Circadian clocks in changing weather and seasons: lessons from the picoalga Ostreococcus tauri.

Daylight is the primary cue used by circadian clocks to entrain to the day/night cycle so as to synchronize physiological processes with periodic environmental changes induced by Earth rotation. However, the temporal daylight pattern is not the same every day due to erratic weather fluctuations or regular seasonal changes. Then, how do circadian clocks operate properly in varying weather and seasons? In this paper, we discuss the strategy unveiled by recent studies of the circadian clock of Ostreococcus tauri, the smallest free-living eukaryotic organism. It combines mechanisms controlling light inputs and clock sensitivity, shaping both the dynamics of the core circadian oscillator and its forcing by light so as to ensure stable and precise synchronization in all weather and seasons.

A eukaryotic LOV-histidine kinase with circadian clock function in the picoalga Ostreococcus.

The marine environment has unique properties of light transmission, with an attenuation of long wavelengths within the first meters of the water column. Marine organisms have therefore evolved specific blue-light receptors such as aureochromes to absorb shorter-wavelength light. Here, we identify and characterize a light, oxygen, or voltage sensing (LOV) containing histidine kinase (LOV-HK) that functions as a new class of eukaryotic blue-light receptor in the pico-phytoplanktonic cell Ostreococcus tauri. This LOV-HK is related to the large family of LOV-HKs found in prokaryotes. Phylogenetic analysis indicates that the LOV domains from LOV-HKs, including O. tauri LOV-HK, and phototropins (phot; plant and green algal LOV serine/threonine kinases) have different evolutionary histories. Photochemical analysis shows that the LOV domain of LOV-HK binds a flavin cofactor and absorbs blue light with a fast photocycle compared with its prokaryotic counterparts. Ostreococcus tauri LOV-HK expression is induced by blue light and is under circadian control. Further, both overexpression and downregulation of LOV-HK result in arrhythmia of the circadian reporter CCA1:Luc under constant blue light. In contrast, photochemical inactivation of O. tauri LOV-HK is without effect, demonstrating its importance for function of the circadian clock under blue light. Overexpression/downregulation of O. tauriLOV-HK alters CCA1 rhythmicity under constant red light, irrespective of LOV-HK's photochemical reactivity, suggesting that O. tauri LOV-HK also participates in regulation of the circadian clock independent of its blue-light-sensing property. Molecular characterization of O. tauri LOV-HK demonstrates that this type of photoreceptor family is not limited to prokaryotes.

Clocks in the green lineage: comparative functional analysis of the circadian architecture of the picoeukaryote ostreococcus.

Biological rhythms that allow organisms to adapt to the solar cycle are generated by endogenous circadian clocks. In higher plants, many clock components have been identified and cellular rhythmicity is thought to be driven by a complex transcriptional feedback circuitry. In the small genome of the green unicellular alga Ostreococcus tauri, two of the master clock genes Timing of Cab expression1 (TOC1) and Circadian Clock-Associated1 (CCA1) appear to be conserved, but others like Gigantea or Early-Flowering4 are lacking. Stably transformed luciferase reporter lines and tools for gene functional analysis were therefore developed to characterize clock gene function in this simple eukaryotic system. This approach revealed several features that are comparable to those in higher plants, including the circadian regulation of TOC1, CCA1, and the output gene Chlorophyll a/b Binding under constant light, the relative phases of TOC1/CCA1 expression under light/dark cycles, arrhythmic overexpression phenotypes under constant light, the binding of CCA1 to a conserved evening element in the TOC1 promoter, as well as the requirement of the evening element for circadian regulation of TOC1 promoter activity. Functional analysis supports TOC1 playing a central role in the clock, but repression of CCA1 had no effect on clock function in constant light, arguing against a simple TOC1 /CCA1 one-loop clock in Ostreococcus. The emergence of functional genomics in a simple green cell with a small genome may facilitate increased understanding of how complex cellular processes such as the circadian clock have evolved in plants.

A phosphate-regulated promoter for fine-tuned and reversible overexpression in Ostreococcus: application to circadian clock functional analysis.

BACKGROUND: The green picoalga Ostreococcus tauri (Prasinophyceae), which has been described as the smallest free-living eukaryotic organism, has minimal cellular ultra-structure and a very small genome. In recent years, O. tauri has emerged as a novel model organism for systems biology approaches that combine functional genomics and mathematical modeling, with a strong emphasis on light regulated processes and circadian clock. These approaches were made possible through the implementation of a minimal molecular toolbox for gene functional analysis including overexpression and knockdown strategies. We have previously shown that the promoter of the High Affinity Phosphate Transporter (HAPT) gene drives the expression of a luciferase reporter at high and constitutive levels under constant light. METHODOLOGY/PRINCIPAL FINDINGS: Here we report, using a luciferase reporter construct, that the HAPT promoter can be finely and reversibly tuned by modulating the level and nature of phosphate in culture medium. This HAPT regulation was additionally used to analyze the circadian clock gene Time of Cab expression 1 (TOC1). The phenotype of a TOC1ox/CCA1:Luc line was reverted from arrhythmic to rhythmic simply by adding phosphate to the culture medium. Furthermore, since the time of phosphate injection had no effect on the phase of CCA1:Luc expression, this study suggests further that TOC1 is a central clock gene in Ostreococcus. CONCLUSIONS/PERSPECTIVES: We have developed a phosphate-regulated expression system that allows fine gene function analysis in Ostreococcus. Recently, there has been a growing interest in microalgae as cell factories. This non-toxic phosphate-regulated system may prove useful in tuning protein expression levels quantitatively and temporally for biotechnological applications.

Insights into the regulation of the core clock component TOC1 in the green picoeukaryote Ostreococcus.

Living organisms such as plants and animals have evolved endogenous clocks in order to anticipate the environmental changes associated with the earth's rotation and to orchestrate biological processes in the course of the 24 hour daily cycle. We have recently identified clock components in the primitive green picoalga Ostreococcus tauri, a promising minimal cellular and genomic model for systems biology approaches. A homologue of the Arabidopsis core clock gene Time of CAB expression-1 (TOC1) was shown to play a central role in Ostreococcus heralding an early emergence of clock components in the green lineage. Here we report the regulation of TOC1 at dusk in response to light and dark cues.