SUMMARYDespite our increasing understanding of the molecular determinants essential for circadian clock function, we still lack a complete picture of the mechanisms contributing to clock progression in plants. Here, we explore the role of REVEILLE8/LHY-CCA1-LIKE5 (RVE8/LCL5) within the Arabidopsis circadian system. RVE8/LCL5 encodes a MYB-like transcription factor similar to CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and ELONGATED HYPOCOTYL (LHY), which are essential regulators of the Arabidopsis circadian clock. Consistent with the sequence similarity, the rhythmic expression of RVE8/LCL5 shows a morning acrophase comparable to that of CCA1 and LHY. Plants mis-expressing RVE8/LCL5 display a variety of circadian phenotypes, including altered circadian gene expression and photoperiodic flowering time. Similar to CCA1, RVE8/LCL5 regulates the expression of the oscillator gene TOC1 (TIMING OF CAB EXPRESSION1) by associating with the TOC1 promoter and by modulating the pattern of histone 3 (H3) acetylation. However, the mechanisms of RVE8/ LCL5 and CCA1 activity in this regulation differ markedly. Indeed, the use of chromatin immunoprecipitation and pharmacological inhibition assays reveals that RVE8/LCL5 favours a hyper-acetylated state of H3 at the TOC1 promoter, which may facilitate the rising phase of TOC1. In contrast, CCA1 represses TOC1 expression by promoting histone deacetylation. Thus, despite the sequence homology and the similar morning phase of expression, RVE8/LCL5 and CCA1 have opposing regulatory functions within the Arabidopsis circadian clock, although CCA1 has a more predominant role. We propose that contrasting chromatin compaction and transcriptional modulation through the opposing activities of RVE8/LCL5 and CCA1 might provide a finetuning mechanism for precisely shaping the TOC1 circadian waveform in Arabidopsis.

Whereas Glc is stored in small-sized hydrosoluble glycogen particles in archaea, eubacteria, fungi, and animal cells, photosynthetic eukaryotes have resorted to building starch, which is composed of several distinct polysaccharide fractions packed into a highly organized semicrystalline granule. In plants, both the initiation of polysaccharide synthesis and the nucleation mechanism leading to formation of new starch granules are currently not understood. Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, defines the tiniest eukaryote with one of the smallest genomes. We show that it accumulates a single starch granule at the chloroplast center by using the same pathway as higher plants. At the time of plastid division, we observe elongation of the starch and division into two daughter structures that are partitioned in each newly formed chloroplast. These observations suggest that in this system the information required to initiate crystalline polysaccharide growth of a new granule is contained within the preexisting polysaccharide structure and the design of the plastid division machinery.Starch and glycogen define the most widespread form of Glc storage in living cells and consist of a-1,4 linked glucan chains with a-1,6 branches (Buléon et al., 1998; Ball and Morell, 2003). Hydrosoluble glycogen particles cannot grow greater than 40 nm in diameter because the structure becomes too crowded with Glc at the periphery of the particle to accommodate the presence of enzymes of glycogen synthesis or degradation (Meléndez et al., 1998). This limitation is due to both the higher branching level of the polymer and to its rather uniform branching pattern. There presently seems to be no other limit to the size of a starch granule than that afforded by the availability of substrate for continuing synthesis. Amylopectin, the major polysaccharide of starch, aggregates into insoluble semicrystalline material because of its asymmetrical distribution of a-1,6 linkages generating clusters of branches responsible for formation of arrays of parallel double helical structures (Buléon et al., 1998). The appearance of starch coincides with the acquisition of photosynthesis by the eukaryotic cell. No such polymer can presently be found in bacteria, archea, fungi, or animal cells. Although starch is clearly associated with the acquisition of photoautotrophy by eukaryotes, glycogen seems to be the predominant form of Glc storage within cyanobacteria and purple nonsulfur bacteria (for review, see Preiss and Romeo, 1989). In addition, nonphotosynthetic eukaryotes accumulating amylopectin-like polymers such as apicomplexa parasite heterotrophic dinoflagellates or others were always subsequently inferred to be derived from a photosynthetic eukaryote ancestor (McFadden et al., 1996). Starch has been found to accumulate in plastids in green algae and land plants, while red algae, glaucophytes, dinoflagellates, apicomplexa parasites, and cryptophytes accumulate an extraplastidial form of so-called floridean starch (Vi...

Ostreococcus tauri (Prasinophyceae) is a marine unicellular green alga which diverged early in the green lineage. The interest of O. tauri as a potential model to study plant cell division is based on its key phylogenetic position, its simple binary division, a very simple cellular organisation and now the availability of the full genome sequence. In addition O. tauri has a minimal yet complete set of cell cycle control genes. Here we show that division can be naturally synchronised by light/dark cycles and that organelles divide before the nucleus. This natural synchronisation, although being only partial, enables the study of the expression of CDKs throughout the cell cycle. The expression patterns of OtCDKA and OtCDKB were determined both at the mRNA and protein levels. The single OtCDKA gene is constantly expressed throughout the cell cycle, whereas OtCDKB is highly regulated and expressed only in S/G2/M phases. More surprisingly, OtCDKA is not phosphorylated at the tyrosine residue, in contrast to OtCDKB which is strongly phosphorylated during cell division. OtCDKA kinase activity appears before the S phase, indicating a possible role of this protein in the G1/S transition. OtCDKB kinase activity occurs later than OtCDKA, and its tyrosine phosphorylation is correlated to G2/M, suggesting a possible control of the mitotic activity. To our knowledge this is the first organism in the green lineage which showed CDKB tyrosine phosphorylation during cell cycle progression.