The Mechanisms of Calcification in Coccolithophores - The molecular basis of calcium and inorganic carbon transport in Emiliania huxleyi

Coccolithophores are calcifying marine phytoplankton that through the fixation of inorganic carbon into calcite and particulate organic carbon play a fundamental role in global carbon cycles. As the CO2 concentration of the surface ocean increases through the anthropogenic release of CO2 by burning fossil fuels both a decrease in pH (ocean acidification) and a increase in dissolved inorganic carbon (ocean carbonation) are taking place. To understand the impact of these ocean changes on coccolithophores it is essential that we rapidly increase our knowledge of the cellular processes underlying coccolithophore physiology. This doctoral thesis focuses on the cellular and molecular processes involved in the transport of Ca2+, inorganic carbon and H+ in relation to calcification and photosynthesis in the coccolithophore species Emiliania huxleyi. The thesis comprises 7 chapters: Chapter 1 is a general introduction to coccolithophore cellular biology and global carbon cycling; Chapters 2-6 are a combination of publications and data chapters; Chapter 7 provides a synthesis of the results placing the presented data into context of coccolithophores in a changing ocean, highlighting future research areas. Chapter 2 is a published review of the current literature on the molecular aspects of calcification in coccolithophores. It identifies key gaps in our knowledge of coccolithophore cellular biology and presents new hypotheses for the transport of substrates to the site of calcification. Chapter 3 investigates some of the proposed hypotheses by examining the role of several candidate Ca2+, H+ and inorganic carbon transport genes in calcifying and non-calcifying cells of E. huxleyi, using quantitative reverse transcriptase PCR. The data provides strong evidence that a putative HCO3- transporter (AEL1), a Ca2+/H+ exchanger (CAX3), a vacuolar H+-ATPase pump (ATPVc/c’) and a gene encoding for a coccolith-associated protein, GPA, play key roles in E. huxleyi biomineralization. CAX3 and AEL1 were chosen for further analysis and were successfully cloned and expressed in Saccharomyces cerevisiae and Human Embryonic Kidney cells (HEK293) respectively (Chapter 6). However complete characterization of CAX3 and AEL1 was unsuccessful. CAX3 failed to complement the Ca2+ sensitive phenotype of a S. cerevisiae mutant, with further expression in a Ca2+ sensitive Escherichia coli mutant resulting in a lethal phenotype. The investigation of HCO3- transport in HEK293 cells expressing AEL1 gave negative results, potentially due to the poor localization of AEL1 to the plasma membrane. The data highlights the importance of developing genetic transformation techniques in coccolithophores to reduce the dependency of using foreign expression systems for the characterization of genes. The influence of the individual carbonate system components (CO2, HCO3-, CO32- and H+) on coccolithophore physiology and genetic response is relatively unknown. Chapter 4 disentangles the individual carbonate system components investigating their influence on calcification, particulate organic carbon fixation and gene expression in E. huxleyi. It identifies, for the first time, the genetic basis of a carbon concentrating mechanism (CCM) in coccolithophores, with the transcription of multiple CCM associated genes up-regulated at low concentrations of HCO3- and CO2. Physiological data combined with expression data indicates that calcification does not function as a CCM under carbon limitation and is instead reduced to allow the redistribution of inorganic carbon from calcification to photosynthesis. Furthermore, the data confirms previous studies that the substrate for calcification is HCO3- and that growth and organic carbon fixation rates are primarily influenced by CO2. The recent sequencing of the E. huxleyi genome has provided vast quantities of genetic data that requires detailed analysis to realise its full potential. Chapter 5 analyses the genome for calcification and photosynthesis related transport genes discovering that E. huxleyi has a diverse range of inorganic carbon, Ca2+ and H+ transporters, from classical plant, animal and bacterial families. The presence of multiple Na+/Ca2+ exchangers, a family of almost exclusively animal transporters indicates that coccolithophores have the potential to use both H+ and Na+ electrochemical gradients to drive secondary transport. Furthermore the identification of green algal CCM genes may provide a strong basis for investigating the evolution of CCMs in eukaryotic algae. The data presented in this thesis provides a significant step in our understanding of coccolithophore physiology at a cellular and molecular level. It offers a solid platform for future research in coccolithophore cell biology an area of research that is essential to comprehend the role of coccolithophores in gobal carbon cycling and how they will respond and adapt to future ocean changes

Phase transition energetics in mesoscale photosynthetic condensates

The pyrenoid is a model two-component biomolecular condensate, vital for efficient photosynthesis in algae. Despite simulations predicting qualitative features of liquid-liquid phase separation driving their formation, the underlying energetics remain unclear. By modelling interactions between Rubisco protein carbon-capturing machinery inside pyrenoids as linker chemical and stretch potentials we explain spectroscopic and single-molecule data over physiological concentrations. This new parametrisation can be used for quantitative predictions in generalized emergent self-assembly of two-component condensates.Comment: v2: correction in the calculations v3: added experimental wor

Thylakoid localized bestrophin-like proteins are essential for the CO2 concentrating mechanism of Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii possesses a CO2 concentratingmechanism (CCM) which helps in successful acclimationto low CO2 conditions. Current models of the CCM postulate that aseries of ion transporters bring HCO3- from outside the cell to thethylakoid lumen, where the carbonic anhydrase CAH3 dehydratesaccumulated HCO3- to CO2, raising the CO2 concentration forRubisco. Previously, HCO3- transporters have been identified atboth the plasma membrane and the chloroplast envelope, butthe transporter thought to be on the thylakoid membrane hasnot been identified. Three paralogous genes (BST1, BST2, BST3)belonging to the bestrophin family have been found to be upregulatedin low CO2 conditions, and their expression is controlledby CIA5, a transcription factor that controls many CCM genes.YFP fusions demonstrate that all three proteins are located onthe thylakoid membrane, and interactome studies indicate thatthey might associate with chloroplast CCM components. A singlemutant defective in BST3 still grows nearly normally on low CO2,indicating that the three bestrophin-like proteins may have redundantfunctions. Therefore, an RNAi approach was adopted to reducethe expression of all three genes at once. RNAi mutants withreduced expression of BST1-3 were unable to grow at low CO2concentrations, exhibited a reduced affinity to inorganic carboncompared to the wild type cells, and showed reduced inorganiccarbon uptake. We propose that these bestrophin-like proteins areessential components of the CCM that deliver HCO3- accumulatedin the chloroplast stroma to CAH3 inside the thylakoid lumen

Assembly of the algal CO2-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif

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

Dissecting the impact of CO2and pH on the mechanisms of photosynthesis and calcification in the coccolithophoreEmiliania huxleyi

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

Introducing an algal carbon-concentrating mechanism into higher plants: location and incorporation of key components.

Many eukaryotic green algae possess biophysical carbon-concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO2 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 H(14) CO3 (-) 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

A spatial interactome reveals the protein organization of the algal CO2 concentrating mechanism

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

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

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