morfogènesi de biomassa lignocel·lulòsica, prenent com a organisme model Arabidopsis thaliana, per, [EN] The development of plant vasculature has been a major advantage in adapting plant species to changing weather situations and many factors that affect their survival. Formation of xylem, phloem, or specialized cells in the transport of nutrients are some of the changes in the phenotypic level experienced by vascular plants. The regulation of these phenotypic variations is integrated into an extensive network of genetic regulation where we could establish 3 levels: a first level of upstream regulatory genes, a second level of perse regulators and a third level of effector genes. This cascade of gene regulation that encodes proteins (e.g. transcription factors, enzymes etc. ) attributes a wide plasticity to the global process of differentiation in xylem. In addition, thanks to current knowledge of the gene sequence, it is possible to search within these regulatory networks for the differences between different ecotypes of the same organism and to better understand its genetic adaptation to the environament by natural selection. Through statistical tools we have established significant relationships between singlenucleotide polymorphisms (SNPs) of a population of the Iberian Peninsula of the Arabidopsis Thaliana organism and climatological variables of the ecosystems of which they are original. Some of these modifications reflect in variants of coding genes and on the other hand may serve to clarify what evolution path has led the accessions of a particular geographical area to have its current genotype, intimately correlated with the historical climatological or geography of an area. The climate crisis that we are facing and research into new alternative energy sources to fossil fuels encourages new biotechnological approaches to biomass use. Understanding the regulation of the morphogenesis of lignocellulosic biomass, taking as a model organism Arabidopsis Thaliana, will broaden the horizon to sustainable wood production and to a better resource ( such as water o

[ES] El complejo de prefoldina se identificó originalmente en humanos como co-chaperona que ayuda a la CCT de chaperonina citosólica en el plegamiento de actina y tubulinas. Es un complejo heteromérico formado por seis proteínas pequeñas no relacionadas, prefoldina 1-6, y se conserva en eucariotas. En Arabidopsis, los mutantes simpoles de dfp presentan defectos graves en la organización de los microtúbulos, con consecuencias directas en la expansión celular de los hipocotilos y la raíz primaria, y en algunos casos, también son defectuosos en la adaptación al estrés ambiental. Sin embargo, queda por determinar en qué medida las diferentes prefoldinas, individualmente o en complejo, contribuyen al desarrollo de la planta y a las respuestas al estrés. El propósito de esta tesis de Master es la caracterización funcional de todos los mutantes de dfp simples, quíntuples y sextuples para tratar de asignar funciones específicas y / o determinar la contribución de cada subunidad prefoldina a diferentes procesos de desarrollo e.g. crecimiento de la raíz, tiempo de floración, tamaño y forma de roseta, etc., y respuesta al estrés, p. ej. Sal y osmótica. Finalmente, los análisis morfológicos y fisiológicos se complementarán con dos enfoques moleculares: (i) una secuencia de ARN para analizar el transcriptoma del mutante sextuple, y (ii) inmunoblots y análisis confocales en mutantes dfp para determinar defectos en la acumulación de tubulina y la organización de los microtúbulos ., [EN] The prefoldin complex was originally identified in humans as a co-chaperone helping the cytosolic chaperonin CCT in the folding of actin and tubulins. It is a highly conserved heterohexameric complex composed of subunits PFD1 to 6. In Arabidopsis, single pfd mutants display severe defects in microtubule organization, with direct consequences in cellular expansion of hypocotyls and primary root, and in some cases, are defective in the adaptation to environmental stress as well. However, it remains to be determined the extent to which the different prefoldins, individually or in complex, contribute to these responses. In this master¿s thesis, we have confirmed that PFD1 and PFD2 act as canonical PFDs, due to the microtubule organization defects observed in the previously uncharacterized pfd1 and pfd2 mutants. Importantly, we have prepared a mutant defective in the six PFD genes, pfdx6. A transcriptomic analysis of the sextuple mutant revealed novel roles for PFD in germination, lateral root development and in response to hormones (auxin and ABA). Our phenotypic analyses of the singles pfd and the pfdx6 mutants has allowed us to discern between processes that depend on the activity of the whole complex and those that are controlled redundantly by more than one PFD subunit. Our results reveal that the PFD complex, although it is not essential for the viability of the plant, it has a relevant role in development processes as well as in the adaptation to the environment.

