Marine Biotechnology

, Volume 11, Issue 3, pp 327–333 | Cite as

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

  • Manami Satoh
  • Koji Iwamoto
  • Iwane Suzuki
  • Yoshihiro Shiraiwa
Original Article


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. 45Ca incorporation into coccoliths was more than 97% of the total 45Ca incorporation by whole cells. In a batch culture, orthophosphate in the medium (final concentration, 28.7 μM) 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°C than at 12°C. The calcification started to increase earlier than AP activity, and the increase was much higher at 12°C than at 20°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 45Ca 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.


Calcification Coccolith production Coccolithophore Cold stress Emiliania huxleyi Phosphate deprivation 



We thank Prof. I. Inouye and his lab at the University of Tsukuba for their kind support in microscopic observations. We also thank the staff at the Radioisotope Center of the University of Tsukuba for their kind help with the radioisotope experiments. This work was supported in part by a Sasakawa Scientific Research Grant from the Japan Science Society to MS (15-365M).


  1. Brassell SC, Eglinton G, Marlowe IT, Pflaumann U, Sarnthein M (1986) Molecular stratigraphy: a new tool for climatic assessment. Nature 320:129–133CrossRefGoogle Scholar
  2. Danbara A, Shiraiwa Y (1999) The requirement of selenium for the growth of marine coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanica and Helladosphaera sp (Prymnesiophyceae). Plant Cell Physiol 40:762–766Google Scholar
  3. de Vrind-de Jong EW, de Vrind JPM (1997) Algal deposition of carbonates and silicates. In: Banfield JF, Nealson KH (eds) Geomicrobiology: Interactions between Microbes and Minerals. The Mineralogical Society of America, Washington, DC, pp 267–307Google Scholar
  4. Dyhrman ST, Palenik B (1999) Phosphate stress in cultures and field populations of the dinoflagellate Prorocentrum minimum detected by a single-cell alkaline phosphatase assay. Appl Environ Microbiol 65:3205–3212PubMedGoogle Scholar
  5. Dyhrman ST, Palenik B (2003) Characterization of ectoenzyme activity and phosphate-regulated proteins in the coccolithophorid Emiliania huxleyi. J Plankton Res 25:1215–1225CrossRefGoogle Scholar
  6. Edvardsen B, Eikrem W, Green JC, Andersen RA, Moon-van der Staay SY, Medlin LK (2000) Phylogenetic reconstructions of the Haptophyta inferred from 18S ribosomal DNA sequences and available morphological data. Phycologia 39:19–35Google Scholar
  7. Fujiwara S, Tsuzuki M, Kawachi M, Minaka N, Inouye I (2001) Molecular phylogeny of the Haptophyta based on the rbcL gene and sequence variation in the spacer region of the RUBISCO operon. J Phycol 37:121–129CrossRefGoogle Scholar
  8. Guy C, Kaplan F, Kopka J, Selbig J, Hincha DK (2008) Metabolomics of temperature stress. Physiol Plant 132:220–235PubMedGoogle Scholar
  9. Hoppe HG (2003) Phosphatase activity in the sea. Hydrobiologia 493:187–200CrossRefGoogle Scholar
  10. Hurry VM, Malmberg G, Gardestöm P, Öquist G (1994) Effect of short-term shift to low temperature and of long-term cold hardening on photosynthesis and ribulose 1,5-bisphosphate carboxylase/oxygenase and sucrose phosphate synthase activity in leaves of winter rye (Secale cereale L.). Plant Physiol 106:983–990PubMedGoogle Scholar
  11. Landry DM, Gaasterland T, Palenik BP (2006) Molecular characterization of a phosphate-regulated cell-surface protein from the coccolithophorid, Emiliania huxleyi (Prymnesiophyceae). J Phycol 42:814–821CrossRefGoogle Scholar
  12. Linschooten C, van Bleijswijk JDL, van Emburg PR, de Vrind JPM, Kemper ES, Westberg P, de Vrind-de Jong EW (1991) Role of the light–dark cycle and medium composition on the production of coccoliths by Emiliania huxleyi (Haptophyceae). J Phycol 27:82–86CrossRefGoogle Scholar
