Reduced calcification decreases photoprotective capability in the coccolithophorid Emiliania huxleyi

Intracellular calcification of coccolithophores generates CO? and consumes additional energy for acquisition of calcium and bicarbonate ions; therefore, it may correlate with photoprotective processes by influencing the energetics. To address this hypothesis, a calcifying Emiliania huxleyi strain (CS-369) was grown semi-continuously at reduced (0.1 mM, LCa) and ambient Ca2? concentrations (10 mM, HCa) for 150 d (>200 generations). The HCa-grown cells had higher photosynthetic and calcification rates and higher contents of Chl a and carotenoids compared with the naked (bearing no coccoliths) LCa-grown cells. When exposed to stressfull levels of photosynthetically active radiation (PAR), LCa-grown cells displayed lower photochemical yield and less efficient non-photochemical quenching (NPQ). When the LCa- or HCa-grown cells were inversely shifted to their counterpart medium, LCa to HCa transfer increased photosynthetic carbon fixation (P), calcification rate (C), the C/P ratio, NPQ and pigment contents, whereas those shifted from HCa to LCa exhibited the opposite effects. Increased NPQ, carotenoids and quantum yield were clearly linked with increased or sustained calcification in E. huxleyi. The calcification must have played a role in dissipating excessive energy or as an additional drainage of electrons absorbed by the photosynthetic antennae. This phenomenon was further supported by testing two non-calcifying strains, which showed insignificant changes in photosynthetic carbon fixation and NPQ when transferred to LCa conditions.     

Characterization of ectoenzyme activity and phosphate-regulated proteins in the coccolithophorid Emiliania huxleyi

Three phosphate-regulated proteins in the coccolithophorid Emilianiahuxleyi were detected by the biotinylation of cell-surface proteins.Two of these phosphate-regulated proteins have reduced denaturedmolecular weights near 110 000 Da (118 078 and 110 541, respectively),while the third, and most abundant, is 69 087 Da. Inductionof the three proteins and the common marker of phosphate stress,alkaline phosphatase activity, occur in the presence of <0.25µM inorganic phosphate in batch culture. Phosphate-regulatedproteins and enzyme activity differed among E. huxleyi strains.Alkaline phosphatase is an enzyme commonly induced by phytoplanktonin response to phosphate stress in order for cells to scavengeinorganic phosphate from organic sources. In E. huxleyi, thisenzyme activity and the phosphate-regulated proteins are rapidlylost when phosphate is added back to phosphate-stressed cultures.This contrasts with the slower loss of alkaline phosphataseactivity in the dinoflagellate Prorocentrum minimum. The presenceof the three phosphate-regulated proteins and enzyme activityappear to differ somewhat among E. huxleyi strains. Based onthese differences between strains, kinetic data, growth experimentsand enzyme activities, the 69 087 Da protein may be a phosphatasewith a high specificity for 5'-nucleotides.     

Polymorphic microsatellite loci in global populations of the marine coccolithophorid Emiliania huxleyi

The marine coccolithophorid Emiliania huxleyi is an important component of the marine carbon cycle because bloom development results in the export of calcium carbonate from the ocean surface to the abyss. Laboratory and field studies demonstrate significant biogeographical, ecological, physiological and morphological plasticity in E. huxleyi and suggest high underlying genetic variability. Here we describe seven polymorphic microsatellite loci from the E. huxleyi genome and their degree of polymorphism in clonal isolates of different geographical origin. Our results indicate a high degree of genetic diversity within E. huxleyi.     

Induction of phase variation events in the life cycle of the marine coccolithophorid Emiliania huxleyi

Emiliania huxleyi is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.     

The effect of growth temperature on the long-chain alkenes composition in the marine coccolithophorid Emiliania huxleyi

The hydrocarbon fraction of a pure culture of Emiliania huxleyi, composed of a mixture of C31, C33, C37 and C38 polyunsaturated n-alkenes, appeared strongly dependent on the growth temperature of the alga between 8 degrees C and 25 degrees C. The total hydrocarbon content increased linearly with decreasing temperatures. C37 and C38 alkenes (which accounted for more than 90% of the total hydrocarbons) showed distinct changes in distribution compared to C31 and C33 alkenes, suggesting different biological syntheses and/or functions for these two groups of compounds. C37 and C38 alkenes and C37 methyl ketones (alkenones) all showed a trend to lower proportions of the two diunsaturated isomers and to higher proportions of the corresponding trienes with decreasing temperature. Unlike the alkenone unsaturation ratio (U37k'), ratios based on the C37 and C38 alkadi- and trienes could be linearly related to the growth temperature of E. huxleyi only between 15 degrees C and 25 degrees C. The modifications in the distribution of alkenes induced by varying temperature appeared, however, to be twice as fast as the modifications undergone by the alkenones. Although structurally and biochemically related, the distinct evolutions of alkenes and alkenones in response to changes in growth temperature might indicate that these two classes of compounds play two distinct physiological functions. The non-systematic linearity of relationships to temperature of parameters based on alkenes distribution suggested that these compounds are of limited use as paleotemperature indicator in the marine environment in contrast with the alkenones.     

