Coccolith Vesicle Research Articles

De novo transcriptome profile of coccolithophorid alga Emiliania huxleyi CCMP371 at different calcium concentrations with proteome analysis.

The haptophyte alga Emiliania huxleyi is the most abundant coccolithophore in the modern ocean and produces elaborate calcite crystals, called coccolith, in a separate intracellular compartment known as the coccolith vesicle. Despite the importance of biomineralization in coccolithophores, the molecular mechanism underlying it remains unclear. Understanding this precise machinery at the molecular level will provide the knowledge needed to enable further manipulation of biomineralization. In our previous study, altering the calcium concentration modified the calcifying ability of E. huxleyi CCMP371. Therefore in this study, we tested E. huxleyi cells acclimated to three different calcium concentrations (0, 0.1, and 10 mM). To understand the whole transcript profile at different calcium concentrations, RNA-sequencing was performed and used for de novo assembly and annotation. The differentially expressed genes (DEGs) among the three different calcium concentrations were analyzed. The functional classification by gene ontology (GO) revealed that ‘intrinsic component of membrane’ was the most enriched of the GO terms at the ambient calcium concentration (10 mM) compared with the limited calcium concentrations (0 and 0.1 mM). Moreover, the DEGs in those comparisons were enriched mainly in ‘secondary metabolites biosynthesis, transport and catabolism’ and ‘signal transduction mechanisms’ in the KOG clusters and ‘processing in endoplasmic reticulum’, and ‘ABC transporters’ in the KEGG pathways. Furthermore, metabolic pathways involved in protein synthesis were enriched among the differentially expressed proteins. The results of this study provide a molecular profile for understanding the expression of transcripts and proteins in E. huxleyi at different calcium concentrations, which will help to identify the detailed mechanism of its calcification.

X-ray nanotomography of coccolithophores reveals that coccolith mass and segment number correlate with grid size

Coccolithophores of the Noëlaerhabdaceae family are covered by imbricated coccoliths, each composed of multiple calcite crystals radially distributed around the periphery of a grid. The factors that determine coccolith size remain obscure. Here, we used synchrotron-based three-dimensional Coherent X-ray Diffraction Imaging to study coccoliths of 7 species of Gephyrocapsa, Emiliania and Reticulofenestra with a resolution close to 30 nm. Segmentation of 45 coccoliths revealed remarkable size, mass and segment number variations, even within single coccospheres. In particular, we observed that coccolith mass correlates with grid perimeter which scales linearly with crystal number. Our results indirectly support the idea that coccolith mass is determined in the coccolith vesicle by the size of the organic base plate scale (OBPS) around which R-unit nucleation occurs every 110–120 nm. The curvation of coccoliths allows inference of a positive correlation between cell nucleus, OBPS and coccolith sizes.

An Extracellular Polysaccharide-Rich Organic Layer Contributes to Organization of the Coccosphere in Coccolithophores

Coccolithophores are globally abundant marine microalgae characterised by their ability to form calcite platelets (coccoliths). The coccoliths are produced internally in a Golgi-derived vesicle. Mature coccoliths are extruded from the cell to form a protective covering on the cell surface, known as the coccosphere. Current evidence indicates that calcite precipitation in the coccolith vesicle is modulated by coccolith-associated polysaccharides (CAPs). Whilst previous research into CAPs has focussed on their roles in calcite precipitation within the coccolith vesicle, little is known of their extracellular roles. Using fluorescent lectins, we visualise the extracellular polysaccharide-rich organic layer associated with external coccoliths and demonstrate that it differs between species in structure and composition. Biochemical analysis of polysaccharide extracted from coccoliths indicated substantial differences between species in monosaccharide composition and uronic acid content. In Coccolithus braarudii our studies indicate that polysaccharide-rich material is extruded with the coccoliths, where it plays a role in the adhesion of the coccoliths to the cell surface and contributes to the overall organisation of the coccosphere. Together, these results highlight the important extracellular roles of CAPs and their contribution to the dynamic nature of the coccosphere.

Formation and mosaicity of coccolith segment calcite of the marine algae Emiliania huxleyi.

