INCOMPLETENESS OF THE COCCOSPHERE AS A POSSIBLE STIMULUS FOR COCCOLITH FORMATION IN PLEUROCHRYSIS CARTERAE(PRYMNESIOPHYCEAE)1

Pleurochrysis carterae is a marine biflagellate that produces calcified structures called coccoliths. The coccoliths are formed inside the cells and released from the latter after formation. The light dependence of calcium incorporation in this species was studied using⁴⁵Ca as a tracer. Cells exposed to a repeating cycle of 16 h of light and 8 h of darkness incorporated calcium in extracellular coccoliths at a more or less constant rate throughout a cycle. The cells divided during the dark periods with a concomitant decrease in size. Their size increased during the light periods Coccolith formation in cells incubated in continuous darkness was greatly reduced and finally ceased. These cells did not divide and did not increase in size. Removal of extracellular coccoliths prior to the calcium incorporation experiments stimulated coccolith formation both in dark-incubated cells and in cells exposed to a repeating light-dark cycle. Cells in the stationary phase of growth ceased producing coccoliths. Calcification could be induced in these cells by removal of the extracellular coccoliths. Based on these findings we suggest that cells of Pleurochrysis carterae tend to produce a complete cover of coccoliths and that the available cell surface is a factor controlling coccolith formation.     

ROLE OF THE LIGHT-DARK CYCLE AND MEDIUM COMPOSITION ON THE PRODUCTION OF COCCOLITHS BY EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Production of coccoliths by cells of Emiliania huxleyi (Lohmann) Hay and Mohler was measured during exposure of the cells to two diel light-dark cycles (16:8 h). During the light period about eight coccoliths per cell were formed at a constant rate of one coccolith per 2 h. Cells divided during the first half of the dark period. No coccolith production took place during the dark period. With electron microscopy we found early-stage, coccolith-production compartments in cells after mitosis while still in the dark. No calcification was observed in these compartments. Cells grown on enriched seawater (Eppley's medium) tended to produce enough coccoliths to cover the cell in a single layer. When these cells reached the stationary phase coccolith production stopped. Coccolith production was induced by removal of extracellular coccoliths. Cells grown on medium containing 2% of the nitrate and phosphate of Eppley's medium tended to produce coccoliths in the stationary phase. This resulted in the formation of multiple layers of coccoliths. The multiple covering was restored after decalcification of stationary cells. Formation of multiple layers of coccoliths may help the cells reach deeper, nutrient-rich water by increasing the sinking rate of the cells.     

Adjustment to Light and Dark Rates of Coccolith Formation

Coccolith formation in Coccolithus huxleyi was investigated in a semiquantitative manner by microscopic enumeration of coccolith-carrying cells appearing, after varying periods in normal growth medium, following an initial removal of all coccoliths by treatment with a decalcifying medium. Coccoliths were formed both in the light and in the dark, but the rate of coccolith formation was very much greater in the light. Time-course experiments on the uptake of carbon-14 in coccoliths demonstrated that after a period in darkness, the cells required half an hour of illumination in order to adjust to the light rate of coccolith formation. Coccoliths were produced at a maximum rate only as long as the illumination lasted, although about one hour in the dark was needed before the transition back to the original dark rate was complete.     

Polysaccharide localization, coccolith formation, and Golgi dynamics in the coccolithophorid Hymenomonas carterae

The periodic acid-thiocarbohydrazide-silver proteinate staining technique according to Thiéry (1967) was employed for visualization of the ultrastructural localization of polysaccharides in the coccolithophorid alga Hymenomonas carterae . Preferential staining was observed in the Golgi apparatus, including constituents and precursors of "scales" and " coccoliths " (scales with a rim of elaborate CaCO3 crystals), which are extruded and become part of the cell wall. Cells fixed in the presence of the polyanion-precipitating agent cetylpyridiniumchloride showed a voluminous coat over the crystalline matter of the coccolith giving the extracellular coccoliths the appearance of being glued together. Soluble acid polysaccharides are thought to occur in the coat. Evidence is presented that the coat and the crystalline matter are produced simultaneously. The excellent stainability of the Golgi apparatus allowed study of its morphology in considerable detail, and permitted a tentative reconstruction of the formation of coccoliths and scales and of the Golgi dynamics in general. The question of whether Golgi cisternae are mobile or static entities is dealt with.     