[EN] The present work describes the effects on iron homeostasis when copper transport was deregulated inArabidopsis thalianaby overexpressing high affinity copper transporters COPT1 and COPT3 (COPTOE). A genome-wide analysis conducted onCOPT1(OE)plants, highlighted that iron homeostasis gene expression was affected under both copper deficiency and excess. Among the altered genes were those encoding the iron uptake machinery and their transcriptional regulators. Subsequently,COPT(OE)seedlings contained less iron and were more sensitive than controls to iron deficiency. The deregulation of copper (I) uptake hindered the transcriptional activation of the subgroup Ib of basic helix-loop-helix (bHLH-Ib) factors under copper deficiency. Oppositely, copper excess inhibited the expression of the master regulatorFITbut activatedbHLH-Ibexpression inCOPT(OE)plants, in both cases leading to the lack of an adequate iron uptake response. As copper increased in the media, iron (III) was accumulated in roots, and the ratio iron (III)/iron (II) was increased inCOPT(OE)plants. Thus, iron (III) overloading inCOPT(OE)roots inhibited local iron deficiency responses, aimed to metal uptake from soil, leading to a general lower iron content in theCOPT(OE)seedlings. These results emphasized the importance of appropriate spatiotemporal copper uptake for iron homeostasis under non-optimal copper supply. The understanding of the role of copper uptake in iron metabolism could be applied for increasing crops resistance to iron deficiency.

[EN] In many perennial plants, seasonal flowering is primarily controlled by environmental conditions, but in certain polycarpic plants, environmental signals are locally gated by the presence of developing fruits initiated in the previous season through an unknown mechanism. Polycarpy is defined as the ability of plants to undergo several rounds of reproduction during their lifetime, alternating vegetative and reproductive meristems in the same individual. To understand how fruits regulate flowering in polycarpic plants, we focused on alternate bearing in Citrus trees that had been experimentally established as fully flowering or nonflowering. We found that the presence of the fruit causes epigenetic changes correlating with the induction of the CcMADS19 floral repressor, which prevents the activation of the floral promoter CiFT2 even in the presence of the floral inductive signals. By contrast, newly emerging shoots display an opposite epigenetic scenario associated with CcMADS19 repression, thereby allowing the activation of CiFT2 the following cold season.

[EN] The plants vascular system consists of a complex network of different types of transport tissue that allows the movement of water and molecules to the organs that are required. For that reason, the vascular development is important to allow the transport of nutrients and so enable the proper plant growth. Phytohormones are one of the factors that take part in the proper vascular development. Strigolactones (SLs) are a group of phytohormones which have been recently involved in the vascular development. Hitherto, it is known that SLs are involved in the regulation of the cellular proliferation. However, it is unknown whether SLs have any effect in the establishment of the vascular pattern. In this work, we pretend to characterize the vascular effects which are induced due to the absence of a proper SLs signaling in Arabidopsis thaliana (A.thaliana), [ES] El sistema vascular de las plantas se trata de una compleja red de tejidos que permite el transporte de agua y moléculas a los órganos donde se necesitan. Por ello, el desarrollo de la vasculatura es necesario para permitir el transporte de nutrientes y con ello promover el correcto desarrollo de la planta. Uno de los factores que intervienen en el correcto desarrollo vascular son las fitohormonas. En concreto, las estrigolactonas (SLs) son una familia de hormonas vegetales que recientemente se han visto implicadas en el desarrollo vascular. Hasta ahora, se sabe que las SLs están implicadas en la regulación de la proliferación celular promovida por el cámbium. Sin embargo, se desconoce si las SLs tienen algún efecto en el establecimiento del patrón de desarrollo de la vasculatura. En este trabajo, se pretende caracterizar de un modo preciso cuales son los efectos a nivel de vasculatura que produce la ausencia de una correcta señalización de SLs en Arabidopsis thaliana (A.thaliana)