Gephyrocapsa huxleyi (Emiliania huxleyi) as a model system for coccolithophore biology

Coccolithophores are the most abundant calcifying organisms in modern oceans and are important primary producers in many marine ecosystems. Their ability to generate a cellular covering of calcium carbonate plates (coccoliths) plays a major role in marine biogeochemistry and the global carbon cycle. Coccolithophores also play an important role in sulfur cycling through the production of the climate-active gas dimethyl sulfide. The primary model organism for coccolithophore research is Emiliania huxleyi, now named Gephyrocapsa huxleyi. G. huxleyi has a cosmopolitan distribution, occupying coastal and oceanic environments across the globe, and is the most abundant coccolithophore in modern oceans. Research in G. huxleyi has identified many aspects of coccolithophore biology, from cell biology to ecological interactions. In this perspective, we summarize the key advances made using G. huxleyi and examine the emerging tools for research in this model organism. We discuss the key steps that need to be taken by the research community to advance G. huxleyi as a model organism and the suitability of other species as models for specific aspects of coccolithophore biology.     

Characterization of NADH: nitrate reductase from the coccolithophorid Emiliania huxleyi (Lohman) Hay & Mohler (Haptophyceae)

Abstract Nitrate reductase was purified from and characterized in a bloom-forming unicellular calcifying alga, Emiliania huxleyi (Haptophyceae). The molecular masses of the native form and the subunit were 514 and 85 kDa, respectively, showing that the enzyme is a hexamer composed of 6 homologous subunits. The K _m values for NADH and NO3− were 40 μM and 104 μM, respectively. Activity of the reduction of nitrate was very high with reduced methylviologen and NADH, but no activity was observed with NADPH or reduced flavin mononucleotide; oxidation of NADH was very high with cytochrome c but did not occur with ferricyanide. These results indicate that Emiliania nitrate reductase is NADH-specific (EC 1.6.6.1), and that among algae and plants its subunit structure and kinetic properties are unique.     

Accumulation and Utilization of Dissolved Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi

The mechanism for utilization of dissolved inorganic carbon(DIC) was investigated in the marine unicellular calcareousalga Emiliania huxleyi, grown with constant aeration. The apparentK_0.5 (DIC), the concentration of DIC which attains one-halfof the maximum velocity of apparent photosynthesis, for photosyntheticevolution of O₂, measured under saturating light, was 5.5 mM(55 µM for CO₂) at pH 8.0 and 25°C. The value of K_0.5was not affected by inhibitors of carbonic anhydrase (CA), andan electrometric assay of CA showed that the enzyme was notinvolved in photosynthesis in this alga. The rate of photosyntheticfixation of ¹⁴C-DIC into acid-stable products was about 20 timeshigher than that into CaCO₃, irrespective of the external concentrationof DIC. In short-term experiments, ¹⁴C-DIC was usually incorporatedinto the internal pool of DIC (IIC) to concentrations up to13 to 16 times higher than that of the external DIC. CO₂ addedexternally was utilized mainly for fixation of CO₂ and accumulationof IIC. By contrast, HCO^-₃ was utilized mainly for productionof CaCO₃ and accumulation of IIC. Incorporation of ¹⁴C intoIIC was partially suppressed by DCMU or in darkness but itstransfer to CaCO₃ was unaffected. These results suggest thataccumulation of IIC in this alga, even under ordinary circumstances,is only partially responsible for increasing the efficiencyof utilization of DIC by photosynthetic fixation but may bemost useful for the production of CaCO₃. (Hydroxyethylidene) bisphosphonic acid, an inhibitor of thegrowth of CaCO₃ crystals, completely suppressed production ofCaCO₃. The accumulation of IIC was also partially suppressed,but photosynthetic fixation of CO₂ was enhanced. In a pulse-chaseexperiment with ¹⁴CDIC, ¹⁴C incorporated into IIC and CaCO₃in darkness was transferred to acid-stable products of photosynthesisin the light. These results suggest that ¹⁴C-DIC in IIC andpre-formed CaCO₃ may be useful sources of carbon for fixationof CO₂. (Received July 2, 1993; Accepted January 10, 1994)     

Characterization of the selenite uptake mechanism in the coccolithophore Emiliania huxleyi (Haptophyta)