CALCIFICATION BY COCCOLITHOPHORIDS: EFFECTS OF pH AND Sr1

Cells of Coccolithus huxleyi which fail to deposit CaCO₃ and form coccoliths often occur as unwanted components in cultures used for studies of calcification. Non-calcified cells generally cannot be made to recalcify, but they can be removed from cultures by treatment at elevated pH or by a method based on faster sinking of calcified cells. Lowering the concentrations of nitrate, phosphate, or trace metals in the medium did not restore calcifying ability of non-calcified cells. However, addition of strontium did promote recalcification of decalcified Cricosphaera carterae grown under calcium limitation. Strontium seemed to promote coccolith attachment to cells rather than to affect calcium uptake or coccolith formation itself.     

A novel highly acidic polysaccharide with inhibitory activity on calcification from the calcified scale "coccolith" of a coccolithophorid alga, Pleurochrysis haptonemofera

Coccolith, a calcified scale with species-specific fine structure produced by marine unicellular coccolithophorid algae, consists of calcium carbonate (CaCO(3)) crystals and a small amount of organic matrices. A novel polysaccharide named coccolith matrix acidic polysaccharide (CMAP) was isolated from the coccolith of a coccolithophorid alga, Pleurochrysis haptonemofera. The structure of CMAP was determined by chemical analysis and NMR spectroscopy including COSY, TOCSY, HMQC, and HMBC to be a polysaccharide composed of the following unit: -->4) l-iduronic acid (alpha1-->2) meso-tartaric acid (3-->1) glyoxylic acid (1-->. It has four carboxyl groups per a disaccharide unit as observed in another polysaccharide PS-2 characterized previously in Pleurochrysis carterae. CMAP showed a strong inhibitory activity on CaCO(3) precipitation. These results suggest that CMAP serves as a regulator in the calcification of the coccolith.     

THE FINE STRUCTURE OF HYMENOMONAS (CRICOSPHAERA) CARTERAE. II. OBSERVATIONS ON SCALE AND COCCOLITH PRODUCTION

The fine structure of the marine coccolithophorid Hymenomonas (Cricosphaera) carterae (Braarud & Fagerland) Manton & Peterfi is reported. Details of the formation of the circular organic body scales are presented. Their formation is shown to be closely linked with the presence of tubules found within the Golgi cisternae. The details of coccolith production are also discussed. The formation of the organic matrix scale appears to be associated with a densely staining organelle, the intracellular coccolith pre-cursor. The precise mechanisms involved in the mineralization of the organic matrix scale is not known but 2 possibilities are discussed. The production of coccoliths in H. carterae is compared with coccolith production in other coccolithophorids that have been investigated finestructurally.     

Polyanion-mediated mineralization — assembly and reorganization of acidic polysaccharides in the Golgi system of a coccolithophorid alga during mineral deposition

Summary Immunolocalization of two highly acidic polysaccharides (PS-1 and PS-2) in a calcifying algaPleurochrysis carterae is described throughout the mineralization process, from before crystal nucleation through the cessation of crystal growth. This unicellular coccolithophorid alga is a useful model for mineralization because it produces calcified scales known as coccoliths in homogeneous cell culture. PS-1 and PS-2 were localized in the crystal coats of mature coccoliths and in electron dense Golgi particles. The polyanions are synthesized in medial Golgi cisternae and co-aggregate with calcium ions into discrete 25 nm particles. Particle-laden vesicles bud from cisternal margins and fuse with a coccolith-forming saccule containing an organic oval-shaped scale which forms the base of the future coccolith. The particles are localized on the base before the onset of mineral deposition and are present in the coccolith saccule throughout the period of crystal (CaCO₃) nucleation and growth. During the final phase of coccolith formation, the particles disappear, and the mature crystals acquire an amorphous coat containing PS-1 and PS-2 polysaccharides which remain with the mineral phase after the coccoliths are extruded from the cell. Postulated mechanisms of polyanion-mediated mineralization are reviewed and their relevance to the calcification of coccoliths is addressed.Abbreviations PS-1 polysaccharide one - PS-2 polysaccharide two - BSA bovine serum albumin - SDS sodium dodecyl sulfate - MES 2-(N-morpholino)-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - DHA 3-deoxy-lyxo-2-heptulosaric acid - TCA trichloroacetic acid     

Isolation and characterization of a novel acidic polysaccharide containing tartrate and glyoxylate residues from the mineralized scales of a unicellular coccolithophorid alga Pleurochrysis carterae.

