A reversible decrease in pH i induced by a brief reduction in extracellular pH o 10 min at pH o 6. As changes in pH o also affect extracellular C i speciation, we used a pulse of NH 4 Cl 10 mM, 10 min, Figure S7 to induce intracellular acidification while maintaining constant pH o .
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This resulted in a Inhibition of calcification continued for up to 2 h post-treatment, suggesting that down-regulation of the calcification machinery operates in response to disruption of pH i Figure 6B. Whilst indirect effects of pH o and NH 4 Cl treatments may contribute to the inhibition of calcification, the similar effects of different treatments imply a direct relationship between pH i homeostasis and calcification. A Calcification rate following manipulation of pH i. Coccolith production by decalcified C.
Upper left shows a scanning electron micrograph of a C. Note the appreciable lag in restoration of the initial calcification rate following acidification of the cytosol. Traces are normalized to initial rate 0— min. Mature coccoliths are arranged on the extracellular surface, surrounding the cell to form a coccosphere A. However, coccolith formation occurs within the intracellular Golgi-derived coccolith vacuole.
At normal seawater pH 8. Other membrane transporters yet to be characterised are likely involved in longer term maintenance of cytoplasmic pH F. In combination with the emerging genomic information, our data provide clear evidence for physiological features that are novel for photosynthetic eukaryotes. Our electrophysiological and molecular analyses lead us to propose the following model Figure 7. H v 1 homologues are not universally present in marine algae, being absent from the genomes of prasinophytes both Ostreococcus and Micromonas and the brown macroalga, Ectocarpus siliculosus , suggesting that these channels play specialised cellular roles in coccolithophores and diatoms.
Interestingly, a protein exhibiting weak similarity to EhH v 1 is also present in the genomes of the moss Physcomitrella patens and other land plants, although in these predicted proteins a conserved arginine in S4 corresponding to human R is replaced by a threonine residue. Analysis of the transmembrane domains of coccolithophore H v 1 proteins indicates that the acidic residues are broadly conserved, along with the arginine residues associated with voltage gating in S4. Understandably, much attention has been paid to the effects of a decrease in calcite saturation on the dissolution of extracellular coccoliths.
An understanding of the combined effects of ocean acidification on both calcite saturation and intracellular pH homeostasis is likely to be critical for unravelling the factors underlying the variation seen in laboratory studies of coccolithophore responses to ocean acidification. Over this time period the oceans have remained supersaturated with regard to calcite, although surface ocean pH likely varied within the range of pH 7.
The sensitivity of calcification to transient changes in cytoplasmic pH is also evident from our results. Before electrophysiological and optical recordings, cells were decalcified with brief EGTA treatment followed by trituration as previously described  , which removed the external calcite coccosphere and body scales. This brief treatment did not affect subsequent cellular calcification and growth rates  , .
The recording chamber volume was 1. Specific ionic compositions of bath and pipette solutions were chosen to give optimal buffering or pH responses and are given in Table S2 and in the figure legends. Liquid junction potentials were calculated using the junction potential tool in Clampex Molecular Devices, Sunnyvale, CA and corrected off-line. Current voltage relations were determined on leak subtracted families by measuring the maximum steady state amplitude averaging between 10 and 50 ms of the current trace.
Reversal potentials were determined by manually measuring the peak tail currents of leak subtracted families of traces and calculating a linear regression versus test voltage. Decalcified C. For each excitation wavelength, the average fluorescence intensity was determined for a region of interest encompassing the whole cell and used to calculate the ratio. Background fluorescence was minimal and was not subtracted. We were unable to achieve a satisfactory calibration for ester-loaded cells using the nigericin technique as BCECF fluorescence was not stable in C.
Location of intracellular coccoliths was determined by imaging in reflectance mode nm excitation of the confocal microscope. To confirm expression and the coding sequence of these genes, 1. Due to the high GC content E. These inserts were subcloned into pcDNA3. For electrophysiology, HEK cells were transiently co-transfected with 1. HEK cells transfected with the GFP-fusions demonstrated localisation to both the plasma membrane and endomembranes for both proteins and exhibited very similar currents to the non-fusion proteins during patch-clamp recordings.
All mutagenesis products were confirmed by DNA sequencing. Amino acid sequences of proteins were aligned using ClustalW. For the phylogenetic analysis, an alignment was constructed based on the conserved residues surrounding the four transmembrane domains.
Maximum likelihood phylogenetic analysis was performed using PhyML within the Bosque software package  , based on the JTT substitution matrix . One hundred bootstrap replicates were performed. The intracellular and extracellular solutions were based on those used by Sasaki et al. Calcification rate in C. The change in grey scale image intensity, which is proportional to production of birefringent calcite, was determined using LSM Image Examiner software Zeiss. Light micrographs of calcified and decalcified C. Top panel are calcified cells.
