The molecular bases of circadian clocks have been studied in animals, fungi, bacteria, and plants, but not in eukaryotic algae. To establish a new model for molecular analysis of the circadian clock, here we identified a large number of components of the circadian system in the eukaryotic unicellular alga Chlamydomonas reinhardtii by a systematic forward genetic approach. We isolated 105 insertional mutants that exhibited defects in period, phase angle, and/or amplitude of circadian rhythms in bioluminescence derived from a luciferase reporter gene in their chloroplast genome. Simultaneous measurement of circadian rhythms in bioluminescence and growth rate revealed that some of these mutants had defects in the circadian clock itself, whereas one mutant had a defect in a specific process for the chloroplast bioluminescence rhythm. We identified 30 genes (or gene loci) that would be responsible for rhythm defects in 37 mutants. Classification of these genes revealed that various biological processes are involved in regulation of the chloroplast rhythmicity. Amino acid sequences of six genes that would have crucial roles in the circadian clock revealed features of the Chlamydomonas clock that have both partially plant-like and original components. The molecular bases of circadian clocks have been studied in animals, fungi, bacteria, and plants (Dunlap 1999;Harmer et al. 2001). Despite the striking biochemical features of circadian clocks (e.g., oscillation with long periodicity [∼24 h] and its temperature compensation) (Bünning 1973), their central components are not conserved between these kingdoms (Dunlap 1999;Harmer et al. 2001). To understand the nature of oscillation mechanisms and the evolutionary history of clock components, it is important to understand circadian clock systems of a wide range of organisms.Circadian rhythms of unicellular algae have been studied extensively (Mittag 2001), but no clock component of eukaryotic algae has yet been identified. Chlamydomonas reinhardtii is one of the best-studied algae in circadian rhythm research. A forward genetic approach to identify circadian clock components of Chlamydomonas was started more than three decades ago (Bruce 1970). Although several clock mutants have been isolated (Bruce 1972(Bruce , 1974Mergenhagen 1984), the genes responsible could not be identified because of limitations of tools for genetic analyses. However, since Chlamydomonas is now one of the most attractive model organisms in molecular genetics (Harris 2001), it is possible to re-establish it as a model for studying the molecular mechanism of the circadian clock. For this purpose, we previously developed bioluminescence reporter strains with a codon-optimized luciferase gene in their chloroplast genomes to enable real-time monitoring of circadian rhythms (Breton and Kay 2006;Matsuo et al. 2006).In this study, we screened ∼16,000 insertional mutants for defects in circadian rhythmicity of the chloroplast bioluminescence reporter, isolated 105 mutants, and identified 30 genes (or gene loci...
Although the circadian clock is a self-sustaining oscillator having a periodicity of nearly 1 d, its period length is not necessarily 24 h. Therefore, daily adjustment of the clock (i.e., resetting) is an essential mechanism for the circadian clock to adapt to daily environmental changes. One of the major cues for this resetting mechanism is light. In the unicellular green alga Chlamydomonas reinhardtii, the circadian clock is reset by blue/green and red light. However, the underlying molecular mechanisms remain largely unknown. In this study, using clock protein-luciferase fusion reporters, we found that the level of RHYTHM OF CHLOROPLAST 15 (ROC15), a clock component in C. reinhardtii, decreased rapidly after light exposure in a circadian-phase-independent manner. Blue, green, and red light were able to induce this process, with red light being the most effective among them. Expression analyses and inhibitor experiments suggested that this process was regulated mainly by a proteasome-dependent protein degradation pathway. In addition, we found that the other clock gene, ROC114, encoding an F-box protein, was involved in this process. Furthermore, we demonstrated that a roc15 mutant showed defects in the phase-resetting of the circadian clock by light. Taken together, these data strongly suggest that the light-induced degradation of ROC15 protein is one of the triggers for resetting the circadian clock in C. reinhardtii. Our data provide not only a basis for understanding the molecular mechanisms of light-induced phase-resetting in C. reinhardtii, but also insights into the phase-resetting mechanisms of circadian clocks in plants.LUCnc | light pulse | phase shift C ircadian rhythms, observed ubiquitously in organisms from prokaryotic cyanobacteria to humans, are generated by the circadian clock, which is thought to rely on transcriptional/translational feedback loops and posttranslational biochemical oscillations of some genes and their protein products called clock genes/proteins (1-4). Clock genes/proteins have been identified in several organisms, including mammals, insects, fungi, land plants, cyanobacteria, and, recently, green algae (1, 2, 5-8). Except for general kinases and phosphatases (9), most of the components of circadian clocks are not conserved among evolutionarily divergent organisms. On the other hand, the components are conserved to some extent between organisms that are relatively close evolutionarily (i.e., mammals and insects, land plants and green algae) (6-8, 10).The clock components in algae were first identified in the model green alga Chlamydomonas reinhardtii. There are the RNAbinding protein complex CHLAMY1 (11), a casein kinase (12), and RHYTHM OF CHLOROPLAST (ROC) proteins, including putative DNA-binding proteins (ROC15, ROC40, ROC66, and ROC75), an F-box protein (ROC114), and a leucine-rich repeat protein (ROC55) (13). The DNA-binding motifs of ROC proteins are homologous to those of Arabidopsis thaliana proteins associated with the circadian clock and photoperiodic flowering (13)...
We discovered a new CK2 inhibitor and revealed its mechanism of action, connecting the circadian clock and cancer regulation.
The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR) proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas.
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