[ES] En las plantas, el crecimiento secundario es el proceso por el cual las raíces, los tallos y el hipocotilo crecen en grosor. Este proceso es muy beneficioso para las plantas porque proporciona el soporte mecánico y la estabilidad que necesitan para crecer y apoyar sus estructuras. Además, desde un punto de vista aplicado, el crecimiento secundaria es de gran relevancia económica y social porque conduce a la formación de madera. El crecimiento secundario está mediado por la actividad de un meristemo llamado cambium vascular que está programado para dar lugar, exclusivamente, a los dos tejidos vasculares: el xilema y el floema. En los últimos años, se han realizado muchos avances en el conocimiento del control hormonal y genético del desarrollo de la vasculatura secundaria. Sin embargo, gran parte de la regulación genética que impulsa este proceso aún se desconoce. El trabajo presentado aquí se basa en un estudio de la variación natural asociada con la transcriptómica en Arabidopsis thaliana (Gutiérrez, resultados no publicados), de los cuales se ha seleccionado 14 genes que potencialmente regulan la actividad del cambium. Este estudio apunta a arrojar luz sobre la función de tales genes. Habiendo sido identificados a través de un enfoque basado en la variación natural, esperamos encontrar, entre los 14 genes probados, nuevos reguladores de crecimiento secundarios que, potencialmente, han sido relevantes a lo largo de la evolución para adaptar el proceso a las condiciones ambientales., [EN] In plants, secondary growth is the process by which roots, stems and hypocotyl grow in thickness. This process is very beneficial for plants because it provides them with the mechanical support and stability that they need to grow and support their structures. In addition, from an applied point of view, secondary growth is of high economic and social relevance because it leads to the formation of wood. The secondary growth is mediated by the activity of a meristem called vascular cambium that is programmed to give rise, exclusively, to the two vascular tissues: the xylem and the phloem. In recent years, many advances have been made on the knowledge of hormonal control and genetic control of the development of secondary vasculature. However, much of the genetic regulation that drives this process is still unknown. The work presented here is based on a study of natural variation associated with transcriptomics in Arabidopsis thaliana (Gutiérrez, unpublished results), from which a selection of 14 genes potentially regulating the cambium activity was obtained. This study aims at shedding light on the function of such genes. Having been identified through an approach based on natural variation, we expect to find, among the 14 tested genes, new secondary growth regulators that, potentially, have been relevant over evolution to adapt the process to the environment conditions.

