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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.
The 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.2
In 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.
Under light/dark cycles, pTOC1:Luc transcriptional (TOC1 promoter driving the expression of firefly luciferase) and TOC1:Luc translational reporter lines (TOC1 gene fused in frame to luciferase) displayed different patterns of luminescence, translational reporters exhibiting steeper decreases of luminescence in darkness compare to transcriptional reporters.
This difference was not seen under constant light, corresponding to free-running conditions of the clock, consistent with TOC1 being regulated at the post-transcriptional level at dusk.4 It is possible therefore, that TOC1 is degraded in the dark in Ostreoccus just as it is in the plant model Arabidopsis. Such a mechanism might explain how the clock adjusts to day length.
To test this hypothesis further, TOC1 transcriptional (pTOC1:Luc) and translational (TOC1:Luc) reporter lines entrained under LD 12:12, were exposed to advanced dawn from the time of dusk of the entraining cycle (time 12; Fig. 1A). For convenience, the time of dawn of the entraining LD cycle was chosen as the reference time. Shortening the night by advancing the time of dawn in early night had a marked effect on the TOC1:Luc signals which dramatically increased in response to light ON (Fig. 1A). As a result, TOC1:Luc profiles exhibited a gradient of amplitude that increased with the time of light ON. In contrast, advancing dawn had no immediate effect on pTOC1:Luc which remained similar in phase and amplitude to that in LD 12:12 except for dawn at time 14, when a transient stabilisation of pTOC1:Luc was observed (Fig. 1B). Comparisons of TOC1:Luc and pTOC1:Luc patterns of luminescence suggest that resetting of TOC1 operates mainly at the post-transcriptional level when the timing of dawn is advanced early in the night (Fig. 1A and B). To further investigate how dusk contributes to the adjustment of TOC1 to day length we next designed experiments in which the timing of dusk was delayed. Under long days (12 to 22 hours of light), the luminescence of TOC1:Luc translational reporter increased with the day length until time 16 to 18 (Fig. 1C). In contrast, only a small and transient activation of TOC1 promoter activity, as reported by pTOC1:Luc, was observed until time 14, suggesting further that under these conditions, the increase in TOC1:Luc luminescence results mainly from a post-transcriptional regulation (Fig. 1D). Together the data from Figure 1 suggests that the light-dependent increase (or dark-dependent decrease) of TOC1 operates mainly at the post-transcriptional level.
To identify the mechanisms involved in TOC1 regulation during early night, we developed a pharmacological approach using transcriptional (cordycepin 3′-deoxyadenosine), translational (emetine dihydrochloride) and proteasome (MG132) inhibitors. The inhibitors were added 30 minutes before time 12 to a representative TOC1:Luc translational reporter line in three different experimental light conditions: standard LD 12:12, long photoperiod (LD 16:8) and constant light (LL). We chose to use only the translational reporter line since the experiments represented in Figure 1 showed little effect of light or dark treatments on pTOC1:Luc transcriptional reporter. Luminescence in Figure 2 reports de novo TOC1:Luc synthesis, including transcription and/or translation. In all conditions, addition of emetine at a 4 µM concentration suddenly stopped the luminescence increase indicating that emetine had effectively inhibited the translation process of TOC1-Luc mRNA present at that time (Fig. 2).
Under LD 12:12 TOC1:Luc luminescence of emetine-treated cells was only slightly lower than the control. The most likely explanation here is that other mechanisms are involved in the regulation of TOC1 at around time 12. In contrast, under long days or in LL, the TOC1:Luc control signal increased dramatically until time 16, well above the level of emetinet-reated cells, suggesting a major translational regulation of TOC1 at that time. Addition of the proteasome inhibitor MG132 at a 20 µM concentration, shortly before time 12 resulted in an immediate increase of TOC1:Luc luminescence above the control level, suggesting that TOC1 is degraded by the proteasome upon transfer to darkness under LD 12:12. In contrast, in LD 16:8 or in LL, the level of TOC1:Luc was higher in control cells suggesting that the degradation of TOC1 by the proteasome is restricted to a time-window around time 12, or that the synthesis of TOC1 between ZT12 and ZT16 counterbalances its degradation. Finally, inhibition of transcription with cordycepin (70 µM) had little effect on TOC1:Luc levels under LD 12:12 indicating that at this time the contribution of TOC1 transcription to the peak of TOC1 is minor (as suggested also from Fig. 1C and D) and that it is overridden by post-transcriptional regulations of TOC1. Under long days, cordycepintreated TOC1:luc line displayed a slightly smaller level of luminescence compared to control, suggesting that the large peak of TOC1:Luc arises also from de novo transcription of TOC1.
In summary our pharmacological approach indicates that (1) the regulation of TOC1 occurs mainly at the post-transcriptional level. (2) TOC1 is degraded by the proteasome upon transfer to darkness around time 12 in LD 12:12 but that this mechanism does not operate at time 16. (3) The translation (and to some extent the transcription) process is essential for the peak of TOC1 that occurs at time 16 under long days and LL.
We have previously demonstrated that TOC1 plays a central role in the circadian clock of Ostreococcus.4 Here we show that TOC1 is tightly regulated at the light/dark transition. As in the land plant Arabidopsis, TOC1 appears to be degraded in early night. On the other hand, the translation of TOC1 contributes considerably to the TOC1 peak under long day conditions. In view of this, future studies should focus on the importance of TOC1 regulation in the clock adjustment to day length and on identifying clock components involved in TOC1 degradation at dusk in the absence of ZEITLUPE and GIGANTEA homologues in Ostreococcus.
Previously published online: www.landesbioscience.com/journals/psb/article/11212