Lower panel cells have been decalcified in buffered EGTA artificial seawater. Note in the lower panel a patch clamp electrode attached to the decalcified cell containing a mature intracellular coccolith. The effect of pH i on C. Whole cell currents from C. The pH of the external ASW solution was 8. Pipette solution was mM K-glutamate pH 7. Hyperpolarisation of the C. Simultaneous patch clamp and pH imaging was performed in order to examine the effect of hyperpolarisation on pH i. A representative of three replicate experiments is shown. Internal and external solutions are as used in Figure 5A P1b, E1.
Determination of in vivo calcification rate by cross-polarized light microscopy. The figure shows the increase in cross-polarized light intensity monitored as decalcified cells produce coccoliths and the resultant calcite accumulates in the field of view. Stills from the time-lapse video illustrate the increase in grey-scale intensity during the 20 h incubation. Initial cross-polarised light intensity level at the start of the plot is due to the presence of internal coccoliths which are not removed by the decalcification protocol.
The birefringence of calcite enables real time imaging of coccolith production. Birefringence in initial images is due to the presence of internal coccoliths which are not removed by the decalcification protocol. Manipulation of intracellular pH in C. Composition of electrophysiology solutions mM. In some experiments pH was adjusted to pH 6. For C. We thank Dr. Abstract Marine coccolithophorid phytoplankton are major producers of biogenic calcite, playing a significant role in the global carbon cycle.
Author Summary The production of calcium carbonate structures by marine organisms has a major influence on the Earth's carbon cycle and is responsible for the eventual formation of sedimentary rocks such as chalk and limestone. Introduction Coccolithophores represent a pan-global group of oceanic phytoplankton, often forming massive monospecific blooms in oceanic waters.
Download: PPT. Figure 1. Figure 2. Figure 3. Conservation of amino acid sequences between H v 1 orthologues. Figure 4. Figure 5. Figure 6. Calcification and pH i regulation in C. Figure 7. Model of the major ion fluxes associated with calcification and pH homeostasis in coccolithophores. Discussion In combination with the emerging genomic information, our data provide clear evidence for physiological features that are novel for photosynthetic eukaryotes.
Intracellular pH Measurement Decalcified C. Supporting Information. Figure S1. Figure S2. Figure S3. Figure S4. Figure S5. Figure S6. Figure S7. Table S1. Table S2. References 1. Saez A. R, et al. View Article Google Scholar 2.
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R Calcification in coccolithophores: a cellular perspective. Coccolithophores: from molecular processes to global impact. Berlin: Springer. Finally, based on global vertical distributions of TA and ancillary data, Feely et al. Hence, CaCO 3 dissolution is at present time acting as a biogeochemical pump for anthropogenic CO 2 , and that could be underestimated since biologically mediated dissolution above the saturation horizon is not accounted for, as discussed in Sabine and Mackenzie Recent investigations during a coccolithophorid bloom in the North Sea, have underlined the importance of the microbial food web in the transformation of dimethylsulfoniopropionate DMSP , the precursor of DMS, produced by phytoplankton Burkill et al.
In vitro DMSP-lyase activity was very high, but there was little evidence for high in situ activity Zubkov et al. The abundance and diversity of these bacteria in marine habitats have been shown to be closely linked to DMS producing phytoplanktonic blooms Gonzalez et al. Responses of ecosystems and coccolithophorid calcification to oceanic acidification Current model projections predict that surface ocean partial pressure of CO 2 pCO 2 levels will double over their pre-industrial values by the middle of this century, with accompanying surface ocean pH changes that are 3 times greater than those experienced during the transition from glacial to interglacial periods Falkowski et al.
In vitro experiments suggest that the repercussions of such acidification could be significant on the phytoplanktonic communities, ecosystems and carbon cycle. Indeed, even if some studies have shown that marine autotrophic communities are often insensitive to pCO 2 changes, several investigations have revealed that some seagrass Zimmerman et al.
It has also been shown that phytoplanktonic assemblages can experience marked shifts in composition under elevated pCO 2 conditions Boyd and Doney, , Tortell et al. In mesocosm experiments, raising CO 2 concentration enhances carbon export of coccolithophores, mainly due to the increase of TEP production and changes in the rain ratio Delille et al.
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Coccolithophores may respond in different ways to fossil fuel emissions and climate change over the next few centuries. Conversely the acidification of seawater could increase in situ CaCO 3 dissolution and thus the buffer capacity of the ocean. Comparison of global bloom maps from remote sensing suggests another possible response to global change. The main drivers of future changes in the coccolithophorid distribution and the associated feedback mechanisms need thus to be better evaluated.
For the time being, the large scale impacts of oceanic acidification on phytoplanktonic communities and carbon cycle particularly in relation to CO 2 sequestration remain to be investigated Gruber et al. References Alldredge A.
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A role for diatom-like silicon transporters in calcifying coccolithophores
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