[EN] Plants are sessile organisms that need to constantly adapt to changing environmental conditions. In the presence of a stress, plants promote the defensive responses at the expense of reducing growth, a process that is known as the growth-defense trade-off. The signaling pathway established by the phytohormone gibberellins (GA) and DELLA proteins is central for the regulation of this trade-off. GA levels respond to environmental and endogenous signals and transmit this information through the degradation of DELLA proteins. DELLAs interact with many other proteins, mainly transcription factors, to inhibit cell growth and division and to activate the defense mechanisms. An ambitious objective of biotechnological interest is to break the balance between growth and defense, that is to obtain plants able to activate an efficient defensive response without compromising growth. We have explored this possibility through the analysis of the interaction between DELLA proteins and SMR1, a negative regulator of cell cycle progression. We first confirmed previous work in the group, showing that DELLA proteins bind to genomic regions close to the SMR1 gene. We also show that DELLAs repress inflorescence growth through the upregulation of SMR1 expression. Thus, the smr1-1 loss-of-function restores flowering-time and partially suppresses dwarfism in gai-1D mutants. Finally, we have optimized protocols and generated the tools necessary to evaluate if the defensive response of gai-1D mutants is affected by enhanced shoot growth in gai-1D;smr1-1 plants., [ES] Para contrarrestar su incapacidad de movimiento, las plantas han desarrollado una estrategia muy eficiente: monitorizar de manera continua las condiciones ambientales y utilizar esta información para optimizar el uso de su maquinaria celular. Un ejemplo claro es que las plantas sometidas a algún tipo de estrés (sequía, alta concentración de sal, temperaturas extremas...) frenan su crecimiento y simultáneamente activan el programa de defensa. Esta respuesta doble está coordinada por las proteínas DELLA que, mediante la interacción con factores de transcripción, inhiben el ciclo celular y activan respuestas que protegen frente al estrés. Con este proyecto querríamos contestar varias preguntas: (1) ¿cómo regulan las DELLA el ciclo celular? (2) ¿se pueden desacoplar las dos respuestas coordinadas por las DELLA? (3) El freno del crecimiento ¿supone por sí mismo una respuesta protectora frente a estrés? Para ello nos centraremos en el desarrollo de la inflorescencia, la región de la planta donde se producen los frutos y que, por tanto, tiene un elevado interés biotecnológico. En el trabajo reciente del laboratorio con la planta modelo Arabidopsis thaliana, hemos encontrado que una mutación en el gen SMR1 (un regulador negativo del ciclo celular) suprime muchos de los defectos en el crecimiento de los mutantes semi-enanos gai-1D (ganancia de función de DELLA). Sin embargo, la respuesta defensiva de las plantas gai-1D smr1-1 apenas parece verse afectada. Estas observaciones sugieren que es posible desacoplar las diferentes funciones DELLA. El proyecto que proponemos profundizará en este análisis a través de 2 objetivos principales: (1) caracterizar SMR1 como una diana directa de las proteínas DELLA y (2) caracterizar el mutante doble gai-1D;smr1-1 como una herramienta para desacoplar el balance entre crecimiento y defensa mediado por las DELLA.

The present work describes the effects on iron homeostasis when copper transport was deregulated in Arabidopsis thaliana by overexpressing high affinity copper transporters COPT1 and COPT3 (COPTOE). A genome-wide analysis conducted on COPT1OE plants, highlighted that iron homeostasis gene expression was affected under both copper deficiency and excess. Among the altered genes were those encoding the iron uptake machinery and their transcriptional regulators. Subsequently, COPTOE seedlings contained less iron and were more sensitive than controls to iron deficiency. The deregulation of copper (I) uptake hindered the transcriptional activation of the subgroup Ib of basic helix-loop-helix (bHLH-Ib) factors under copper deficiency. Oppositely, copper excess inhibited the expression of the master regulator FIT but activated bHLH-Ib expression in COPTOE plants, in both cases leading to the lack of an adequate iron uptake response. As copper increased in the media, iron (III) was accumulated in roots, and the ratio iron (III)/iron (II) was increased in COPTOE plants. Thus, iron (III) overloading in COPTOE roots inhibited local iron deficiency responses, aimed to metal uptake from soil, leading to a general lower iron content in the COPTOE seedlings. These results emphasized the importance of appropriate spatiotemporal copper uptake for iron homeostasis under non-optimal copper supply. The understanding of the role of copper uptake in iron metabolism could be applied for increasing crops resistance to iron deficiency.

