Photosynthesis Research

, Volume 109, Issue 1–3, pp 205–221 | Cite as

Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana

  • Masaaki Tachibana
  • Andrew E. Allen
  • Sae Kikutani
  • Yuri Endo
  • Chris Bowler
  • Yusuke MatsudaEmail author
Regular Paper


It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the β-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1:YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two β-CA genes, P. tricornutum likely contains five α- and two γ-CA genes, whereas T. pseudonana has three α-, five γ-, four δ-, and one ζ-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO2 conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO2 and 5% CO2. However, mRNA levels of one putative α-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative α-CA (CA-1), ζ-CA (CA-3) and δ-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO2. Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm. These results suggest that CAs are constitutively present in the four chloroplastic membrane systems in P. tricornutum and that CO2 responsive CAs occur in the pyrenoid of P. tricornutum, and in the stroma and periplasm of T. pseudonana.


Marine diatom Carbonic anhydrase Inorganic carbon concentrating mechanism Pyrenoid 



Ribulose-1,5-bisphosphate carboxylase/oxygenase


Carbonic anhydrase


Chloroplast endoplasmic reticulum


Periplastidial compartment


Blob-like structure


Chloroplast envelope


Inorganic carbon concentrating mechanism


Transmission electron microscopy



We thank Ms. Nobuko Higashiuchi and Ms. Megumi Fujii for their technical assistance and Ms. Miyabi Inoue for her skilful secretarial assistance. This research was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Kwansei Gakuin University, Research Center for Environmental Bioscience), and by Steel Industry Foundation for the Advancement of Environmental Protection Technology (to Y. M.), and by funding from the National Science Foundation (NSF-MCB-1024913, NSF OCE-0727997, and NSF OCE-0722374) to AEA.

Supplementary material

11120_2011_9634_MOESM1_ESM.eps (978 kb)
Supplementary material 1 (EPS 978 kb)
11120_2011_9634_MOESM2_ESM.doc (60 kb)
Supplementary material 2 (DOC 59 kb)
11120_2011_9634_MOESM3_ESM.doc (62 kb)
Supplementary material 3 (DOC 62 kb)
11120_2011_9634_MOESM4_ESM.doc (40 kb)
Supplementary material 4 (DOC 39 kb)
11120_2011_9634_MOESM5_ESM.doc (45 kb)
Supplementary material 5 (DOC 45 kb)