Coccolithophores belong to the most abundant calcium carbonate mineralizing organisms. Coccolithophore biomineralization is a complex and highly regulated process, resulting in a product that strongly differs in its intricate morphology from the abiogenically produced mineral equivalent. Moreover, unlike extracellularly formed biological carbonate hard tissues, coccolith calcite is neither a hybrid composite, nor is it distinguished by a hierarchical microstructure. This is remarkable as the key to optimizing crystalline biomaterials for mechanical strength and toughness lies in the composite nature of the biological hard tissue and the utilization of specific microstructures. To obtain insight into the pathway of biomineralization of Emiliania huxleyi coccoliths, we examine intracrystalline nanostructural features of the coccolith calcite in combination with cell ultrastructural observations related to the formation of the calcite in the coccolith vesicle within the cell. With TEM diffraction and annular dark-field imaging, we prove the presence of planar imperfections in the calcite crystals such as planar mosaic block boundaries. As only minor misorientations occur, we attribute them to dislocation networks creating small-angle boundaries. Intracrystalline occluded biopolymers are not observed. Hence, in E.huxleyi calcite mosaicity is not caused by occluded biopolymers, as it is the case in extracellularly formed hard tissues of marine invertebrates, but by planar defects and dislocations which are typical for crystals formed by classical ion-by-ion growth mechanisms. Using cryo-preparation techniques for SEM and TEM, we found that the membrane of the coccolith vesicle and the outer membrane of the nuclear envelope are in tight proximity, with a well-controlled constant gap of ~4nm between them. We describe this conspicuous connection as a not yet described interorganelle junction, the "nuclear envelope junction". The narrow gap of this junction likely facilitates transport of Ca2+ ions from the nuclear envelope tothe coccolith vesicle. On the basis of our observations, we propose that formation of the coccolith utilizes the nuclear envelope-endoplasmic reticulum Ca2+ -store of the cell for the transport of Ca2+ ions from the external medium to the coccolith vesicle and that E.huxleyi calcite forms by ion-by-ion growth rather than by a nanoparticle accretion mechanism.

Extreme strontium concentrations reveal specific biomineralization pathways in certain coccolithophores with implications for the Sr/Ca paleoproductivity proxy.

The formation of the coccolith biominerals by a group of marine algae (the Coccolithophores) offers fascinating research avenues both from the biological and geological sides. It is surprising how biomineralisation by a key phytoplanktonic group remains underconstrained, yet is influential on ocean alkalinity and responsible for the built up of our paleoclimatic archive over the last 200 Myrs. Here, we report two close relative coccolith taxa exhibiting substantial bioaccumulation of strontium: Scyphosphaera and Pontosphaera grown in the laboratory or retrieved from Pliocene sediments. This strontium enrichment relative to calcium is one order of magnitude greater than reported in other coccoliths of the orders Isochrysidales and Coccolithales, and extends well beyond established controls on Sr/Ca ratios by temperature and growth rate. We discuss this prominent vital effect in relation with possible specific uptake of strontium relative to calcium from the extracellular environment to the coccolith vesicle in coccolithophores excreting very large scale coccoliths. The report of Sr-rich biominerals challenges our current understanding of the cellular acquisition and intracellular trafficking of alkaline earth cations in phytoplanktonic calcifying eukaryotic algae. The presence of Sr-rich coccolith species in the geological record has to be quantitatively considered in future Sr/Ca-based palaeoceanographic reconstruction.

The uronic acid content of coccolith-associated polysaccharides provides insight into coccolithogenesis and past climate.

Unicellular phytoplanktonic algae (coccolithophores) are among the most prolific producers of calcium carbonate on the planet, with a production of ∼1026 coccoliths per year. During their lith formation, coccolithophores mainly employ coccolith-associated polysaccharides (CAPs) for the regulation of crystal nucleation and growth. These macromolecules interact with the intracellular calcifying compartment (coccolith vesicle) through the charged carboxyl groups of their uronic acid residues. Here we report the isolation of CAPs from modern day coccolithophores and their prehistoric predecessors and we demonstrate that their uronic acid content (UAC) offers a species-specific signature. We also show that there is a correlation between the UAC of CAPs and the internal saturation state of the coccolith vesicle that, for most geologically abundant species, is inextricably linked to carbon availability. These findings suggest that the UAC of CAPs reports on the adaptation of coccolithogenesis to environmental changes and can be used for the estimation of past CO2 concentrations.

Incorporation of zinc into the coccoliths of the microalga Emiliania huxleyi.

