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Summary 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: One way that circadian rhythms impact plant productivity is through their regulation of metabolism. Here, we explore this topic with a focus on the mechanisms of circadian control of metabolism, the circadian regulation of photoassimilate exudation into the rhizosphere, and shared and differing aspects of the circadian regulation of metabolism across photosynthetic organisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

As a ubiquitous picophytoplankton in the ocean and an early-branching green alga, Ostreococcus tauri is a model prasinophyte species for studying the functional evolution of the light-harvesting systems in photosynthesis. Here, we report the structure and function of the O. tauri photosystem I (PSI) supercomplex in low light conditions, where it expands its photon-absorbing capacity by assembling with the light-harvesting complexes I (LHCI) and a prasinophyte-specific light-harvesting complex (Lhcp). The architecture of the supercomplex exhibits hybrid features of the plant-type and the green algal-type PSI supercomplexes, consisting of a PSI core, an Lhca1-Lhca4-Lhca2-Lhca3 belt attached on one side and an Lhca5-Lhca6 heterodimer associated on the other side between PsaG and PsaH. Interestingly, nine Lhcp subunits, including one Lhcp1 monomer with a phosphorylated amino-terminal threonine and eight Lhcp2 monomers, oligomerize into three trimers and associate with PSI on the third side between Lhca6 and PsaK. The Lhcp1 phosphorylation and the light-harvesting capacity of PSI were subjected to reversible photoacclimation, suggesting that the formation of OtPSI-LHCI-Lhcp supercomplex is likely due to a phosphorylation-dependent mechanism induced by changes in light intensity. Notably, this supercomplex did not exhibit far-red peaks in the 77 K fluorescence spectra, which is possibly due to the weak coupling of the chlorophyll a603-a609 pair in OtLhca1-4.

The physicochemical properties of starches from different botanical origin, such as viscosity, gelatinization temperature, solubility and degradability depend on the amylose/amylopectin ratio and the length and frequency of the α-1,6-glycosidic bonds in amylopectin. These branches depend on the action of the starch branching enzymes (SBEs), which are highly structurally conserved in plants. We recently identified a novel gene from Ostreococcus tauri (OsttaSBEIII) which codes for a protein showing starch branching activity (OsttaSBEIII) and with a different structure than other known SBEs from plants, containing two in-tandem carbohydrate binding modules (CBM41-CBM48) at its N-terminus. OsttaSBEIII overexpression in A. thaliana plants resulted in a higher starch content and smaller granules with an increased degradability. OEOsttaSBE lines showed also an increase in the expression and activity of starch degradative enzymes and a higher content of glucose and inorganic phosphate, which suggests a remodeling of the granule structure in response to the expression of OsttaSBEIII. These results allow us to propose the use of OsttaSBEIII as a new strategy to obtain starches with greater degradability that would be useful for different biotechnological applications.

IntroductionPhotosynthetic organisms respond to nitrogen (N) deprivation with the slowdown of photosynthesis and electron transport resulting in the balance the carbon (C)/N ratio. Under this extreme condition, organisms trigger complex mechanisms to keep growing using different N sources and recycling N containing molecules. In particular, phytoplankton are able to uptake L-arginine (L-Arg) as an organic N source. L-Arg can be assimilated mainly by the arginase, arginine deimidase, arginine decarboxylase or L-amino oxidase pathways.ResultsWe analyzed the effect of different N sources on the growth of the green algae Ostreococcus tauri. N starvation caused an inhibition of culture growth and a decrease in chlorophyll content. The addition of L-Arg to an N-deprived medium promotes a sustained growth rate of O. tauri culture and the increase of chlorophyll levels. The transcript level of genes involved in N uptake and metabolism were increased in N-starved condition while the addition of L-Arg as the sole N source reduced their induction. Since the O. tauri genome lacks the classical pathways to metabolize L-Arg, another enzyme/s may be responsible for L-Arg catabolism. Previously, we characterized the nitric oxide synthase (NOS) enzyme from O. tauri (OtNOS) which oxidizes L-Arg producing nitric oxide (NO) and citrulline. The NOS inhibitor L-NAME blocks the effect promoted by L-Arg on N-deprived O. tauri growth. Besides, NO level increased in O. tauri cells growing in L-Arg containing medium, suggesting the participation of OtNOS enzyme in L-Arg metabolism during N starvation.DiscussionOur hypothesis suggests that, after NOS-dependent Arg degradation, non-enzymatic oxidation of NO produces N oxides (mainly NO2-) that are re-incorporated to the N primary metabolism. As expected, N deprivation increases the lipid content in Ostreococcus. The addition of L-Arg or NO2- as the sole N sources showed a similar increase in lipid content to N deprivation. In summary, our results demonstrate that L-Arg is able to function as N source in Ostreococcus. The evidences on an alternative pathway of N supply and metabolism in a photosynthetic microorganism are discussed. These results could also allow the development of biotechnological tools for increasing lipid production for industry.

