, Volume 233, Issue 2, pp 261–273 | Cite as

Mannitol-1-phosphate dehydrogenase activity in Ectocarpus siliculosus, a key role for mannitol synthesis in brown algae

  • Sylvie Rousvoal
  • Agnès Groisillier
  • Simon M. Dittami
  • Gurvan Michel
  • Catherine Boyen
  • Thierry Tonon
Original Article


Mannitol represents a major end product of photosynthesis in brown algae (Phaeophyceae), and is, with the β-1,3-glucan laminarin, the main form of carbon storage for these organisms. Despite its importance, little is known about the genes and enzymes responsible for the metabolism of mannitol in these seaweeds. Taking benefit of the sequencing of the Ectocarpus siliculosus genome, we focussed our attention on the first step of the synthesis of mannitol (reduction of the photo-assimilate fructose-6-phosphate), catalysed by the mannitol-1-phosphate dehydrogenase (M1PDH). This activity was measured in algal extracts, and was shown to be regulated by NaCl concentration in the reaction medium. Genomic analysis revealed the presence of three putative M1PDH genes (named EsM1PHD1, EsM1PDH2 and EsM1PDH3). Sequence comparison with orthologs demonstrates the modular architecture of EsM1PHD1 and EsM1PDH2, with an additional N-terminal domain of unknown function. In addition, gene expression experiments carried out on samples harvested through the diurnal cycle, and after several short-term saline and oxidative stress treatments, showed that EsM1PDH1 is the most highly expressed of these genes, whatever the conditions tested. In order to assess the activity of the corresponding protein, this gene was expressed in Escherichia coli. Cell-free extracts prepared from bacteria containing EsM1PDH1 displayed higher M1PDH activity than bacteria transformed with an empty plasmid. Further characterisation of recombinant EsM1PDH1 activity revealed its very narrow substrate specificity, salt regulation, and sensitivity towards an inhibitor of SH-enzymes.


Brown algae Ectocarpus Enzymatic activity Mannitol Primary metabolism 



Analysis of variance


Artificial seawater


Culture collection of algae and protozoa


Database of expressed sequence tags




Ethylenediaminetetraacetic acid


Ethylene glycol tetraacetic acid




Hydrophobic cluster analysis


4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid




Hydrogen peroxide


Luria–Bertani medium






Mannitol-1-phosphate dehydrogenase




3-(N-morpholino)propanesulfonic acid


National Center for Biotechnology Information




Practical salinity unit





SD received funding from the European community’s Sixth Framework Program (contract n° MESTCT 2005-020737). Part of this work was performed within the framework of the ‘Marine Genomics Europe’ NoE (Network of Excellence) (European Commission contract No. GOCE-CT-2004-505403).


