Circadian clocks are biological signalling networks which have a period of ~24 hours under constant environmental conditions. They have been identified in a wide range of organisms, from cyanobacteria to mammals and through the temporal co-ordination of biological processes are believed to increase individual fitness. The mechanisms which generate these self-sustained rhythms, the pathways of entrainment and the target outputs of the clock are all areas of great interest to circadian biologists. The plant circadian clock is believed to comprise of interlocking feedback loops of transcription and translation. The morning MYB-transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) bind to the promoter of TIMING OF CAB2 1 (TOC1) and repress its expression, as well as their own. As levels of CCA1 and LHY fall, TOC1 is expressed and activates the expression of its repressors. This is a simplified version of the known clock components and the current model contains this core loop as well as an interlocked morning and evening loop, which also incorporates some post-translational modification (Chapter 1). Understanding the plant circadian network and its entrainment are the topics of this thesis. The study has focused on two plant species, the land plant Arabidopsis thaliana and the picoeukaryotic marine algae Ostreococcus tauri. In both of these species light-mediated entrainment of the clock has been investigated (Chapter 8), as well as the core circadian mechanism. In A. thaliana the role of a circadian associated gene, EARLY FLOWERING 3 has been a particular focus for investigation, through both experimentation and mathematical models (Chapters 4 and 5). In O. tauri the responses to light signals have been tested, as have the circadian responses to pharmacological manipulation (Chapters 6, 7 and 8). The work presented identifies a role for ELF3 in the repression of circadian genes and also links it with the regulation of protein stability. Likewise, in O. tauri the regulation of protein stability is identified to be a key mechanism for sustaining circadian rhythms. As well as investigating the clock in plants, certain photoreceptors have been characterised in S. cerevisiae with the aim of linking them to a synthetic oscillator. Together the work presented in this thesis provides evidence for the circadian community to aid with the understanding of circadian rhythms in plants, and possibly other organisms.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25181号 / 医博第5067号 / 新制||医||1071(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 武藤 学, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

La température ambiante joue un rôle direct dans le fonctionnement et le développement de la plante. L'augmentation des températures ambiantes mondiales pose un défi important aux espèces de plantes sauvages et cultivées. La plupart des espèces de plantes ajustent leur cycle de reproduction et leur développement pour optimiser leur survie et leur forme par temps ambiant élevé (Barnabás et al., 2008; Fitter et Fitter, 2002; Willis et al., 2008). Ces adaptations conduisent généralement à des hypocotyles allongés, à une réduction du nombre de feuilles au moment de la floraison, à une transition accélérée des phases de croissance végétative à reproductive, à une réduction du nombre de graines, à des gousses plus petites et à une diminution de la surface foliaire. Face au changement climatique rapide, en particulier à l'augmentation de la température permissive à la croissance ambiante, il est urgent de régler la thermoréponse des usines pour adapter les installations aux changements climatiques et garantir la production alimentaire future.L'expression de PIF4 est contrôlée par le complexe du soir (EC), un complexe à 3 protéines comprenant ELF3, ELF4 et LUX, en fonction de la température. La CE est capable de réprimer l'expression de PIF4 en se liant à des motifs spécifiques sur le promoteur de PIF4 à une température de croissance ambiante inférieure. Cependant, cette répression de l'expression de PIF4 est éliminée à une température ambiante de croissance plus élevée, ce qui entraîne un vieillissement prématuré des plantes.RésultatsLes trois protéines de la CE ont été produites en utilisant une stratégie d’expression différente, ELF4 et LUX dans E. coli, et ELF3 dans des cellules d’insecte. Les méthodes de purification des protéines et les tampons ont été optimisés pour obtenir des ELF3 et LUX stables. Une chromatographie d'exclusion de taille a été réalisée pour vérifier les états oligomères des protéines. LUX étant la seule protéine de la CE à posséder un domaine de liaison à l'ADN connu, elle a été utilisée pour effectuer des tests de déplacement de la mobilité afin de comprendre l'affinité de liaison de LUX pour ses motifs ADN cibles obtenus à partir de tests de puces de liaison de protéines. D'après les tests de décalage de mobilité, il a été observé que le domaine de liaison à l'ADN (DBD) seul pouvait se lier aux motifs d'ADN de son substrat à des concentrations molaires nano alors qu'une quantité de protéine excédentaire de 10 fois était nécessaire pour que la longueur totale de LUX obtienne la même quantité de liaison. Des pistes de cristallisation à haut débit ont été réalisées pour LUX et LUX-DBD pleine longueur avec deux motifs de liaison différents. Aucun cristal n'a été obtenu pour le LUX complet, alors que des cristaux ont été obtenus pour le LUX DBD avec les deux motifs d'ADN. La structure de LUX DBD en complexe avec ses motifs cibles a été résolue par diffraction aux rayons X. À partir de la structure Crystal, il a été constaté que LUX DBD avait adopté une structure à 3 hélices, la seconde hélice étant responsable de la lecture de la base. Fait intéressant, il a été observé qu'un résidu d'arginine présent au niveau de l'hélice n-terminale