The role of ROC75 as a daytime component of the circadian oscillator in Chlamydomonas reinhardtii

The circadian clocks in chlorophyte algae have been studied in two model organisms, Chlamydomonas reinhardtii and Ostreococcus tauri. These studies revealed that the chlorophyte clocks include some genes that are homologous to those of the angiosperm circadian clock. However, the genetic network architectures of the chlorophyte clocks are largely unknown, especially in C. reinhardtii. In this study, using C. reinhardtii as a model, we characterized RHYTHM OF CHLOROPLAST (ROC) 75, a clock gene encoding a putative GARP DNA-binding transcription factor similar to the clock proteins LUX ARRHYTHMO (LUX, also called PHYTOCLOCK 1 [PCL1]) and BROTHER OF LUX ARRHYTHMO (BOA, also called NOX) of the angiosperm Arabidopsis thaliana. We observed that ROC75 is a day/subjective day-phase-expressed nuclear-localized protein that associates with some night-phased clock genes and represses their expression. This repression may be essential for the gating of reaccumulation of the other clock-related GARP protein, ROC15, after its light-dependent degradation. The restoration of ROC75 function in an arrhythmic roc75 mutant under constant darkness leads to the resumption of circadian oscillation from the subjective dawn, suggesting that the ROC75 restoration acts as a morning cue for the C. reinhardtii clock. Our study reveals a part of the genetic network of C. reinhardtii clock that could be considerably different from that of A. thaliana. Author summary Circadian clock genes are not well-conserved during evolution, and the evolutionary history of circadian clocks is largely unknown. Green plants consist of two main clades: the Streptophyta (includes land plants and a part of freshwater green algae), and the Chlorophyta (includes a wide range of marine and freshwater green algae). Although the circadian clocks in chlorophyte algae include some components that are homologous to those of the circadian clock in flowering plants, it is not fully understood whether the chlorophyte clocks tick in the same manner as the flowering plant clocks. In this study, we characterized ROC75, a clock-related putative transcription factor, in the freshwater green alga Chlamydomonas reinhardtii. We found that ROC75 is a daytime repressor for the circadian clock in this alga, in contrast to its homologous protein, LUX (PCL1), which is known as a nighttime repressor for the flowering plant clock. Our study can serve as a step toward understanding the evolutionary history of circadian clocks in gre