The coccolithophore Emiliania huxleyi is covered with elaborated calcite plates, the so-called coccoliths, which are produced inside the cells. We investigated the incorporation of zinc into the coccoliths of E. huxleyi by applying different zinc and calcium amounts via the culture media and subsequently analyzing the zinc content in the cells and the Zn/Ca ratio of the coccoliths. To investigate the Zn/Ca ratio of coccoliths built in the manipulated media, the algae have first to be decalcified, i.e. coccolith free. We used a newly developed decalcification method to obtain 'naked' cells for cultivation. E. huxleyi proliferated and produced new coccoliths in all media with manipulated Zn/Ca ratios. The cells and the newly built coccoliths were investigated regarding their zinc content and their Zn/Ca ratio, respectively. High zinc amounts were taken up by the algae. The Zn/Ca ratio of the coccoliths was positively correlated to the Zn/Ca ratio of the applied media. The unique feature of the coccoliths was maintained also at high Zn/Ca ratios. We suggest the following pathway of the zinc ions into the coccoliths: first, the zinc ions are bound to the cell surface, followed by their transportation into the cytoplasm. Obviously, the zinc ions are removed afterwards into the coccolith vesicle, where the zinc is incorporated into the calcite coccoliths which are then extruded. The incorporation of toxic zinc ions into the coccoliths possibly due to a new function of the coccoliths as detoxification sites is discussed.

Coccolith-Derived Isotopic Proxies in Palaeoceanography: Where Geologists Need Biologists

Coccolithophore biominerals, the coccoliths, represent an important part of the Meso-Cenozoic sedimentary archive. Geochemical analyses of coccoliths can be used to unravel climatic fluctuations in the oceanic realm, but such reconstructions are complicated due to the problem of the “vital effect”. This concept refers to the modulation in the record of the physico-chemistry of seawater in calcite due to algal physiology. For decades, it was thought that the magnitude of the vital effect was species-specific and constant for a given species. Recent studies aiming at a mechanistic understanding of these processes point towards a plastic and environmental-dependent interplay between the physiology of coccolithophores and isotopic composition in coccolith calcite. This “mobilis in mobili” relationship opens the door to the possibility to explore the vital effects as palaeoenvironmental proxies undertaking an interspecies approach. New physiological parameters, such as the quantification of calcification rates, pH, and calcium and carbon pools in the coccolith vesicle would further help geologists to constrain the vital effect. Emerging “non-traditional” isotope systems will also contribute to refine the transfer functions between coccolith geochemistry, vital effect, and palaeoenvironments.

Constraints on the vital effect in coccolithophore and dinoflagellate calcite by oxygen isotopic modification of seawater

In this study, we show that there are independent controls of 18O/16O and 13C/12C fractionation in coccolithophore and dinoflagellate calcite due to the contrasting kinetics of each isotope system. We demonstrate that the direction and magnitude of the oxygen isotope fractionation with respect to equilibrium is related to the balance between calcification rate and the replenishment of the internal pool of dissolved inorganic carbon (DIC). As such, in fast growing cells, such as those of Emiliania huxleyi and Gephyrocapsa oceanica (forming the so-called “heavy group”), calcification of the internal carbon pool occurs faster than complete isotopic re-adjustment of the internal DIC pool with H2O molecules. Hence, coccoliths reflect the heavy oxygen isotope signature of the CO2 overprinting the whole DIC pool. Conversely, in large and slow growing cells, such as Coccolithus pelagicus ssp. braarudii, complete re-equilibration is achieved due to limited influx of CO2 leading to coccoliths that are precipitated in conditions close to isotopic equilibrium (“equilibrium group”). Species exhibiting the most negative oxygen isotope composition, such as Calcidiscus leptoporus (“light group”), precipitate coccolith under increased pH in the coccolith vesicle, as previously documented by the “carbonate ion effect”. We suggest that, for the carbon isotope system, any observed deviation from isotopic equilibrium is only “apparent”, as the carbon isotopic composition in coccolith calcite is controlled by a Rayleigh fractionation originating from preferential incorporation of 12C into organic matter. Therefore, species with low PIC/POC ratios as E. huxleyi and G. oceanica are shifted towards positive carbon isotope values as a result of predominant carbon fixation into the organic matter. By contrast, cells with higher PIC/POC as C. braarudii and C. leptoporus maintain, to some extent, the original negative isotopic composition of the CO2. The calcareous dinoflagellate Thoracosphaera heimii exhibits different behaviour for both isotopic systems, in particular with respect to its very negative carbon isotope composition, owing to coeval intra and extracellular biomineralisation in this group. In this study, we also investigate the sensitivity of 18O/16O fractionation to varying ambient oxygen isotope composition of the medium for inorganic, coccolithophore, and dinoflagellate calcite precipitated under controlled laboratory conditions. The varying responses of different taxa to increased oxygen isotope composition of the growth medium may point to a potential bias in sea surface temperature reconstructions that are based on the oxygen isotopic compositions of sedimentary calcite, especially during times of changing seawater oxygen isotopic composition. Overall, this study represents an important step towards establishing a mechanistic understanding of the “vital effect” in coccolith and dinoflagellate calcite, and provides valuable information for interpreting the geochemistry of the calcareous nannofossils in the sedimentary record, at both monospecific and interspecies levels.

