The green lineage is reportedly 1,500 million years old, evolving shortly after the endosymbiosis event that gave rise to early photosynthetic eukaryotes. In this study, we unveil the complete genome sequence of an ancient member of this lineage, the unicellular green alga Ostreococcus tauri (Prasinophyceae). This cosmopolitan marine primary producer is the world's smallest free-living eukaryote known to date. Features likely reflecting optimization of environmentally relevant pathways, including resource acquisition, unusual photosynthesis apparatus, and genes potentially involved in C4 photosynthesis, were observed, as was downsizing of many gene families. Overall, the 12.56-Mb nuclear genome has an extremely high gene density, in part because of extensive reduction of intergenic regions and other forms of compaction such as gene fusion. However, the genome is structurally complex. It exhibits previously unobserved levels of heterogeneity for a eukaryote. Two chromosomes differ structurally from the other eighteen. Both have a significantly biased G؉C content, and, remarkably, they contain the majority of transposable elements. Many chromosome 2 genes also have unique codon usage and splicing, but phylogenetic analysis and composition do not support alien gene origin. In contrast, most chromosome 19 genes show no similarity to green lineage genes and a large number of them are specialized in cell surface processes. Taken together, the complete genome sequence, unusual features, and downsized gene families, make O. tauri an ideal model system for research on eukaryotic genome evolution, including chromosome specialization and green lineage ancestry.genome heterogeneity ͉ genome sequence ͉ green alga ͉ Prasinophyceae ͉ gene prediction

Circadian rhythms are ubiquitous in eukaryotes, and co-ordinate numerous aspects of behaviour, physiology and metabolism, from sleep/wake cycles in mammals to growth and photosynthesis in plants1,2. This daily timekeeping is thought to be driven by transcriptional/translational feedback loops, whereby rhythmic expression of clock gene products regulates expression of associated genes in approximately 24-hour cycles. The specific transcriptional components differ between phylogenetic kingdoms3. The unicellular pico-eukaryotic alga, Ostreococcus tauri, possesses a naturally minimised clock, which includes many features that are shared with higher eukaryotes (plants), such as a central negative feedback loop that involves the morning-expressed CCA1 and evening-expressed TOC1 genes4. Given that recent observations in animals and plants have revealed prominent post-translational contributions to timekeeping5, a reappraisal of the transcriptional contribution to oscillator function is overdue. Here we show that non-transcriptional mechanisms are sufficient to sustain circadian timekeeping in the eukaryotic lineage, though they normally function in conjunction with transcriptional components. We identify oxidation of peroxiredoxin proteins as a transcription-independent rhythmic biomarker, which is also rhythmic in mammals6. Moreover we show that pharmacological modulators of the mammalian clockwork have the same effects on rhythms in Ostreococcus. Post-translational mechanisms, and at least one rhythmic marker, appear to be better conserved than transcriptional clock regulators. It is plausible that the oldest oscillator components are non-transcriptional in nature, as in cyanobacteria7, and are conserved across kingdoms.

Biological rhythms that allow organisms to adapt to the solar cycle are generated by endogenous circadian clocks. In higher plants, many clock components have been identified and cellular rhythmicity is thought to be driven by a complex transcriptional feedback circuitry. In the small genome of the green unicellular alga Ostreococcus tauri, two of the master clock genes Timing of Cab expression1 (TOC1) and Circadian Clock-Associated1 (CCA1) appear to be conserved, but others like Gigantea or Early-Flowering4 are lacking. Stably transformed luciferase reporter lines and tools for gene functional analysis were therefore developed to characterize clock gene function in this simple eukaryotic system. This approach revealed several features that are comparable to those in higher plants, including the circadian regulation of TOC1, CCA1, and the output gene Chlorophyll a/b Binding under constant light, the relative phases of TOC1/CCA1 expression under light/ dark cycles, arrhythmic overexpression phenotypes under constant light, the binding of CCA1 to a conserved evening element in the TOC1 promoter, as well as the requirement of the evening element for circadian regulation of TOC1 promoter activity. Functional analysis supports TOC1 playing a central role in the clock, but repression of CCA1 had no effect on clock function in constant light, arguing against a simple TOC1 /CCA1 one-loop clock in Ostreococcus. The emergence of functional genomics in a simple green cell with a small genome may facilitate increased understanding of how complex cellular processes such as the circadian clock have evolved in plants.

