Evidence of a putative CO2 delivery system to the chromatophore in the photosynthetic amoeba Paulinella.

The photosynthetic amoeba, Paulinella provides a recent (ca. 120 Mya) example of primary plastid endosymbiosis. Given the extensive data demonstrating host lineage-driven endosymbiont integration, we analysed nuclear genome and transcriptome data to investigate mechanisms that may have evolved in Paulinella micropora KR01 (hereinafter, KR01) to maintain photosynthetic function in the novel organelle, the chromatophore. The chromatophore is of α-cyanobacterial provenance and has undergone massive gene loss due to Muller's ratchet, but still retains genes that encode the ancestral α-carboxysome and the shell carbonic anhydrase, two critical components of the biophysical CO2 concentrating mechanism (CCM) in cyanobacteria. We identified KR01 nuclear genes potentially involved in the CCM that arose via duplication and divergence and are upregulated in response to high light and downregulated under elevated CO2. We speculate that these genes may comprise a novel CO2 delivery system (i.e., a biochemical CCM) to promote the turnover of the RuBisCO carboxylation reaction and counteract photorespiration. We posit that KR01 has an inefficient photorespiratory system that cannot fully recycle the C2 product of RuBisCO oxygenation back to the Calvin-Benson cycle. Nonetheless, both these systems appear to be sufficient to allow Paulinella to persist in environments dominated by faster-growing phototrophs.

Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome.

BACKGROUND: In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties are originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools. RESULTS: In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays. CONCLUSIONS: Our results provide the first window into the 5-hmU and chromatin accessibility landscapes in dinoflagellates.

Alternative electron pathways of photosynthesis power green algal CO2 capture.

Microalgae contribute to about half of global net photosynthesis, which converts sunlight into the chemical energy (ATP and NADPH) used to transform CO2 into biomass. Alternative electron pathways of photosynthesis have been proposed to generate additional ATP that is required to sustain CO2 fixation. However, the relative importance of each alternative pathway remains elusive. Here, we dissect and quantify the contribution of cyclic, pseudo-cyclic and chloroplast-to-mitochondria electron flows for their ability to sustain net photosynthesis in the microalga Chlamydomonas reinhardtii. We show that (i) each alternative pathway can provide sufficient additional energy to sustain high CO2 fixation rates, (ii) the alternative pathways exhibit cross-compensation, and (iii) the activity of at least one of the three alternative pathways is necessary to sustain photosynthesis. We further show that all pathways have very different efficiencies at energizing CO2 fixation, with the chloroplast-mitochondria interaction being the most efficient. Overall, our data lay bioenergetic foundations for biotechnological strategies to improve CO2 capture and fixation.

Dramatic Changes in Mitochondrial Subcellular Location and Morphology Accompany Activation of the CO2 Concentrating Mechanism.

Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for ~50% of global photosynthesis, compartmentalize their Rubisco into a specialized structure known as the pyrenoid when the cells experience limiting CO2 conditions; this compartmentalization appears to be a component of the CO2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO2 limiting conditions, although a role for this reorganization in CCM function remains unclear. We used the green microalgae Chlamydomonas reinhardtii to monitor changes in the position and ultrastructure of mitochondrial membranes as cells transition between high CO2 (HC) and Low/Very Low CO2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral location, become wedged between the plasma membrane and chloroplast envelope, and mitochondrial membranes orient in parallel tubular arrays that extend from the cell's apex to its base. We show that these ultrastructural changes require protein and RNA synthesis, occur within 90 min of shifting cells to VLC conditions, correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membrane, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, which is involved in membrane-microtubule interactions. Furthermore, blocking mitochondrial electron transport in VLC acclimated cells reduces the cell's affinity for inorganic carbon. Overall, our results suggest that CIA5-dependent mitochondrial repositioning/reorientation functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

Protocol for mapping the three-dimensional organization of dinoflagellate genomes.

Dinoflagellate genomes often are very large and difficult to assemble, which has until recently precluded their analysis with modern functional genomic tools. Here, we present a protocol for mapping three-dimensional (3D) genome organization in dinoflagellates and using it for scaffolding their genome assemblies. We describe steps for crosslinking, nuclear lysis, denaturation, restriction digest, ligation, and DNA shearing and purification. We then detail procedures sequencing library generation and computational analysis, including initial Hi-C read mapping and 3D-DNA scaffolding/assembly correction. For complete details on the use and execution of this protocol, please refer to Marinov et al.1.