The characterization of mineral-associated polyanions from the unicellular alga Pleurochrysis carterae is described. This species is useful for the study of mineralization, because it produces calcified scales known as coccoliths in homogeneous cell culture. Three acidic polysaccharides (PS-1, PS-2, and PS-3) were extracted from the coccoliths with EDTA and were separated and purified by differential precipitation with magnesium ions and chromatography on DEAE-cellulose. PS-1 and PS-3 are predominantly polymers of galacturonic acid containing lesser amounts of other monosaccharides. PS-2 has an unusual structure. Chemical, enzymatic, and two-dimensional NMR analyses demonstrate that the repeating unit of PS-2 is [----4)D-glucuronate(beta 1----2)meso-tartrate(3----1)glyoxylate(1-]n. Thus PS-2 has a high density of negatively charged groups available for calcium ion binding, similar to the phosphoprotein polyanions of other species. Polysaccharides containing tartrate and/or glyoxylate have not been previously described; these residues may be introduced into PS-2 by a postpolymerization process involving oxidative cleavage of glucuronate or mannuronate residues.     

COCCOLITH PRODUCTION AND DETACHMENT BY EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

Laboratory experiments were performed with the prymnesiophyte Emiliania huxleyi (Lohm.) Hay and Mohler, strain 88E, to quantify calcification per cell, coccolith detachment, and effects of coccolith production on optical scattering of individual cells. ¹⁴C incorporation into attached and detached coccoliths was measured using a bulk filtration technique. ¹⁴C-labeled cells also were sorted using a flow cytometer and analyzed for carbon incorporation into attached coccoliths. The difference between the bulk and flow cytometer analyses provided a ¹⁴C-based estimate of the rate of production of detached coccoliths. Coccolith production and detachment were separated in time in batch cultures, with most detachment happening well after calcification had stopped. Accumulation of coccoliths was maximum at the end of logarithmic growth with 50–80 coccoliths per cell (three to five complete layers of coccoliths around the cells). Net accretion rates of coccoliths were on the order of 7 coccoliths· cell^?1·d^?1 while net detachment rates were as high as 15 coccoliths· cell^?1·d^?1 for stationary phase cells. Equal numbers of coccoliths were attached and detached early in logarithmic growth, and as cells aged, the numbers of detached coccoliths exceeded the attached ones by a factor of 6. Our results demonstrate pronounced charges of forward angle light scatter and 90° light scatter of cells as they grow logarithmically and enter stationary phase. Counts of loose coccoliths in batch cultures are consistent with the detachment of coccoliths in layers rather than individual coccoliths.     

Discrimination of the Cell Surface of the Coccolithophorid Pleurochrysis haptonemofera from Light Scattering and Fluorescence After Fluorescein-Isothiocyanate-Labeled Lectin Staining Measured by Flow Cytometry

We investigated the extent of calcification on the cell surface of the coccolithophorid Pleurochrysis haptonemofera using flow cytometry. Side scattering (SSC) by coccolith-bearing cells was higher than that by naked cells, suggesting the difference was due to scattering of the laser beam by the coccoliths. SSC of coccolith-bearing cells under acidic conditions corresponded well to the extracellular Ca content, although SSC could not be used to detect a delicate change in the coccolith thickness. The increase in SSC during the reproduction of coccoliths after decalcification was consistent with the increase in the number of coccoliths on the cell surface. The fluorescence after fluorescein-isothiocyanate-labeled lectin staining suggests that α-d-mannose, α-d-glucose, d-galactose, d-N-acetylgalactosamine, or derivatives of them are included in the coccoliths. Measurement of SSC and fluorescence after fluorescein-isothiocyanate-labeled lectin staining enabled rapid and quantitative determination of the status on the cell surface and isolation of desirable cells for physiological studies by cell sorting. Received May 22, 2001; accepted July 30, 2001.     

Incorporation of zinc into the coccoliths of the microalga <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>

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.     

Isolation and some characterization of an acidic polysaccharide with anti-calcification activity from coccoliths of a marine alga, Pleurochrysis carterae.