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. [ABSTRACT FROM AUTHOR]

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. • The NO synthase (NOS) from photosynthetic microorganisms were expressed in E. coli. • Expression of NOS increases bacterial growth and tolerance to nitrosative stress. • NOS expression contribute to bacterial growth using Arg as sole N source in the presence of IPTG. • In silico analyses suggest that MH4 could be used as redox NOS cofactor in E. coli. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

One of the major challenges in viral ecology is to assess the impact of viruses in controlling the abundance of specific hosts in the environment. To this end, techniques that enable the detection and quantification of virus-host interactions at the single-cell level are essential. With this goal in mind, we implemented virus fluorescence in situ hybridization (VirusFISH) using as a model the marine picoeukaryote Ostreococcus tauri and its virus Ostreococcus tauri virus 5 (OtV5). VirusFISH allowed the visualization and quantification of the proportion of infected cells during an infection cycle in experimental conditions. We were also able to quantify the abundance of free viruses released during cell lysis, discriminating OtV5 from other mid-level fluorescence phages in our non-axenic infected culture that were not easily distinguishable with flow cytometry. Our results showed that although the major lysis of the culture occurred between 24 and 48 h after OtV5 inoculation, some new viruses were already produced between 8 and 24 h. With this work, we demonstrate that VirusFISH is a promising technique to study specific virus-host interactions in non-axenic cultures and establish a framework for its application in complex natural communities.

One of the major challenges in viral ecology is to assess the impact of viruses in controlling the abundance of specific hosts in the environment. To this end, techniques that enable the detection and quantification of virus-host interactions at the single-cell level are essential. With this goal in mind, we implemented virus fluorescence in situ hybridization (VirusFISH) using as a model the marine picoeukaryote Ostreococcus tauri and its virus Ostreococcus tauri virus 5 (OtV5). VirusFISH allowed the visualization and quantification of the proportion of infected cells during an infection cycle in experimental conditions. We were also able to quantify the abundance of free viruses released during cell lysis, discriminating OtV5 from other mid-level fluorescence phages in our non-axenic infected culture that were not easily distinguishable with flow cytometry. Our results showed that although the major lysis of the culture occurred between 24 and 48 h after OtV5 inoculation, some new viruses were already produced between 8 and 24 h. With this work, we demonstrate that VirusFISH is a promising technique to study specific virus-host interactions in non-axenic cultures and establish a framework for its application in complex natural communities. [ABSTRACT FROM AUTHOR]