  1. Armbrust EV et al (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306:79–86PubMedCrossRefGoogle Scholar
  2. Badger MR, Price GD (1989) Carbonic anhydrase activity associated with the cyanobacterium Synechococcus PCC7942. Plant Physiol 89:51–60PubMedCrossRefGoogle Scholar
  3. Badger MR, Price GD (1994) The role of carbonic anhydrase in photosynthesis. Ann Rev Plant Physiol Plant Mol Biol 45:369–392CrossRefGoogle Scholar
  4. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795PubMedCrossRefGoogle Scholar
  5. Bowler C et al (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456:239–244PubMedCrossRefGoogle Scholar
  6. Burkhardt S, Amoroso G, Riebesell U, Sültemeyer D (2001) CO2 and HCO3 uptake in marine diatoms acclimated to different CO2 concentrations. Limnol Oceanogr 46:1378–1391CrossRefGoogle Scholar
  7. Cavalier-Smith T (2000) Membrane heredity and early chloroplast evolution. Trends Plant Sci 5:174–182PubMedCrossRefGoogle Scholar
  8. Colman B, Rotatore C (1995) Photosynthetic inorganic carbon uptake and accumulation in two marine diatoms. Plant Cell Env 18:919–924CrossRefGoogle Scholar
  9. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016PubMedCrossRefGoogle Scholar
  10. Eriksson M, Karlsson J, Ramazanov Z, Gardeström P, Samuelsson G (1996) Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 93:12031–12034PubMedCrossRefGoogle Scholar
  11. Eriksson M, Villand P, Gardeström P, Samuelsson G (1998) Induction and regulation of expression of a low-CO2-induced mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol 116:637–641PubMedCrossRefGoogle Scholar
  12. Falkowski PG, Barber RT, Smetacek VV (1998) Biogeochemical controls and feedbacks on ocean primary production. Science 281:200–207PubMedCrossRefGoogle Scholar
  13. Field CB, Behrenfeld MJ, Randerson JT, Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281:237–240PubMedCrossRefGoogle Scholar
  14. Fukuzawa H, Suzuki E, Komukai Y, Miyachi S (1992) A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942. Proc Natl Acad Sci USA 89:4437–4441PubMedCrossRefGoogle Scholar
  15. Funke RP, Kovar JL, Weeks DP (1997) Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1. Plant Physiol 114:237–244PubMedCrossRefGoogle Scholar
  16. Gibbs SP (1981) The chloroplast endoplasmic reticulum: structure, function and evolutionary significance. Int Rev Cytol 72:49–99CrossRefGoogle Scholar
  17. Giordano M, Norici A, Forssen M, Eriksson M, Raven JA (2003) An anaplerotic role for mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol 132:2126–2134PubMedCrossRefGoogle Scholar
  18. Gould SB, Sommer MS, Hadfi K, Zauner S, Kroth PG, Maier UG (2006a) Protein targeting into the complex plastid of cryptophytes. J Mol Evol 62:674–681PubMedCrossRefGoogle Scholar
  19. Gould SB, Sommer MS, Kroth PG, Gile GH, Keeling PJ, Maier UG (2006b) Nucleus-to-nucleus gene transfer and protein retargeting into a remnant cytoplasm of cryptophytes and diatoms. Mol Biol Evol 23:2413–2422PubMedCrossRefGoogle Scholar
  20. Gruber A, Vugrinec S, Hempel F, Gould SB, Maier UG, Kroth PG (2007) Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif. Plant Mol Biol 64:519–530PubMedCrossRefGoogle Scholar
  21. Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can J Microbiol 8:229–239PubMedCrossRefGoogle Scholar
  22. Harada H, Matsuda Y (2005) Identification and characterization of a new carbonic anhydrase in the marine diatom Phaeodactylum tricornutum. Can J Bot 83:909–916CrossRefGoogle Scholar
  23. Harada H, Nakatsuma D, Ishida M, Matsuda Y (2005) Regulation of the expression of intracellular β-carbonic anhydrase in response to CO2 and light in the marine diatom Phaeodactylum tricornutum. Plant Physiol 139:1041–1050PubMedCrossRefGoogle Scholar
  24. Harrison PJ, Waters RE, Taylor FJR (1980) A broad spectrum artificial sea water medium for coastal and open ocean phytoplankton. J Phycol 16:28–35Google Scholar
  25. Hempel F, Bullmann L, Lau J, Zauner S, Maier UG (2009) ERAD-derived preprotein transport across the second outermost plastid membrane of diatoms. Mol Biol Evol 26:1781–1790PubMedCrossRefGoogle Scholar
  26. Hewett-Emmett D, Tashian RE (1996) Functional diversity, conservation, and convergence in the evolution of the α-, β-, and γ-carbonic anhydrase gene families. Mol Phylogenet Evol 5:50–77PubMedCrossRefGoogle Scholar
  27. Holdsworth ES, Colbeck J (1976) The pattern of carbon fixation in the marine unicellular alga Phaeodactylum tricornutum. Mar Biol 38:189–199CrossRefGoogle Scholar
