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 DNAbinding 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.

Chloroplast genomes in land plants include approximately 20 intron-containing genes. Most of the introns are similar to the group II introns found in fungi, algae and some bacteria, but no self-splicing has been reported. To analyze splicing reactions in chloroplasts, we developed a tobacco chloroplast-based in vitro system. We optimized the splicing reaction using atpF precursor mRNA (pre-mRNA). Our system requires a high ATP concentration, whereas ATP is unnecessary for self-splicing group II introns. Self-splicing group II introns possess two exon-binding sites (EBS1 & 2) complementary to two intron-binding sites (IBS1 & 2) in the 3′ end of 5′ exons, which are involved in 5′ splice-site selection. Using our in vitro system and atpF pre-mRNA, we analyzed short sequences corresponding to the above EBSs and IBSs. Mutation analyses revealed that EBS1-IBS1 pairing is essential while EBS2-IBS2 pairing is important but not crucial for splicing. The first 3′ exon nucleotide determines the 3′ splice-sites of self-splicing introns. However, mutations to this nucleotide in atpF pre-mRNA did not affect splicing. This result suggests that the mechanism underlying chloroplast pre-mRNA splicing differs partly from that mediating the self-splicing of group II introns.

Light plays a major role in resetting the circadian clock, allowing the organism to synchronize with the environmental day and night cycle. In Chlamydomonas the light-induced degradation of the circadian clock protein, RHYTHM OF CHLOROPLAST 15 (ROC15), is considered one of the key events in resetting the circadian clock. Red/violet and blue light signals have been shown to reach the clock via different molecular pathways; however, many of the participating components of these pathways are yet to be elucidated. Here, we used a forward genetics approach using a reporter strain that expresses a ROC15-luciferase fusion protein. We isolated a mutant that showed impaired ROC15 degradation in response to a wide range of visible wavelengths and impaired light-induced phosphorylation of ROC15. These results suggest that the effects of different wavelengths converge before acting on ROC15 or at ROC15 phosphorylation. Furthermore, the mutant showed a weakened phase resetting in response to light, but its circadian rhythmicity remained largely unaffected under constant light and constant dark conditions. Surprisingly, the gene disrupted in this mutant was found to encode a protein that possessed a very weak similarity to the Arabidopsis thaliana EARLY FLOWERING 3 (ELF3). Our results suggest that this protein is involved in the many different light signaling pathways to the Chlamydomonas circadian clock. However, it may not influence the transcriptional oscillator of Chlamydomonas to a great extent. This study provides an opportunity to further understand the mechanisms underlying light-induced clock resetting and explore the evolution of the circadian clock architecture in Viridiplantae.