Molecular characterization of light input to the circadian clock of the microalga Ostrococcus tauri

Les microalgues du phytoplancton sont exposées à des variations fréquentes et rapides de la qualité et de l'intensité spectrale en milieu marin. On peut donc supposer qu'il existe des mécanismes de photoperception spécifiques aux microalgues, différents de ceux identifiés chez les organismes terrestres. L'importance de l'horloge circadienne dans la transmission de l'information lumineuse et notamment la photopériode a largement été caractérisée chez plusieurs organismes modèles terrestres. Le principal objectif de ma thèse était d'étudier les régulations des gènes de l'horloge en réponse à la lumière, chez la microalgue Ostreococcus tauri. Le développement récent des techniques de génomique fonctionnelle chez cette microalgue eucaryote l'a promue comme un nouvel organisme modèle pour l'étude de mécanismes complexes tels que horloge circadienne. Mon étude s'est focalisée sur la caractérisation d'une voie de signalisation de type système à deux composants susceptible de transmettre le signal lumineux vers l'oscillateur central de l'horloge. J'ai étudié les régulations des principaux acteurs de l'horloge d'Ostreococcus par la lumière, et en particulier celles du gène TOC1. J'ai aussi caractérisé la protéine LOV-HK, un nouveau type de photorécepteur à la lumière bleue chez les eucaryotes, dont l'activité est requise pour le bon fonctionnement de l'horloge d'Ostreococcus. L'importance des régulations transcriptionnelles de TOC1 et de LOV-HK, ainsi que leurs fonctions dans l'oscillateur central ont été abordées par l'utilisation d'un promoteur inductible. Enfin, j'ai montré que LOV-HK et plus globalement l'horloge régulent la croissance cellulaire et la biomasse, démontrant leur rôle central dans le contrôle de la physiologie d'Ostreococcus tauri.Light quality and intensity change frequently in the water column. Therefore marine microalgae are exposed to large changes in light spectrum. Photoperception mechanisms in microalgae are expected to differ from those of land plants since the marine environment has unique properties of light transmission. The focus of my PhD project concerns two mains topics, circadian clock regulation and photoperception in the microalga Ostreococcus tauri. In recent years, O. tauri has emerged as a promising model organism using functional genomics approaches to study complex processes such as the circadian clock regulations. My study was focused on the involvement of a two components system in light transmission to the circadian clock of Ostreococcus. I have studied the molecular mechanisms underlying the regulation of the core clock component TOC1. I have also characterized a novel eukaryotic blue light photoreceptor called LOV-HK, which regulates circadian clock function in Ostreococcus. Using an inducible promoter system to modulate the levels of TOC1 and LOV-HK, I have analyzed the importance of their transcriptional regulations in the clock. Finally, I have shown that LOV-HK and more generally the circadian clock, regulates cell growth and biomass in Ostreococcus tauri

Analysis of <i>TOC1</i> circadian function.

<p>(<b>A</b>) Experimental protocol. Luciferase reporter lines CCA1:Luc and TOC1ox/CCA1:Luc were released in constant light at Time 0 after an initial synchronization by a 6 hour dark pulse. Pi was added at a 30 µM final concentration at three different times (t1 = 98, t2 = 104 and t3 = 108 hours). Arrows indicate the three times of Pi addition relative to the phases of CCA1:Luc control line (<b>B</b>). For the three times of Pi addition, a rhythmic expression pattern of CCA1:Luc was recovered. The phase of CCA1:Luc peak of expression relative to the time of Pi injection was determined on the second peak of CCA1:Luc (e.g. Phase 1 corresponds to the time t1 of Pi addition). (<b>C</b>) The phase of CCA1:Luc peak of expression is plotted as a function of the time of Pi addition. Note that for the three times, no circadian gating of CCA1:Luc response is observed (N = 3, ±SD).</p

Clocks in the Green Lineage: Comparative Functional Analysis of the Circadian Architecture of the Picoeukaryote Ostreococcus[W]

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

Map and nucleotide sequence of the <i>High Affinity Phosphate Transporter</i> promoters.

<p>(<b>A</b>) Scheme of the <i>O. tauri HAPT</i> gene. <i>HAPT</i> is localized on the atypical Chromosome II (ChrII) downstream of 667 bp-long repeated sequences. The gene structure (promoter, introns, exons) is supported by ESTs. (<b>B</b>) 119 bp-long <i>HAPT</i> putative promoter. A perfectly conserved Phosphate response element (P1BS) is located at position −76. (<b>C</b>) <i>HAPT</i> is one of the few genes conserved in <i>O. tauri</i> and <i>O. lucimarinus</i> viruses. The putative promoter sequences of these two viruses exhibit a conserved TATA Box at position −30 but no P1BS sequence.</p

Effect of phosphate on cell growth and pHAPT:Luc promoter activity.