Marine phytoplankton, comprising cyanobacteria, micro- and pico-algae are key to photosynthesis, oxygen production and carbon assimilation on Earth. The unicellular green picoalga Ostreococcus tauri holds a key position at the base of the green lineage of plants, which makes it an interesting model organism. O. tauri has adapted to survive in low levels of nitrogen and phosphorus in the open ocean and also during rapid changes in the levels of these nutrients in coastal waters. In this study, we have employed untargeted proteomic and lipidomic strategies to investigate the molecular responses of O. tauri to low-nitrogen and low-phosphorus environments. In the absence of external nitrogen, there was an elevation in the expression of ammonia and urea transporter proteins together with an accumulation of triglycerides. In phosphate-limiting conditions, the expression levels of phosphokinases and phosphate transporters were increased, indicating an attempt to maximise scavenging opportunities as opposed to energy conservation conditions. The production of betaine lipids was also elevated, highlighting a shift away from phospholipid metabolism. This finding was supported by the putative identification of betaine synthase in O. tauri. This work offers additional perspectives on the complex strategies that underpin the adaptive processes of the smallest known free-living eukaryote to alterations in environmental conditions.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step for the synthesis of glycogen in cyanobacteria and starch in green algae and plants. The enzyme from cyanobacteria is homotetrameric (α4), while that from green algae and plants is heterotetrameric (α2β2). These ADP-Glc PPases are allosterically regulated by 3-phosphoglycerate (3PGA, activator) and inorganic orthophosphate (Pi, inhibitor). Previous studies on the cyanobacterial and plant enzymes showed that 3PGA binds to two highly conserved Lys residues located in the C-terminal domain. We observed that both Lys residues are present in the small (α) subunit of the Ostreococcus tauri enzyme; however, one of these Lys residues is replaced by Arg in the large (β) subunit. In this work, we obtained the K443R and R466K mutants of the O. tauri small and large subunits, respectively, and co-expressed them together or with their corresponding wild type counterparts. Our results show that restoring the Lys residue in the large subunit enhanced 3PGA affinity, whereas introduction of an Arg residue in the small subunit reduced 3PGA affinity of the heterotetramers. Inhibition kinetics also showed that heterotetramers containing the K443R small subunit mutant were less sensitive to Pi inhibition, but only minor changes were observed for those containing the R466K large subunit mutant, suggesting a leading role of the small subunit for Pi inhibition of the heterotetramer. We conclude that, during evolution, the ADP-Glc PPase large subunit from green algae and plants acquired mutations in its regulatory site. The rationale for this could have been to accommodate sensitivity to particular metabolic needs of the cell or tissue.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step for the synthesis of glycogen in cyanobacteria and starch in green algae and plants. The enzyme from cyanobacteria is homotetrameric (α4), while that from green algae and plants is heterotetrameric (α2β2). These ADP-Glc PPases are allosterically regulated by 3-phosphoglycerate (3PGA, activator) and inorganic orthophosphate (Pi, inhibitor). Previous studies on the cyanobacterial and plant enzymes showed that 3PGA binds to two highly conserved Lys residues located in the C-terminal domain. We observed that both Lys residues are present in the small (α) subunit of the Ostreococcus tauri enzyme; however, one of these Lys residues is replaced by Arg in the large (β) subunit. In this work, we obtained the K443R and R466K mutants of the O. tauri small and large subunits, respectively, and co-expressed them together or with their corresponding wild type counterparts. Our results show that restoring the Lys residue in the large subunit enhanced 3PGA affinity, whereas introduction of an Arg residue in the small subunit reduced 3PGA affinity of the heterotetramers. Inhibition kinetics also showed that heterotetramers containing the K443R small subunit mutant were less sensitive to Pi inhibition, but only minor changes were observed for those containing the R466K large subunit mutant, suggesting a leading role of the small subunit for Pi inhibition of the heterotetramer. We conclude that, during evolution, the ADP-Glc PPase large subunit from green algae and plants acquired mutations in its regulatory site. The rationale for this could have been to accommodate sensitivity to particular metabolic needs of the cell or tissue. [ABSTRACT FROM AUTHOR]

Ostreococcus tauri is the smallest free-living unicellular organism with one copy of each core cell cycle genes in its genome. There is a growing interest in this green algae due to its evolutionary origin. Since O. tauri is diverged early in the green lineage, relatively close to the ancestral eukaryotic cell, it might hold a key phylogenetic position in the eukaryotic tree of life. In this study, we focus on the regulatory network of its cell division cycle. We propose a mathematical modelling framework to integrate the existing knowledge of cell cycle network of O. tauri. We observe that feedback loop regulation of both G1/S and G2/M transitions in O. tauri is conserved, which can make the transition bistable. This is essential to make the transition irreversible as shown in other eukaryotic organisms. By performing sequence analysis, we also predict the presence of the Greatwall/PP2A pathway in the cell cycle of O. tauri. Since O. tauri cell cycle machinery is conserved, the exploration of the dynamical characteristic of the cell division cycle will help in further understanding the regulation of cell cycle in higher eukaryotes. [ABSTRACT FROM AUTHOR]

The efficiency with which absorbed light is converted to net growth is a key property for estimating global carbon production. We previously showed that, despite considerable evolutionary distance, Dunaliella tertiolecta (Chlorophyceae) and Thalassiosira weissflogii (Bacillariophyceae) share a common strategy of photosynthetic energy utilization and nearly identical light energy conversion efficiencies. These findings suggested that a single model might be appropriate for describing relationships between measures of phytoplankton production. This conclusion was further evaluated for Ostreococcus tauri RCC1558 and Micromonas pusilla RCC299 (Chlorophyta, Prasinophyceae), two picoeukaryotes with contrasting geographic distributions and swimming abilities. Nutrient-dependent photosynthetic efficiencies in O. tauri were similar to the previously studied larger algae. Specifically, absorption-normalized gross oxygen and carbon production and net carbon production were independent of nutrient limited growth rate. In contrast, all measures of photosynthetic efficiency were strongly dependent on nutrient availability in M. pusilla. This marked difference was accompanied by a diminished relationship between Chla:C and nutrient limited growth rate and a remarkably greater efficiency of gross-to-net energy conversion than the other organisms studied. These results suggest that the cost-benefit of decoupling pigment concentration from nutrient availability enables motile organisms to rapidly exploit more frequent encounters with micro-scale nutrient patches in open ocean environments.

Circadian clocks in eukaryotes involve both transcriptional-translational feedback loops, post-translational regulation, and metabolic, non-transcriptional oscillations. We recently identified the involvement of circadian oscillations in the intracellular concentrations of magnesium ions ([Mg2+]i) that were conserved in three eukaryotic kingdoms. [Mg2+]i in turn contributes to transcriptional clock properties of period and amplitude, and can function as a zeitgeber to define phase. However, the mechanism—or mechanisms—responsible for the generation of [Mg2+]i oscillations, and whether these are functionally conserved across taxonomic groups, remain elusive. We employed the cellular clock model Ostreococcus tauri to provide a first study of an MgtE domain-containing protein in the green lineage. OtMgtE shares homology with the mammalian SLC41A1 magnesium/sodium antiporter, which has previously been implicated in maintaining clock period. Using genetic overexpression, we found that OtMgtE contributes to both timekeeping and daily changes in [Mg2+]i. However, pharmacological experiments and protein sequence analyses indicated that critical differences exist between OtMgtE and either the ancestral MgtE channel or the mammalian SLC41 antiporters. We concluded that even though MgtE domain-containing proteins are only distantly related, these proteins retain a shared role in contributing to cellular timekeeping and the regulation of [Mg2+]i.

Green algae have a highly complex and diverse set of cryptochrome photoreceptor candidates including members of the following subfamilies: plant, plant-like, animal-like, DASH and cryptochrome photolyase family 1 (CPF1). While some green algae encode most or all of them, others lack certain members. Here we present an overview about functional analyses of so far investigated cryptochrome photoreceptors from the green algae Chlamydomonas reinhardtii (plant and animal-like cryptochromes) and Ostreococcus tauri (CPF1) with regard to their biological significance and spectroscopic properties. Cryptochromes of both algae have been demonstrated recently to be involved to various extents in circadian clock regulation and in Chlamydomonas additionally in life cycle control. Moreover, CPF1 even performs light-driven DNA repair. The plant cryptochrome and CPF1 are UVA/blue light receptors, whereas the animal-like cryptochrome responds to almost the whole visible spectrum including red light. Accordingly, plant cryptochrome, animal-like cryptochrome and CPF1 differ fundamentally in their structural response to light as revealed by their visible and infrared spectroscopic signatures, and in the role of the flavin neutral radical acting as dark form or signaling state. [ABSTRACT FROM AUTHOR]

Calcium-dependent protein kinases (CDPKs) regulate plant development and many stress signalling pathways through the complex cytosolic [Ca 2+ ] signalling. The genome of Ostreococcus tauri ( Ot ), a model prasinophyte organism that is on the base of the green lineage, harbours three sequences homologous to those encoding plant CDPKs with the three characteristic conserved domains (protein kinase, autoregulatory/autoinhibitory, and regulatory domain). Phylogenetic and structural analyses revealed that putative Ot CDPK proteins are closely related to CDPKs from other Chlorophytes. We functionally characterised the first marine picophytoeukaryote CDPK gene ( OtCDPK 1) and showed that the expression of the three OtCDPK genes is up-regulated by nitrogen depletion. We conclude that CDPK signalling pathway might have originated early in the green lineage and may play a key role in prasinophytes by sensing macronutrient changes in the marine environment. [ABSTRACT FROM AUTHOR]

Viruses are important evolutionary drivers of host ecology and evolution. The marine picoplankton Ostreococcus tauri has three known resistance types that arise in response to infection with the Phycodnavirus OtV5: susceptible cells (S) that lyse following viral entry and replication; resistant cells (R) that are refractory to viral entry; and resistant producers (RP) that do not all lyse but maintain some viruses within the population. To test for evolutionary costs of maintaining antiviral resistance, we examined whether O. tauri populations composed of each resistance type differed in their evolutionary responses to several environmental drivers (lower light, lower salt, lower phosphate and a changing environment) in the absence of viruses for approximately 200 generations. We did not detect a cost of resistance as measured by life-history traits (population growth rate, cell size and cell chlorophyll content) and competitive ability. Specifically, all R and RP populations remained resistant to OtV5 lysis for the entire 200-generation experiment, whereas lysis occurred in all S populations, suggesting that resistance is not costly to maintain even when direct selection for resistance was removed, or that there could be a genetic constraint preventing return to a susceptible resistance type. Following evolution, all S population densities dropped when inoculated with OtV5, but not to zero, indicating that lysis was incomplete, and that some cells may have gained a resistance mutation over the evolution experiment. These findings suggest that maintaining resistance in the absence of viruses was not costly. [ABSTRACT FROM AUTHOR]

Light, oxygen and voltage (LOV) proteins detect blue light by formation of a covalent 'photoadduct' between the flavin chromophore and the neighboring conserved cysteine residue. LOV proteins devoid of this conserved photoactive cysteine are unable to form this 'photoadduct' upon light illumination, but they can still elicit functional response via the formation of neutral flavin radical. Recently, tryptophan residue has been shown to be the primary electron donors to the flavin excited state. Photoactive cysteine (Cys42) and tryptophan (Trp68) residues in the LOV1 domain of phototropin1 of Ostreococcus tauri (OtLOV1) was mutated to alanine and threonine respectively. Effect of these mutations have been studied using molecular dynamics simulation and spectroscopic techniques. Molecular dynamics simulation indicated that W68T did not affect the structure of OtLOV1 protein, but C42A leads to some structural changes. An increase in the fluorescence lifetime and quantum yield values was observed for the Trp68 mutant. An increase in the fluorescence lifetime and quantum yield of Trp68 mutant compared to the wild type protein suggests that Trp68 residue participates in quenching of the flavin excited state followed by photoexcitation. Enhanced photo-physical properties of Trp68 OtLOV1 mutant might enable its use for the optogenetic and microscopic applications. • Ostreococcus tauri LOV1 domain of phototropin1 is a Flavin binding fluorescent protein. • Simulation study suggest that C42A mutation leads to slight structural changes. • Mutation of Trp68 to Threonine accelerated the dark state recovery. • A mutant of Cys42 to Alanine and Trp68 to Threonine resulted in an increased fluorescence lifetime of the LOV1 domain. [ABSTRACT FROM AUTHOR]

Microalgal-bacterial interactions are commonly found in marine environments and are well known in diatom cultures maintained in laboratory. These interactions also exert strong effects on bacterial and algal diversity in the oceans. Small green eukaryote algae of the class Mamiellophyceae (Chlorophyta) are ubiquitous and some species, such as Ostreococcus spp., are particularly important in Mediterranean coastal lagoons, and are observed as dominant species during phytoplankton blooms in open sea. Despite this, little is known about the diversity of bacteria that might facilitate or hinder O. tauri growth. We show, using rDNA 16S sequences, that the bacterial community found in O. tauri RCC4221 laboratory cultures is dominated by γ-proteobacteria from the Marinobacter genus, regardless of the growth phase of O. tauri RCC4221, the photoperiod used, or the nutrient conditions (limited in nitrogen or phosphorous) tested. Several strains of M. algicola were detected, all closely related to strains found in association with taxonomically distinct organisms, particularly with dinoflagellates and coccolithophorids. These sequences were more distantly related to M. adhaerens, M. aquaeoli and bacteria usually associated to euglenoids. This is the first time, to our knowledge, that distinct Marinobacter strains have been found to be associated with a green alga in culture.

