As plants are sessile organisms, processes controlling plant growth and development must react to fluctuations in the external environment to aid plant survival. However, as the climate of the Earth changes and becomes more extreme, plants become less able to develop to their optimal capacity and this can have an adverse effect on crop yield and biofuel feedstock production. Thus, it is becoming increasingly important to understand the molecular mechanisms used by plants to respond to external stimuli. One important system that plants utilise in their response to environmental fluctuations is the circadian clock. The circadian clock is a time-measuring device that buffers the timing of plant growth and development against fluctuations in the local environment, such as temperature, light quality and light intensity. Importantly, the circadian clock is also able to measure day-length (photoperiod). Thus, plant development and growth is co-ordinated with photoperiod that is closely linked to seasonal changes. A key example of this is the time taken for a plant to flower. Flowering of Arabidopsis thaliana occurs specifically in long-days (LDs) of spring/summer months. Thus, the circadian clock is a key regulator promoting flowering in LD conditions. In conjunction with experimental studies, mathematical modelling has proven to be a successful method of elucidating the mechanisms that underlie complex biological systems. One example of this 'systems biology' approach is in uncovering the components that make up the Arabidopsis circadian clock mechanism. Previous research in our group has also led to the development of a model describing photoperiodic flowering that is tentatively linked to the circadian clock mechanism. In this thesis I shall develop on these models to highlight five key results: 1. using rhythmic PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 mRNA as an example, I shall show that multiple circadian regulators are required to describe rhythmic transcription of target genes across multiple photoperiods; 2. the stabilisation of CONSTANS (CO) protein by the blue light-signalling component FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) is required to for flowering in LDs and has a relatively larger impact on photoperiodic flowering than FKF1-dependent degradation of CYCLING DOF FACTOR 1 (CDF1), an inhibitor of flowering; 3. multiple components of the circadian clock play specific post-translational roles in photoperiodic flowering to promote the acceleration of flowering specifically in LDs; 4. temperature regulation of photoperiodic flowering can be explained through an interaction between CO and PIF proteins, limiting the effects of temperature to a specific time-window in a 24-hour day; 5. red light- and temperature-control of the circadian clock can be explained by altering the post-translational regulation of circadian clock components.

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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.

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defense responses to pathogens and pests. In contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. We cloned and characterized three maize LOXs ZmLOX10 ZmLOX11 and ZmLOX12. Both ZmLOX10 and ZmLOX11 apeared to be 13-LOX, whereas ZmLOX12 is a unique 9-LOX. Whereas leaf was the preferential site of ZmLOX10 expression, ZmLOX11 was strongly expressed in silks. Induction of these ZmLOX10 and ZmLOX12 by wounding and defense-related compounds suggested their role in plant resistance mechanisms against pests and pathogens. Abscisic acid, however, was the only inducer of ZmLOX11 in leaves. Higher increase in ZmLOX10 transcripts in maize infected by fungus Cochliobolus carbonum implicated this gene in resistance responses to necrotrophic pathogens. In addition, ZmLOX10 was shown to be the first reported LOX to be regulated by a circadian clock. It was found that ZmLOX10 was also inducible by low temperatures. Phenotypical studies of wild type and mutant near isogenic lines showed that expression of ZmLOX12, specific to underground organs, was required for pathogenesis of F. verticillioides on maize mesocotyls.

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defense responses to pathogens and pests. In contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. We cloned and characterized three maize LOXs ZmLOX10 ZmLOX11 and ZmLOX12. Both ZmLOX10 and ZmLOX11 apeared to be 13-LOX, whereas ZmLOX12 is a unique 9-LOX. Whereas leaf was the preferential site of ZmLOX10 expression, ZmLOX11 was strongly expressed in silks. Induction of these ZmLOX10 and ZmLOX12 by wounding and defense-related compounds suggested their role in plant resistance mechanisms against pests and pathogens. Abscisic acid, however, was the only inducer of ZmLOX11 in leaves. Higher increase in ZmLOX10 transcripts in maize infected by fungus Cochliobolus carbonum implicated this gene in resistance responses to necrotrophic pathogens. In addition, ZmLOX10 was shown to be the first reported LOX to be regulated by a circadian clock. It was found that ZmLOX10 was also inducible by low temperatures. Phenotypical studies of wild type and mutant near isogenic lines showed that expression of ZmLOX12, specific to underground organs, was required for pathogenesis of F. verticillioides on maize mesocotyls.