Photosynthesis and other factors affecting the establishment and maintenance of cnidarian-dinoflagellate symbiosis.

Coral growth depends on the partnership between the animal hosts and their intracellular, photosynthetic dinoflagellate symbionts. In this study, we used the sea anemone Aiptasia, a laboratory model for coral biology, to investigate the poorly understood mechanisms that mediate symbiosis establishment and maintenance. We found that initial colonization of both adult polyps and larvae by a compatible algal strain was more effective when the algae were able to photosynthesize and that the long-term maintenance of the symbiosis also depended on photosynthesis. In the dark, algal cells were taken up into host gastrodermal cells and not rapidly expelled, but they seemed unable to reproduce and thus were gradually lost. When we used confocal microscopy to examine the interaction of larvae with two algal strains that cannot establish stable symbioses with Aiptasia, it appeared that both pre- and post-phagocytosis mechanisms were involved. With one strain, algae entered the gastric cavity but appeared to be completely excluded from the gastrodermal cells. With the other strain, small numbers of algae entered the gastrodermal cells but appeared unable to proliferate there and were slowly lost upon further incubation. We also asked if the exclusion of either incompatible strain could result simply from their cells' being too large for the host cells to accommodate. However, the size distributions of the compatible and incompatible strains overlapped extensively. Moreover, examination of macerates confirmed earlier reports that individual gastrodermal cells could expand to accommodate multiple algal cells. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Draft genome of Chloroflexus sp. MS-CIW-1, of the Chloroflexus sp. MS-G group from Mushroom Spring, Yellowstone National Park.

Chloroflexus sp. MS-CIW-1 was isolated from a phototrophic mat in Mushroom Spring, an alkaline hot spring in Yellowstone National Park, WY, USA. We report the draft genome of 4.8 Mb consisting of 6 contigs with 3755 protein-coding genes and a GC content of 54.45%.

Chloroplast Methyltransferase Homolog RMT2 is Involved in Photosystem I Biogenesis.

Oxygen (O2), a dominant element in the atmosphere and essential for most life on Earth, is produced by the photosynthetic oxidation of water. However, metabolic activity can cause accumulation of reactive O2 species (ROS) and severe cell damage. To identify and characterize mechanisms enabling cells to cope with ROS, we performed a high-throughput O2 sensitivity screen on a genome-wide insertional mutant library of the unicellular alga Chlamydomonas reinhardtii. This screen led to identification of a gene encoding a protein designated Rubisco methyltransferase 2 (RMT2). Although homologous to methyltransferases, RMT2 has not been experimentally demonstrated to have methyltransferase activity. Furthermore, the rmt2 mutant was not compromised for Rubisco (first enzyme of Calvin-Benson Cycle) levels but did exhibit a marked decrease in accumulation/activity of photosystem I (PSI), which causes light sensitivity, with much less of an impact on other photosynthetic complexes. This mutant also shows increased accumulation of Ycf3 and Ycf4, proteins critical for PSI assembly. Rescue of the mutant phenotype with a wild-type (WT) copy of RMT2 fused to the mNeonGreen fluorophore indicates that the protein localizes to the chloroplast and appears to be enriched in/around the pyrenoid, an intrachloroplast compartment present in many algae that is packed with Rubisco and potentially hypoxic. These results indicate that RMT2 serves an important role in PSI biogenesis which, although still speculative, may be enriched around or within the pyrenoid.

Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome.

In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties were originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools. In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays.

Symbiont Identity Impacts the Microbiome and Volatilome of a Model Cnidarian-Dinoflagellate Symbiosis.

