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Boreal and temperate woody perennials are highly adapted to their local climate, which delimits the length of the growing period. Moreover, seasonal control of growth-dormancy cycles impacts tree productivity and geographical distribution. Therefore, traits related to phenology are of great interest to tree breeders and particularly relevant in the context of global warming. The recent application of transcriptional profiling and genetic association studies to poplar species has provided a robust molecular framework for investigating molecules with potential links to phenology. The environment dictates phenology by modulating the expression of endogenous molecular switches, the identities of which are currently under investigation. This review outlines the current knowledge of these molecular switches in poplar and covers several perspectives concerning the environmental control of growth-dormancy cycles. In the process, we highlight certain genetic pathways which are affected by short days, low temperatures and cold-induced signaling.

Additional file 2: Table S2. Description of GoldenBraid parts used in this work.

Background Winter dormancy is an adaptive mechanism that allows trees from temperate and cold regions to survive the harsh conditions of this season. Critical steps of this process are strongly influenced by environmental cues, mainly daylength and temperature. The mechanism that integrates these signals is the circadian clock. Despite the importance of the correct functioning of the clock for the healthy state of the plant [1], low temperatures cause the disruption of the circadian clock in trees, which consists in a transcriptional activation followed by an arrhythmic expression [2-5]. In this work we uncover winter-associated regulators of the circadian clock in poplar. Methods Firstly, we made a transcriptional fusion with the promoter of LHY2, a circadian clock gene, and the luciferase gene. This construct was used to generate transgenic poplars (717-1B4, INRA clone). With these events we characterized the expression of this promoter under different conditions of photoperiod and temperature. To this aim we have set up a circadian luminiscence assay registering luciferase activity from leaf discs with a luminometer. Then we carried out a Yeast One Hybrid (Y1H) screening with a library enriched in winter-associated factors and using this promoter as bait. Candidate regulators are tested in vivo using Golden Braid technology [6] and transient assays in poplar, by which we overexpressed and silenced the candidate genes. Results and Conclusions Here we present the characterization of the Populus tremula x alba LHY2 promoter under three different photoperiod conditions. Our results indicate the selected promoter region contains the circadian elements as well as the luciferase activity shows the expected expression under both long and short days. In the Y1H screening, we found several candidates that are classified either as transcription factors or chromatin remodelers. We will discuss the possible role of these proteins as regulators of the poplar circadian clock.

Background Woody species are highly adapted to their habitats. In response to environmental cues woody perennials trigger self-protective developmental programmes, in which signal transduction, transcriptional reprogramming and epigenetic regulation could participate in defining the winter dormancy state. Winter dormancy is the mechanism used by perennial plants to survive the harsh conditions of winter in temperate and cold regions and determines the geographical distribution of tree species (Chuine and Beaubien 2001; Horvath et al. 2003; Allona et al. 2008). Epigenetic control of winter dormancy in woody plants is barely known. Among the important epigenetic marks, 5-methyl cytosine (5mC) regulates gene expression in animals and plants. Global changes in 5mC DNA methylation have been shown in the transition of developmental stages in plants such as chestnut bud set and burst, flowering in azalea, aging in pine trees among other. However, the mechanism and the enzymes involved in the modification of the methylome and its control over those development processes remain to be identified. Our previous results showed higher DNA methylation and less acetylated Lys 8 of histone H4 global levels in poplar stem during winter dormancy compared to active growing season (Conde et al. 2013). In this study we focus in the understanding of the molecular mechanism behind these changes in DNA methylation profile and their role in the control of winter dormancy. Methods Analysis of the 5-methyl cytosine levels by the application of the immunofluorescence-based method set up in our lab, in stem vibratome sections cut from hybrid poplar (Populus tremula x alba) growing in the field at different stages of winter dormancy process. To develop a protocol for buds paraffin wax embedding to analyze the level of 5-methyl cytosine by applying our immunofluorescence-based method in poplar apex microtome sections in diferents stages of winter dormancy. RT-PCR analysis to determine the profile of gene expresion at diferent stages of winter dormancy involved in modification of DNA methylation profile. Hybrid poplar transformation to obtain transgenic lines with modified expression of a demethylase and phenological experiments with selected lines. Results and Conclusions The immunolocalization assays performed in poplar stem sections showed that DNA methylation leves fall suddenly when trees coming from the dormant state are near to restore the growing season. We have determined the spatial distribution of DNA methylation changes in this organ. We have identified two poplar homologs to Arabidopsis DME gene: PtaDML8/PtaDML10. The DME protein promotes global DNA demethylation along the genome during endosperm development. Our RT-PCR analyses indicate that the expression of PtaDML8/PtaDML10 genes increases significantly when trees are near to restart growing after winter dormancy. The phenologycal assays showed that PtaDML8/PtaDML10 knockdown plants have a delayed in resuming of growth after dormancy. Taken together, we hypothesize that an active control of the 5mC DNA methylation might play a key role in winter dormancy and that 5mC demethylases would be crucial in this process.