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.T he red algae, together with the glaucophytes and the Chloroplastida, are members of the Archaeplastida, the phylogenetic group formed during the primary endosymbiosis event that gave rise to the first photosynthetic eukaryote. Red algal genomes, both plastid and nuclear, also contributed, via secondary endosymbiosis, to several other eukaryotic lineages, including

BackgroundPicoeukaryotes represent an important, yet poorly characterized component of marine phytoplankton. The recent genome availability for two species of Ostreococcus and Micromonas has led to the emergence of picophytoplankton comparative genomics. Sequencing has revealed many unexpected features about genome structure and led to several hypotheses on Ostreococcus biology and physiology. Despite the accumulation of genomic data, little is known about gene expression in eukaryotic picophytoplankton.ResultsWe have conducted a genome-wide analysis of gene expression in Ostreococcus tauri cells exposed to light/dark cycles (L/D). A Bayesian Fourier Clustering method was implemented to cluster rhythmic genes according to their expression waveform. In a single L/D condition nearly all expressed genes displayed rhythmic patterns of expression. Clusters of genes were associated with the main biological processes such as transcription in the nucleus and the organelles, photosynthesis, DNA replication and mitosis.ConclusionsLight/Dark time-dependent transcription of the genes involved in the main steps leading to protein synthesis (transcription basic machinery, ribosome biogenesis, translation and aminoacid synthesis) was observed, to an unprecedented extent in eukaryotes, suggesting a major input of transcriptional regulations in Ostreococcus. We propose that the diurnal co-regulation of genes involved in photoprotection, defence against oxidative stress and DNA repair might be an efficient mechanism, which protects cells against photo-damage thereby, contributing to the ability of O. tauri to grow under a wide range of light intensities.

SummaryTo identify molecular players implicated in cytokinesis and division plane determination, the Arabidopsis thaliana genome was explored for potential cytokinesis genes. More than 100 open reading frames were selected based on similarity to yeast and animal cytokinesis genes, cytoskeleton and polarity genes, and Nicotiana tabacum genes showing cell cycle-controlled expression. The subcellular localization of these proteins was determined by means of GFP tagging in tobacco Bright Yellow-2 cells and Arabidopsis plants. Detailed confocal microscopy identified 15 proteins targeted to distinct regions of the phragmoplast and the cell plate. EB1-and MAP65-like proteins were associated with the plus-end, the minus-end, or along the entire length of microtubules. The actin-binding protein myosin, the kinase Aurora, and a novel cell cycle protein designated T22, accumulated preferentially at the midline. EB1 and Aurora, in addition to other regulatory proteins (homologs of Mob1, Sid1, and Sid2), were targeted to the nucleus, suggesting that this organelle operates as a coordinating hub for cytokinesis.

Seasonality in marine microorganisms has been classically observed in phytoplankton blooms, and more recently studied at the community level in prokaryotes, but rarely investigated at the scale of individual microbial taxa. Here we test if specific marine eukaryotic phytoplankton, bacterial and archaeal taxa display yearly rhythms at a coastal site impacted by irregular environmental perturbations. Our seven-year study in the Bay of Banyuls (North Western Mediterranean Sea) shows that despite some fluctuating environmental conditions, many microbial taxa displayed significant yearly rhythms. The robust rhythmicity was found in both autotrophs (picoeukaryotes and cyanobacteria) and heterotrophic prokaryotes. Sporadic meteorological events and irregular nutrient supplies did, however, trigger the appearance of less common non-rhythmic taxa. Among the environmental parameters that were measured, the main drivers of rhythmicity were temperature and day length. Seasonal autotrophs may thus be setting the pace for rhythmic heterotrophs. Similar environmental niches may be driving seasonality as well. The observed strong association between Micromonas and SAR11, which both need thiamine precursors for growth, could be a first indication that shared nutritional niches may explain some rhythmic patterns of cooccurrence.

TPLATE was previously identified as a potential cytokinesis protein targeted to the cell plate. Disruption of TPLATE in Arabidopsis thaliana leads to the production of shriveled pollen unable to germinate. Vesicular compartmentalization of the mature pollen is dramatically altered, and large callose deposits accumulate near the intine cell wall layer. Green fluorescent protein (GFP)-tagged TPLATE expression under the control of the pollen promoter Lat52 complements the phenotype. Downregulation of TPLATE in Arabidopsis seedlings and tobacco (Nicotiana tabacum) BY-2 suspension cells results in crooked cell walls and cell plates that fail to insert into the mother wall. Besides accumulating at the cell plate, GFP-fused TPLATE is temporally targeted to a narrow zone at the cell cortex where the cell plate connects to the mother wall. TPLATE-GFP also localizes to subcellular structures that accumulate at the pollen tube exit site in germinating pollen. Ectopic callose depositions observed in mutant pollen also occur in RNA interference plants, suggesting that TPLATE is implicated in cell wall modification. TPLATE contains domains similar to adaptin and b-COP coat proteins. These data suggest that TPLATE functions in vesicle-trafficking events required for site-specific cell wall modifications during pollen germination and for anchoring of the cell plate to the mother wall at the correct cortical position.