[ES] Las auxinas son unas fitohormonas muy importantes para la planta en múltiples aspectos, desde regular la arquitectura de la raíz o el tallo, hasta participar en el desarrollo vascular o en la respuesta a los tropismos. Su función depende en gran medida de su gradiente de concentración, establecido gracias a una familia de proteínas transportadoras conocida como PINFORMED (PIN). La localización de dichos transportadores en la membrana plasmática será determinante para formar el gradiente. En esta familia destaca PIN1, el principal contribuidor al gradiente. PIN3, por su parte, participa en respuesta a gravitropismos y fototropismos, relocalizándose en la membrana para cambiar el flujo de auxinas y, por tanto, el gradiente., [EN] Auxins are phytohormones involved in a lot of important processes for the plant, from the architecture regulation of roots and stems, to the participation on vascular development or tropic responses. The function highly depends on the concentration gradient, established due to a carrier protein family known as PINFORMED (PIN). Their location on the plasma membrane is crucial to originate this gradient. Above all the family members, we can remark PIN1, the main contributor to the different concentration of auxins along the plant. Moreover, PIN3 respond to gravitropisms and phototropisms, being relocated on the membrane to change the auxin flux and, in consequence, the gradient.

[ES] El crecimiento secundario es un proceso central en el desarrollo de las plantas que resulta en el engrosamiento de tallos y raíces. Desde un punto de vista biológico, el crecimiento secundario es de gran importancia porque proporciona el soporte mecánico y la estabilidad que las plantas necesitan para crecer y sostener sus propias estructuras. Por otra parte, desde un punto de vista productivo, el crecimiento secundario es una fuente directa de biomasa con aplicaciones biotecnológicas. Este proceso está regulado por un grupo de células meristemáticas, denominadas cambium, las cuales están localizadas cerca del cortex. Estos meristemos vasculares son los responsables del desarrollo del tejido vascular, produciendo xilema (hacia adentro) y floema (hacia afuera). La acumulación de anillos vasculares causa la aparición del crecimiento secundario. Si bien el conocimiento disponible sobre su control hormonal es extenso, actualmente desconocemos gran parte de su regulación genética. El trabajo aquí expuesto pretende aprovechar la variación natural presente en Arabidopsis thaliana para identificar nuevos potenciales genes reguladores del cambium mediante un análisis transcriptómico sobre 8 accesiones con distintos niveles de crecimiento secundario., [EN] Secondary growth is a central process in the development of plants that results in the thickening of stems and roots. From a biological point of view, secondary growth is of great importance because it provides the mechanical support and stability that plants need. On the other hand, from a productive point of view, secondary growth is a direct source of biomass with biotechnological applications. This process is regulated by a group of meristematic cells called cambium which are located near of the cortex. They are responsible for the development of vascular tissue: xylem (inwards) and phloem (outwards). The accumulation of these vascular rings causes the appearance of secondary growth. While hormonal regulation of the cambium activity has been widely studied, currently, we do not have enough knowledge about genetic regulation of this process. The present work expects to identify new potential cambium regulatory genes through a transcriptomic approach with 8 accessions with different secondary growth levels using the natural variation present in Arabidopsis thaliana.

[ES] Las plantas son organismos sésiles que necesitan monitorizar constantemente las condiciones ambientales y, en consecuencia, ajustar el uso de su maquinaria celular para sobrevivir y lograr el éxito reproductivo. De ahí que las plantas sometidas a diferentes tipos de estrés abiótico, como la sequía, restrinjan el crecimiento y promuevan los mecanismos de defensa. Esta doble respuesta está controlada por las proteínas DELLA que, a través de la interacción con factores de transcripción, inhiben la progresión del ciclo celular a la vez que activan las respuestas a estrés. Este proyecto ha explorado la posibilidad de separar estas funciones de las proteínas DELLA en crecimiento y defensa. Datos preliminares en el grupo no publicados muestran que la pérdida de función en el gen SMR1, un regulador negativo del ciclo celular, suprime parcialmente el defecto en la elongación del tallo de los mutantes semienanos gai-1D. Por lo tanto, hemos caracterizado con detalle el crecimiento de la inflorescencia en las plantas gai-1D; smr1-1 y RGAp:GFP-rga¿17;smr1-1, y analizado su respuesta a condiciones extremas de estrés por sequía. Además, hemos generado un conjunto de líneas testigo SMR1 para examinar el patrón de expresión de SMR1 en el tallo de la inflorescencia y para estudiar cómo está regulado por las proteínas DELLA., [EN] Plants are sessile organisms that need to continuously monitor the environmental conditions and accordingly adjust the use of their cellular machinery in order to survive and achieve reproductive success. Hence plants subjected to different types of abiotic stress, such as drought, restrict growth and promote defence mechanisms. This dual response is controlled by DELLA proteins that, through the interaction with transcription factors, inhibit cell cycle progression and simultaneously activate stress responses. This project has explored the possibility of separating these DELLA functions in growth and defence. Preliminary unpublished data in the group shows that loss-of-function in the SMR1 gene, a negative regulator of the cell cycle, partially suppresses the defect in stem elongation of gai-1D semi-dwarf mutants. We have characterized with detail inflorescence growth in gai-1D;smr1-1 and RGAp:GFP- rga¿17;smr1-1 plants and analyzed their response to extreme drought stress conditions. In addition, we have generated a set of SMR1 reporter lines to examine SMR1 expression pattern in the inflorescence stem and to study how it is regulated by DELLA proteins.