Winter dormancy is an adaptive mechanism that allows trees from temperate and cold regions to survive the harsh conditions of this season. This process is strongly influenced by daylength and temperature, which are signals that are integrated by the circadian clock. Low temperatures cause the disruption of the circadian clock in trees, which consists in a transcriptional activation followed by an arrhythmic expression. In this work we uncover winter-associated regulators of the circadian clock in poplar. We generated transgenic poplars that express a transcriptional fusion between the promoter of a circadian clock gene and the luciferase gene. Setting up a circadian luminiscence assay by which we register the luciferase activity from leaf discs with a luminometer, we have characterized the expression of this promoter under different photoperiods. Then, we carried out a Yeast One Hybrid (Y1H) screening with a library enriched in winter-associated factors using this promoter as bait. Candidate regulators are tested in vivo by transient assays in poplar, overexpressing and silencing the candidate genes. Our results indicate the selected promoter region contains the circadian elements as well as the luciferase activity shows the expected expression under both long and short days. In the Y1H screening, we found several candidates that are classified either as transcription factors or chromatin remodelers. We discuss the putative role of these proteins as regulators of the poplar circadian clock.

As plants are sessile organisms, the decision to grow or to stop growing is fundamental for their survival. To survive to the harsh condition of the winter, temperate and boreal trees undergo a self-protective developmental reprograming known as winter dormancy. Its mechanism involves an intricate interplay between endogenous growth regulators and the winter external signal, such as shortening of photoperiod and cold temperatures. Currently, only few factors including circadian and developmental transcription factors and hormonal signaling molecules have been shown to modulate the extension of the dormancy period in woody plants. In our laboratory, the circadian controlled transcription factor RAV promotes sylleptic branching and reduces dormancy length when RAV is ectopic overexpressed in poplar (1). Comparative mRNA profiling of RAV overexpressing versus wild type revealed a transcriptional rearrangement happens as effect of constitutive high level of RAV, giving rise to phenology. However, to define RAV mode of action as a transcriptional regulator and how RAV activity trigger a modification of dormancy-growth cycle, its primary targets genes should be investigated. At this particular, the translational fusion of a given transcription factor to the rat glucorticoid receptor domain (GR) have been widely used in Arabidopsis and recently in poplar (2). In this work we show the standardization of a methodology required to identify primary target genes for a transcription factor in poplar. This will be useful to future exploration of RAV1 gene network and its mode of action in spatio-temporal studies at tissue level during growth-dormancy cycle in poplar.