  28. Johnston AM, Raven JA (1996) Inorganic carbon accumulation by the marine diatom Phaeodactylum tricornutum. Eur J Phycol 31:285–290CrossRefGoogle Scholar
  29. Karlsson J, Clarke AK, Chen ZY, Hugghins SY, Park YI, Husic HD, Moroney JV, Samuelsson G (1998) A novel α-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2. EMBO J 17:1208–1216PubMedCrossRefGoogle Scholar
  30. Keeling PJ (2004) Diversity and evolutionary history of plastids and their hosts. Am J Bot 91:1481–1493PubMedCrossRefGoogle Scholar
  31. Kilian O, Kroth PG (2005) Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids. Plant J 41:175–183PubMedCrossRefGoogle Scholar
  32. Kitao Y, Matsuda Y (2009) Formation of macromolecular complexes of carbonic anhydrases in the chloroplast of a marine diatom by the action of the C-terminal helix. Biochem J 419:681–688PubMedCrossRefGoogle Scholar
  33. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580PubMedCrossRefGoogle Scholar
  34. Kroth PG (2002) Protein transport into secondary plastids and the evolution of primary and secondary plastids. Int Rev Cytol 221:191–255PubMedCrossRefGoogle Scholar
  35. Kroth PG et al (2008) A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis. PLoS One 3:e1426PubMedCrossRefGoogle Scholar
  36. Kuchitsu K, Tsuzuki M, Miyachi S (1988) Characterization of the pyrenoid isolated from unicellular green alga Chlamydomonas reinhardtii: particulate form of RuBisCO protein. Protoplasma 144:17–24CrossRefGoogle Scholar
  37. Kuchitsu K, Tsuzuki M, Miyachi S (1991) Polypeptide composition and enzyme activities of the pyrenoid and its regulation by CO2 concentration in unicellular green algae. Can J Bot 69:1062–1069CrossRefGoogle Scholar
  38. Lane TW, Morel FMM (2000) A biological function for cadmium in marine diatoms. Proc Natl Acad Sci USA 97:4627–4631PubMedCrossRefGoogle Scholar
  39. Lane TW, Saito MA, George GN, Pickering IJ, Prince RC, Morel FMM (2005) A cadmium enzyme from marine diatom. Nature 435:42PubMedCrossRefGoogle Scholar
  40. Larkin MA et al (2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  41. Lichtlé C, McKay RML (1992) Immunogold localization of photosystem I and photosystem II light-harvesting complexes in cryptomonad thylakoids. Biol Cell 74:187–194CrossRefGoogle Scholar
  42. Long BM, Badger MR, Whitney SM, Price GD (2007) Analysis of carboxysomes from Synechococcus PCC7942 reveals multiple Rubisco complexes with carboxysomal proteins CcmM and CcaA. J Biol Chem 282:29323–29335PubMedCrossRefGoogle Scholar
  43. Matsuda Y, Hara T, Colman B (2001) Regulation of the induction of bicarbonate uptake by dissolved CO2 in the marine diatom, Phaeodactylum tricornutum. Plant Cell Env 24:611–620CrossRefGoogle Scholar
  44. McGinn PJ, Morel FMM (2008a) Expression and Inhibition of the carboxylating and decarboxylating enzymes in the photosynthetic C4 pathway of marine diatoms. Plant Physiol 146:300–309PubMedCrossRefGoogle Scholar
  45. McGinn PJ, Morel FMM (2008b) Expression and regulation of carbonic anhydrases in the marine diatom Thalassiosira pseudonana and in natural phytoplankton assemblages from Great Bay, New Jersey. Physiol Plant 133:78–91PubMedCrossRefGoogle Scholar
  46. Mckay RML, Gibbs SP, Vaughn KC (1991) Rubisco activase is present in the pyrenoid of green algae. Protoplasma 162:38–45CrossRefGoogle Scholar
  47. Mitra M, Lato SM, Ynalvez RA, Xiao Y, Moroney JV (2004) Identification of a new chloroplast carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol 135:173–182PubMedCrossRefGoogle Scholar
  48. Montsant A, Jabbari K, Maheswari U, Bowler C (2005) Comparative genomics of the pennate diatom Phaeodactylum tricornutum. Plant Physiol 137:500–513PubMedCrossRefGoogle Scholar
  49. Moroney JV, Chen ZY (1998) The role of the chloroplast in inorganic carbon uptake by eukaryotic algae. Can J Bot 76:1025–1034Google Scholar
  50. Moroney JV, Bartlett SG, Samuelsson G (2001) Carbonic anhydrases in plants and algae. Plant Cell Env 24:141–153CrossRefGoogle Scholar
  51. Moustafa A, Beszteri B, Maier UG, Bowler C, Valentin K, Bhattacharya D (2009) Genomic footprints of a cryptic plastid endosymbiosis in diatoms. Science 324:1724–1726PubMedCrossRefGoogle Scholar
  52. Nassoury N, Fritz L, Morse D (2001) Circadian changes in ribulose-1,5-bisphosphate carboxylase/oxygenase distribution inside individual chloroplasts can account for the rhythm in dinoflagellate carbon fixation. Plant Cell 13:923–934PubMedCrossRefGoogle Scholar
