Cytosolic free calcium (Ca2+cyt) is a ubiquitous second messenger in eukaryotes. In Arabidopsis thaliana, diurnal or circadian (diel) rhythms in [Ca2+]cyt have been widely documented. There is evidence to suggest that these diel [Ca2+]cyt oscillations modulate different signalling pathways, including photoperiodic signal transduction, gating responses to endogenous and environmental stimuli and feed-back entrainment of the core circadian clock itself. However, direct evidence for the role of Ca2+ in clock entrainment or as an output from the clock is lacking, and the question of the functional role of diel [Ca2+]cyt oscillations remains open. The role of diel [Ca2+]cyt rhythms in A. thaliana and their relationship relative to the central molecular oscillator was investigated. While it was found that diel [Ca2+]cyt oscillations persist throughout the life cycle of A. thaliana, I found no indication that diel [Ca2+]cyt rhythms are involved in photoperiodic signalling. Furthermore, I demonstrated that normal diel [Ca2+]cyt oscillations persist even in the absence of a functioning core circadian clock, indicating that, contrary to the accepted view, diel [Ca2+]cyt oscillations are not directly controlled by the core circadian clock, but are more probably generated by a non-transcriptional oscillator. In silico analysis of the amino-acid sequences of the 12 core clock proteins revealed that TOC1 contains a putative EF-hand and may therefore provide a route into the molecular oscillator for diel [Ca2+]cyt signals. The TOC1 sequence was altered to eliminate the Ca2+ coordinating residues but attempts to express this protein in E. coli, N. benthamiana and Baculovirus were unsuccessful. Complementation of the A. thaliana toc1-1 mutant with transgenes containing the endogenous TOC1 promoter sequence upstream of the wild type or the altered TOC1 sequences were also unsuccessful. A series of experiments were conducted to provide empirical data for Boolean Logic models of circadian rhythmicity that would enable further characterisation of the potential link between diel [Ca2+]cyt oscillations and TOC1.

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.

From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana), a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)) that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies) putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1) in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partlyconserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of thethree-loop network typical of the circadian clock in angiosperms were present beforethe split of gymnosperms and angiosperms.