Trees have developed the mechanism of winter dormancy to survive the harsh conditions of winter in temperate and cold regions. It determines the geographical distribution of tree species and the long of the growing period. Epigenetic control of winter dormancy in woody plants is barely known. 5-methyl cytosine (5mC) is an important epigenetic mark that regulates gene expression in animals and plants. The profile of DNA methylation changes in the transition of developmental stages in plants such as chestnut bud set and burst, flowering in azalea, aging in pine trees among other. So far, the enzymes involved in the modification of the methylome and the molecular mechanism behind the control over those development processes remain to be understood. Our previous results showed higher DNA methylation and less acetylated Lys 8 of histone H4 global levels in poplar stem during winter dormancy compared to active growing season. Analysis of the 5-methyl cytosine levels by the application of the immunofluorescence-based method set up in our lab showed that global DNA methylation decrease significantly when trees are near to restore the growing season after the winter dormancy. We have identified two poplar homologs to Arabidopsis DME gene: PtaDML8/PtaDML10. DME protein promotes global DNA demethylation along the genome during the endosperm development. Our RT-PCR analyses indicate that the expression of PtaDML8/PtaDML10 genes increases significantly when trees are near to restart growing after the winter. Functional characterization showed that PtaDML8/PtaDML10 knockdown plants have a delayed in resuming of growth after dormancy. Taken together, we hypothesize that an active control of the 5mC DNA methylation play a key role in winter dormancy and that 5mC demethylases would be crucial for poplar dormant-growth transition

Winter dormancy is the mechanism used by perennial plants to survive the harsh conditions of winter in temperate and cold regions and determines the geographical distribution of tree species (Chuine and Beaubien 2001; Horvath et al. 2003; Allona et al. 2008). Epigenetic control of winter dormancy in woody plants is barely known. Among the important epigenetic marks, 5-methyl cytosine (5mC) regulates gene expression in animals and plants. Global changes in 5mC DNA methylation have been shown in the transition of developmental stages in plants such as chestnut bud set and burst, flowering in azalea, aging in pine trees among other. However, the mechanism and the enzymes involved in the modification of the methylome and its control over those development processes remain to be identified. Our previous results showed higher DNA methylation and less acetylated Lys 8 of histone H4 global levels in poplar stem during winter dormancy compared to active growing season (Conde et al. 2013). Analysis of the 5-methyl cytosine levels by the application of the immunofluorescece-based method set up in our lab showed that DNA methylation leves fall suddenly when trees are near to restore the growing season coming from the dormant state. We have identified two poplar homologs to Arabidopsis DME gene: PtaDML8/PtaDML10. DME protein promotes global DNA demethylation along the genome during the endosperm development. Our RT-PCR analyses indicate that the expression of PtaDML8/PtaDML10 genes increases significantly when trees are near to restart growing after winter dormancy. The phenologycal assays showed that PtaDML8/PtaDML10 knockdown plants have a delayed in resuming of growth after dormancy. Taken together, we hypothesize that an active control of the 5mC DNA methylation might play a key role in winter dormancy and that 5mC demethylases would be crucial in this process.

Complex I of Arabidopsis includes five structurally related subunits representing gamma-type carbonic anhydrases termed CA1, CA2, CA3, CAL1, and CAL2. The position of these subunits within complex I was investigated. Direct analysis of isolated subcomplexes of complex I by liquid chromatography linked to tandem mass spectrometry allowed the assignment of the CA subunits to the membrane arm of complex I. Carbonate extraction experiments revealed that CA2 is an integral membrane protein that is protected upon protease treatment of isolated mitoplasts, indicating a location on the matrix-exposed side of the complex. A structural characterization by single particle electron microscopy of complex I from the green alga Polytomella and a previous analysis from Arabidopsis indicate a plant-specific spherical extra-domain of about 60 A in diameter, which is attached to the central part of the membrane arm of complex I on its matrix face. This spherical domain is proposed to contain a heterotrimer of three CA subunits, which are anchored with their C termini to the hydrophobic arm of complex I. Functional implications of the complex I-integrated CA subunits are discussed.