[EN] The growth of the microorganism is remarked by the integration and coordination of different types of development. In plants, the secondary growth represents the radial growth of the roots and stems in thickness. This process is very important because contributes to the mechanical support and stability for a good length growth and properly maintain their organs. In addition, the secondary growth is the source of a huge amount of biomass that is the origin of the wood of the trees. Even it is so important it is unknown its genetic and molecular regulation. In our Lab a previous experiment was performed with the GWAS technique. It was identified new regulatory gens in the secondary growth in Arabidopsis thaliana where was found the PIN3 gen. This gen encode the protein that transports auxins. The auxins promotes the secondary growth, in parallel, the PIN3 has been associated to the response as gravity or light. In other way, it is also proved that the weight generated by the own plant triggers a good amount of vascular tissue, so the line of investigation could be to identify if PIN3 is causing this response. The aim of this study is to identify if PIN3 is a key player in the weight of the plant subjected to extra load for Col0 and mutant plants of PIN3. It is also a case of study the contribution of PIN3 to this response., El desarrollo de los organismos multicelulares está marcado por la integración y coordinación de diversos procesos de crecimiento. En las plantas, el crecimiento secundario es el proceso mediante el cual los tallos y las raíces crecen radialmente (en grosor). Este proceso es de vital importancia porque proporciona el soporte mecánico y la estabilidad que las plantas necesitan para poder expandir su crecimiento longitudinal y sostener sus órganos. Además, el crecimiento secundario es la fuente directa de una gran cantidad de biomasa, ya que es el origen de la madera en los árboles. Pese a la gran importancia del crecimiento secundario, actualmente, se conoce muy poco acerca de su regulación genética y molecular. Utilizando una estrategia basada en genómica comparada y asociaciones de variación genómica con variación fenotípica en diversos ecotipos de la planta modelo Arabidopsis thaliana, nuestro laboratorio ha podido identificar nuevos genes reguladores del proceso (Milhinhos et al.,2016; resultados no publicados),entre los cuales se encuentra el gen PIN3, que codifica una proteína de transporte de auxinas. Por una parte, se sabe que las auxinas promocionan el crecimiento secundario y, por otra, PIN3 ha sido previamente asociado a respuestas al medio ambiente tales como la luz o la gravedad. Por lo tanto, nuestra hipótesis de partida es que PIN3 modularía el crecimiento secundario dosificando los niveles de auxinas en función de las condiciones ambientales. El objetivo de este proyecto es probar nuestra hipótesis mediante la caracterización de la función concreta de PIN3 durante el crecimiento secundario. Para ello utilizará una estrategia genética, molecular y bioquímica que permitirá entender (i) qué condiciones medioambientales condicionan la actividad de PIN3 durante el crecimiento secundario, así como (ii) cuál es el mecanismo molecular mediante el cual dicha actividad es modulada.