B/Ca in coccoliths and relationship to calcification vesicle pH and dissolved inorganic carbon concentrations

Coccolithophorid algae are microscopic but prolific calcifiers in modern and ancient oceans. When the pH of seawater is modified, as may occur in the future due to ocean acidification, different species and strains of coccolithophorids have exhibited diverse calcification responses in laboratory culture. Since their biomineralization is a completely intracellular process, it is unclear why their response should be affected by extracellular seawater pH. Variations in the B/Ca in coccoliths are potential indicators of pH shifts in the intracellular coccolith vesicle where calcification occurs, because B/Ca in abiogenic calcites increases at higher pH due to the greater abundance of borate ions, the only B species incorporated into calcite. We used a SIMS ion probe to measure B/Ca of coccoliths from three different strains of Emiliania huxleyi and one strain of Coccolithus braarudii braarudii cultured under different seawater pH conditions to ascertain if the B/Ca can be used to elucidate how coccolithophorids respond to changing ocean pH. These data are interpreted with the aid of a conceptual model of cellular boron acquisition by coccolithophorids. Based on uptake in other plants, we infer that boron uptake by coccolithophorid cells is dominated by passive uptake of boric acid across the lipid bilayer. Subsequently, in the alkaline coccolith vesicle (C.V.), boron speciates according to the C.V. pH, and borate is incorporated into the coccolith. At increasing seawater pH, the relative abundance of the neutral boric acid in seawater decreases, lowering the potential B flux into the cell. Homeostasis or constant pH of the coccolith vesicle results in a decrease of the B/Ca in the coccolith with increasing seawater pH. In contrast, if coccolith vesicle pH increases with increasing seawater pH, then the B/Ca will increase as the fraction of borate in the coccolith vesicle increases. The coccolith B/Ca is also expected to depend inversely on the dissolved inorganic carbon (DIC) concentration in the coccolith vesicle. The B/Ca in cultured coccoliths is much lower than that of foraminifera or corals and limits precision in the analysis. Modest variations in DIC or pH of the coccolith vesicle can account for the observed trends in B/Ca in cultured coccoliths. The model shows that paired measurements of B/Ca and B isotopic composition of the calcite could distinguish between regulation of pH or DIC in the coccolith vesicle.

Dynamics of formation and secretion of heterococcoliths by Coccolithus pelagicus ssp. braarudii

The formation and secretion of heterococcoliths by the non-motile life phase of the coccolithophore Coccolithus pelagicus was investigated using electron microscopy and time-lapse bright field imaging. Coccolithogenesis in C. pelagicus exhibited sequential mineralization of single coccoliths in Golgi-derived and nuclear-associated vesicles, a pattern similar to the formation of heterococcoliths in Emiliania huxleyi. Our TEM data show that only on maturation does the single coccolith vesicle migrate away from the nucleus before secretion. A reticular body, distinct from the Golgi body, was also clearly visible at the distal surface of the developing coccolith vesicle, suggesting this is a common structural feature in placolith cells that mineralize and secrete coccoliths one at a time. Time-lapse imaging revealed that the coccolith secretion process is rapid, taking 60–190 seconds, and involves considerable contractile activity to eject and position the coccolith on the surface of the cell. An intact flagellar root apparatus was discovered at the anterior pole of this non-motile cell from which polarized secretion of coccoliths occurs, which may indicate a novel role for such cytoskeletal structures. Freeze-fracture preparations revealed columnar deposits and adhesions linking the scales and coccolith baseplates to the cell, across the periplasmic space providing points of attachment for cellular movement. Rotatory movements of the cell relative to external coccoliths were exhibited by all actively calcifying cells. These movements enable the cell, while exhibiting morphologically polarized secretion, to locate and secrete a mature coccolith in a spatially well-defined manner. Finally, the time-lapse imaging approach described here provides an opportunity to quantify the regulation of coccolith production in single cells with high temporal resolution allowing responses of calcification to rapidly fluctuating environmental conditions such as light–dark transitions to be examined in detail, which has not been possible with bulk calcification studies.