12 Pàg., The adaptation and survival of boreal and temperate perennials relies on the precise demarcation of the growing season. Seasonal growth and development are defined by day length and temperature signals. Under long-day conditions in spring, poplar FLOWERING LOCUS T2 (FT2) systemically induces shoot growth. In contrast, FT2 downregulation induced by autumnal short days triggers growth cessation and bud set. However, the molecular role of FT2 in local and long-range signaling is not entirely understood. In this study, the CRISPR/Cas9 editing tool was used to generate FT2 loss of function lines of hybrid poplar. Results indicate that FT2 is essential to promote shoot apex development and restrict internode elongation under conditions of long days. The application of bioactive gibberellins (GAs) to apical buds in FT2 loss of function lines was able to rescue bud set. Expression analysis of GA sensing and metabolic genes and hormone quantification revealed that FT2 boosts the 13-hydroxylation branch of the GA biosynthesis pathway in the shoot apex. Paclobutrazol treatment of WT leaves led to limited internode growth in the stem elongation zone. In mature leaves, FT2 was found to control the GA 13-hydroxylation pathway by increasing GA2ox1 and reducing GA3ox2 expression, causing reduced GA1 levels. We here show that in poplar, the FT2 signal promotes shoot apex development and restricts internode elongation through the GA 13-hydroxylation pathway., The study was supported by grants PGC2018-093922- B-I00 awarded to IA and MP and SEV-2016-0672 (2017-2021) to the CBGP “Severo Ochoa Programme for Centres of Excellence in R&D” from the Agencia Estatal de Investigación of Spain. DG-S has a FPI fellowship (PRE2019-089312) from Ministerio de Ciencia e Innovacion de España

17 Pág., Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown. By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) and two demethylases DEMETER-LIKE (DML) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor. We compared the ectomycorrhizas formed between transgenic and wild-type (WT) trees and analyzed their methylomes and transcriptomes. The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi-ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX-dml and RNAi-ddm1) and 120 genes/1441 TEs in the fungal genome suggesting a host-induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes (cis and trans effects) in common with 17 being differentially methylated (cis). Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate-mediated pathways, and immune response but also terpenoid metabolism., MDS and JV received PhD grants from the MRES and Conseil Régional Région Centre Val de Loire, respectively. The LBLGC and the LEE/CNRGH benefit from the support of the ANR EPITREE (ANR-17-CE32-0009-01, https://www6.inrae.fr/epitree-project_eng/) to SM and JT. We thank RTP3E CNRS and IHPE for the access to the bioinformatic platform (http://galaxy.univ-perp.fr/, Christian Chaparro, France). CVF, AK, FM, FG, and LF benefit from the support of the Genomic Science Program (project ‘Plant-Microbe Interface’), US Department of Energy, Office of Science, Biological and Environmental Research under the contract DE-AC05-00OR22725 and the Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems (ARBRE; grant ANR-11-LABX-0002-01). The work (proposal: 10.46936/10.25585/60001022) conducted by the US Department of Energy JGI (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy operated under Contract no. DE-AC02-05CH11231.

The aim of this study is to design a biohybrid neural interface capable of performing synapses with a motor neuron. Following the technology of graphene-based field-effect transistors (gFET), a device capable of generating an electrical signal when the neurotransmitter acetylcholine (ACh) is released has been developed. This device consists of two gold electrodes acting as a drainer and transistor source with a graphene gate. Graphene is a biocompatible material sensitive to pH changes, changing its conductive properties depending on the concentration of protons in the medium. Acetylcholinesterase (AChE) is an enzyme present in neurons of the peripheral nervous system and is capable of hydrolyzing ACh into acetate and choline, releasing protons. The method of detection of ACh is based on the catalytic activity of AChE. To immobilize the AChE enzyme in the gold electrodes of the gFET, a Silanization by Activated Steam (AVS) process has been studied to functionalize titanium sheets metallized with gold. This technique coats the gold surface with a high density of primary amino groups. Utilizing the chemistry of the crosslinkers it is intended to immobilize the enzyme covalently and permanently Immobilization of AChE by crosslinkers N-(3 Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was achieved by a stable but non-durable amide bond. On the other hand, immobilization by the crosslinker sulfosuccinimidil 6-(3'-(2-pyridyldithio) propionamido) hexanoate (sulfo-LC-SPDP) was not achieved, but it has been shown that its binding to AChE does not prevent the catalytic activity of the enzyme.