  13. Los DA, Ray MK, Murata N (1997) Differences in the control of the temperature-dependent expression of four genes for desaturases in Synechocystis sp. PCC 6803. Mol Microbiol 25:1167–1175PubMedCrossRefGoogle Scholar
  14. Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27:31–36CrossRefGoogle Scholar
  15. Okada H, McIntyre A (1979) Seasonal distribution of modern coccolithophores in the western North Atlantic Ocean. Mar Biol 54:319–328CrossRefGoogle Scholar
  16. Paasche E (1998) Roles of nitrogen and phosphorous in coccolith formation in Emiliania huxleyi (Prymnesiophyceae). Eur J Phycol 33:33–42CrossRefGoogle Scholar
  17. Paasche E (2002) A review of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae), with particular reference to growth, coccolith formation, and calcification–photosynthesis interactions. Phycologia 40:503–529CrossRefGoogle Scholar
  18. Paasche E, Brubak S (1994) Enhanced calcification in the coccolithophorid Emiliania huxleyi (Haptophyceae) under phosphorus limitation. Phycologia 33:324–330Google Scholar
  19. Perry MJ (1972) Alkaline phosphatase activity in subtropical Central North Pacific waters using a sensitive fluorometric method. Mar Biol 15:113–119CrossRefGoogle Scholar
  20. Pienaar RN (1994) Ultrastructure and calcification of coccolithophores. In: Winter A, Siesser WG (eds) Coccolithophores. Cambridge University Press, Cambridge, UK, pp 13–37Google Scholar
  21. Prahl FG, Wakeham SG (1987) Calibration of unsaturation patterns in long-chain ketone compositions for palaeotemperature assessment. Nature 330:367–369CrossRefGoogle Scholar
  22. Rao IM, Terry N (1989) Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet: I. Changes in growth, gas exchange, and Calvin cycle enzymes. Plant Physiol 90:814–819PubMedCrossRefGoogle Scholar
  23. Reichardt W, Overbeck J, Steubing L (1967) Free dissolved enzymes in lake waters. Nature 216:1345–1347CrossRefGoogle Scholar
  24. Riegman R, Stolte W, Noordeloos AAM, Slezak D (2000) Nutrient uptake, and alkaline phosphatase (EC 3: 1: 3: 1) activity of Emiliania huxleyi (Prymnesiophyceae) during growth under N and P limitation in continuous cultures. J Phycol 36:87–96CrossRefGoogle Scholar
  25. Shiraiwa Y (2003) Physiological regulation of carbon fixation in the photosynthesis and calcification of coccolithophorids. Comp Biochem Physiol 136:775–783CrossRefGoogle Scholar
  26. Sorrosa JM, Satoh M, Shiraiwa Y (2005) Low temperature stimulates cell enlargement and intracellular calcification of coccolithophorids. Mar Biotechnol 7:128–133PubMedCrossRefGoogle Scholar
  27. van Bleijswijk JDL, Kempers RS, Veldhuis MJ (1994) Cell and growth characteristics of types A and B of Emiliania huxleyi (Prymnesiophyceae) as determined by flow cytometry and chemical analyses. J Phycol 30:230–241CrossRefGoogle Scholar
  28. Wada H, Gombos Z, Murata N (1994) Contribution of membrane lipids to the ability of the photosynthetic machinery to tolerate temperature stress. Proc Natl Acad Sci USA 91:4273–4277PubMedCrossRefGoogle Scholar
  29. Westbroek P, Young JR, Linschooten K (1989) Coccolith production (biomineralization) in the marine algae Emiliania huxleyi. J Protozool 36:368–373Google Scholar
  30. Winter A, Jordan RW, Roth PH (1994) Biogeography of living coccolithophores in ocean waters. In: Winter A, Siesser WG (eds) Coccolithophores. Cambridge University Press, Cambridge, UK, pp 161–177Google Scholar
  31. Xu Y, Wahlund TM, Feng L, Shaked Y, Morel FMM (2006) A novel alkaline phosphatase in the coccolithophore Emiliania huxleyi (Prymnesiophyceae) and its regulation by phosphorus. J Phycol 42:835–844CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Manami Satoh
    • 1
  • Koji Iwamoto
    • 1
  • Iwane Suzuki
    • 1
  • Yoshihiro Shiraiwa
    • 1
  1. 1.Graduate School of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan

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