  1. Akazaki H, Kawai F, Chida H, Matsumoto Y, Hirayama M, Hoshikawa K, Unzai S, Hakamata W, Nishio T, Park SY, Oku T (2008) Cloning, expression and purification of cytochrome c6 from the brown alga Hizikia fusiformis and complete X-ray diffraction analysis of the structure. Acta Cryst F64:674–680Google Scholar
  2. Bartsch I, Wiencke C, Bischof K, Buchholz CM, Buck BH, Eggert A, Feuerpfeil P, Hanelt D, Jacobsen S, Karez R et al (2008) The genus Laminaria sensu lato: recent insights and developments. Eur J Phycol 43:1–86CrossRefGoogle Scholar
  3. Callebaut I, Labesse G, Durand P, Poupon A, Canard L, Chomilier J, Henrissat B, Mornon JP (1997) Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives. Cell Mol Life Sci 53:621–645CrossRefPubMedGoogle Scholar
  4. Charrier B, Coelho SM, Le Bail A, Tonon T, Michel G, Potin P, Kloareg B, Boyen C, Peters AF, Cock JM (2008) Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research. New Phytol 177:319–332CrossRefPubMedGoogle Scholar
  5. Cock JM, Sterck L, Rouzé P, Scornet D, Allen AA, Amoutzias G, Anthouard V, Artiguenave F, Aury JM, Badger JH et al (2010) The Ectocarpus genome and the independent evolution of multicellularity in the brown algae. Nature 465:617–621CrossRefPubMedGoogle Scholar
  6. Davidson IR, Reed RH (1985) The physiological significance of mannitol accumulation in brown algae: the role of mannitol as a compatible cytoplasmic solute. Phycologia 24:449–457CrossRefGoogle Scholar
  7. de Franco PO, Rousvoal S, Tonon T, Boyen C (2009) Whole genome survey of the glutathione transferase family in the brown algal model Ectocarpus siliculosus. Mar Genom 1:135–148CrossRefGoogle Scholar
  8. Dittami SM, Scornet D, Petit JL, Ségurens B, Da Silva C, Corre E, Dondrup M, Glatting KH, König R, Sterck L et al (2009) Global expression analysis of the brown alga Ectocarpus siliculosus (Phaeophyceae) reveals large-scale reprogramming of the transcriptome in response to abiotic stress. Genome Biol 10:R66CrossRefPubMedGoogle Scholar
  9. Eggert A, Raimund S, Van Den Daele K, Karsten U (2006) Biochemical characterization of mannitol metabolism in the unicellular red alga Dixoniella grisea (Rhodellophyceae). Eur J Phycol 41:405–413CrossRefGoogle Scholar
  10. Eggert A, Raimund S, Michalik D, West J, Karsten U (2007) Ecophysiological performance of the primitive red alga Dixoniella grisea (Rhodellophyceae) to irradiance, temperature and salinity stress: growth responses and the osmotic role of mannitol. Phycologia 46:22–28CrossRefGoogle Scholar
  11. Gravot A, Dittami SM, Rousvoal S, Lugan R, Eggert A, Collén J, Boyen C, Bouchereau A, Tonon T (2010) Diurnal oscillations of metabolite abundance and gene analysis provide new insights into central metabolic processes of the brown alga Ectocarpus siliculosus. New Phytol 188:98–110CrossRefPubMedGoogle Scholar
  12. Groisillier A, Hervé C, Jeudy A, Rebuffet E, Pluchon PF, Chevolot Y, Flament D, Geslin C, Morgado IM, Power D et al (2010) MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms. Microb Cell Fact 9:45CrossRefPubMedGoogle Scholar
  13. Heesch S, Cho GY, Peters AF, Le Corguillé G, Falentin C, Boutet G, Coëdel S, Jubin C, Samson G, Corre E et al (2010) A sequence-tagged genetic map for the brown alga Ectocarpus siliculosus provides large-scale assembly of the genome sequence. New Phytol 188:42–51CrossRefPubMedGoogle Scholar
  14. Hellebust JA (1976) Effect of salinity on photosynthesis and mannitol synthesis in the green flagellate Platymonas suecica. Can J Bot 54:1735–1741CrossRefGoogle Scholar
  15. Hervé C, de Franco PO, Groisillier A, Tonon T, Boyen C (2008) New members of the glutathione transferase family discovered in red and brown algae. Biochem J 412:535–544CrossRefPubMedGoogle Scholar
  16. Ikawa T, Watanabe T, Nisizawa K (1972) Enzymes involved in the last steps of the biosynthesis of mannitol in brown algae. Plant Cell Physiol 13:1017–1029Google Scholar
  17. Iwamoto K, Shiraiwa Y (2005) Salt-regulated mannitol metabolism in algae. Mar Biotech 7:407–415CrossRefGoogle Scholar
  18. Iwamoto K, Kawanobe H, Shiraiwa Y, Ikawa T (2001) Purification and characterization of mannitol-l-phosphatase in the red alga Caloglossa continua (Ceramiales, Rhodophyta). Mar Biotech 3:493–500CrossRefGoogle Scholar
  19. Iwamoto K, Kawanobe H, Ikawa T, Shiraiwa Y (2003) Characterization of salt-regulated mannitol-1-phosphate dehydrogenase in the red alga Caloglossa continua. Plant Physiol 133:893–900CrossRefPubMedGoogle Scholar
  20. Jiang W, Wu LF, Tomich J, Saier MH Jr, Niehaus WG (1990) Corrected sequence of the mannitol (mtl) operon in Escherichia coli. Mol Microbiol 4:2003–2006CrossRefPubMedGoogle Scholar
  21. Karsten U, West JA (1993) Ecophysiological studies on six species of the mangrove red algal genus Caloglossa. Aust J Plant Physiol 20:729–739CrossRefGoogle Scholar
  22. Karsten U, West JA, Mostaert AS, King RJ, Barrow KD, Kirst GO (1992) Mannitol in the red alga genus Caloglossa (Harvey). Agardh J J Plant Physiol 140:292–297Google Scholar
  23. Karsten U, Barrow KD, Nixdorf O, West JA, King RJ (1997) Characterization of mannitol metabolism in the mangrove red alga Caloglossa leprieurii (Montagne). J Agardh Planta 201:173–178CrossRefGoogle Scholar
  24. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066CrossRefPubMedGoogle Scholar
  25. Kirst GO (1975) Correlation between content of mannitol and osmotic stress in the brackish-water alga Platymonas subcordiformis. Z Pflanzenphysiol 76:316–325Google Scholar
  26. Kirst GO (1989) Salinity tolerance of eukaryotic marine algae. Annu Rev Plant Physiol Plant Mol Biol 40:21–53Google Scholar
  27. Le Bail A, Dittami SM, de Franco PO, Rousvoal S, Cock JM, Tonon T, Charrier B (2008) Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus. BMC Mol Biol 9:75CrossRefPubMedGoogle Scholar
  28. Lesk AM (1995) NAD-binding domains of dehydrogenases. Curr Opin Struc Biol 5:775–783CrossRefGoogle Scholar
  29. Michel G, Tonon T, Scornet D, Cock JM, Kloareg B (2010) Central and storage carbon metabolism of the brown alga Ectocarpus siliculosus: insights into the origin and evolution of storage carbohydrates in Eukaryotes. New Phytol 188:67–81CrossRefPubMedGoogle Scholar
  30. Mostaert AS, Karsten U, King RJ (1995) Inorganic ions and mannitol in the red alga Caloglossa leprieurii (Ceramiales, Rhodophyta): response to salinity change. