Phase-resetting mechanism of the circadian clock in Chlamydomonas reinhardtii.

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.

Pentatricopeptide repeat proteins involved in plant organellar RNA editing.

C-to-U RNA editing has been widely observed in organellar RNAs in terrestrial plants. Recent research has revealed the significance of a large, plant-specific family of pentatricopeptide repeat (PPR) proteins for RNA editing and other RNA processing events in plant mitochondria and chloroplasts. PPR protein is a sequence-specific RNA-binding protein that identifies specific C residues for editing. Discovery of the RNA recognition code for PPR motifs, including verification and prediction of the individual RNA editing site and its corresponding PPR protein, expanded our understanding of the molecular function of PPR proteins in plant organellar RNA editing. Using this knowledge and the co-expression database, we have identified two new PPR proteins that mediate chloroplast RNA editing. Further, computational target assignment using the PPR RNA recognition codes suggests a distinct, unknown mode-of-action, by which PPR proteins serve a function beyond site recognition in RNA editing.

De novo activated transcription of inserted foreign coding sequences is inheritable in the plant genome.

The manner in which inserted foreign coding sequences become transcriptionally activated and fixed in the plant genome is poorly understood. To examine such processes of gene evolution, we performed an artificial evolutionary experiment in Arabidopsis thaliana. As a model of gene-birth events, we introduced a promoterless coding sequence of the firefly luciferase (LUC) gene and established 386 T2-generation transgenic lines. Among them, we determined the individual LUC insertion loci in 76 lines and found that one-third of them were transcribed de novo even in the intergenic or inherently unexpressed regions. In the transcribed lines, transcription-related chromatin marks were detected across the newly activated transcribed regions. These results agreed with our previous findings in A. thaliana cultured cells under a similar experimental scheme. A comparison of the results of the T2-plant and cultured cell experiments revealed that the de novo-activated transcription concomitant with local chromatin remodelling was inheritable. During one-generation inheritance, it seems likely that the transcription activities of the LUC inserts trapped by the endogenous genes/transcripts became stronger, while those of de novo transcription in the intergenic/untranscribed regions became weaker. These findings may offer a clue for the elucidation of the mechanism by which inserted foreign coding sequences become transcriptionally activated and fixed in the plant genome.