A switch from diurnal to nocturnal activity in S. ehrenbergi is accompanied by an uncoupling of light input and the circadian clock

The subterranean mole rat Spalax ehrenbergi superspecies represents an extreme example of adaptive visual and neuronal reorganization. Despite its total visual blindness, its daily activity rhythm is entrainable to light-dark cycles, indicating that it can confer light information to the clock. Although most individuals are active during the light phase under laboratory conditions (diurnal animals), some individuals switch their activity period to the night (nocturnal animals). Similar to other rodents, the Spalax circadian clock is driven by a set of clock genes, including the period (sPer) genes. In this work, we show that diurnal mole rats express the Per genes sPer1 and sPer2 with a peak during the light period. Light can synchronize sPer gene expression to an altered light-dark cycle and thereby reset the clock. In contrast, nocturnal Spalax express sPer2 in the dark period and sPer1 in a biphasic manner, with a light-dependent maximum during the day and a second light-independent maximum during the night. Although sPer1 expression remains light inducible, this is not sufficient to reset the molecular clockwork. Hence, the strict coupling of light, Per expression, and the circadian clock is lost. This indicates that Spalax can dissociate the light-driven resetting pathway from the central clock oscillator.     

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.     

TIME FOR COFFEE encodes a nuclear regulator in the Arabidopsis thaliana circadian clock

The plant circadian clock is required for daily anticipation of the diurnal environment. Mutation in Arabidopsis thaliana TIME FOR COFFEE (TIC) affects free-running circadian rhythms. To investigate how TIC functions within the circadian system, we introduced markers for the evening and morning phases of the clock into tic and measured evident rhythms. The phases of evening clock genes in tic were all advanced under light/dark cycles without major expression level defects. With regard to morning-acting genes, we unexpectedly found that TIC has a closer relationship with LATE ELONGATED HYPOCOTYL (LHY) than with CIRCADIAN CLOCK ASSOCIATED1, as tic has a specific LHY expression level defect. Epistasis analysis demonstrated that there were no clear rhythms in double mutants of tic and evening-acting clock genes, although double mutants of tic and morning-acting genes exhibited a similar free-running period as tic. We isolated TIC and found that its mRNA expression is continuously present over the diurnal cycle, and the encoded protein appears to be strictly localized to the nucleus. Neither its abundance nor its cellular distribution was found to be clock regulated. We suggest that TIC encodes a nucleus-acting clock regulator working close to the central oscillator.     

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.Key words: Ostreococcus, circadian, clock, plants, microalgaeThe circadian clock is an autonomous timer, which provides for living organisms a means to measure time internally. As such the clock has two fundamental properties: (1) it allows the organism to anticipate daily predictable environmental changes such as light and temperature cycles; (2) it coordinates key physiological processes during the 24 hour daily cycle. As a result, internal time given by the clock and external time given by the photoperiod exhibit a stable phase relationship for a wide range of photoperiods during the course of the year. The clock is therefore also involved in regulating annual rhythms such as flowering (also called photoperiodism) and many clock mutants have been identified on the basis of abnormalities in the timing of flowering. Other clock genes, such as TOC1 were identified through screens for defects in the rhythmic expression of circadian-regulated genes such as the Light-harvesting complex (LHCB/CAB) gene.²In plants, the clock appears as a complex circuit relying on interconnected feedback loops, which are being studied through a combination of experimental and modelling approaches.³ However, circadian studies are complicated by cell-autonomous and tissue specific clockworks in multicellular plants. We have recently implemented tools for gene function, analysis in the very simple green cell Ostreococcus tauri.⁴ Amongst known core clock genes we identified two homologues of the plant clock genes TOC1 and CCA1 (Circadian Clock Associated 1). Furthermore we found that a conserved CCA1 binding site was required for the circadian expression of TOC1. TOC1 appears to play a more central role than CCA1 in the Ostreococcus clock since only TOC1 knock-down abolishes circadian function in constant light.⁴ In plants the dark-dependent degradation of TOC1 relies on the F box protein ZEITLUPE which is stabilized by GIGANTEA in blue light.^5,6 We identified no ZEITLUPE AND GIGANTEA homologues in Ostreococcus. We chose, therefore to investigate the regulation of TOC1 in Ostreococcus at dusk since there are similarities between TOC1 functions and patterns of expression in Ostreococcus and Arabidopsis.     