servait de pince pour interagir avec le motif ADN cible.En utilisant les tests de mutagenèse dirigée et de décalage de mobilité, il a été confirmé que cette arginine présente à la position 146 de la protéine est essentielle pour déterminer l'affinité. Il a été constaté que l'affinité de liaison était réduite d'un facteur 5 lorsque cet acide aminé était modifié. En outre pour comprendre son effet in planta. Les lignes de lux mutantes ont été complétées par une longueur totale de type sauvage et une version substituée par Arg14Ala de la longueur totale de LUX. Grâce à ces expériences, nous avons pu montrer que la complémentation complète du mutant R146A n’était pas observée, alors que la version de type sauvage était capable de compléter complètement le phénotype du mutant / Ambient temperature plays a direct role in plant functioning and development. Increase in global ambient temperatures poses a significant challenge to wild and cultivated plant species. Most plant species adjust reproductive timing and development to optimize survival and fitness in higher ambient temperatures (Barnabás et al., 2008; Fitter and Fitter, 2002; Willis et al., 2008). These adaptations generally lead to elongated hypocotyls, fewer leaves at time of flowering, accelerated transition from vegetative to reproductive growth phases, fewer seeds, smaller seed pods and decreased leaf area. In the face of rapid climate change, specifically increased ambient growth permissive temperatures, tuning plant thermoresponse is urgently needed to engineer plants for adaptation to climate change and for securing future food production.PIF4 expression is controlled by evening complex (EC), a 3 protein complex comprising of ELF3, ELF4 and LUX, in a temperature dependent manner. The EC is able to repress PIF4 expression by binding to specific motifs at the promoter of PIF4 at lower ambient growth temperature. However this repression of PIF4 expression is removed at higher ambient growth temperature leading to premature ageing of plantsResultsAll three proteins of EC were produced using different expression strategy, ELF4 and LUX in E. coli while ELF3 in insect cells. Protein purification methods and buffers were optimized to obtain stable ELF3 ELF4 and LUX. Size exclusion chromatography was done to verify oligomeric states of the proteins. Since LUX is the only protein in the EC which has known DNA binding domain, it was used for doing mobility shift assays to understand the binding affinity of LUX for its target DNA Motifs which were obtained from protein binding microarrays assays. From the mobility shift assays, it was observed that the DNA binding domain (DBD) alone could bind to its substrate DNA motifs at Nano molar concentrations while 10 fold excesses amount of protein was required for the full length LUX to obtain same amount of binding. High throughput crystallization trails were carried out for full length LUX and LUX- DBD with two different binding motifs. No crystals were obtained for full length LUX while Crystals were obtained for LUX DBD with both the DNA Motifs. Structure for LUX DBD in complex with its target motifs were solved through X-Ray diffraction. Using site directed mutagenesis and Mobility shift assays it was confirmed that this Arginine present at the 146 position of the protein is critical for determining affinity. It was found that the binding affinity was reduced by a factor of 5 when this amino acid was changed. Further to understand its effect in planta.. With this experiments we were able to show that with the R146A mutant full complementation wasn’t observed while the wildtype version was able to completely complement the mutant phenotype.To understand temperature based dynamics of the complex, ELF3, which is the most intrinsically disordered protein of the three proteins that constitute the complex, was studied for structural variation through CD spectroscopy and DLS experiments. From these experiments it was found that ELF3 attains a β-sheet like confirmation at higher temperature while a more globular confirmation at lower temperatures. It was found that this activity of ELF is reversible allowing for flexibility of the whole complex. We found that there were prion like domains in ELF3 protein which were primarily responsible for transition to β-sheet structure at higher temperature.In Order to engineer plants that could survive at higher ambient temperature, we decided to mutate promoter elements of PIF4 through CRISPR/Cas9 to obtain plants that can survive higher temperature.

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorder is a disease caused by prenatal alcohol exposure. It is characterized by craniofacial abnormalities, growth retardation, central nervous system defects, learning disabilities and a variety of other minor defects. Even though it affects 2-5% of individuals born every year, very little is known about the mechanisms that cause it. The zebrafish (Danio rerio) presents as an interesting and efficient model for studying this disease. This study provides some insight into the mechanisms underlying observed FASD phenotypes and, more specifically, the transcription factor elf3, which is downregulated in response to ethanol exposure during early embryonic development. Here we show a number of elf3 target genes that are downregulated during early development in response to ethanol exposure. We also give some insight into the expression pattern of elf3 in relation to zygotic genome activation. Translation blocking morpholino oligonucleotides were used to implicate Elf3 in epiboly movements during gastrulation and zebrafish tail development. Taken together these results help to strengthen the zebrafish as a model for FASD in addition to giving greater insight into both the expression pattern and role of Elf3 during development.