Orchestrated transcription of biological processes in the marine picoeukaryote Ostreococcus exposed to light/dark cycles

Background: Picoeukaryotes 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. Results: We 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. Conclusions: Light/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

Robustness of circadian clocks to daylight fluctuations: hints from the picoeucaryote Ostreococcus tauri

The development of systemic approaches in biology has put emphasis on identifying genetic modules whose behavior can be modeled accurately so as to gain insight into their structure and function. However most gene circuits in a cell are under control of external signals and thus quantitative agreement between experimental data and a mathematical model is difficult. Circadian biology has been one notable exception: quantitative models of the internal clock that orchestrates biological processes over the 24-hour diurnal cycle have been constructed for a few organisms, from cyanobacteria to plants and mammals. In most cases, a complex architecture with interlocked feedback loops has been evidenced. Here we present first modeling results for the circadian clock of the green unicellular alga Ostreococcus tauri. Two plant-like clock genes have been shown to play a central role in Ostreococcus clock. We find that their expression time profiles can be accurately reproduced by a minimal model of a two-gene transcriptional feedback loop. Remarkably, best adjustment of data recorded under light/dark alternation is obtained when assuming that the oscillator is not coupled to the diurnal cycle. This suggests that coupling to light is confined to specific time intervals and has no dynamical effect when the oscillator is entrained by the diurnal cycle. This intringuing property may reflect a strategy to minimize the impact of fluctuations in daylight intensity on the core circadian oscillator, a type of perturbation that has been rarely considered when assessing the robustness of circadian clocks

Microarray data can predict diurnal changes of starch content in the picoalga Ostreococcus

<p>Abstract</p> <p>Background</p> <p>The storage of photosynthetic carbohydrate products such as starch is subject to complex regulation, effected at both transcriptional and post-translational levels. The relevant genes in plants show pronounced daily regulation. Their temporal RNA expression profiles, however, do not predict the dynamics of metabolite levels, due to the divergence of enzyme activity from the RNA profiles.</p> <p>Unicellular phytoplankton retains the complexity of plant carbohydrate metabolism, and recent transcriptomic profiling suggests a major input of transcriptional regulation.</p> <p>Results</p> <p>We used a quasi-steady-state, constraint-based modelling approach to infer the dynamics of starch content during the 12 h light/12 h dark cycle in the model alga <it>Ostreococcus tauri</it>. Measured RNA expression datasets from microarray analysis were integrated with a detailed stoichiometric reconstruction of starch metabolism in <it>O. tauri </it>in order to predict the optimal flux distribution and the dynamics of the starch content in the light/dark cycle. The predicted starch profile was validated by experimental data over the 24 h cycle. The main genetic regulatory targets within the pathway were predicted by <it>in silico </it>analysis.</p> <p>Conclusions</p> <p>A single-reaction description of starch production is not able to account for the observed variability of diurnal activity profiles of starch-related enzymes. We developed a detailed reaction model of starch metabolism, which, to our knowledge, is the first attempt to describe this polysaccharide polymerization while preserving the mass balance relationships. Our model and method demonstrate the utility of a quasi-steady-state approach for inferring dynamic metabolic information in <it>O. tauri </it>directly from time-series gene expression data.</p

Temperature Acclimation of the Picoalga Ostreococcus tauri Triggers Early Fatty-Acid Variations and Involves a Plastidial ?3-Desaturase

Alteration of fatty-acid unsaturation is a universal response to temperature changes. Marine microalgae display the largest diversity of polyunsaturated fatty-acid (PUFA) whose content notably varies according to temperature. The physiological relevance and the molecular mechanisms underlying these changes are however, still poorly understood. The ancestral green picoalga Ostreococcus tauri displays original lipidic features that combines PUFAs from two distinctive microalgal lineages (Chlorophyceae, Chromista kingdom). In this study, optimized conditions were implemented to unveil early fatty-acid and desaturase transcriptional variations upon chilling and warming. We further functionally characterized the O. tauri ω3-desaturase which is closely related to ω3-desaturases from Chromista species. Our results show that the overall omega-3 to omega-6 ratio is swiftly and reversibly regulated by temperature variations. The proportion of the peculiar 18:5 fatty-acid and temperature are highly and inversely correlated pinpointing the importance of 18:5 temperature-dependent variations across kingdoms. Chilling rapidly and sustainably up-regulated most desaturase genes. Desaturases involved in the regulation of the C18-PUFA pool as well as the Δ5-desaturase appear to be major transcriptional targets. The only ω3-desaturase candidate, related to ω3-desaturases from Chromista species, is localized at chloroplasts in Nicotiana benthamiana and efficiently performs ω3-desaturation of C18-PUFAs in Synechocystis sp. PCC6803. Overexpression in the native host further unveils a broad impact on plastidial and non-plastidial glycerolipids illustrated by the alteration of omega-3/omega-6 ratio in C16-PUFA and VLC-PUFA pools. Global glycerolipid features of the overexpressor recall those of chilling acclimated cells.Développement d'une infrastructure française distribuée pour la métabolomique dédiée à l'innovatio