En los inicios de la enfermedad pandémica por coronavirus (COVID-19), causada por el síndrome respiratorio agudo severo coronavirus 2 (SARS-CoV-2) presenta una gravedad variable desde episodios asintomáticos hasta muy graves. El COVID-19, de la familia Coronaviridie, afecta principalmente a las vías respiratorias que produce tos seca, fiebre, mialgia, dolor de cabeza, fatiga hasta una neumonía intersticial e insuficiencia respiratoria grave. Sin embargo, algunos de estos pacientes registraron manifestaciones cutáneas y lesiones del sistema vascular fuera de la esfera respiratoria como síndromes autoinflamatorios severos (fiebre mediterránea familiar, dermatosis neutrofílicas), erupciones vesiculares, urticarianas y maculopapulares, livedo, necrosis y otras formas de vasculitis o síndromes DRESS conocidos por reacciones adversas hacia fármacos. Se desconocen los mecanismos por el que se lleva cabo estas lesiones cutáneas que se han ido relacionando con mayor frecuencia dentro del cuadro infeccioso COVID-19. Estos síntomas pueden estar provocados por una infección viral, una respuesta adversa hacia el fármaco o ambos. Además, estos signos clínicos pueden empeorar acorde a la susceptibilidad de los pacientes ligada a los antígenos leucocitarios humanos (HLA) que pueden provocar una tormenta inmune que causa un daño multiorgánico e incluso la muerte. El objetivo principal de este Trabajo de Fin de Grado consiste en estudiar la importancia del papel de la respuesta celular en las lesiones cutáneas inducidos por virus y por fármacos a través de la prueba de transformación de linfocitos (LTT). Estos basados en la capacidad de las células T de memoria del paciente específicas del fármaco para que proliferen una vez que hayan sido estimuladas por un antígeno. Los resultados obtenidos muestran si los linfocitos han proliferado sobre los diferentes tratamientos genéricos que se usaron en la primera oleada COVID-19: Azitromicina, Amoxicilina, Ácido Clavulánico, Hidroxicloroquina, Lopinavir y Ritonavir. De modo que en presencia de proliferación nos puede aportar información para acercarnos el origen de las lesiones cutáneas descritas de los pacientes. Aunque aún desconocemos el mecanismo molecular, los datos obtenidos en este trabajo permiten apoyar la idea de que los ensayos LTT no se correlaciona con las pruebas cutáneas y de provocación y parece tan solo asociarse con la exposición hacia fármacos.