Coccolith is a calcified scale with species-specific fine structure produced by marine unicellular coccolithophorid algae, and consists of calcium carbonate crystals and organic matrices. EDTA-soluble organic materials extracted from coccoliths of Pleurochrysis carterae showed anti-calcification activity. They were separated by anion-exchange HPLC, and two fractions, fractions A and B, were obtained. Fraction B, which was more active than fraction A, was further separated into six consecutive fractions, B1-B6, by second anion-exchange HPLC. 1H NMR spectral analyses of these fractions suggested that a novel acidic polysaccharide, designated CMAP, existed throughout B1-B6 and that the latter four fractions mainly contained another acidic polysaccharide, PS-2, characterized previously. Since PS-2 did not show anti-calcification activity, CMAP was found to be the active principle.     

Structural and physiological effects of calcium and magnesium in Emiliania huxleyi (Lohmann) Hay and Mohler

In organisms which perform both photosynthesis and calcification, the fact that calcification proceeds faster in the light than in the dark has led to the long-established view that photosynthesis and calcification are closely coupled. It is now clear that calcification does not promote photosynthesis, but an enhancement of calcification by photosynthesis could still explain why calcification is faster in the light. To test this, the kinetics of the two processes were monitored over a wide range of calcium concentrations (0-50 mM) in the coccolithophore Emiliania huxleyi. The addition of 50 mM calcium strongly inhibited both processes, but when incubated in lower concentrations, rates of calcification increased up to 20 mM calcium whilst those of photosynthesis remained constant over the same range of calcium concentrations. So, rates of calcification are able to rise without a concomitant increase in photosynthetic rates. In addition, calcification rate and coccolith morphology responded similarly to changes in calcium concentrations; low calcification rates were associated with poor coccolith structure (undercalcification) and high calcification rates with perfectly formed coccoliths. Calcium concentration thus strongly influences calcification affecting both crystal structure and rate of calcite deposition. A similar structural analysis of coccoliths from cells grown in different magnesium concentrations showed that this ion is also essential for calcification, since strong signs of coccolith malformation and undercalcification were apparent at both low and high magnesium concentrations. In contrast with the calcium results, coccoliths were flawless only in the normal seawater concentration of 58 mM magnesium. We conclude that photosynthesis and calcification are not closely coupled and that calcification depends on a precise balance of both calcium and magnesium concentrations.     

Isolation and Some Characterization of an Acidic Polysaccharide with Anti-calcification Activity from Coccoliths of a Marine Alga,Pleurochrysis carterae

Coccolith is a calcified scale with species-specific fine structure produced by marine unicellular coccolithophorid algae, and consists of calcium carbonate crystals and organic matrices. EDTA-soluble organic materials extracted from coccoliths of Pleurochrysis carterae showed anti-calcification activity. They were separated by anion-exchange HPLC, and two fractions, fractions A and B, were obtained. Fraction B, which was more active than fraction A, was further separated into six consecutive fractions, B1-B6, by second anion-exchange HPLC. ¹H NMR spectral analyses of these fractions suggested that a novel acidic polysaccharide, designated CMAP, existed throughout B1-B6 and that the latter four fractions mainly contained another acidic polysaccharide, PS-2, characterized previously. Since PS-2 did not show anti-calcification activity, CMAP was found to be the active principle.     

Coccolith production inHymenomonas carterae

Summary The formation of coccoliths inHymenomonas carterae was studied. It was found that the Golgi body was directly involved in the production of the baseplate scale, the organic matrix membrane and the deposition of calcium carbonate to produce the coccolith. The ICP body is no longer regarded as being involved in coccolith production but more with the getting rid of excess coccoliths within the cell. Because the ICP body contains acid hydrolases it is suggested that this organelle now be referred to as a residual body.Financial assistance from the Council for Scientific and Industrial Research is gratefully acknowledged.     

CALCIFICATION AND ITS INHIBITION IN COCCOLITHOPHORIDS1,2

The protein synthesis inhibitors cycloheximide and chloramphenicol and the transport inhibitors LaCl₃, oligoniycin, and ethacrynic acid were found to inhibit reversibly calcium uptake, coccolith formation, and cell division in the coccolithophorid Cricosphacra (Hymenomonas) carterae. With some, compounds, recovery of calcification was retarded, and in the case of oligomycin, incomplete. Glycerol, 0.5 M, partially reversed the inhibitory effect of chloramphenicol on calcification but not on division. Ouabain was without significant effect on calcium uptake but slowed division in 3 of 5 experiments. Ruthenium red inhibited, neither calcification nor division. In the absence of light, calcification did not occur.     