Circadian rhythms persist without transcription in a eukaryote

Circadian rhythms are ubiquitous in eukaryotes, and coordinate numerous aspects of behaviour, physiology and metabolism, from sleep/wake cycles in mammals to growth and photosynthesis in plants. This daily timekeeping is thought to be driven by transcriptionaltranslational feedback loops, whereby rhythmic expression of clock- gene products regulates the expression of associated genes in approximately 24-hour cycles. The specific transcriptional components differ between phylogenetic kingdoms. The unicellular pico-eukaryotic alga Ostreococcus tauri possesses a naturally minimized clock, which includes many features that are shared with plants, such as a central negative feedback loop that involves the morning-expressed CCA1 and evening-expressed TOC1 genes. Given that recent observations in animals and plants have revealed prominent post-translational contributions to timekeeping, 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, although 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 mammals. Moreover we show that pharmacological modulators of the mammalian clock mechanism have the same effects on rhythms in Ostreococcus. Post-translational mechanisms, and at least one rhythmic marker, seem to be better conserved than transcriptional clock regulators. It is plausible that the oldest oscillator components are non-transcriptional in nature, as in cyanobacteria, and are conserved across kingdoms

Cycle cellulaire et polarisation du zygote Fucus régulations et interactions

The correct orchestration of cell cycle and differentiation is required for the development of all organisms. In plants, there is little data on the molecular mechanisms involved in the coordination of both of these processes. The zygote of the brown alga Fucus is a model to study plant early embryogenesis. Polarisation of the Fucus zygote occurs after fertilization and is concomitant with the first mitotic cell cycle. Little is known about cell cycle, and altough molecular actors involved in polarization have been identified, transduction pathways controlling the establishment of polarity remain to be determined. In this context, we are interested in the regulation of cell cycle and polarisation, as well as, in interconnection between these two processes. We have demonstrated, that the first cell cycle of Fucus zygotes encompasses four well-defined phases G1, G2, S and M, and is under the tight control of cell-cycle dependent-kinase-related proteins (CDK). Usual functional checkpoints are found, ensuring that mitosis will not occur in the presence of incomplete DNA replication or of mitotic spindle defects. Two CDKs containing the hallmark PSTAIRE, p32 and p34, are translated from maternal mRNAs, after fertilization, and their progressive synthesis correlates with an increase in CDK activity until G2 phase, where the transcription of a positive regulator is further required to achieve CDK activation before mitosis. Both of these CDKs appear to be negatively regulated by tyrosine phosphorylation, during normal cell cycle progression and following activation of the DNA replication checkpoint. Since p34 is the major target of the CDK inhibitor purvalanol, it appears to play a major role in cell cycle control. We have also demonstrated that, at the G1/S transition, p34 controls the formation of the embryonic axis, which is achieved during S phase. On the other hand, we have shown that axis formation is essential for embryo patterning and is under the control of tyrosine-kinases-related proteins, whose inactivation delayed but do not hampered cell division. Thus, whereas axis formation is cell-cycle dependent, a putative control of the cell-cycle control by polarisation events remains to be shown.L'orchestration correcte de la différenciation et du cycle cellulaire est nécessaire au développement de tout organisme. Chez les végétaux, il n'existe que peu de données sur les mécanismes moléculaires mis en jeu pour coordonner ces deux événements. Le zygote de l'algue brune Fucus est un modèle de l'embryogenèse précoce des végétaux. La polarisation du zygote de Fucus s'effectue après la fécondation et coïncide avec le premier cycle cellulaire mitotique. Des acteurs moléculaires impliqués dans la polarisation zygotique ont été identifiés mais les voies de transduction contrôlant l'établissement de la polarité restent à préciser. Par ailleurs les connaissances sur le cycle cellulaire du Fucus sont très réduites. Dans ce contexte, nous avons voulu savoir comment la polarisation et le cycle cellulaire sont régulés et s'il existe des interactions entre ces deux événements. Nous avons montré que le cycle cellulaire présente quatre phases G1, S, G2 et M bien définies, possède les mécanismes de surveillance usuels, qui rendent la mitose dépendante de la réplication et de l'assemblage correct du fuseau, et que sa progression (transitions G1/S, G2/M, sortie de mitose, cytodiérèse) dépend étroitement de l'activité de protéines de types kinases dépendantes des cylines (CDK). Deux CDK à motif PSTAIRE, p32 et p34, sont traduites à partir ARNm maternels, après la fécondation, et leur synthèse progressive correspond à l'augmentation de l'activité kinase des CDK, dont le pic mitotique requiert la transcription d'un régulateur positif. Ces deux CDK sont négativement régulées par phosphorylation sur tyrosine, lors de la progression normale et lors de l'activation du point de contrôle de la réplication de l'ADN. P34 est spécifiquement liée par l'inhibiteur de CDK, purvalanol, et jouerait un rôle majeur dans le contrôle du cycle cellulaire. Nous avons montré que, à la transition G1/S, la CDK p34 contrôle également la formation de l'axe de polarité embryonnaire, qui s'effectue durant la phase S. D'autre part, nous avons mis en évidence que la formation de l'axe est essentielle au modelage de l'embryon et qu'elle est régulée par des protéines de type kirosine-kinases. Ces kinases ne sont pas nécessaires à la division. Aussi, la formation de l'axe de polarité est sous le contrôle du cycle cellulaire, la réciproque reste à démontrer

