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Phase separation underpins many biologically important cellular events such as RNA metabolism, signaling and CO2 fixation. However, determining the composition of a phase-separated organelle is often challenging due to its sensitivity to environmental conditions, which limits the application of traditional proteomics techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase-separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID-based proximity labeling technique in which proximal proteins in Chlamydomonas chloroplasts are labeled by biotin radicals generated from the TurboID-tagged protein. By fusing two core pyrenoid components with the TurboID tag, we generated a high-confidence pyrenoid proxiome that contains most known pyrenoid proteins, in addition to new pyrenoid candidates. Fluorescence protein tagging of seven previously uncharacterized TurboID-identified proteins showed that six localized to a range of sub-pyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox-sensitive iron-sulfur cluster metabolism. This developed pipeline can be used to investigate a broad range of biological processes in Chlamydomonas, especially at a temporally resolved sub-organellar resolution.

Exploration of life in extreme environments allows the discovery of intriguing organisms with extraordinary biotechnological potential. An example of extreme environments is represented by hot springs, where harsh conditions (pH < 1; Temperature > 50°C; high concentrations of metals) are prohibitive for most living organisms, except for archaea, bacteria and a few eukaryotes like the unicellular red alga Galdieria. Phylogenetic analysis based on a few plastid and nuclear genes highlighted the intricate genetic structure of Galdieria and the hypothesis of diverging clades within the G. sulphuraria species. To resolve enigmatic relationships between lineages, we used plastid, mitochondrial and nuclear genome-scale data obtained from numerous strains from around the world. The resulting phylogenomic analysis identified: i) the divergence of each of the mitochondrial, plastid, and nuclear genomes into the same six clear lineages; ii) the independent evolution of the lineages; iii) the incongruent interlineages relationships between the three genomes. Differential evolutionary pressure between the strains and the genomes were also highlighted by synonymous and non-synonymous substitutions.

The regulated abundance and spatial distribution of proteins determines cellular structure and function. The discovery of green fluorescent protein (GFP) and fusing it to a target protein to determine subcellular localization revolutionized cell biology. Most localization studies involve introducing additional copies of a target gene genetically fused to GFP and under the control of a constitutive promoter, resulting in the expression of the GFP-fusion protein at non-native levels. Here we have developed a single vector CRISPR/Cas9 guided GFP knock-in strategy in the diatomThalassiosira pseudonana. This enables precise and scarless knock-in of GFP at the endogenous genomic location to create GFP fusion proteins under their nativecisandtransregulatory elements with knock-in efficiencies of over 50%. We show that a previously uncharacterized bestrophin-like protein localizes to the CO2-fixing pyrenoid and demonstrate that by measuring GFP fluorescence we can track relative protein abundance in response to environmental change. To enable endogenous tagging, we developed a Golden Gate Molecular Cloning system for the rapid assembly of episomes for transformation intoThalassiosira pseudonanavia bacterial conjugation. In addition, this versatile toolbox enables CRISPR/Cas9 gene editing, provides a broad range of validated fluorophores and enables future large-scale functional studies in diatoms.Significance statementFluorescent protein (FP) tagging is a widely utilized technique for understanding the spatial distribution of proteins. However, introducing extra gene copies under constitutive promoters that randomly integrate into the genome can result in non-biologically relevant expression levels, unwanted genomic mutations and localization artefacts. To overcome this, we developed a novel single vector system capable of CRISPR/Cas9-guided endogenous GFP tagging in a globally important model diatom. This allows scarless GFP knock-in at precise genomic locations resulting in GFP fusions regulated by native promoters/terminators, which facilitates accurate localization and determination of relative protein abundance. Moreover, the developed modular cloning framework is user-friendly and opens the door for high throughput large-scale studies, including FP tagging, knock-out, and knock-in.

Extremophiles, while typically bacteria and archaea, are also found in the eukaryotic domain of life. The eukaryote Galdieria sulphuraria is a thermoacidophilic red alga belonging to the class Cyanidiophyceae, an especially unique class as it comprises the basal clade of eukaryotic extremophiles. Galdieria species can grow both photosynthetically and heterotrophically on a variety of carbon sources, thriving down to pH 0 and temperatures up to 56 °C, while tolerating high levels of reactive oxygen species and high levels of heavy metals. Here we report whole-genome sequencing of three G. sulphuraria strains, uncovering a compact (13.1 Mb – 16.0 Mb) nuclear genome with 72-73 chromosomes, dependent on the strain.Comparative analyses of the macro synteny revealed significant structural rearrangement between G. sulphuraria isolates and the genome shows signs of sexual recombination. This, along with the large number of nuclear chromosomes compared to the genome size, reveals a mechanism of intrinsic adaptability in this eukaryotic extremophile, uncovering how G. sulphuraria can thrive in a rapidly changing extreme environment.

The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3− from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3− to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3− transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO2 conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO2, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1–3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3− accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.

Significance Chloroplasts are of vital importance in photosynthetic eukaryotic organisms. Like mitochondria, they contain their own genomes. Nevertheless, most chloroplast proteins are encoded by nuclear genes, translated in the cytosol, and must cross the chloroplast envelope membranes to reach their proper destinations inside the organelle. Despite its fundamental role, our knowledge of the machinery catalyzing chloroplast protein import is incomplete and controversial. Here, we address the evolutionary conservation, composition, and function of the chloroplast protein import machinery using the green alga Chlamydomonas reinhardtii as a model system. Our findings help clarify the current debate regarding the composition of the chloroplast protein import machinery, provide evidence for cross-compartmental coordination of its biogenesis, and open promising avenues for its structural characterization., In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons—termed TOC and TIC—located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.

Approximately one-third of the Earth's photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid and how the organelle's subcompartments-membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath-are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we found that pyrenoid proteins share a sequence motif. We show that the motif is necessary and sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix-starch sheath interface suggests that the motif holds the pyrenoid's three subcompartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multilayered phase-separated organelle.

Pyrenoids are non-membrane bound organelles found in chloroplasts of algae and hornwort plants that can be seen by light-microscopy. Pyrenoids are formed by liquid-liquid phase separation (LLPS) of Rubisco, the primary CO

Approximately one-third of the Earth’s photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid, and how the organelle’s sub-compartments - membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath - are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we discovered that pyrenoid proteins share a sequence motif. We show that the motif is sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix-starch sheath interface suggests that the motif holds the pyrenoid’s three sub-compartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multi-layered phase-separated organelle.One Sentence SummaryA ubiquitous Rubisco-binding motif targets proteins to the pyrenoid and holds together the pyrenoid’s three sub-compartments.

A protein motif mediates targeting of proteins to the pyrenoid and appears to link the pyrenoid’s three subcompartments., Approximately one-third of the Earth’s photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid and how the organelle’s subcompartments—membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath—are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we found that pyrenoid proteins share a sequence motif. We show that the motif is necessary and sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix–starch sheath interface suggests that the motif holds the pyrenoid’s three subcompartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multilayered phase-separated organelle.

The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research., A high-throughput system was developed to clone large, complex genes at high frequency and perform mutant complementation and protein tagging with a range of fluorophores in Chlamydomonas reinhardtii.

Significance Models of the CO2 concentrating mechanism (CCM) of green algae and diatoms postulate that chloroplast CO2 is generated from HCO3− brought into the acidic thylakoid lumen and converted to CO2 by specific thylakoid carbonic anhydrases. However, the identity of the transporter required for thylakoid HCO3− uptake has remained elusive. In this work, 3 bestrophin-like proteins, BST1–3, located on the thylakoid membrane have been found to be essential to the CCM of Chlamydomonas. Reduction in expression of BST1–3 markedly reduced the inorganic carbon affinity of the alga. These proteins are prime candidates to be thylakoid HCO3− transporters, a critical currently missing step of the CCM required for future engineering efforts of the Chlamydomonas CCM into plants to improve photosynthesis., The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3− from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3− to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3− transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO2 conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO2, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1–3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3− accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.

Summary Many eukaryotic green algae possess biophysical carbon‐concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO 2 concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H14 CO 3 ‐ uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild‐type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species.

Pyrenoids are non-membrane bound organelles found in chloroplasts of algae and hornwort plants that can be seen by light-microscopy. Pyrenoids are formed by liquid-liquid phase separation (LLPS) of Rubisco, the primary CO2 fixing enzyme, with an intrinsically disordered multivalent Rubisco-binding protein. Pyrenoids are the heart of algal and hornwort biophysical CO2 concentrating mechanisms, which accelerate photosynthesis and mediate about 30% of global carbon fixation. Even though LLPS may underlie the apparent convergent evolution of pyrenoids, our current molecular understanding of pyrenoid formation comes from a single example, the model alga Chlamydomonas reinhardtii. In this review, we summarise current knowledge about pyrenoid assembly, regulation and structural organization in Chlamydomonas and highlight evidence that LLPS is the general principle underlying pyrenoid formation across algal lineages and hornworts. Detailed understanding of the principles behind pyrenoid assembly, regulation and structural organization within diverse lineages will provide a fundamental understanding of this biogeochemically important organelle and help guide ongoing efforts to engineer pyrenoids into crops to increase photosynthetic performance and yields. 2

Cells and organelles are not homogeneous but include microcompartments that alter the spatiotemporal characteristics of cellular processes. The effects of microcompartmentation on metabolic pathways are however difficult to study experimentally. The pyrenoid is a microcompartment that is essential for a carbon concentrating mechanism (CCM) that improves the photosynthetic performance of eukaryotic algae. Using Chlamydomonas reinhardtii, we obtained experimental data on photosynthesis, metabolites, and proteins in CCM-induced and CCM-suppressed cells. We then employed a computational strategy to estimate how fluxes through the Calvin-Benson cycle are compartmented between the pyrenoid and the stroma. Our model predicts that ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco, and 3-phosphoglycerate (3PGA), its product, diffuse in and out of the pyrenoid, respectively, with higher fluxes in CCM-induced cells. It also indicates that there is no major diffusional barrier to metabolic flux between the pyrenoid and stroma. Our computational approach represents a stepping stone to understanding microcompartmentalized CCM in other organisms., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe; 1122

Approximately 30‐40% of global CO2 fixation occurs inside a poorly understood non‐membrane‐bound organelle called the pyrenoid, which is found in the chloroplasts of most eukaryotic algae. The pyrenoid matrix, which is densely packed with the CO2‐fixing enzyme Rubisco, is thought to be crystalline. Here, we show that the pyrenoid matrix of the unicellular green alga Chlamydomonas reinhardtii is not crystalline, but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that the pyrenoid is inherited both by fission and de novo assembly. A simple model inspired by this system predicts the existence of a "magic number" effect that influences phase separation in liquid‐like organelles. This view of the pyrenoid matrix as a phaseseparated compartment provides a new paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the fundamental principles that govern the architecture and inheritance of liquid‐like organelles.

Approximately one-third of global CO 2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO 2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates, and inorganic carbon flux components. We identify three previously undescribed protein layers of the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.

Approximately 30%–40% of global CO 2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO 2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a “magic number” effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.

Contents Summary I. Introduction 54 II. Recent advances in our understanding of the Chlamydomonas CCM 55 III. Current gaps in our understanding of the Chlamydomonas CCM 58 IV. Approaches to rapidly advance our understanding of the Chlamydomonas CCM 58 V. Engineering a CCM into higher plants 58 VI. Conclusion and outlook 59 Acknowledgements 60 References 60 SUMMARY: To meet the food demands of a rising global population, innovative strategies are required to increase crop yields. Improvements in plant photosynthesis by genetic engineering show considerable potential towards this goal. One prospective approach is to introduce a CO

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2 -fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 . Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 . We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.

International audience; Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

Biomineralization in the marine phytoplankton Emiliania huxleyi is a stringently controlled intracellular process. The molecular basis of coccolith production is still relatively unknown although its importance in global biogeochemical cycles and varying sensitivity to increased pCO2 levels has been well documented. This study looks into the role of several candidate Ca2+, H+ and inorganic carbon transport genes in E. huxleyi, using quantitative reverse transcriptase PCR. Differential gene expression analysis was investigated in two isogenic pairs of calcifying and non-calcifying strains of E. huxleyi and cultures grown at various Ca2+ concentrations to alter calcite production. We show that calcification correlated to the consistent upregulation of a putative HCO3- transporter belonging to the solute carrier 4 (SLC4) family, a Ca2+/H+ exchanger belonging to the CAX family of exchangers and a vacuolar H+-ATPase. We also show that the coccolith-associated protein, GPA is downregulated in calcifying cells. The data provide strong evidence that these genes play key roles in E. huxleyi biomineralization. Based on the gene expression data and the current literature a working model for biomineralization-related ion transport in coccolithophores is presented.

A number of studies are providing increasing genomic and transcriptomic information on the molecular components of transport, and biochemical control in the cell biology of calcification in coccolithophores. In this review we summarise recent evidence for molecular components involved in the trans-cellular transport of Ca2+, inorganic carbon and H+ between the external medium and the intracellular calcification compartment. We present new hypotheses for the transport of substrates to the site of calcification and for the removal of products, highlighting key gaps in our current knowledge. We also discuss how a cellular and molecular approach will improve abilities to understand and predict responses and adaptation to changing ocean chemistry of this important group of microorganisms.