Action Spectrum of Coccolith Formation

The action spectrum of coccolith formation in Coccolithus huxleyi was determined by measuring the uptake of carbon-14 in coccoliths in four-hour experiments as a function of light intensity at each of seven wavelengths. An action spectrum | of photosynthetic carbon assimilation was obtained at the same time. The coccolith action spectrum had peaks at wavelengths of about 440 nm and 670 nm. probably corresponding to the regions of maximum cellular absorption and carbon assimilation. However, blue light appeared to be relatively more efficient in coccolith formation than in carbon assimilation. The results suggest that light-dependent coccolith formation may be catalyzed by two photochemical reactions, one mediated by chloroplast pigments and the other by some pigment absorbing specifically in the blue part of the spectrum.     

Effects of Ca and Mg on Growth and Calcification of the Coccolithophorid Pleurochrysis haptonemofera: Ca Requirement for Cell Division in Coccolith-Bearing Cells and for Normal Coccolith Formation with Acidic Polysaccharides

The effects of Ca²⁺ and Mg²⁺ on cellular growth and calcification in Pleurochrysis haptonemofera were investigated. In the presence of a normal concentration of Mg²⁺, coccolith-bearing cells (C-cells) required more than 0.5 mM Ca²⁺ for growth, while naked cells could grow even with 0.5 mM Ca²⁺. The calcification rate of C-cells, which was determined using decalcified cells, was significantly repressed with less than or equal to 0.5 mM Ca²⁺. Although the calcification rate did not change so much with 5–30 mM Ca²⁺, it decreased with higher concentrations of Ca²⁺, as well as C-cell-specific growth repression. Under these conditions, Ca²⁺ affected the rate of coccolith formation, but neither the coccolith morphology nor total amounts and ratios of divalent cations and acidic polysaccharides (Ph-PS-1, -2, and -3) were included in coccoliths. These findings suggest that sufficient calcification is required for the division of C-cells. Under low Ca²⁺ and high Mg²⁺ conditions, coccoliths with an abnormal morphology, having immature shield elements, were synthesized. Composition analysis of the coccoliths revealed high Mg/Ca and low Ph-PS-2/(Ph-PS-1 and -3) ratios, as compared with those under low Ca²⁺ and normal Mg²⁺ conditions, suggesting that the abnormal morphology is due to a change in the crystal type and/or acidic polysaccharide composition.     

Cold Stress Stimulates Intracellular Calcification by the Coccolithophore, Emiliania huxleyi (Haptophyceae) Under Phosphate-Deficient Conditions

Intracellular calcification by the coccolith-producing haptophyte Emiliania huxleyi (NIES 837) is regulated by various environmental factors. This study focused on the relationship between cold and phosphate-deficient stresses to elucidate how those factors control coccolith production. (45)Ca incorporation into coccoliths was more than 97% of the total (45)Ca incorporation by whole cells. In a batch culture, orthophosphate in the medium (final concentration, 28.7 muM) was rapidly depleted within 3 days, and then extracellular alkaline phosphatase (AP) activity, an indicator of phosphate deprivation, increased during the stationary growth phase. The increase in AP activity was slightly higher at 20 degrees C than at 12 degrees C. The calcification started to increase earlier than AP activity, and the increase was much higher at 12 degrees C than at 20 degrees C. Such enhancement of calcification was suppressed by the addition of phosphate, while AP activity was also suppressed after a transient increase. These results suggest that phosphate deprivation is a trigger for calcification and that a rather long induction period is needed for calcification compared to the increase in AP activity. While calcification was greatly stimulated by cold stress, other cellular activities such as growth, phosphate utilization, and the induction of AP activity were suppressed. The stimulation of coccolith production by cold stress was minimal under phosphate-sufficient conditions. The high calcification activity estimated by (45)Ca incorporation was confirmed by morphological observations of coccoliths on the cell surface under bright-field and polarization microscopy. These results indicate that phosphate deprivation is the primary factor for stimulating coccolith production, and cold stress is a secondary acceleration factor that stimulates calcification under conditions of phosphate deprivation.