Cell Biology, Biochemistry and Genomics of Coccolithophore Biomineralization.

AbstractThe coccoliths that form the outer cell covering of the unicellular coccolithophores are assembled in a coccolith vesicle. The coccolith vesicle maintains the conditions and protein carriers sufficient to import the raw materials (Ca2+ and CO32-) for calcite formation and, furthermore, to initiate and sustain mineralization. We have isolated the coccolith vesicle membrane on which a proton-pumping, vacuolar ATPase has been localized. The V-ATPase has been characterized and its membrane-spanning subunit c has been cloned and sequenced. We used the highly conserved sequence for designing and constructing probes and primers that allowed us to analyze expression patterns to show a direct relationship between expression of the gene and calcification. Our collaborative work has now turned to a genomics approach that will lead us to additional calcification-specific genes.

EFFECTS OF NITROGEN AND PHOSPHORUS AVAILABILITY ON THE EXPRESSION OF THE COCCOLITH‐VESICLE V‐ATPASE (SUBUNIT C) Of PLEUROCHRYSIS (HAPTOPHYTA)1

The coccolithophores Emiliania and Pleurochrysis demonstrate increased coccolith production when growth is reduced by nitrate or phosphate limitation. The function of enhanced coccolith production under these conditions and its regulation have not been resolved. Studies at the molecular level are ideally suited to determine the exact relationship between calcification and other cellular functions. In a previous study we provided evidence for the presence of a vacuolar H+‐ATPase on coccolith vesicle membranes of P. carterae. These trans‐Golgi–derived vesicles are the sites of coccolith production, with each vesicle containing one coccolith. In this study, expression of the vap gene, encoding subunit c of the vacuolar H+‐ATPase, was investigated. Our objective was to explore potential relationships between vap expression, nutrient‐dependent growth, and calcification. Specifically, we monitored vap expression relative to two genes, fcp and pcna, whose expression was previously shown to vary with growth conditions; fcp encodes a fucoxanthin chl a/c‐binding protein, and pcna encodes the proliferating cell nuclear antigen. Relative to the expression of pcna and fcp, vap expression was highest at nutrient concentrations where growth curves and chl a patterns indicated arrest of cell division. Our results indicate that the level of vap expression does not decrease when cell growth diminishes.

A COCCOLITHOPHORID CALCIFYING VESICLE WITH A VACUOLAR‐TYPE ATPASE PROTON PUMP: CLONING AND IMMUNOLOCALIZATION OF THE V0 SUBUNIT c

Coccolithophorid calcification is subcellular. It relies on a single Golgi apparatus to produce coccoliths consisting of an organic baseplate and calcite. The calcification reaction is initiated in a calcifying vesicle derived from the trans‐most Golgi. We have cloned a subunit of a V (vacuolar)‐ATPase (EC 3.6.1.3., ATP phosphohydrolase) from a Pleurochrysis (Haptophyceae) cDNA library of transcripts expressed during calcification. Degenerate PCR primers were developed after alignment of the higher plant V‐ATPase subunit c genes to identify conserved consensus sequences. The library was screened with a homologous probe obtained by PCR. The cloned gene is found as a single copy on the P. cartarae (strain 136) genome and includes a 495‐base pair open reading frame encoding a 164 amino acid polypeptide and deduced molecular mass of 16.2 kDa. Its deduced amino acid sequence shows a close relationship to subunit c of the Vo domain of the vacuolar proton‐pumping ATPase of higher plants. An in vitro‐synthesized oligopeptide corresponding to the L2 extramembrane domain was used for rabbit immunization. Affinity‐purified antiserum detected a polypeptide band with an apparent molecular mass of 24 kDa in immunoblots of highly enriched coccolith vesicle membranes. Immunofluoresence microscopy showed antibody specificity for the membranes of isolated coccolith vesicles. This work provides support for the existence of an authentic, vacuolar‐type, proton‐pumping ATPase on coccolith vesicle membranes in a calcifying coccolithophorid.