The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses.

Emiliania huxleyi virus 86 (EhV-86) belongs to the family Phycodnaviridae, a group of viruses that infect a wide range of freshwater and marine eukaryotic algae. Phycodnaviridae is one of the five families that belong to a large and phylogenetically diverse group of viruses known as nucleocytoplasmic large dsDNA viruses (NCLDVs). To date, our understanding of algal NCLDV entry is based on the entry mechanisms of members of the genera Chlorovirus and Phaeovirus, both of which consist of non-enveloped viruses that 'inject' their genome into their host via a viral inner-membrane host plasma membrane fusion mechanism, leaving an extracellular viral capsid. Using a combination of confocal and electron microscopy, this study demonstrated for the first time that EhV-86 differs from its algal virus counterparts in two fundamental areas. Firstly, its capsid is enveloped by a lipid membrane, and secondly, EhV-86 enters its host via either an endocytotic or an envelope fusion mechanism in which an intact nucleoprotein core still encapsulated by its capsid is seen in the host cytoplasm. Real-time fluorescence microscopy showed that viral internalization and virion breakdown took place within the host on a timescale of seconds. At around 4.5 h post-infection, virus progeny were released via a budding mechanism during which EhV-86 virions became enveloped with host plasma membrane. EhV-86 therefore appears to have an infection mechanism different from that employed by other algal NCLDVs, with entry and exit strategies showing a greater analogy to animal-like NCLDVs.

Marine phytoplankton have developed the remarkable ability to tightly regulate the concentration of free calcium ions in the intracellular cytosol at a level of ~ 0.1 μmol L−1 in the presence of seawater Ca2+ concentrations of 10 mmol L−1. The low cytosolic calcium ion concentration is of utmost importance for proper cell signalling function. While the regulatory mechanisms responsible for the tight control of intracellular Ca2+ concentration are not completely understood, phytoplankton taxonomic groups appear to have evolved different strategies, which may affect their ability to cope with changes in seawater Ca2+ concentrations in their environment on geological timescales. For example, the Cretaceous (145 to 66 Ma), an era known for the high abundance of coccolithophores and the production of enormous calcium carbonate deposits, exhibited seawater calcium concentrations up to 4 times present-day levels. We show that calcifying coccolithophore species (Emiliania huxleyi, Gephyrocapsa oceanica and Coccolithus braarudii) are able to maintain their relative fitness (in terms of growth rate and photosynthesis) at simulated Cretaceous seawater calcium concentrations, whereas these rates are severely reduced under these conditions in some non-calcareous phytoplankton species (Chaetoceros sp., Ceratoneis closterium and Heterosigma akashiwo). Most notably, this also applies to a non-calcifying strain of E. huxleyi which displays a calcium sensitivity similar to the non-calcareous species. We hypothesize that the process of calcification in coccolithophores provides an efficient mechanism to alleviate cellular calcium poisoning and thereby offered a potential key evolutionary advantage, responsible for the proliferation of coccolithophores during times of high seawater calcium concentrations. The exact function of calcification and the reason behind the highly ornate physical structures of coccoliths remain elusive.

SummaryAssembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.

Coccolithophores are important calcifying phytoplankton predicted to be impacted by changes in ocean carbonate chemistry caused by the absorption of anthropogenic CO2. However, it is difficult to disentangle the effects of the simultaneously changing carbonate system parameters (CO2, bicarbonate, carbonate and protons) on the physiological responses to elevated CO2. Here, we adopted a multifactorial approach at constant pH or CO2 whilst varying dissolved inorganic carbon (DIC) to determine physiological and transcriptional responses to individual carbonate system parameters. We show that Emiliania huxleyi is sensitive to low CO2 (growth and photosynthesis) and low bicarbonate (calcification) as well as low pH beyond a limited tolerance range, but is much less sensitive to elevated CO2 and bicarbonate. Multiple up-regulated genes at low DIC bear the hallmarks of a carbon-concentrating mechanism (CCM) that is responsive to CO2 and bicarbonate but not to pH. Emiliania huxleyi appears to have evolved mechanisms to respond to limiting rather than elevated CO2. Calcification does not function as a CCM, but is inhibited at low DIC to allow the redistribution of DIC from calcification to photosynthesis. The presented data provides a significant step in understanding how E. huxleyi will respond to changing carbonate chemistry at a cellular level.