<p>(<b>A</b>) The effect of β-glycerophosphate (Po for organic phosphate), NaH₂PO₄ (Pi for inorganic phosphate) or combined Pi+Po was monitored on cell growth as recorded by flow cytometry. <i>Ostreococcus</i> cells were grown in Keller medium containing 10 µM β-glycerophosphate (K_Po), Keller medium containing 10 µM NaH₂PO₄ instead of β-glycerophosphate (K_Pi), and Keller Medium supplemented with 10 µM NaH₂PO₄K_Po+Pi). (<b>B</b>) Cells were grown in ASW containing various concentrations of Pi or Po. Cell growth concentration was determined after 60 hours in culture. Similar dose response concentrations were obtained even though cell concentrations were slightly lower in ASW_(Pi) (N = 3, ±SD). (<b>C</b>) <i>In vitro</i> luminescence measurement of accumulated luciferase per cell over 60 hours in a pHAPT:Luc reporter line (N = 3, ±SD). A dose dependent inhibition was observed for both Po and Pi but the inhibition by Pi occurred at lower concentration.</p

Reversion of circadian clock <i>TOC1</i>-overexpression phenotype by modulating the HAPT promoter activity.

<p>(<b>A</b>) TOC1ox/CCA1:Luc cells overexpressing <i>TOC1</i> under <i>HAPT</i> promoter display an arrhythmic profile of CCA1:Luc luminescence under constant light when grown in ASW containing 10 µM Po (standard Keller Medium). Pi was added at the time 0 of transfer to constant light (after an initial 6 hour dark pulse) to repress the HAPT promoter activity. For [Pi]≥5 µM, TOC1ox/CCA1:Luc cells recovered rhythmic expression of CCA1:Luc. (<b>B</b>) Quantitative effect of phosphate on TOC1ox/CCA1:Luc rhythmicity in constant light. To assess rhythmicity, relative amplitude error (RAE) estimation was performed over at least 3 days of recording, using the FFT-NLLS program from BRASS. For all [Pi]≥5 µM, robust rhythms were observed (RAE<0.4, N = 3), the period variations were below 1 hour and no dose-dependent effect was observed. (<b>C</b>) Addition of Pi to a 30 µM final concentration at time 98 restored a rhythmic pattern of CCA1:Luc in <i>TOC1</i> overexpressing line. Time 0 corresponds to time of Pi addition.</p

Time-course inhibition of <i>HAPT</i> promoter activity by inorganic phosphate.

<p>(<b>A</b>) <i>In vivo</i> kinetics of luciferase activity of pHAPT:Luc cells grown in ASW+10 µM Po before being exposed to various Pi concentrations for 28 hours from time 0. Maximal inhibition was observed for Pi concentrations above or equal to 50 µM (N = 3, ±SD). (<b>B</b>) Dose dependent inhibition of pHAPT:Luc <i>in vivo</i> luminescence by inorganic NaH₂PO₄. Luminescence measured 28 hours after Pi is reported as a function of Pi concentration. At this time a 50% inhibition is observed for [Pi]∼5 µM (N = 3, ±SD). (<b>C</b>) Corresponding luciferase mRNA levels measured at various times after Pi addition at a 50 µM concentration. Transcript amounts were quantified by real time quantitative RT-PCR and normalized to EF1α. Time 0 corresponds to the time of Pi addition. Control cells were grown in ASW 10 µM Po without Pi.</p

A plastidial DEAD box RNA helicase plays a critical role in high light acclimation by modulating ribosome biogenesis in Chlamydomonas reinhardtii

ABSTRACT Photosynthetic organisms have developed sophisticated strategies to fine-tune light energy conversion to meet the metabolic demand, thereby optimizing growth in fluctuating light environments. Although mechanisms such as energy dissipation, photosynthetic control, or the photosystem II (PSII) damage and repair have been widely studied, little is known about the regulation of protein synthesis capacity during light acclimation. By screening a Chlamydomonas reinhardtii insertional mutant library using chlorophyll fluorescence imaging, we isolated a high chlorophyll fluorescence mutant ( hf 0 ) defected in a gene encoding a putative plastid targeted DEAD-box RNA helicase called CreRH22. CreRH22 is rapidly induced upon illumination and belongs to the GreenCut, a set of proteins specific to photosynthetic organisms. While photosynthesis is slightly affected in the mutant under low light (LL), exposure to high light (HL) induces a marked decrease in both PSII and PSI, and a strong alteration of the light-induced gene expression pattern. These effects are explained by the inability of hf 0 to increase plastid ribosome amounts under HL. We conclude that CreRH22, by promoting ribosomal RNA precursor maturation in a light-dependent manner, enables the assembly of extra-ribosomes required to synthesize photosystem subunits at a higher rate, a critical step in the acclimation of algae to HL