  53. Nielsen H, Krogh A (1998) Prediction of signal peptides and signal anchors by a hidden Markov model. In: Proceedings of the sixth international conference on intelligent systems for molecular biology (ISMB 6). AAAI Press, Menlo Park, California, pp 122–130Google Scholar
  54. Peña KL, Castel SE, de Araujo C, Espie GS, Kimber MS (2010) Structural basis of the oxidative activation of the carboxysomal γ-carbonic anhydrase, CcmM. Proc Natl Acad Sci USA 107:2455–2460PubMedCrossRefGoogle Scholar
  55. Poulsen N, Chesley PM, Kröger N (2006) Molecular genetic manipulation of the diatom Thalassiosira pseudonana (bacillariophyceae). J Phycol 42:1059–1065CrossRefGoogle Scholar
  56. Price GD, Badger MR (1989) Expression of human carbonic anhydrase in the cyanobacterium Synechococcus PCC7942 creates a high CO2-requiring phenotype: evidence for a central role for carboxysomes in the CO2 concentrating mechanism. Plant Physiol 91:505–513PubMedCrossRefGoogle Scholar
  57. Pronina NA, Semenenko VE (1984) Localization of membrane bound and soluble forms of carbonic anhydrase in the Chlorella cell. Fiziol Rast (Moscow) 31:241–251Google Scholar
  58. Raven JA (1997) CO2-concentrating mechanisms: a direct role for thylakoid lumen acidification? Plant Cell Env 20:147–154CrossRefGoogle Scholar
  59. Raven JA (2001) A role for mitochondrial carbonic anhydrase in limiting CO2 leakage from low CO2-grown cells of Chlamydomonas reinhardtii. Plant Cell Env 24:261–265CrossRefGoogle Scholar
  60. Reinfelder JR, Kraepiel AML, Morel FMM (2000) Unicellular C4 photosynthesis in a marine diatom. Nature 407:996–999PubMedCrossRefGoogle Scholar
  61. Reinfelder JR, Milligan AJ, Morel FMM (2004) The role of the C4 pathway in carbon accumulation and fixation in a marine diatom. Plant Physiol 135:2106–2111PubMedCrossRefGoogle Scholar
  62. Roberts SB, Lane TW, Morel FMM (1997) Carbonic anhydrase in the marine diatom Thalassiosira weissflogii (Bacillariophyceae). J Phycol 33:845–850CrossRefGoogle Scholar
  63. Roberts K, Granum E, Leegood RC, Raven JA (2007) C3 and C4 pathways of photosynthetic carbon assimilation in marine diatoms are under genetic, not environmental, control. Plant Physiol 145:230–235PubMedCrossRefGoogle Scholar
  64. Rost B, Riebesell U, Burkhardt S, Sültemeyer D (2003) Carbon acquisition of bloom-forming marine phytoplankton. Limnol Oceanogr 48:55–67CrossRefGoogle Scholar
  65. Satoh D, Hiraoka Y, Colman B, Matsuda Y (2001) Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom Phaeodactylum tricornutum. Plant Physiol 126:1459–1470PubMedCrossRefGoogle Scholar
  66. Smith KS, Ferry JG (2000) Prokaryotic carbonic anhydrases. FEMS Microbiol Rev 24:335–366PubMedCrossRefGoogle Scholar
  67. So AK, Espie GS, Williams EB, Shively JM, Heinhorst S, Cannon GC (2004) A novel evolutionary lineage of carbonic anhydrase (ε class) is a component of the carboxysome shell. J Bacteriol 186:623–630PubMedCrossRefGoogle Scholar
  68. Sonnhammer EL, von Heijne G, Krogh A (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6:175–182PubMedGoogle Scholar
  69. Tanaka Y, Nakatsuma D, Harada H, Ishida M, Matsuda Y (2005) Localization of soluble β-carbonic anhydrase in the marine diatom Phaeodactylum tricornutum. Sorting to the chloroplast and cluster formation on the girdle lamellae. Plant Physiol 138:207–217PubMedCrossRefGoogle Scholar
  70. Tripp BC, Smith K, Ferry JG (2001) Carbonic anhydrase: new insights for an ancient enzyme. J Biol Chem 276:48615–48618PubMedCrossRefGoogle Scholar
  71. Xu Y, Feng L, Jeffrey PD, Morel FMM (2008) Structure and metal exchange in the cadmium carbonic anhydrase of marine diatoms. Nature 452:56–61PubMedCrossRefGoogle Scholar
  72. Yamano T, Tsujikawa T, Hatano K, Ozawa S, Takahashi Y, Fukuzawa H (2010) Light and low-CO2-dependent LCIB-LCIC complex localization in the chloroplast supports the carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Cell Physiol 51:1453–1468PubMedCrossRefGoogle Scholar
  73. Zaslavskaia LA, Lippmeier JC, Kroth PG, Grossman AR, Apt KE (2000) Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. J Phycol 36:379–386CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Masaaki Tachibana
    • 1
  • Andrew E. Allen
    • 2
    • 3
  • Sae Kikutani
    • 1
  • Yuri Endo
    • 1
  • Chris Bowler
    • 2
  • Yusuke Matsuda
    • 1
    Email author
  1. 1.Department of Bioscience, School of Science and TechnologyKwansei Gakuin UniversitySandaJapan
  2. 2.Environmental and Evolutionary Genomics Section, Institut de Biologie de l’Ecole Normale Supérieure (IBENS)CNRS UMR8197 INSERM U1024, Ecole Normale SupérieureParisFrance
  3. 3.J. Craig Venter InstituteSan DiegoUSA

Personalised recommendations