Prasinoviruses are large dsDNA viruses commonly found in aquatic systems worldwide, where they can infect and lyse unicellular prasinophyte algae such as Ostreococcus. Host susceptibility is virus strain-specific, but resistance of susceptible Ostreococcus tauri strains to a virulent virus arises frequently. In clonal resistant lines that re-grow, viruses are usually present for many generations, and genes clustered on chromosome 19 show physical rearrangements and differential expression. Here, we investigated changes occurring during the first two weeks after inoculation of the prasinovirus OtV5. By serial dilutions of cultures at the time of inoculation, we estimated the frequency of resistant cells arising in virus-challenged O. tauri cultures to be 10−3–10−4 of the inoculated population. Re-growing resistant cells were detectable by flow cytometry 3 days post-inoculation (dpi), visible re-greening of cultures occurred by 6 dpi, and karyotypic changes were visually detectable at 8 dpi. Resistant cell lines showed a modified spectrum of host-virus specificities and much lower levels of OtV5 adsorption.

As a ubiquitous picophytoplankton in the ocean and an early-branching green alga, Ostreococcus tauri is a model prasinophyte species for studying the functional evolution of the light-harvesting systems in photosynthesis. Here, we report the structure and function of the O. tauri photosystem I (PSI) supercomplex in low light conditions, where it expands its photon-absorbing capacity by assembling with the light-harvesting complexes I (LHCI) and a prasinophyte-specific light-harvesting complex (Lhcp). The architecture of the supercomplex exhibits hybrid features of the plant-type and the green algal-type PSI supercomplexes, consisting of a PSI core, an Lhca1-Lhca4-Lhca2-Lhca3 belt attached on one side and an Lhca5-Lhca6 heterodimer associated on the other side between PsaG and PsaH. Interestingly, nine Lhcp subunits, including one Lhcp1 monomer with a phosphorylated amino-terminal threonine and eight Lhcp2 monomers, oligomerize into three trimers and associate with PSI on the third side between Lhca6 and PsaK. The Lhcp1 phosphorylation and the light-harvesting capacity of PSI were subjected to reversible photoacclimation, suggesting that the formation of Ot PSI-LHCI-Lhcp supercomplex is likely due to a phosphorylation-dependent mechanism induced by changes in light intensity. Notably, this supercomplex did not exhibit far-red peaks in the 77 K fluorescence spectra, which is possibly due to the weak coupling of the chlorophyll a 603- a 609 pair in Ot Lhca1-4., Competing Interests: AI, JS, XS, EK, AW, MY, CN, CS, KM, ZL, JM No competing interests declared, (© 2023, Ishii, Shan et al.)

Most organisms anticipate daily environmental variations and orchestrate cellular functions thanks to a circadian clock which entrains robustly to the day/night cycle, despite fluctuations in light intensity due to weather or seasonal variations. Marine organisms are also subjected to fluctuations in light spectral composition as their depth varies, due to differential absorption of different wavelengths by sea water. Studying how light input pathways contribute to circadian clock robustness is therefore important.Ostreococcus tauri, a unicellular picoplanktonic marine green alga with low genomic complexity and simple cellular organization, has become a promising model organism for systems biology. Functional and modeling approaches have shown that a core circadian oscillator based on orthologs of Arabidopsis TOC1 and CCA1 clock genes accounts for most experimental data acquired under a wide range of conditions. Some evidence points at putative light input pathway(s) consisting of a two-component signaling system (TCS) controlled by the only two histidine kinases (HK) of O. tauri. LOVHK is a blue light photoreceptor under circadian control, that is required for circadian clock function. An involvement of Rhodopsin-HK (RhodHK) is also conceivable since rhodopsin photoreceptors mediate blue to green light input in animal circadian clocks.Here, we probe the role of LOVHK and RhodHK in mediating light input to the TOC1-CCA1 oscillator using a mathematical model incorporating the TCS hypothesis. This model agrees with clock gene expression time series representative of multiple environmental conditions in blue or green light, characterizing entrainment by light/dark cycles, free-running in constant light, and resetting. Experimental and theoretical results indicate that both blue and green light can reset O. tauri circadian clock. Moreover, our mathematical analysis suggests that Rhod-HK is a blue-green light receptor and drives the clock together with LOV-HK.