Las variaciones ambientales determinan el crecimiento estacional, las transiciones en el desarrollo y el comportamiento reproductivo de plantas y animales. Se conoce muy poco de cómo los organismos integran la información estacional a nivel molecular. En plantas perennes como el chopo, los ciclos anuales de crecimiento-dormancia garantizan la supervivencia y la reproducción sexual. Estos ciclos también determinan varias características de gran importancia económica, como la producción de biomasa y el rendimiento de los cultivos. El conocimiento de los mecanismos moleculares que dan origen a los eventos fenológicos anuales permite el diseño de estrategias biotecnológicas para mejorar la adaptación de los arboles frente al cambio climático. En los últimos años, se han realizado importantes avances en este campo de estudio. Se ha demostrado que varios reguladores de la floración de Arabidopsis participan en la regulación de la transición del período de crecimiento al período de dormancia en chopo. Estos genes actúan como interruptores sensibles a los cambios medioambientales controlando diversos aspectos de la fisiología y el desarrollo de los árboles. Sin embargo, se sabe poco sobre los mecanismos que participan en la aclimatación del chopo a las condiciones ambientales del otoño. Así mismo, todavía no se ha identificado la conexión entre la aclimatación otoñal del chopo y la regulación estomática. En esta tesis, se investiga el papel funcional del factor de transcripción RAV1, un ortólogo del represor floral TEMPRANILLO de Arabidopsis, durante la transición del período de crecimiento al período de dormancia invernal. Mostramos que RAV1 no reprime a FT2 ni desempeña un papel en la regulación del cese de crecimiento. Por lo tanto, la regulación fotoperiódica del crecimiento apical en chopo muestra características conservadas y diferentes con respecto a su función en la floración de Arabidopsis. Hemos identificado los genes controlados a nivel transcripcional por RAV1, lo que revela su implicación en la regulación estacional de la respuesta de la planta al estrés ambiental. También demostramos que RAV1 controla la expresión de los genes RESPIRATORY BURST OXIDASE HOMOLOG D1 (RbohD1) y ETHYLENE RESPONSE FACTOR 72-1 (ERF72-1), que muestran una estrecha coexpresión con RAV1 en condiciones de días cortos y baja temperatura, además de durante la inducción otoñal. Las líneas de sobreexpresión de RAV1 mostraron niveles más altos de conductancia estomática, fotosíntesis y biomasa respecto a las plantas silvestres (WT). Por el contrario, las plantas con niveles de expresión mas bajos de RAV1 (RAV1kd) no mostraron diferencias con respecto al ecotipo silvestre. Además, demostramos que la sobreexpresión de RAV1 producía mas especies reactivas de oxigeno (ROS) en las células estomáticas y un cierre más rápido de las estomas después del tratamiento con la hormona ABA. Además, el tratamiento con el inhibidor DPI redujo parcialmente el cierre estomático dependiente de ABA, implicando de esta manera a la enzima RBOH en este proceso. Las medidas de conductancia estomática a lo largo del día en condiciones de día corto y baja temperatura demostraron que las plantas que sobreexpresan RAV1 tienen un movimiento estomático diurno más rápido y eficaz en comparación con las plantas WT y RAV1kd. En este trabajo, proponemos que la activación otoñal del RAV1 aumenta la actividad estomática diurna que contribuye a la aclimatación del chopo a las condiciones del otoño. Por otra parte, observamos que la transcripción de RbohD muestra ritmos diurnos en chopo y Arabidopsis. Es importante destacar que los ritmos diurnos del gen RbohD en Arabidopsis, generados por la integración de señales cíclicas endógenas y ambientales, contribuyen a la magnitud de la respuesta de defensa contra el ataque de patógenos. ----------ABSTRACT---------- Environmental variations determine seasonal growth, developmental transitions and reproductive behavior in plants and animals. How these organisms integrate seasonal information at the molecular level remain poorly understood. In perennial plants such as poplar, growth-dormancy cycles guarantee survival and sexual reproduction. They also determine several economically important traits, such as tree annual growth rate and yield. Understanding the molecular mechanisms underlying tree annual phenological events supports the design of strategies for tree biotechnology facing plant adaptation and acclimation to climate change. In the last years, important advances have been made in this field of study. Several regulators of Arabidopsis flowering have been shown to participate in the regulation of poplar growth to dormancy transition. These genes act as environmental sensitive switches controlling diverse aspects of tree annual development. One aspect little investigated is the regulation of poplar acclimation during autumnal season. Moreover, the link between poplar autumnal acclimation and stomatal regulation has not yet been identified. In this thesis, the functional role of RAV1 transcription factor, a poplar ortholog to the Arabidopsis floral repressor TEMPRANILLO/RELATED TO ABI3 AND VP1 (TEM/RAV1), is investigated during the transition from growth to dormancy period. We found that RAV1 neither represses FT2 nor plays a role in the regulation of growth cessation, therefore photoperiodic regulation of shoot growth in poplar show conserved and distinct features respect to Arabidopsis flowering. Through a reverse genetic approach, we identified genes transcriptionally controlled by RAV1, revealing its implication in the seasonal regulation of plant response to environmental stresses. We found that RAV1 control the expression of RESPIRATORY BURST OXIDASE HOMOLOG D1 and ETHYLENE RESPONSE FACTOR 72-1, showing a tightly coexpression under short days and low ambient temperature and that showed autumnal induction. RAV1 overexpressing lines showed higher levels of stomatal conductance, photosynthesis, and biomass than wild-type and RAV1 knockdown plants. We showed that an increased guard cell-ROS burst in hybrid poplar RAV1 overexpressing plants is associated with faster stomatal closure after ABA treatment. DPI treatment partially reduced ABA-dependent stomatal closure implicating the RBOH enzyme. Time course changes in stomatal conductance under short days and low ambient temperatura conditions indicated that RAV1 overexpressing plants showed faster diurnal stomatal movement in comparison with wild-type and RAV1 knock down lines. We propose that seasonal activation of RAV1 increases the diurnal stomatal activity contributing to poplar acclimation during autumn. Moreover, we found that RbohD transcription shows diurnal rhythms in poplar and Arabidopsis. Importantly, RbohD gene diurnal rhythms in Arabidopsis, generated by integrating endogenous and environmental cyclic signals, contribute to the magnitude of defense response against pathogen attack.