Coccolith crystals ofPleurochrysis carterae: Crystallographic faces, organization, and development

The crystallographic and morphological configuration of the mineral ring associated with the coccoliths ofPleurochrysis carterae was determined by transmission electron microscopy and electron diffraction. Mature Pleurochrysis coccoliths consist of an oval organic base plate, a distal rim of interlocking calcite crystals, and a narrow ribbon of organic material which tethers the mineral ring to the base plate. Crystals of two distinct forms (R and V units) alternate about the rim in a quasi regular manner; their crystallographicc-axes are aligned parallel to and inclined about 63° to the coccolith plane, respectively. The mineral ring has four platelike elements: the distal-shield and outer-tube elements which form the V unit, and the proximal-shield and inner-tube elements which form the R units. The platy surfaces of both tube elements correspond to the common (10\(\bar 1\)4) rhombohedral faces of calcite, and the plates of the proximal-shield element are prismatic (2\(\overline {11}\)0) faces. The plates of the distal-shield element are rather curved and their orientation does not correspond to a favorable calcite face; however, for convenience they are described as approximately (\(\bar 1\)108) faces, faces which rarely, if ever, develop in inorganic sources of calcite. During coccolith development the earliest habits observed for both V and R units correspond to rectangular parallelepipeds. Outgrowth from the initial V unit begins by expansion of (10\(\bar 1\)4) faces which form the platy surfaces of the outer-tube element. Throughout this period of development the mineral ring is flexible, at least in an isolated state. Subsequent outgrowth of the inner-tube and proximal-shield elements from the initial R unit produces a rigid interlocking ring. The unusual (\(\bar 1\)108) faces of the distal-shield element develop after the crystals are locked in place. Organic structures in intimate association with the mineral phase during its nucleation and growth include the coccolith ribbon, the calcium-polyanion particles, and the membrane of the coccolith vesicle. These structures are described in reference to their putative functions in regulating the development of V and R units.

V‐ AND P‐TYPE Ca2+‐STIMULATED ATPases IN A CALCIFYING STRAIN OF PLEUROCHRYSIS SP. (HAPTOPHYCEAE)

Coccolithophorids are marine unicellular algae characterized by their ability to carry out controlled, subcellular calcification. The biochemical and kinetic features of membrane‐bound Ca2+‐stimulated ATPases have been examined. Membranes and organelles from axenic cultures of Pleurochrysis sp. (CCMP299) were isolated by means of sucrose density centrifugation. High levels of Ca2+‐stimulated ATPase were detected in chloroplasts, Golgi apparatus, plasma membrane, and coccolith vesicles. The sensitivity of the enzyme activity in the organelles and membranes was assessed with pharmacologic agents that are known to be specific for the several isoforms of Ca2+‐stimulated ATPase. The Ca2+‐stimulated ATPase activity in the Golgi and coccolith vesicle preparations was sensitive to nitrate, thiocyanate, and sodium azide and insensitive to vanadate, cyclopiazonic acid, and thapsigargin. ATP‐dependent H+ movement, but not 45Ca2+ transport, across the coccolith vesicle was demonstrated. The Ca2+‐stimulated ATPase in the plasma membrane preparation was sensitive to vanadate. ATP‐dependent, vanadate‐sensitive efflux of 45Ca2+ was demonstrated for microsomal material derived from gradient‐isolated plasma membrane. Polypeptides from isolated Golgi and coccolith vesicle preparations cross‐reacted to an antibody raised against a subunit of the oat root proton pump, whereas polypeptides from the chloroplast preparations did not cross‐react. These findings show that a V‐type Ca2+‐stimulated ATPase is located on the coccolith vesicle membrane and a P‐type Ca2+‐stimulated ATPase is located on the plasma membrane.

Costs and benefits of calcification in coccolithophorids

Calcification in coccolithophorids requires major intracellular fluxes of inorganic carbon and calcium. This paper summarises the major cellular fluxes of substrates and products of calcification described in a simple four compartment model (cytosol, Golgi, coccolith vesicle and chloroplast). Measurements of the cytosolic and intra-coccolith vesicle pH and electrical potentials across the plasma membrane and coccolith vesicle membrane allow calculations of the proton electrochemical gradients across these membranes and estimates of the free carbonate and calcium concentrations in the coccolith vesicle. Calcification may provide a relatively low cost route for elevating the concentration of carbon dioxide in the chloroplast. This may have benefits in terms of the nutrient requirements for photosynthesis and growth. In particular, a close relationship appears to exist between calcification and the availability of phosphorus which may correlate with the occurrence of large scale blooms of Emiliania huxleyi in the North Atlantic.

