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The slow kinetics and poor substrate specificity of the key photosynthetic CO₂-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from Phaeodactylum tricornutum. PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid–liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo–electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1–Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.

A phase-separated, liquid-like organelle called the pyrenoid mediates CO₂ fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model alga Chlamydomonas that has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix. We find that the mutant’s phenotypes are due to disruption of a gene, which we call StArch Granules Abnormal 1 (SAGA1) because starch sheath granules, or plates, in mutants lacking SAGA1 are more elongated and thinner than those of wild type. SAGA1 contains a starch binding motif, suggesting that it may directly regulate starch sheath morphology. SAGA1 localizes to multiple puncta and streaks in the pyrenoid and physically interacts with the small and large subunits of the carbon-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a major component of the liquid-like pyrenoid matrix. Our findings suggest a biophysical mechanism by which starch sheath morphology affects pyrenoid number and CO₂-concentrating mechanism function, advancing our understanding of the structure and function of this biogeochemically important organelle. More broadly, we propose that the number of phase-separated organelles can be regulated by imposing constraints on their surface area.

Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency 1 . The α- and β-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)—a complex of eight large (RbcL) and eight small (RbcS) subunits—and carbonic anhydrase 2 – 4 . As HCO 3 − can diffuse through the proteinaceous carboxysome shell but CO 2 cannot 5 , carbonic anhydrase generates high concentrations of CO 2 for carbon fixation by Rubisco 6 . The shell also prevents access to reducing agents, generating an oxidizing environment 7 – 9 . The formation of β-carboxysomes involves the aggregation of Rubisco by the protein CcmM 10 , which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain 8 followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain 11 . It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS 2 – 4 . Here we have reconstituted the Rubisco–CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid–liquid phase separation in eukaryotes 12 , 13 . Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC1 14 . Understanding carboxysome biogenesis will be important for efforts to engineer CO 2 -concentrating mechanisms in plants 15 – 19 . The structure of a Rubisco–CcmM complex sheds light on the formation of carboxysomes in cyanobacteria.

Membraneless organelles containing the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are a common feature of organisms utilizing CO₂ concentrating mechanisms to enhance photosynthetic carbon acquisition. In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while some algae and hornworts have evolved Rubisco condensates known as pyrenoids. In both cases, CO₂ fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the improved function of carboxysomes to the generation of elevated CO₂ within the organelle via a colocalized carbonic anhydrase (CA) and inwardly diffusing HCO₃⁻, which have accumulated in the cytoplasm via dedicated transporters. Here, we present a concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction. We evaluate this in a reaction–diffusion compartment model to investigate functional advantages these protons may provide Rubisco condensates and carboxysomes, prior to the evolution of HCO₃⁻ accumulation. Our model highlights that diffusional resistance to reaction species within a condensate allows Rubisco-derived protons to drive the conversion of HCO₃⁻ to CO₂ via colocalized CA, enhancing both condensate [CO₂] and Rubisco rate. Protonation of Rubisco substrate (RuBP) and product (phosphoglycerate) plays an important role in modulating internal pH and CO₂ generation. Application of the model to putative evolutionary ancestors, prior to contemporary cellular HCO₃⁻ accumulation, revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO₂ and low light environments.

The rising concentration of global atmospheric carbon dioxide (CO2) has severely affected our planet’s homeostasis. Efforts are being made worldwide to curb carbon dioxide emissions, but there is still no strategy or technology available to date that is widely accepted. Two basic strategies are employed for reducing CO2 emissions, viz. (i) a decrease in fossil fuel use, and increased use of renewable energy sources; and (ii) carbon sequestration by various biological, chemical, or physical methods. This review has explored microalgae’s role in carbon sequestration, the physiological apparatus, with special emphasis on the carbon concentration mechanism (CCM). A CCM is a specialized mechanism of microalgae. In this process, a sub-cellular organelle known as pyrenoid, containing a high concentration of Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco), helps in the fixation of CO2. One type of carbon concentration mechanism in Chlamydomonas reinhardtii and the association of pyrenoid tubules with thylakoids membrane is represented through a typical graphical model. Various environmental factors influencing carbon sequestration in microalgae and associated techno-economic challenges are analyzed critically.

Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts ( Phaeocystis ) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition.

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.

Carbon-concentrating mechanisms (CCMs) are a widespread phenomenon in photosynthetic organisms. In vascular plants, the evolution of CCMs ([C44-carbon compound] and crassulacean acid metabolism [CAM]) is associated with significant shifts, most often to hot, dry and bright, or aquatic environments. If and how CCMs drive distributions of other terrestrial photosynthetic organisms, remains little studied. Lichens are ecologically important obligate symbioses between fungi and photosynthetic organisms. The primary photosynthetic partner in these symbioses can include CCM-presenting cyanobacteria (as carboxysomes), CCM-presenting green algae (as pyrenoids) or green algae lacking any CCM. We use an extensive dataset of lichen communities from eastern North America, spanning a wide climatic range, to test the importance of CCMs as predictors of lichen ecology and distribution. We show that the presence or absence of CCMs leads to opposite responses to temperature and precipitation in green algal lichens, and different responses in cyanobacterial lichens. These responses contrast with our understanding of lichen physiology, whereby CCMs mitigate carbon limitation by water saturation at the cost of efficient use of vapor hydration. This study demonstrates that CCM status is a key functional trait in obligate lichen symbioses, equivalent in importance to its role in vascular plants, and central for studying present and future climate responses.

Photosynthetic efficiencies in plants are restricted by the CO₂-fixing enzyme Rubisco but could be enhanced by introducing a CO₂-concentrating mechanism (CCM) from green algae, such as Chlamydomonas reinhardtii (hereafter Chlamydomonas). A key feature of the algal CCM is aggregation of Rubisco in the pyrenoid, a liquid-like organelle in the chloroplast. Here we have used a yeast two-hybrid system and higher plants to investigate the protein–protein interaction between Rubisco and essential pyrenoid component 1 (EPYC1), a linker protein required for Rubisco aggregation. We showed that EPYC1 interacts with the small subunit of Rubisco (SSU) from Chlamydomonas and that EPYC1 has at least five SSU interaction sites. Interaction is crucially dependent on the two surface-exposed α-helices of the Chlamydomonas SSU. EPYC1 could be localized to the chloroplast in higher plants and was not detrimental to growth when expressed stably in Arabidopsis with or without a Chlamydomonas SSU. Although EPYC1 interacted with Rubisco in planta, EPYC1 was a target for proteolytic degradation. Plants expressing EPYC1 did not show obvious evidence of Rubisco aggregation. Nevertheless, hybrid Arabidopsis Rubisco containing the Chlamydomonas SSU could phase separate into liquid droplets with purified EPYC1 in vitro, providing the first evidence of pyrenoidlike aggregation for Rubisco derived from a higher plant.

Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts. This microcompartment enhances the photosynthetic CO2-fixing activity of the Calvin-Benson cycle enzyme Rubisco, suppresses an energetically wasteful oxygenase activity of Rubisco, and mitigates limiting CO2 availability in aquatic environments. Hence, the pyrenoid is functionally analogous to the carboxysomes in cyanobacteria. However, a comprehensive analysis of pyrenoid functions based on its protein composition is lacking. Here we report a proteomic characterization of the pyrenoid in the green alga Chlamydomonas reinhardtii. Pyrenoid-enriched fractions were analyzed by quantitative mass spectrometry. Contaminant proteins were identified by parallel analyses of pyrenoid-deficient mutants. This pyrenoid proteome contains 190 proteins, many of which function in processes that are known or proposed to occur in pyrenoids: e.g. the carbon concentrating mechanism, starch metabolism or RNA metabolism and translation. Using radioisotope pulse labeling experiments, we show that pyrenoid-associated ribosomes could be engaged in the localized synthesis of the large subunit of Rubisco. New pyrenoid functions are supported by proteins in tetrapyrrole and chlorophyll synthesis, carotenoid metabolism or amino acid metabolism. Hence, our results support the long-standing hypothesis that the pyrenoid is a hub for metabolism. The 81 proteins of unknown function reveal candidates for new participants in these processes. Our results provide biochemical evidence of pyrenoid functions and a resource for future research on pyrenoids and their use to enhance agricultural plant productivity. Data are available via ProteomeXchange with identifier PXD004509.