Microalgal–bacterial interactions are commonly found in marine environments and are well known in diatom cultures maintained in laboratory. These interactions also exert strong effects on bacterial and algal diversity in the oceans. Small green eukaryote algae of the class Mamiellophyceae (Chlorophyta) are ubiquitous and some species, such as Ostreococcus spp., are particularly important in Mediterranean coastal lagoons, and are observed as dominant species during phytoplankton blooms in open sea. Despite this, little is known about the diversity of bacteria that might facilitate or hinder O. tauri growth. We show, using rDNA 16S sequences, that the bacterial community found in O. tauri RCC4221 laboratory cultures is dominated by g-proteobacteria from the Marinobacter genus, regardless of the growth phase of O. tauri RCC4221, the photoperiod used, or the nutrient conditions (limited in nitrogen or phosphorous) tested. Several strains of Marinobacter algicola were detected, all closely related to strains found in association with taxonomically distinct organisms, particularly with dinoflagellates and coccolithophorids. These sequences were more distantly related to M. adhaerens, M. aquaeoli and bacteria usually associated to euglenoids. This is the first time, to our knowledge, that distinct Marinobacter strains have been found to be associated with a green alga in culture. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In algae, the biosynthesis of docosahexaenoic acid (22:6ω3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5ω3; EPA) to 22:5ω3, which is required as a substrate for the final Δ4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana, from the fish Oncorhynchus mykiss, from the frog Xenopus laevis, and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens. The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana: one specific for the elongation of (Δ6-)C18-PUFAs and one specific for (Δ5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss, X. laevis, and C. intestinalis accepted both C18- and C20-PUFAs. By coexpression of the Δ6- and Δ5-elongases from T. pseudonana and O. tauri, respectively, with the Δ5- and Δ4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast.This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops.

Viruses are important evolutionary drivers of host ecology and evolution. The marine picoplankton Ostreococcus tauri has three known resistance types that arise in response to infection with the Phycodnavirus OtV5: susceptible cells (S) that lyse following viral entry and replication; resistant cells (R) that are refractory to viral entry; and resistant producers (RP) that do not all lyse but maintain some viruses within the population. To test for evolutionary costs of maintaining antiviral resistance, we examined whether O. tauri populations composed of each resistance type differed in their evolutionary responses to several environmental drivers (lower light, lower salt, lower phosphate and a changing environment) in the absence of viruses for approximately 200 generations. We did not detect a cost of resistance as measured by life-history traits (population growth rate, cell size and cell chlorophyll content) and competitive ability. Specifically, all R and RP populations remained resistant to OtV5 lysis for the entire 200-generation experiment, whereas lysis occurred in all S populations, suggesting that resistance is not costly to maintain even when direct selection for resistance was removed, or that there could be a genetic constraint preventing return to a susceptible resistance type. Following evolution, all S population densities dropped when inoculated with OtV5, but not to zero, indicating that lysis was incomplete, and that some cells may have gained a resistance mutation over the evolution experiment. These findings suggest that maintaining resistance in the absence of viruses was not costly.

Rhythmic behavior is essential for plants; for example, daily (circadian) rhythms control photosynthesis and seasonal rhythms regulate their life cycle. The core of the circadian clock is a genetic network that coordinates the expression of specific clock genes in a circadian rhythm reflecting the 24-hour day/night cycle.Circadian clocks exhibit stochastic noise due to the low copy numbers of clock genes and the consequent cell-to-cell variation: this intrinsic noise plays a major role in circadian clocks by inducing more robust oscillatory behavior. Another source of noise is the environment, which causes variation in temperature and light intensity: this extrinsic noise is part of the requirement for the structural complexity of clock networks.Advances in experimental techniques now permit single-cell measurements and the development of single-cell models. Here we present some modeling studies showing the importance of considering both types of noise in understanding how plants adapt to regular and irregular light variations. Stochastic models have proven useful for understanding the effect of regular variations. By contrast, the impact of irregular variations and the interaction of different noise sources are less studied.