Calcification rate in Emiliania huxleyi Lohmann in response to light, nitrate and availability of inorganic carbon

SUMMARYThe relationship between photosynthesis and calcification was investigated in a high‐calcifying strain of Emiliania huxleyi. At pH 8.3 and a photon flux density of 50 μmol m−2 s−1 calcification and photosynthetic 14CO2 fixation were carbon saturated at 1 mM DIC (dissolved inorganic carbon) but at a photon flux density of 300 μmol m−2 s−1 calcification and photosynthetic 14CO2 fixation were not saturated at the DIC concentration of sea‐water, 2 mM. When HCO3− provides the bulk of inorganic carbon the stoichiometry between photosynthetic 14CO2 fixation and calcification was 1:1. In the high‐calcifying strain of E. huxleyi the stoichiometry between photosynthetic 14CO2 fixation and O2 evolution was 2:1 but in a low calcifying strain the stoichiometry was 1:1. High nitrate concentrations, i.e. 1000 μM were required to inhibit calcification. The optimum pH for calcification and photosynthetic 14CO2 fixation was 7.8. From the results a model is proposed in which a molecule of HCO3− is the precursor of calcite in the coccolith vesicle with the extrusion of H+ into the cytosol, while another HCO3− provides CO2 in the chloroplast with the extrusion of OH−. The interaction of these processes maintains cytoplasmic pH near neutrality.

Calcification and utilization of inorganic carbon by the coccolithophorid Emiliania huxleyi Lohmann

summaryThe relationship between inorganic‐carbon dependent photosynthetic oxygen evolution and calcification was investigated in high‐ and low‐calcifying strains of Emiliania huxleyi showing a ten‐fold difference in calcification rate. Unlike the low‐calcifying strain calcifying cultures showed a four‐fold increase in inorganic carbon (1 mM) dependent photosynthetic oxygen over the pH range 5‐8.3 resulting in a 20 fold difference in photosynthetic rate between the two strains at pH 8.3. Calcifying cells have a high affinity for HCO3−, the concentration of dissolved inorganic carbon [DIC] required for half‐maximal rate of photosynthetic O2 evolution (K0.5[DIC]) being 200 μM at pH 8‐3. In mid‐exponential phase cultures the stoichiometry between 14CO2 fixation and calcification was 1 : 1 so it is likely that the high photosynthetic rate at pH 8.3 is sustained by 14CO2, released from H 14CO2 during calcification.Measurement of bicarbonate transport by the silicone‐oil‐layer centrifugal filtering technique demonstrated a rapid uptake and achievement of equilibrium (less than 3 s) between the intracellular and external inorganic carbon concentrations in low and high‐calcifying cells. Subsequent metabolism of the 14C intracellular inorganic carbon pool did not occur in low‐calcifying cells suggesting the block in calcification occurs either in transport into or within the coccolith vesicle.

Ultrastructural polysaccharide localization in calcifying and naked cells of the coccolithophoridEmiliania huxleyi

Emiliania huxleyi is a marine coccolithophorid which produces coccoliths,i.e., particles consisting of calcite and macromolecular organic material. The coccoliths are formed intracellulary in specialized organelles which comprise a coccolith vesicle (CV) and a reticular body (RB), together forming the CV/RB system or calcifying system. After termination of calcification, the coccolith is extruded and incorporated into the coccosphere,i.e., one or several layers of extracellular coccoliths surrounding the cell. Apart from the coccolith-producing cells (C cells) ofE. huxleyi, there are naked cells (N cells) which seem to have lost the capacity to produce coccoliths but are very similar to the C cells in other morphological respects. Biochemical studies have revealed that polysaccharides may play a regulatory role in calcification. The aim of the present study was to determine the localization of polysaccharides in both C and N cells electron microscopically. For this purpose, a cytochemical staining technique according toThiery (1967) was applied. The CV/RB system of C cells was conspicuously stained. Due to the excellent stainability of this system, a putative succession of morphological stages during coccolithogenesis could be described. The staining pattern of the N cells closely resembled that of the C cells. It was found, however, that the “calcifying” system of N and C cells differed in both morphology and position. It is suggested that the divergent morphology of the “calcifying” system of N cells accounts for its failure to produce coccoliths.