The marine coccolithophore Emiliania huxleyi (Haptophyta) requires selenium as an essential element for growth, and the active species absorbed is selenite, not selenate. This study characterized the selenite uptake mechanism using ??Se as a tracer. Kinetic analysis of selenite uptake showed the involvement of both active and passive transport processes. The active transport was suppressed by 0.5 mM vanadate, a membrane-permeable inhibitor of H?-ATPase, at pH 8.3. When the pH was lowered from 8.3 to 5.3, the selenite uptake activity greatly increased, even in the presence of vanadate, suggesting that the H? concentration gradient may be a motive force for selenite transport. [??Se]Selenite uptake at selenite-limiting concentrations was hardly affected by selenate, sulfate and sulfite, even at 100 μM. In contrast, 3 μM orthophosphate increased the K(m) 5-fold. These data showed that HSeO??, a dominant selenite species at acidic pH, is the active species for transport through the plasma membrane and transport is driven by ΔpH energized by H?-ATPase. Kinetic analysis showed that the selenite uptake activity was competitively inhibited by orthophosphate. Furthermore, the active selenite transport mechanism was shown to be induced de novo under Se-deficient conditions and induction was suppressed by the addition of either sufficient selenite or cycloheximide, an inhibitor of de novo protein synthesis. These results indicate that E. huxleyi cells developed an active selenite uptake mechanism to overcome the disadvantages of Se limitation in ecosystems, maintaining selenium metabolism and selenoproteins for high viability.     

Responses of the Emiliania huxleyi Proteome to Ocean Acidification

Ocean acidification due to rising atmospheric CO₂ is expected to affect the physiology of important calcifying marine organisms, but the nature and magnitude of change is yet to be established. In coccolithophores, different species and strains display varying calcification responses to ocean acidification, but the underlying biochemical properties remain unknown. We employed an approach combining tandem mass-spectrometry with isobaric tagging (iTRAQ) and multiple database searching to identify proteins that were differentially expressed in cells of the marine coccolithophore species Emiliania huxleyi (strain NZEH) between two CO₂ conditions: 395 (∼current day) and ∼1340 p.p.m.v. CO₂. Cells exposed to the higher CO₂ condition contained more cellular particulate inorganic carbon (CaCO₃) and particulate organic nitrogen and carbon than those maintained in present-day conditions. These results are linked with the observation that cells grew slower under elevated CO₂, indicating cell cycle disruption. Under high CO₂ conditions, coccospheres were larger and cells possessed bigger coccoliths that did not show any signs of malformation compared to those from cells grown under present-day CO₂ levels. No differences in calcification rate, particulate organic carbon production or cellular organic carbon: nitrogen ratios were observed. Results were not related to nutrient limitation or acclimation status of cells. At least 46 homologous protein groups from a variety of functional processes were quantified in these experiments, of which four (histones H2A, H3, H4 and a chloroplastic 30S ribosomal protein S7) showed down-regulation in all replicates exposed to high CO₂, perhaps reflecting the decrease in growth rate. We present evidence of cellular stress responses but proteins associated with many key metabolic processes remained unaltered. Our results therefore suggest that this E. huxleyi strain possesses some acclimation mechanisms to tolerate future CO₂ scenarios, although the observed decline in growth rate may be an overriding factor affecting the success of this ecotype in future oceans.     

Intragenomic Spread of Plastid-Targeting Presequences in the Coccolithophore Emiliania huxleyi

Nucleus-encoded plastid-targeted proteins of photosynthetic organisms are generally equipped with an N-terminal presequence required for crossing the plastid membranes. The acquisition of these presequences played a fundamental role in the establishment of plastids. Here, we report a unique case of two non-homologous proteins possessing completely identical presequences consisting of a bipartite plastid-targeting signal in the coccolithophore Emiliania huxleyi. We further show that this presequence is highly conserved in five additional proteins that did not originally function in plastids, representing de novo plastid acquisitions. These are among the most recent cases of presequence spreading from gene to gene and shed light on important evolutionary processes that have been usually erased by the ancient history of plastid evolution. We propose a mechanism of acquisition involving genomic duplications and gene replacement through non-homologous recombination that may have played a more general role for equipping proteins with targeting information.     

Increased coccolith production by Emiliania huxleyi cultures enriched with dissolved inorganic carbon

Cells of Emiliania huxleyi grown on Eppley's medium enriched with dissolved inorganic carbon (DIC) developed multiple layers of coccoliths. The maximum diameter of cells grown in the presence of 13.2 mM DIC was 12.3 m, whereas that of cells grown in the presence of 1.5 mM DIC was 8.0 m. Although enrichment of Eppley's medium with DIC increased both coccolith production and cell growth, coccolith production was enhanced to a greater extent than cell growth. The enrichment of Eppley's medium with DIC was used to enhance production of coccolith particles by E. huxleyi. Repeated-batch culture, in which DIC, Ca²⁺, nitrate and phosphate concentrations in the medium were maintained by replacing the culture medium, was carried out in a closed photobioreactor. During repeated-batch culture, a maximum coccolith yield of 560 mg/l for 2 days and a maximum biomass yield of 810 mg/l for 2 days were achieved. Enrichment and maintenance of DIC is therefore an efficient method for the production of large quantities of coccoliths.