The symbiosis between cnidarians and dinoflagellates underpins the success of reef-building corals in otherwise nutrient-poor habitats. Alterations to symbiotic state can perturb metabolic homeostasis and thus alter the release of biogenic volatile organic compounds (BVOCs). While BVOCs can play important roles in metabolic regulation and signalling, how the symbiotic state affects BVOC output remains unexplored. We therefore characterised the suite of BVOCs that comprise the volatilome of the sea anemone Exaiptasia diaphana ('Aiptasia') when aposymbiotic and in symbiosis with either its native dinoflagellate symbiont Breviolum minutum or the non-native symbiont Durusdinium trenchii. In parallel, the bacterial community structure in these different symbiotic states was fully characterised to resolve the holobiont microbiome. Based on rRNA analyses, 147 unique amplicon sequence variants (ASVs) were observed across symbiotic states. Furthermore, the microbiomes were distinct across the different symbiotic states: bacteria in the family Vibrionaceae were the most abundant in aposymbiotic anemones; those in the family Crocinitomicaceae were the most abundant in anemones symbiotic with D. trenchii; and anemones symbiotic with B. minutum had the highest proportion of low-abundance ASVs. Across these different holobionts, 142 BVOCs were detected and classified into 17 groups based on their chemical structure, with BVOCs containing multiple functional groups being the most abundant. Isoprene was detected in higher abundance when anemones hosted their native symbiont, and dimethyl sulphide was detected in higher abundance in the volatilome of both Aiptasia-Symbiodiniaceae combinations relative to aposymbiotic anemones. The volatilomes of aposymbiotic anemones and anemones symbiotic with B. minutum were distinct, while the volatilome of anemones symbiotic with D. trenchii overlapped both of the others. Collectively, our results are consistent with previous reports that D. trenchii produces a metabolically sub-optimal symbiosis with Aiptasia, and add to our understanding of how symbiotic cnidarians, including corals, may respond to climate change should they acquire novel dinoflagellate partners.

Chlamydomonas: Fast tracking from genomics.

Elucidating biological processes has relied on the establishment of model organisms, many of which offer advantageous features such as rapid axenic growth, extensive knowledge of their physiological features and gene content, and the ease with which they can be genetically manipulated. The unicellular green alga Chlamydomonas reinhardtii has been an exemplary model that has enabled many scientific breakthroughs over the decades, especially in the fields of photosynthesis, cilia function and biogenesis, and the acclimation of photosynthetic organisms to their environment. Here, we discuss recent molecular/technological advances that have been applied to C. reinhardtii and how they have further fostered its development as a "flagship" algal system. We also explore the future promise of this alga in leveraging advances in the fields of genomics, proteomics, imaging, and synthetic biology for addressing critical future biological issues.

Light-independent regulation of algal photoprotection by CO2 availability.

Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO2 conditions. When light energy exceeds CO2 fixation capacity, Chlamydomonas reinhardtii activates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO2 Concentrating Mechanism (CCM). How light and CO2 signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO2 concentrations and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controlling LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO2 and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO2 levels.

Restricting electron flow at cytochrome b6f when downstream electron acceptors are severely limited.

Photosynthetic organisms frequently experience abiotic stress that restricts their growth and development. Under such circumstances, most absorbed solar energy cannot be used for CO2 fixation and can cause the photoproduction of reactive oxygen species (ROS) that can damage the photosynthetic reaction centers of PSI and PSII, resulting in a decline in primary productivity. This work describes a biological "switch" in the green alga Chlamydomonas reinhardtii that reversibly restricts photosynthetic electron transport (PET) at the cytochrome b6f (Cyt b6f) complex when the capacity for accepting electrons downstream of PSI is severely limited. We specifically show this restriction in STARCHLESS6 (sta6) mutant cells, which cannot synthesize starch when they are limited for nitrogen (growth inhibition) and subjected to a dark-to-light transition. This restriction represents a form of photosynthetic control that causes diminished electron flow to PSI and thereby prevents PSI photodamage but does not appear to rely on a ΔpH. Furthermore, when electron flow is restricted, the plastid alternative oxidase (PTOX) becomes active, functioning as an electron valve that dissipates some excitation energy absorbed by PSII and allows the formation of a proton motive force (PMF) that would drive some ATP production (potentially sustaining PSII repair and nonphotochemical quenching [NPQ]). The restriction at the Cyt b6f complex can be gradually relieved with continued illumination. This study provides insights into how PET responds to a marked reduction in availability of downstream electron acceptors and the protective mechanisms involved.

Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light sensitive.

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

The Influence of Symbiosis on the Proteome of the Exaiptasia Endosymbiont Breviolum minutum.