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. Editor's suggested further reading in BioEssays: Another place, another timer: Marine species and the rhythms of life Abstract     

The metabolic sensor AKIN10 modulates the Arabidopsis circadian clock in a light‐dependent manner

Plants generate rhythmic metabolism during the repetitive day/night cycle. The circadian clock produces internal biological rhythms to synchronize numerous metabolic processes such that they occur at the required time of day. Metabolism conversely influences clock function by controlling circadian period and phase and the expression of core‐clock genes. Here, we show that AKIN10, a catalytic subunit of the evolutionarily conserved key energy sensor sucrose non‐fermenting 1 (Snf1)‐related kinase 1 (SnRK1) complex, plays an important role in the circadian clock. Elevated AKIN10 expression led to delayed peak expression of the circadian clock evening‐element GIGANTEA (GI) under diurnal conditions. Moreover, it lengthened clock period specifically under light conditions. Genetic analysis showed that the clock regulator TIME FOR COFFEE (TIC) is required for this effect of AKIN10. Taken together, we propose that AKIN10 conditionally works in a circadian clock input pathway to the circadian oscillator.     

LIGHT-REGULATED WD1 and PSEUDO-RESPONSE REGULATOR9 form a positive feedback regulatory loop in the Arabidopsis circadian clock

In Arabidopsis thaliana, central circadian clock genes constitute several feedback loops. These interlocking loops generate an ~24-h oscillation that enables plants to anticipate the daily diurnal environment. The identification of additional clock proteins can help dissect the complex nature of the circadian clock. Previously, LIGHT-REGULATED WD1 (LWD1) and LWD2 were identified as two clock proteins regulating circadian period length and photoperiodic flowering. Here, we systematically studied the function of LWD1/2 in the Arabidopsis circadian clock. Analysis of the lwd1 lwd2 double mutant revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that activities of LWD1/2 promoters are rhythmic and depend on functional PSEUDO-RESPONSE REGULATOR9 (PRR9) and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7, and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5, and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.     

Diurnal regulation of plant growth

Life occurs in an ever-changing environment. Some of the most striking and predictable changes are the daily rhythms of light and temperature. To cope with these rhythmic changes, plants use an endogenous circadian clock to adjust their growth and physiology to anticipate daily environmental changes. Most studies of circadian functions in plants have been performed under continuous conditions. However, in the natural environment, diurnal outputs result from complex interactions of endogenous circadian rhythms and external cues. Accumulated studies using the hypocotyl as a model for plant growth have shown that both light signalling and circadian clock mutants have growth defects, suggesting strong interactions between hypocotyl elongation, light signalling and the circadian clock. Here, we review evidence suggesting that light, plant hormones and the circadian clock all interact to control diurnal patterns of plant growth.     

The brown clock: circadian rhythms in stramenopiles

Circadian clocks allow organisms to anticipate environmental changes associated with the diurnal light/dark cycle. Circadian oscillators have been described in plants and green algae, cyanobacteria, animals and fungi, however, little is known about the circadian clocks of photosynthetic eukaryotes outside the green lineage. Stramenopiles are a diverse group of secondary endosymbionts whose plastid originated from a red alga. Photosynthetic stramenopiles, which include diatoms and brown algae, play key roles in biogeochemical cycles and are important components of marine ecosystems. Genome annotation efforts indicated the presence of a novel type of oscillator in these organisms and the first circadian clock component in a stramenopile has been recently discovered. This review summarizes the phenotypic characterization of circadian rhythms in stramenopiles and current efforts to determine the mechanisms of this ‘brown clock’. The elucidation of this brown clock will enable a deeper understanding of the role of self-sustained oscillations in the adaptation to life in marine environments.