The cyanobacterium Synechococcus elongatus builds a circadian clock on an oscillator comprised of three proteins, KaiA, KaiB, and KaiC, which can recapitulate a circadian rhythm of KaiC phosphorylation in vitro. The molecular structures of all three proteins are known, and the phosphorylation steps of KaiC, the interaction dynamics among the three Kai proteins, and a weak ATPase activity of KaiC have all been characterized. A mutant of a clock gene in the input pathway, cikA, has a cell division defect, and the circadian clock inhibits the cell cycle for a short period of time during each cycle. However, the interaction between the circadian cycle and the cell cycle and the molecular mechanisms underlying it have been poorly understood. In addition, the subcellular localization of clock proteins and possible localization dynamics, which are critical in the timing circuit of eukaryotic clock systems and might also shed light on the interaction between circadian cycle and cell cycle, have remained largely unknown. A combination of genetics, cell biology, and microscopy techniques has been employed to investigate both questions. This work showed that the cell division defect of a cikA mutant is a function of the circadian clock. High ATPase activity of KaiC coincides with the inhibition of cytokinesis by the circadian clock. CikA likely represses KaiC's ATPase activity through an unknown protein, which in cikA's absence stimulates both the ATPase and autokinase activities independently of KaiA or KaiB. SasA-RpaA acts as an output in the control of cell division, and the localization of FtsZ is the target, although it still remains to be seen how RpaA, directly or indirectly, inhibits FtsZ localization. The project also showed that clock proteins are localized to the cell poles. KaiC is targeted to the cell pole in a phosphorylation-dependent manner. KaiB and CikA are also found at the poles independently of KaiC. KaiA likely only localizes to the cell pole during the dephosphorylation phase, which is dependent on both KaiB and KaiC, specifically on the phosphorylation of KaiC at S431. Overall, significant progress was made in both areas and this project sheds light on how the circadian oscillator operates in cyanobacterial cells and interacts with another fundamental cellular function.

The circadian clock system is essential for mammals to adapt to environmental conditions such as light-dark cycles and to manage the optimal timing for cyclical physiological processes, including sleep-wakefulness, fasting-feeding and multiple aspects of metabolism. The circadian timing system is arranged in hierarchical fashion, with the master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus, acting as the pace-maker and maintaining synchrony among clocks found in every organ system throughout the body. The core molecular clock consists of two interconnected transcriptional-translational feedback loops comprising core clock components: Brain and Muscle Arnt-Like protein1 (BMAL1), Circadian Locomoter Output Cycles Kaput (CLOCK), Period (PER), Cryptochrome (CRY), Nuclear Receptor family1 D1 (REV-ERB) and Retinoic acid-related Orphan Receptor (ROR). Circadian clock disruptions, through environmental changes to light-dark cycles or through genetic modification of core clock genes cause metabolic disturbances. Aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, mediates systemic metabolism and toxicity of a range of environmental contaminants. Epidemiological studies have established a positive correlation between exposure to dioxins and other synthetic organic chemicals and metabolic diseases such as diabetes and dyslipidemia. Animal research have supported these findings by showing that AhR activation has detrimental effects on glucose and lipid homeostasis. Mechanisms for AhR-mediated metabolic dysfunction remain unknown. Coincidently, both AhR and many core clock components, for example BMAL1 and CLOCK, belong to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domain family. Previous studies have linked AhR signaling to circadian rhythm. Importantly, activation of the AhR can impair transcriptional activity of the CLOCK: BMAL1 heterodimer in cultured cells. However, because the AhR is differentially expressed among the body’s tissues, its activation may have distinctive, tissue-specific effects on the hierarchical circadian clock oscillators in vivo, which have not been investigated. Therefore, this dissertation is designed to examine the short-term and long-term effects of AhR activation on circadian clocks and downstream clock-regulated metabolic pathways. Specifically, this dissertation is aimed to explore how acute and chronic activation of AhR affects rhythmic aspects of behavior, as well as clock-controlled glucose and lipid metabolism. In the acute AhR activation model, a single dose of the synthetic AhR agonist, β-Naphthoflavone (BNF), was administered to C57Bl/6J wild type mice. Circadian behavior was monitored before and after acute AhR activation. Circadian expression of core clock genes, as well as key metabolic genes in the liver, skeletal muscle and adipose tissue were examined. Compared to the vehicle group, BNF-treated mice displayed a transient loss of behavioral rhythmicity and delayed activity onset, which suggest that acute activation of AhR acts directly on the central clock, the suprachiasmatic nucleus of the hypothalamus. In contrast, circadian oscillations of core clock genes were not eliminated in the peripheral tissues (liver, skeletal muscle and adipose tissue), but changes were observed in their rhythmic amplitude or phase. Rhythms of key enzymes related to glucose and lipid metabolic pathways in the liver and adipose were decreased while those in the skeletal muscle were increased. These results indicate that acute AhR activation affects the central clock and peripheral clock differently. Moreover, acute AhR activation significantly dampened the rhythm of genes involved in lipogenesis, lipolysis and lipid storage. In the chronic AhR activation model, C57Bl6/J mice were exposed to BNF for a month to explore whether long-term AhR activation can cause bigger disruption of circadian clocks and lead to metabolic dysfunction in vivo. Unexpectedly, general circadian behavior was maintained although after each dose of BNF there was a consistent, transient loss of behavioral rhythmicity and significant phase delay (about 30 minutes) in BNF-treated mice. Liver and skeletal muscle clocks were not significantly altered after 4 doses of BNF, and the in-phase oscillations of core clock genes in liver and skeletal muscle suggested a functional SCN as well as the two peripheral clocks. However, the adipose clock was significantly disrupted. Altered clock-regulated rhythms in lipid metabolism genes are associated with impaired lipid storage functions in white adipose tissues and deregulated plasma lipids in BNF-treated mice. The results of acute and chronic AhR activation support a significant interaction of AhR with the circadian clock system. Although future studies are needed to elucidate how AhR signaling specifically interacts with the clock in different cell types, the current research establishes a model for studying the crosstalk between AhR and circadian rhythm and provides new perspectives into the mechanisms of metabolic diseases correlated with exposure to synthetic organic chemicals.

