Phase-resetting mechanism of the circadian clock in Chlamydomonas reinhardtii

Abstract

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)...

CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas

“…In a previous study, we developed a ROC15-LUC reporter strain that express a fusion protein of ROC15 and firefly luciferase to monitor fluctuations in ROC15 protein levels in vivo [21]. Taking advantage of the reporter strain and a high throughput bioluminescence monitoring system [22,23], we screened mutants showing abnormal diurnal rhythms through ROC15-LUC reporter bioluminescence.…”

“…Mutations in ROC15 affect period length and phase of the circadian clock [14]. Under the daily light/dark cycle, expression levels of ROC15 protein show a diurnal rhythm: ROC15 protein levels increase during the night, decline rapidly at dawn due to light-induced proteasomal degradation, and are kept low during the day [21]. Remarkably, red light is highly effective for degradation of ROC15, as well as the resetting of circadian clock [21].…”

“…Under the daily light/dark cycle, expression levels of ROC15 protein show a diurnal rhythm: ROC15 protein levels increase during the night, decline rapidly at dawn due to light-induced proteasomal degradation, and are kept low during the day [21]. Remarkably, red light is highly effective for degradation of ROC15, as well as the resetting of circadian clock [21]. In addition, since a ROC15 gene mutant shows severe defect in the resetting of circadian clock by light, the light induced degradation of ROC15 is thought to be a key event for the resetting of the Chlamydomonas circadian clock [21].…”

“…Remarkably, red light is highly effective for degradation of ROC15, as well as the resetting of circadian clock [21]. In addition, since a ROC15 gene mutant shows severe defect in the resetting of circadian clock by light, the light induced degradation of ROC15 is thought to be a key event for the resetting of the Chlamydomonas circadian clock [21]. However, photoreceptors and signaling pathways for this process remain unidentified.…”

Clocks in Algae

“…protein is involved in clock resetting upon blue, green and particularly red light input, in a circadian-phase dependent manner (76) . C. reinhardtii has two isoforms of a plant-like cryptochrome, Chlamydomonas Photolyase Homologue1 (CPH1), both of which accumulate in the dark and are rapidly degraded by red or blue light-induced proteolysis (77) .…”