[EN] Comparison of the proteins of thermophilic, mesophilic, and psychrophilic prokaryotes has revealed several features characteristic to proteins adapted to high temperatures, which increase their thermostability. These characteristics include a profusion of disulfide bonds, salt bridges, hydrogen bonds, and hydrophobic interactions, and a depletion in intrinsically disordered regions. It is unclear, however, whether such differences can also be observed in eukaryotic proteins or when comparing proteins that are adapted to temperatures that are more subtly different. When an organism is exposed to high temperatures, a subset of its proteins is overexpressed (heat-induced proteins), whereas others are either repressed (heat-repressed proteins) or remain unaffected. Here, we determine the expression levels of all genes in the eukaryotic model system Arabidopsis thaliana at 22 and 37 degrees C, and compare both the amino acid compositions and levels of intrinsic disorder of heat-induced and heat-repressed proteins. We show that, compared to heat-repressed proteins, heat-induced proteins are enriched in electrostatically charged amino acids and depleted in polar amino acids, mirroring thermophile proteins. However, in contrast with thermophile proteins, heat-induced proteins are enriched in intrinsically disordered regions, and depleted in hydrophobic amino acids. Our results indicate that temperature adaptation at the level of amino acid composition and intrinsic disorder can be observed not only in proteins of thermophilic organisms, but also in eukaryotic heat-induced proteins; the underlying adaptation pathways, however, are similar but not the same.

[EN] Ovule formation is a complex developmental process in plants, with a strong impact on the production of seeds. Ovule primordia initiation is controlled by a gene network, including components of the signaling pathways of auxin, brassinosteroids and cytokinins. By contrast, gibberellins (GAs) and DELLA proteins, the negative regulators of GA signaling, have never been shown to be involved in ovule initiation. Here, we provide molecular and genetic evidence that points to DELLA proteins as novel players in the determination of ovule number in Arabidopsis and in species of agronomic interest, such as tomato and rapeseed, adding a new layer of complexity to this important developmental process. DELLA activity correlates positively with ovule number, acting as a positive factor for ovule initiation. In addition, ectopic expression of a dominant DELLA in the placenta is sufficient to increase ovule number. The role of DELLA proteins in ovule number does not appear to be related to auxin transport or signaling in the ovule primordia. Possible crosstalk between DELLA proteins and the molecular and hormonal network controlling ovule initiation is also discussed.

[ES] El sistema vascular de las plantas consiste en una compleja red de tejidos que permiten el transporte de agua y moléculas hacia los órganos donde son requeridas. Por ello, el desarrollo vascular es importante para un correcto crecimiento de las plantas. Uno de los factores que intervienen en este proceso, a nivel de señalización celular, son las hormonas vegetales. En concreto las estrigolactonas (SLs) son una familia de fitohormonas que hasta ahora eran conocidas por sus efectos sobre la rizosfera y sobre la ramificación, pero que recientemente se han implicado también en el desarrollo vascular. Hasta ahora, se sabe que las SLs están implicadas en la regulación de la proliferación celular en el cambium vascular, pero se ignora si además regulan la adquisición de patrones específicos en la vasculatura. En este trabajo, se pretende caracterizar de un modo preciso cuales son los efectos a nivel de vasculatura que produce la ausencia de D14, receptor de SLs, en Arabidopsis thaliana (A.thaliana)., [EN] The plants vascular system consists in a complex network of transport tissue that allows the mobilization of diverse substances to the organs where they are required. Consequently, the vascular development is important for a proper plant growth. Phytohormones play a pivotal role in vascular development. Strigolactones (SLs) are a group of phytohormones mainly known for its effects on the rhizosphere and the branching, but which have been involved in the vascular development recently. Till now, it is known that SLs are involved in the regulation of the cellular proliferation, but it remains unclear whether they are also involved in the regulation of the vasculature pattern. In this work, we aim at shedding new light in this question by studying the effects on vasculature development of D14, the SLs receptor. To that end, we present work performed in mutants lacking D14 in the model system Arabidopsis thaliana (A.thaliana).