As sessile organism, plants require adaptive mechanisms to fit its development with their local environment. The adaptative versatility is especially meaningful for woody perennials trees from temperate and boreal regions, long-life organisms that have to deal with the harsh winter conditions year after year. With the aim to survive to this season, perennials have evolved the vegetative growth-dormancy phenological transition. During winter dormancy plants enter in a growth quiescent state resistant to freezing and drought stress. In poplar, the shortening of daylength leads to shoot apical growth cessation that is the gateway of dormancy establishment. During last decade, molecular components mediating this photoperiodic control of growth cessation have started to emerge, however understanding the mechanism involved require deeper analysis. Previous studies performed in the laboratory, using poplar as the experimental system, proved that the photoperiod integrator and growth inductor FLOWERING LOCUS T 2 (FT2), is quantitatively repressed in response to the extension of the night. An investigation of the photoperiodic repression route was initiated revealing that the clock gene LATE ELONGATED HYPOCOTYL 2 (LHY2) is involved. Moreover, it was identified the phytochrome signalling pathway as the responsible for perceiving and transferring the photoperiodic external information to LHY2. These results have led to hypothesize that a novel pathway senses the progressive extension of the night and eventually represses FT2 expression, causing poplar shoot apical growth cessation. The purpose of this work is to provide new experimental evidences that support this model, focusing on understanding the photoperiodic control of LHY2 transcription. This study evidences that night break assays, where poplars were subjected to a red-light pulse in the middle of the night, release the repression of FT2 and subsequently the bud set establishment triggered under short days. Moreover, in these conditions LHY2 expression, as well as the other growth repressors acting up/downstream of LHY2 are downregulated, supporting the model hypothesized. Furthermore, it is shown that red-light illumination inhibits LHY2 activation at the end of the night, pointing that phytochrome signalling pathway could control its transcription. Remarkably, it is demonstrated that the ELONGATED HYPOCOTYL 5 chromosome 10 (HY5_10) gene, whose homologue in Arabidopsis is known to act downstream of the phytochromes and physically interact with PHYTOCHROME-INTERACTING FACTOR 1 (PIF1), binds to cis-regulatory elements of the LHY2 promoter, previously identified as essentials for its correct photoperiodic transcriptional activation. Thus, a new model of LHY2 transcriptional regulation based on PIF1/HY5_10 ratio is proposed.