Drosophila melanogaster is an invaluable model organism for the study of basic neuroscience. Using two previously characterized mating behaviours (Longer- and Shorter-Mating Duration), this research aims to further our knowledge of the neural circuit involved in each, and shed light on the mechanism by which four SIFamide producing neurons are involved in both. We also seek to investigate the involvement of core circadian clock genes in interval timing mechanisms. To do so, we investigated the populations of SIFamide receptor expressing neurons necessary for each behaviour and studied the contribution of circadian clock genes within the SIFamide signalling pathway. Our main experimental approach consisted of population specific knock-downs of the SIFamide receptor, the impact of which was assessed using a simple behavioural assay. This approach was complemented by rescue experiments and feminization of neurons. Finally, our investigation of the circadian clock was mediated by circadian gene knock-downs in SIFamide expressing neurons. Our results show that SIFamide signalling for each mating behaviour is mediated by segregated signalling to different, non male-specific SIFamide receptor expressing neuronal populations. We further demonstrate that SIFamide expressing neurons are not involved in the interval timing mechanism of these mating behaviours via core circadian gene contribution. This work presents preliminary results towards the investigation of a novel model of decision-making via neuronal signalling.

Circadian clocks are internal timekeepers that aid survival by allowing organisms, from photosynthetic cyanobacteria to humans, to anticipate predictable daily changes in the environment and make appropriate adjustments to their cellular biochemistry and behaviour. Whilst many of the molecular cogs and gears of circadian clocks are known, the complex interactions of clock components in time and space that generate a reliable internal measure of external time are still under investigation. Computational modelling has aided our understanding of the molecular mechanisms of circadian clocks, nevertheless it remains a major challenge to integrate the large number of clock components and their interactions into a single, comprehensive model that is able to account for the full breadth of clock properties. An important property of circadian clocks is their ability to maintain a constant period over a range of temperatures. Temperature compensation of circadian period is the least understood characteristic of circadian clocks. To investigate possible mechanisms underlying temperature compensation, I first constructed a comprehensive dynamic model of the Neurospora crassa circadian clock that incorporates its key components and their transcriptional and post-transcriptional regulation. The model is based on a compilation of published and new experimental data and incorporates facets of previously described Neurospora clock models. Light components were also incorporated into the model to test it and to reproduce our knowledge of light response of the clock. Also, experiments were carried out to investigate the unknown mechanisms of light response, such as the molecular mechanisms supporting the correct timing of conidiation after light to dark transfer. The model accounts for a wide range of clock characteristics including: a periodicity of 21.6 hours, persistent oscillation in constant conditions, resetting by brief light pulses, and entrainment to full photoperiods. Next, I carried out robustness tests and response coefficient analysis to identify components that strongly influence the period and amplitude of the molecular oscillations. These data measure the influence of the parameters in the model and were beneficial for making and testing predictions in the model. Thermodynamic properties were then introduced into reactions that experimental observations suggested might be temperature sensitive. This analysis indicated that temperature compensation can be achieved if nuclear localisation of a key clock component, FRQ, decreases with increasing temperature. Experiments have been carried out to validate this hypothesis and simulations were made to explore other possible mechanisms. However, from my experimental data and modelling results, the restriction of FRQ nuclear localisation might not be the only mechanism required to achieve temperature compensation. In conclusion, temperature compensation is most likely a complex property and may involve a combination of multiple mechanisms regulating clock component activity over a range of temperatures.

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23148号 / 薬博第848号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 土居 雅夫, 教授 中山 和久, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM