Circadian regulation of metabolism across photosynthetic organisms.

Circadian regulation produces a biological measure of time within cells. The daily cycle in the availability of light for photosynthesis causes dramatic changes in biochemical processes in photosynthetic organisms, with the circadian clock having crucial roles in adaptation to these fluctuating conditions. Correct alignment between the circadian clock and environmental day-night cycles maximizes plant productivity through its regulation of metabolism. Therefore, the processes that integrate circadian regulation with metabolism are key to understanding how the circadian clock contributes to plant productivity. This forms an important part of exploiting knowledge of circadian regulation to enhance sustainable crop production. Here, we examine the roles of circadian regulation in metabolic processes in source and sink organ structures of Arabidopsis. We also evaluate possible roles for circadian regulation in root exudation processes that deposit carbon into the soil, and the nature of the rhythmic interactions between plants and their associated microbial communities. Finally, we examine shared and differing aspects of the circadian regulation of metabolism between Arabidopsis and other model photosynthetic organisms, and between circadian control of metabolism in photosynthetic and non-photosynthetic organisms. This synthesis identifies a variety of future research topics, including a focus on metabolic processes that underlie biotic interactions within ecosystems.

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

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.

The blue-green light-dependent state transition in the marine phytoplankton Ostreococcus tauri.

We explored the adaptive mechanisms of Ostreococcus tauri, a marine picophytoplankton with a ubiquitous ocean presence. We aimed to understand its photosynthetic acclimation, as featured in the cryo-EM structure of its photosystem I (PSI) supercomplex. This structure revealed a unique composition involving a phosphorylated Lhcp trimer bound to the PSI core along with two additional Lhcp trimers, suggesting potential state transitions for photoacclimation. To investigate this hypothesis, we conducted a series of biochemical and physiological experiments. We analyzed absorption spectra to differentiate between PSI and PSII, particularly focusing on blue-green wavelengths, and examined the effects of specific excitation of Lhcp with green light, including its phosphorylation and the formation of the PSI-LHCI-Lhcp supercomplex. Our experiments clarified the distinctive effects attributable to absorption by pigments associated with Lhcp. Exciting Lhcp with green light induced its phosphorylation, leading to the formation of the PSI-LHCI-Lhcp supercomplex. Notably, the functional antenna size of PSI could reversibly expand in response to green light, demonstrating its state transition capability. These findings not only highlight the unique photosynthetic acclimation adapted to the marine environment but also suggest a possible ancestral role of state transitions in green plants, given the phylogenetic position of Prasinophyceae.

Genetic and physiological responses to light quality in a deep ocean ecotype of Ostreococcus, an ecologically important photosynthetic picoeukaryote.

Phytoplankton are exposed to dramatic variations in light quality when cells are carried by upwelling or downwelling currents or encounter sediment. We investigated the potential impact of light quality changes in Ostreococcus, a key marine photosynthetic picoeukaryote, by analysing changes in its transcriptome, pigment content, and photophysiology after acclimation to monochromatic red, green, or blue light. The clade B species RCC809, isolated from the deep euphotic zone of the tropical Atlantic Ocean, responded to blue light by accelerating cell division at the expense of storage reserves and by increasing the relative level of blue-light-absorbing pigments. It responded to red and green light by increasing its potential for photoprotection. In contrast, the clade A species OTTH0595, which originated from a shallow water environment, showed no difference in photosynthetic properties and minor differences in carotenoid contents between light qualities. This was associated with the loss of candidate light-quality responsive promoter motifs identified in RCC809 genes. These results demonstrate that light quality can have a major influence on the physiology of eukaryotic phytoplankton and suggest that different light quality environments can drive selection for diverse patterns of responsiveness and environmental niche partitioning.

Homologs of Ancestral CNNM Proteins Affect Magnesium Homeostasis and Circadian Rhythmicity in a Model Eukaryotic Cell.

Biological rhythms are ubiquitous across organisms and coordinate key cellular processes. Oscillations of Mg2+ levels in cells are now well-established, and due to the critical roles of Mg2+ in cell metabolism, they are potentially fundamental for the circadian control of cellular activity. The identity of the transport proteins responsible for sustaining Mg2+ levels in eukaryotic cells remains hotly debated, and several are restricted to specific groups of higher eukaryotes. Here, using the eukaryotic minimal model cells of Ostreococcus tauri, we report two homologs of common descents of the Cyclin M (CNNM)/CorC protein family. Overexpression of these proteins leads to a reduction in the overall magnesium content of cells and a lengthening of the period of circadian gene expression rhythms. However, we observed a paradoxical increase in the magnesium content of the organelle fraction. The chemical inhibition of Mg2+ transport has a synergistic effect on circadian period lengthening upon the overexpression of one CNNM homolog, but not the other. Finally, both homologs rescue the deleterious effect of low extracellular magnesium on cell proliferation rates. Overall, we identified two CNNM proteins that directly affect Mg2+ homeostasis and cellular rhythms.

CBM20CP, a novel functional protein of starch metabolism in green algae.

Ostreococcus tauri is a picoalga that contains a small and compact genome, which resembles that of higher plants in the multiplicity of enzymes involved in starch synthesis (ADP-glucose pyrophosphorylase, ADPGlc PPase; granule bound starch synthase, GBSS; starch synthases, SSI, SSII, SSIII; and starch branching enzyme, SBE, between others), except starch synthase IV (SSIV). Although its genome is fully sequenced, there are still many genes and proteins to which no function was assigned. Here, we identify the OT_ostta06g01880 gene that encodes CBM20CP, a plastidial protein which contains a central carbohydrate binding domain of the CBM20 family, and a coiled coil domain at the C-terminus that lacks catalytic activity. We demonstrate that CBM20CP has the ability to bind starch, amylose and amylopectin with different affinities. Furthermore, this protein interacts with OsttaSSIII-B, increasing its binding to starch granules, its catalytic efficiency and promoting granule growth. The results allow us to postulate a functional role for CBM20CP in starch metabolism in green algae. KEY MESSAGE: CBM20CP, a plastidial protein that has a modular structure but lacks catalytic activity, regulates the synthesis of starch in Ostreococcus tauri.

Nitric oxide synthases from photosynthetic organisms improve growth and confer nitrosative stress tolerance in E. coli. Insights on the pterin cofactor.

Nitric oxide synthase (NOS) catalyzes NO formation from the substrate l-arginine (Arg). Previously, NOS with distinct biochemical properties were characterized from two photosynthetic microorganisms, the unicellular algae Ostreococcus tauri (OtNOS) and the cyanobacteria Synechococcus PCC 7335 (SyNOS). In this work we studied the effect of recombinant OtNOS and SyNOS expressed under IPTG-induced promoter in E. coli, a bacterium that lacks NOS. Results show that OtNOS and SyNOS expression promote E. coli growth in a nutrient replete medium and allow to better metabolize Arg as N source. In LB medium, OtNOS induces the expression of the NO dioxygenase hmp in E. coli, in accordance with high NO levels visualized with the probe DAF-FM DA. In contrast, SyNOS expression does not induce hmp and show a slight increase of NO production compared to OtNOS. NOS expression reduces ROS production and increases viability of E. coli cultures growing in LB. A strong nitrosative stress provoked by the addition of 1 mM of the NO donors sodium nitroprusside (SNP) and nitrosoglutathione (GSNO) inhibits bacterial growth rate. Under these conditions, the expression of OtNOS or SyNOS counteracts NO donor toxicity restoring bacterial growth. Finally, using bioinformatic tools and ligand docking analyses, we postulate that tetrahydromonapterin (MH4), an endogenous pterin found in E. coli, could act as cofactor required for NOS catalytic activity. Our findings could be useful for the development of biotechnological applications using NOS expression to improve growth in NOS-lacking bacteria.

Growth strategies of a model picoplankter depend on social milieu and pCO2.

Phytoplankton exist in genetically diverse populations, but are often studied as single lineages (single strains), so that interpreting single-lineage studies relies critically on understanding how microbial growth differs with social milieu, defined as the presence or absence of conspecifics. The properties of lineages grown alone often fail to predict the growth of these same lineages in the presence of conspecifics, and this discrepancy points towards an opportunity to improve our understanding of the factors that affect lineage growth rates. We demonstrate that different lineages of a marine picoplankter modulate their maximum lineage growth rate in response to the presence of non-self conspecifics, even when resource competition is effectively absent. This explains why growth rates of lineages in isolation do not reliably predict their growth rates in mixed culture, or the lineage composition of assemblages under conditions of rapid growth. The diversity of growth strategies observed here are consistent with lineage-specific energy allocation that depends on social milieu. Since lineage growth is only one of many traits determining fitness in natural assemblages, we hypothesize that intraspecific variation in growth strategies should be common, with more strategies possible in ameliorated environments that support higher maximum growth rates, such as high CO2 for many marine picoplankton.

Crystal structures of NAD+-linked isocitrate dehydrogenase from the green alga Ostreococcus tauri and its evolutionary relationship with eukaryotic NADP+-linked homologs.

NAD+-linked isocitrate dehydrogenases (NAD-IDHs) catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate. Previously, we identified a novel phylogenetic clade including NAD-IDHs from several algae in the type II subfamily, represented by homodimeric NAD-IDH from Ostreococcus tauri (OtIDH). However, due to its lack of a crystalline structure, the molecular mechanisms of the ligand binding and catalysis of OtIDH are little known. Here, we elucidate four high-resolution crystal structures of OtIDH in a ligand-free and various ligand-bound forms that capture at least three states in the catalytic cycle: open, semi-closed, and fully closed. Our results indicate that OtIDH shows several novel interactions with NAD+, unlike type I NAD-IDHs, as well as a strictly conserved substrate binding mode that is similar to other homologs. The central roles of Lys283' in dual coenzyme recognition and Lys234 in catalysis were also revealed. In addition, the crystal structures obtained here also allow us to understand the catalytic mechanism. As expected, structural comparisons reveal that OtIDH has a very high structural similarity to eukaryotic NADP+-linked IDHs (NADP-IDHs) within the type II subfamily rather than with the previously reported NAD-IDHs within the type I subfamily. It has also been demonstrated that OtIDH exhibits substantial conformation changes upon ligand binding, similar to eukaryotic NADP-IDHs. These results unambiguously support our hypothesis that OtIDH and OtIDH-like homologs are possible evolutionary ancestors of eukaryotic NADP-IDHs in type II subfamily.

Prasinovirus Attack of Ostreococcus Is Furtive by Day but Savage by Night.

Prasinoviruses are large DNA viruses that infect diverse genera of green microalgae worldwide in aquatic ecosystems, but molecular knowledge of their life cycles is lacking. Several complete genomes of both these viruses and their marine algal hosts are now available and have been used to show the pervasive presence of these species in microbial metagenomes. We have analyzed the life cycle of Ostreococcus tauri virus 5 (OtV5), a lytic virus, using transcriptome sequencing (RNA-Seq) from 12 time points of healthy or infected Ostreococcus tauri cells over a day/night cycle in culture. In the day, viral gene transcription remained low while host nitrogen metabolism gene transcription was initially strongly repressed for two successive time points before being induced for 8 h, but during the night, viral transcription increased steeply while host nitrogen metabolism genes were repressed and many host functions that are normally reduced in the dark appeared to be compensated either by genes expressed from the virus or by increased expression of a subset of 4.4% of the host's genes. Some host cells underwent lysis progressively during the night, but a larger proportion were lysed the following morning. Our data suggest that the life cycles of algal viruses mirror the diurnal rhythms of their hosts.IMPORTANCE Prasinoviruses are common in marine environments, and although several complete genomes of these viruses and their hosts have been characterized, little is known about their life cycles. Here we analyze in detail the transcriptional changes occurring over a 27-h-long experiment in a natural diurnal rhythm, in which the growth of host cells is to some extent synchronized, so that host DNA replication occurs late in the day or early in the night and cell division occurs during the night. Surprisingly, viral transcription remains quiescent over the daytime, when the most energy (from light) is available, but during the night viral transcription activates, accompanied by expression of a few host genes that are probably required by the virus. Although our experiment was accomplished in the lab, cyclical changes have been documented in host transcription in the ocean. Our observations may thus be relevant for eukaryotic phytoplankton in natural environments.

Protective Action of Ostreococcus tauri and Phaeodactylum tricornutum Extracts towards Benzo[a]Pyrene-Induced Cytotoxicity in Endothelial Cells.

Marine microalgae are known to be a source of bioactive molecules of interest to human health, such as n-3 polyunsaturated fatty acids (n-3 PUFAs) and carotenoids. The fact that some of these natural compounds are known to exhibit anti-inflammatory, antioxidant, anti-proliferative, and apoptosis-inducing effects, demonstrates their potential use in preventing cancers and cardiovascular diseases (CVDs). Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is an ubiquitous environmental pollutant known to contribute to the development or aggravation of human diseases, such as cancer, CVDs, and immune dysfunction. Most of these deleterious effects are related to the activation of the polycyclic aromatic hydrocarbon receptor (AhR). In this context, two ethanolic microalgal extracts with concentrations of 0.1 to 5 µg/mL are tested, Ostreoccoccus tauri (OT) and Phaeodactylum tricornutum (PT), in order to evaluate and compare their potential effects towards B[a]P-induced toxicity in endothelial HMEC-1 cells. Our results indicate that the OT extract can influence the toxicity of B[a]P. Indeed, apoptosis and the production of extracellular vesicles were decreased, likely through the reduction of the expression of CYP1A1, a B[a]P bioactivation enzyme. Furthermore, the B[a]P-induced expression of the inflammatory cytokines IL-8 and IL1-ß was reduced. The PT extract only inhibited the expression of the B[a]P-induced cytokine IL-8 expression. The OT extract therefore seems to be a good candidate for counteracting the B[a]P toxicity.

Comparative Analysis of Culture Conditions for the Optimization of Carotenoid Production in Several Strains of the Picoeukaryote Ostreococcus.

Microalgae are promising sources for the sustainable production of compounds of interest for biotechnologies. Compared to higher plants, microalgae have a faster growth rate and can be grown in industrial photobioreactors. The microalgae biomass contains specific metabolites of high added value for biotechnology such as lipids, polysaccharides or carotenoid pigments. Studying carotenogenesis is important for deciphering the mechanisms of adaptation to stress tolerance as well as for biotechnological production. In recent years, the picoeukaryote Ostreococcustauri has emerged as a model organism thanks to the development of powerful genetic tools. Several strains of Ostreococcus isolated from different environments have been characterized with respect to light response or iron requirement. We have compared the carotenoid contents and growth rates of strains of Ostreococcus (OTTH595, RCC802 and RCC809) under a wide range of light, salinity and temperature conditions. Carotenoid profiles and productivities varied in a strain-specific and stress-dependent manner. Our results also illustrate that phylogenetically related microalgal strains originating from different ecological niches present specific interests for the production of specific molecules under controlled culture conditions.

Central role for ferritin in the day/night regulation of iron homeostasis in marine phytoplankton.

In large regions of the open ocean, iron is a limiting resource for phytoplankton. The reduction of iron quota and the recycling of internal iron pools are among the diverse strategies that phytoplankton have evolved to allow them to grow under chronically low ambient iron levels. Phytoplankton species also have evolved strategies to cope with sporadic iron supply such as long-term storage of iron in ferritin. In the picophytoplanktonic species Ostreococcus we report evidence from observations both in the field and in laboratory cultures that ferritin and the main iron-binding proteins involved in photosynthesis and nitrate assimilation pathways show opposite diurnal expression patterns, with ferritin being maximally expressed during the night. Biochemical and physiological experiments using a ferritin knock-out line subsequently revealed that this protein plays a central role in the diel regulation of iron uptake and recycling and that this regulation of iron homeostasis is essential for cell survival under iron limitation.

Connectivity among Photosystem II centers in phytoplankters: Patterns and responses.

Fast Repetition and Relaxation chlorophyll fluorescence induction is used to estimate the effective absorption cross section of PSII (σPSII), to analyze phytoplankton acclimation and electron transport. The fitting coefficient ρ measures excitation transfer from closed PSII to remaining open PSII upon illumination, which could theoretically generate a progressive increase in σPSII for the remaining open PSII. To investigate how ρ responds to illumination we grew marine phytoplankters with diverse antenna structures (Prochlorococcus, Synechococcus, Ostreococcus and Thalassiosira pseudonana) under limiting or saturating growth light. Initial ρ varied with growth light in Synechococcus and Thalassiosira. With increasing actinic illumination PSII closed progressively and ρ decreased for all four taxa, in a pattern explicable as an exponential decay of ρ with increasing distance between remaining open PSII reaction centers. This light-dependent down-regulation of ρ allows the four phytoplankters to limit the effect of increasing light upon σPSII. The four structurally distinct taxa showed, however, distinct rates of response of ρ to PSII closure, likely reflecting differences in the spacing or orientation among their PSII centers. Following saturating illumination recovery of ρ in darkness coincided directly with PSII re-opening in Prochlorococcus. Even after PSII had re-opened in Synechococcus a transition to State II slowed dark recovery of ρ. In Ostreococcus sustained NPQ slowed dark recovery of ρ. In Thalassiosira dark recovery of ρ was slowed, possibly by a light-induced change in PSII spacing. These patterns of ρ versus PSII closure are thus a convenient probe of comparative PSII spacings.

A lycopene ß-cyclase/lycopene ε-cyclase/light-harvesting complex-fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α-carotene and ß-carotene at different ratios.

Biosynthesis of asymmetric carotenoids such as α-carotene and lutein in plants and green algae involves the two enzymes lycopene ß-cyclase (LCYB) and lycopene ε-cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C-terminal light-harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α-carotene and ß-carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C-terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α-carotene-to-ß-carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α-carotene or lutein in bacteria or fungi.

Ostreococcus tauri is a new model green alga for studying iron metabolism in eukaryotic phytoplankton.

BACKGROUND: Low iron bioavailability is a common feature of ocean surface water and therefore micro-algae developed original strategies to optimize iron uptake and metabolism. The marine picoeukaryotic green alga Ostreococcus tauri is a very good model for studying physiological and genetic aspects of the adaptation of the green algal lineage to the marine environment: it has a very compact genome, is easy to culture in laboratory conditions, and can be genetically manipulated by efficient homologous recombination. In this study, we aimed at characterizing the mechanisms of iron assimilation in O. tauri by combining genetics and physiological tools. Specifically, we wanted to identify and functionally characterize groups of genes displaying tightly orchestrated temporal expression patterns following the exposure of cells to iron deprivation and day/night cycles, and to highlight unique features of iron metabolism in O. tauri, as compared to the freshwater model alga Chalamydomonas reinhardtii. RESULTS: We used RNA sequencing to investigated the transcriptional responses to iron limitation in O. tauri and found that most of the genes involved in iron uptake and metabolism in O. tauri are regulated by day/night cycles, regardless of iron status. O. tauri lacks the classical components of a reductive iron uptake system, and has no obvious iron regulon. Iron uptake appears to be copper-independent, but is regulated by zinc. Conversely, iron deprivation resulted in the transcriptional activation of numerous genes encoding zinc-containing regulation factors. Iron uptake is likely mediated by a ZIP-family protein (Ot-Irt1) and by a new Fea1-related protein (Ot-Fea1) containing duplicated Fea1 domains. The adaptation of cells to iron limitation involved an iron-sparing response tightly coordinated with diurnal cycles to optimize cell functions and synchronize these functions with the day/night redistribution of iron orchestrated by ferritin, and a stress response based on the induction of thioredoxin-like proteins, of peroxiredoxin and of tesmin-like methallothionein rather than ascorbate. We briefly surveyed the metabolic remodeling resulting from iron deprivation. CONCLUSIONS: The mechanisms of iron uptake and utilization by O. tauri differ fundamentally from those described in C. reinhardtii. We propose this species as a new model for investigation of iron metabolism in marine microalgae.

Functional characterization of Ostreococcus tauri phototropin.

Phototropins (phots) regulate a range of adaptive processes in plants that serve to optimize photosynthetic efficiency and promote growth. Light sensing by Arabidopsis thaliana phots is predominantly mediated by the Light, Oxygen and Voltage sensing 2 (LOV2) flavin-binding motif located within the N-terminus of the photoreceptor. Here we characterize the photochemical and biochemical properties of phot from the marine picoalga Ostreococcus tauri phototropin (Otphot) and examine its ability to replace phot-mediated function in Arabidopsis. Photochemical properties of Otphot rely on both LOV1 and LOV2. Yet, biochemical analysis indicates that light-dependent receptor autophosphorylation is primarily dependent on LOV2. As found for Arabidopsis phots, Otphot associates with the plasma membrane and partially internalizes, albeit to a limited extent, in response to blue-light irradiation. Otphot is able to elicit a number of phot-regulated processes in Arabidopsis, including petiole positioning, leaf expansion, stomatal opening and chloroplast accumulation movement. However, Otphot is unable to restore phototropism and chloroplast avoidance movement. Consistent with its lack of phototropic function in Arabidopsis, Otphot does not associate with or trigger dephosphorylation of the phototropic signalling component Non-Phototropic Hypocotyl 3 (NPH3). Taken together, these findings indicate that the mechanism of action of plant and evolutionarily distant algal phots is less well conserved than previously thought.

Label-free quantitative analysis of the casein kinase 2-responsive phosphoproteome of the marine minimal model species Ostreococcus tauri.

Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild-type and CK2-overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2-responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. All MS data have been deposited in the ProteomeXchange with identifier PXD000975 (http://proteomecentral.proteomexchange.org/dataset/PXD000975).

Efficient gene targeting and removal of foreign DNA by homologous recombination in the picoeukaryote Ostreococcus.

With fewer than 8000 genes and a minimalist cellular organization, the green picoalga Ostreococcus tauri is one of the simplest photosynthetic eukaryotes. Ostreococcus tauri contains many plant-specific genes but exhibits a very low gene redundancy. The haploid genome is extremely dense with few repeated sequences and rare transposons. Thanks to the implementation of genetic transformation and vectors for inducible overexpression/knockdown this picoeukaryotic alga has emerged in recent years as a model organism for functional genomics analyses and systems biology. Here we report the development of an efficient gene targeting technique which we use to knock out the nitrate reductase and ferritin genes and to knock in a luciferase reporter in frame to the ferritin native protein. Furthermore, we show that the frequency of insertion by homologous recombination is greatly enhanced when the transgene is designed to replace an existing genomic insertion. We propose that a natural mechanism based on homologous recombination may operate to remove inserted DNA sequences from the genome.