[EN] COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (alpha-, beta-, beta' -, gamma-, delta-, epsilon- and xi-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of gamma and delta, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of alpha 2-COP, an Arabidopsis alpha-COP isoform. The alpha 2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24 delta 5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The alpha 2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the alpha 2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the alpha 2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of alpha 2-COP affects the expression of secretory pathway genes.

[EN] EjFT1 and EjFT2 genes were isolated and sequenced from leaves of loquat. EjFT1 is involved in bud sprouting and leaf development, and EjFT2 in floral bud induction. Loquat [Eriobotrya japonica (Thunb.) Lindl.] is an evergreen species belonging to the family Rosaceae, such as apple and pear, whose reproductive development, in contrast with these species, is a continuous process that is not interrupted by winter dormancy. Thus, the study of the mechanism of flowering in loquat has the potential to uncover the environmental and genetic networks that trigger flowering more accurately, contributing for a better understanding of the Rosaceae floral process. As a first step toward understanding the molecular mechanisms controlling flowering, extensive defoliation and defruiting assays, together with molecular studies of the key FLOWERING LOCUS T (FT) gene, were carried out. FT exhibited two peaks of expression in leaves, the first one in early to mid-May, the second one in mid-June. Two FT genes, EjFT1 and EjFT2, were isolated and sequenced and studied their expression. Expression of EjFT1 and EjFT2 peaks in mid-May, at bud sprouting. EjFT2 expression peaks again in mid-June, coinciding with the floral bud inductive period. Thus, when all leaves of the tree were continuously removed from early to late May vegetative apex differentiated into panicle, but when defoliation was performed from early to late June apex did not differentiate. On the other hand, fruit removal advanced EjFT1 expression in old leaves and the sooner the fruit detached, the sooner the bud sprouted. Accordingly, results strongly suggest that EjFT1 might be related to bud sprouting and leaf development, while EjFT2 might be involved in floral bud induction. An integrative model for FT functions in loquat is discussed.

[EN] Floral induction is the necessary process for a plant to be capable to differentiate the meristem into reproductive structures. In Arabidopsis thaliana, it has been shown that the protein encoded by FT gene is produced in the leaves and transported to the meristem, acting as the messenger to floral bud induction. This gene seems to act universally since its, ortologues have been found in all the species studied. Our aim was to find it (them) in loquat (Eriobotrya japonica), and to sequence and characterize it (them). Two ortologues (EjFT1 and EjFT2), very similar two those found in the subfamily Maloideae species, have been found, diverging in function and differing in temporal expression, both expressing at the beginning of the vegetative growth, and EjFT2 also at the floral bud inductive period. Results suggest that EjFT2 might act as the floral bud inductor, and EjFT1 might have a wider functionality, as found in other species., [ES] La inducción floral es el proceso necesario para que una planta pueda diferenciar el meristemo en estructuras reproductivas. En Arabidopsis thaliana se ha demostrado que la proteína codificada por el gen FT actúa como el mensajero de la inducción floral, produciéndose en las hojas y transportándose hasta el meristemo. Este gen parece actuar de forma universal y se han encontrado ortólogos en todas las especies estudiadas. El objetivo de este trabajo es determinar la existencia de ortólogo(s) de FT en el níspero japonés (Eriobotrya japonica), secuenciarlo(s) y caracterizarlo(s). Se han encontrado dos ortólogos (EjFT1 y EjFT2) muy similares a los existentes en otras especies de la subfamilia Maloideae, pero con patrones de expresión distintos entre sí. Así, ambos se expresan al inicio del crecimiento vegetativo y EjFT2 lo hace también en el momento de la inducción floral. Este resultado sugiere que EjFT2 puede ser el responsable de la inducción floral en esta especie, y EjFT1 tendría una funcionalidad más amplia, como ocurre en algunas otras especies.