SPHINGOLIPIDS AND Δ8-SPHINGOLIPID DESATURASE FROM THE PICOALGA OSTREOCOCCUS TAURI AND INVOLVEMENT IN TEMPERATURE ACCLIMATION

SUMMARY Sphingolipids are crucial components of cell membranes. Sphingolipid Δ8-unsaturation is more specific to plants and is involved in the regulation of stress responses. The structure and functions of sphingolipids in microalgae are still poorly understood. Ostreococus tauri is a minimal microalga at the base of the green lineage, and is therefore a key organism for understanding lipid evolution. The present work reports the characterisation as well as the temperature regulation of sphingolipids and Δ8-sphingolipid desaturase from O. tauri . Complex sphingolipids are glycosylceramides with unique glycosyl moieties encompassing hexuronic acid residues, reminiscent of bacterial glucuronosylceramides, with up to three additional hexose residues. In contrast, the ceramide backbones show limited variety, with dihydroxylated C18/C18:1 EΔ8 sphingoid bases and C16:0 fatty-acyl chain being the main compounds. The sphingolipid Δ8-desaturase from O. tauri , although phylogenetically related to plant homologues has a substrate preference similar to the diatom homologue. Both sphingolipid Δ8-desaturase transcripts and sphingolipid Δ8-unsaturation are regulated in a temperature- dependent manner being higher at 14°C than 24°C. Overexpressing the sphingolipid Δ8- desaturase in O. tauri at 24°C results in higher sphingolipid unsaturation and impairs the increase in cell size, structure and chlorophyll. In particular, the cell-size defect is not detected in cells acclimated to 14°C and is furthermore suppressed upon transfer from 24°C to 14°C. Our work provides the first functional evidence for the involvement of sphingolipid Δ8-unsaturation for temperature acclimation in microalgae, suggesting that this function is an ancestral feature in the green lineage

Characterization of Unique Eukaryotic Sphingolipids with Temperature-Dependent Δ8-Unsaturation from the Picoalga Ostreococcus tauri

International audienceAbstract Sphingolipids (SLs) are ubiquitous components of eukaryotic cell membranes and are found in some prokaryotic organisms and viruses. They are composed of a sphingoid backbone that may be acylated and glycosylated. Assembly of various sphingoid base, fatty acyl and glycosyl moieties results in highly diverse structures. The functional significance of variations in SL chemical diversity and abundance is still in the early stages of investigation. Among SL modifications, Δ8-desaturation of the sphingoid base occurs only in plants and fungi. In plants, SL Δ8-unsaturation is involved in cold hardiness. Our knowledge of the structure and functions of SLs in microalgae lags far behind that of animals, plants and fungi. Original SL structures have been reported from microalgae. However, functional studies are still missing. Ostreococcus tauri is a minimal microalga at the base of the green lineage and is therefore a key organism for understanding lipid evolution. In the present work, we achieved the detailed characterization of O. tauri SLs and unveiled unique glycosylceramides as sole complex SLs. The head groups are reminiscent of bacterial SLs, as they contain hexuronic acid residues and can be polyglycosylated. Ceramide backbones show a limited variety, and SL modification is restricted to Δ8-unsaturation. The Δ8-SL desaturase from O. tauri only produced E isomers. Expression of both Δ8-SL desaturase and Δ8-unsaturation of sphingolipids varied with temperature, with lower levels at 24°C than at 14°C. Overexpression of the Δ8-SL desaturase dramatically increases the level of Δ8 unsaturation at 24°C and is paralleled by a failure to increase cell size. Our work provides the first characterization of O. tauri SLs and functional evidence for the involvement of SL Δ8-unsaturation for temperature acclimation in microalgae, suggesting that this function is an ancestral feature in the green lineage