Background: Starch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C. Results: In this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIIIA, SSIII-B and SSIII-C contain two, three and no starch-binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII-B, presenting three N-terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3-D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch-binding domains. In addition, adsorption assays showed that OsttaSSIII-A D2 and SSIII-B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII-B D1 is also important for starch binding. Conclusions: The results presented here suggest that differences between OsttaSSIII-A and SSIII-B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications. [ABSTRACT FROM AUTHOR]

Most organisms anticipate daily environmental variations and orchestrate cellular functions thanks to a circadian clock which entrains robustly to the day/night cycle, despite fluctuations in light intensity due to weather or seasonal variations. Marine organisms are also subjected to fluctuations in light spectral composition as their depth varies, due to differential absorption of different wavelengths by sea water. Studying how light input pathways contribute to circadian clock robustness is therefore important. Ostreococcus tauri, a unicellular picoplanktonic marine green alga with low genomic complexity and simple cellular organization, has become a promising model organism for systems biology. Functional and modeling approaches have shown that a core circadian oscillator based on orthologs of Arabidopsis TOC1 and CCA1 clock genes accounts for most experimental data acquired under a wide range of conditions. Some evidence points at putative light input pathway(s) consisting of a two-component signaling system (TCS) controlled by the only two histidine kinases (HK) of O. tauri. LOV-HK is a blue light photoreceptor under circadian control, that is required for circadian clock function. An involvement of Rhodopsin-HK (Rhod-HK) is also conceivable since rhodopsin photoreceptors mediate blue to green light input in animal circadian clocks. Here, we probe the role of LOV-HK and Rhod-HK in mediating light input to the TOC1-CCA1 oscillator using a mathematical model incorporating the TCS hypothesis. This model agrees with clock gene expression time series representative of multiple environmental conditions in blue or green light, characterizing entrainment by light/dark cycles, free-running in constant light, and resetting. Experimental and theoretical results indicate that both blue and green light can reset O. tauri circadian clock. Moreover, our mathematical analysis suggests that Rhod-HK is a blue-green light receptor and drives the clock together with LOV-HK. [ABSTRACT FROM AUTHOR]

Background: Cost effective next generation sequencing technologies now enable the production of genomic datasets for many novel planktonic eukaryotes, representing an understudied reservoir of genetic diversity. O. tauri is the smallest free-living photosynthetic eukaryote known to date, a coccoid green alga that was first isolated in 1995 in a lagoon by the Mediterranean sea. Its simple features, ease of culture and the sequencing of its 13 Mb haploid nuclear genome have promoted this microalga as a new model organism for cell biology. Here, we investigated the quality of genome assemblies of Illumina GAIIx 75 bp paired-end reads from Ostreococcus tauri, thereby also improving the existing assembly and showing the genome to be stably maintained in culture. Results: The 3 assemblers used, ABySS, CLCBio and Velvet, produced 95% complete genomes in 1402 to 2080 scaffolds with a very low rate of misassembly. Reciprocally, these assemblies improved the original genome assembly by filling in 930 gaps. Combined with additional analysis of raw reads and PCR sequencing effort, 1194 gaps have been solved in total adding up to 460 kb of sequence. Mapping of RNAseq Illumina data on this updated genome led to a twofold reduction in the proportion of multi-exon protein coding genes, representing 19% of the total 7699 protein coding genes. The comparison of the DNA extracted in 2001 and 2009 revealed the fixation of 8 single nucleotide substitutions and 2 deletions during the approximately 6000 generations in the lab. The deletions either knocked out or truncated two predicted transmembrane proteins, including a glutamate-receptor like gene. Conclusion: High coverage (>80 fold) paired-end Illumina sequencing enables a high quality 95% complete genome assembly of a compact ~13 Mb haploid eukaryote. This genome sequence has remained stable for 6000 generations of lab culture. [ABSTRACT FROM AUTHOR]