El movimiento de traslación de la Tierra junto a la inclinación de su eje de rotación, hacen que las latitudes medias del planeta presenten cuatro estaciones en el año. Las variaciones climáticas que estas estaciones generan, provocan que plantas y animales desarrollen procesos fenológicos. Algunos de estos procesos están controlados por el reloj circadiano, un complejo sistema cuya función consiste en programar muchas de las funciones fisiológicas diarias de un organismo a la mejor hora del día. Además, este sistema integra la información de variables climáticas y no climáticas, como la temperatura y el fotoperiodo, transmitiendo dicha información al resto del organismo. En las plantas, estos cambios en el ambiente provocan adaptaciones locales. La dormancia invernal es uno de estos procesos fenológicos influida principalmente por el fotoperiodo y la temperatura. En especies de árboles de latitudes templadas y boreales, este proceso tiene la función de asegurar la supervivencia del árbol al invierno. En algunas especies forestales como el chopo, la dormancia invernal es inducida por el acortamiento del fotoperiodo, el cual se produce de manera gradual desde el verano hasta el invierno. Por tanto, el acortamiento de los días se da tanto retrasando el amanecer como adelantando el anochecer. Hasta el momento, los ciclos de crecimientodormancia han sido explicados teniendo en cuenta únicamente el adelantamiento de las noches. Es por ello que desconocemos si la extensión de las noches tiene alguna función en la parada del crecimiento asociada a la entrada en dormancia, y si el reloj circadiano contribuye de alguna manera a esta posible función. En esta tesis abordaremos esta hipótesis usando el chopo como organismo de estudio. En primer lugar, adaptamos un sistema automático de medida de la actividad luciferasa midiendo dicha actividad en hojas de plantas transgénicas de chopo que expresaban un reportero transcripcional de un gen del reloj circadiano. Además, basándonos en trabajos previos, pusimos a punto la técnica de la expresión transiente en chopo, lo que nos permitió realizar ensayos de transactivación. Esta metodología, junto con el uso de la mutagénesis dirigida, el análisis transcriptómico y la genética molecular, nos permitió revelar una nueva ruta de señalización que traduce la información de la extensión de las noches y que reprime la expresión génica del principal integrador del fotoperiodo y promotor del crecimiento, FLOWERING LOCUS T (FT), todo ello a través de un gen del reloj circadiano. ----------ABSTRACT---------- Mid-latitudes present four seasons along the year, which are caused by the translational movement of the Earth along with its tilted axis. The climate variations associated to the seasons provoke the existence of phenological events in plants and animals. Some of these events are controlled in some extent by the circadian clock, a complex mechanism which function is to set the best time for most of the daily physiological functions, but that also to translate the information of the main climate and non-climate related cues, such as temperature and photoperiod, to the rest of the organism. In the case of plants, that situation generates local adaptations. Winter dormancy is one of these phenological events which is mainly influenced by photoperiod and temperature. For boreal and temperate trees, this process ensures that trees survive the winter and are able to resume their growth after that season. In some tree species, as poplar, the entrance in dormancy is triggered by short photoperiod. From summer to winter, photoperiod starts decreasing in a gradual manner, both delaying the dawn and advancing the dusk. So far, the growth-dormancy cycle is explained by the progressive advance of nights. Then, we still do not know whether the progressive extension of nights have an impact upon dormancy entry and whether the circadian clock plays a role on it. In this thesis, we deal with that hypothesis using poplar as the experimental system. To this aim, we first adapted an automatic methodology to register luciferase activity from transgenic poplar leaves, which express a transcriptional reporter of a circadian clock gene. We also set up the transient expression assay in poplar taking advantage of previous work, to carry out transactivation studies. These methodologies along with the use of directed mutagenesis, transcriptomic analyses and molecular genetics, allowed us to unravel a novel signalling pathway which senses the progressive extension of the nights and represses the gene expression of the photoperiod integrator and growth promoting factor, FLOWERING LOCUS T (FT), via a circadian clock gene.