The cellular mechanisms responsible for the regulation of nutrient exchange, immune response, and symbiont population growth in the cnidarian-dinoflagellate symbiosis are poorly resolved. Here, we employed liquid chromatography-mass spectrometry to elucidate proteomic changes associated with symbiosis in Breviolum minutum, a native symbiont of the sea anemone Exaiptasia diaphana ('Aiptasia'). We manipulated nutrients available to the algae in culture and to the holobiont in hospite (i.e., in symbiosis) and then monitored the impacts of our treatments on host-endosymbiont interactions. Both the symbiotic and nutritional states had significant impacts on the B. minutum proteome. B. minutum in hospite showed an increased abundance of proteins involved in phosphoinositol metabolism (e.g., glycerophosphoinositol permease 1 and phosphatidylinositol phosphatase) relative to the free-living alga, potentially reflecting inter-partner signalling that promotes the stability of the symbiosis. Proteins potentially involved in concentrating and fixing inorganic carbon (e.g., carbonic anhydrase, V-type ATPase) and in the assimilation of nitrogen (e.g., glutamine synthase) were more abundant in free-living B. minutum than in hospite, possibly due to host-facilitated access to inorganic carbon and nitrogen limitation by the host when in hospite. Photosystem proteins increased in abundance at high nutrient levels irrespective of the symbiotic state, as did proteins involved in antioxidant defences (e.g., superoxide dismutase, glutathione s-transferase). Proteins involved in iron metabolism were also affected by the nutritional state, with an increased iron demand and uptake under low nutrient treatments. These results detail the changes in symbiont physiology in response to the host microenvironment and nutrient availability and indicate potential symbiont-driven mechanisms that regulate the cnidarian-dinoflagellate symbiosis.

Symbiosis induces unique volatile profiles in the model cnidarian Aiptasia.

The establishment and maintenance of the symbiosis between a cnidarian host and its dinoflagellate symbionts is central to the success of coral reefs. To explore the metabolite production underlying this symbiosis, we focused on a group of low molecular weight secondary metabolites, biogenic volatile organic compounds (BVOCs). BVOCs are released from an organism or environment, and can be collected in the gas phase, allowing non-invasive analysis of an organism's metabolism (i.e. 'volatilomics'). We characterised volatile profiles of the sea anemone Aiptasia (Exaiptasia diaphana), a model system for cnidarian-dinoflagellate symbiosis, using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. We compared volatile profiles between: (1) symbiotic anemones containing their native symbiont, Breviolum minutum; (2) aposymbiotic anemones; and (3) cultured isolates of B. minutum. Overall, 152 BVOCs were detected, and classified into 14 groups based on their chemical structure, the most numerous groups being alkanes and aromatic compounds. A total of 53 BVOCs were differentially abundant between aposymbiotic anemones and B. minutum cultures; 13 between aposymbiotic and symbiotic anemones; and 60 between symbiotic anemones and cultures of B. minutum. More BVOCs were differentially abundant between cultured and symbiotic dinoflagellates than between aposymbiotic and symbiotic anemones, suggesting that symbiosis may modify symbiont physiology more than host physiology. This is the first volatilome analysis of the Aiptasia model system and provides a foundation from which to explore how BVOC production is perturbed under environmental stress, and ultimately the role they play in this important symbiosis.

Intelligent image-activated sorting of Chlamydomonas reinhardtii by mitochondrial localization.

Organelle positioning in cells is associated with various metabolic functions and signaling in unicellular organisms. Specifically, the microalga Chlamydomonas reinhardtii repositions its mitochondria, depending on the levels of inorganic carbon. Mitochondria are typically randomly distributed in the Chlamydomonas cytoplasm, but relocate toward the cell periphery at low inorganic carbon levels. This mitochondrial relocation is linked with the carbon-concentrating mechanism, but its significance is not yet thoroughly understood. A genotypic understanding of this relocation would require a high-throughput method to isolate rare mutant cells not exhibiting this relocation. However, this task is technically challenging due to the complex intracellular morphological difference between mutant and wild-type cells, rendering conventional non-image-based high-event-rate methods unsuitable. Here, we report our demonstration of intelligent image-activated cell sorting by mitochondrial localization. Specifically, we applied an intelligent image-activated cell sorting system to sort for C. reinhardtii cells displaying no mitochondrial relocation. We trained a convolutional neural network (CNN) to distinguish the cell types based on the complex morphology of their mitochondria. The CNN was employed to perform image-activated sorting for the mutant cell type at 180 events per second, which is 1-2 orders of magnitude faster than automated microscopy with robotic pipetting, resulting in an enhancement of the concentration from 5% to 56.5% corresponding to an enrichment factor of 11.3. These results show the potential of image-activated cell sorting for connecting genotype-phenotype relations for rare-cell populations, which require a high throughput and could lead to a better understanding of metabolic functions in cells.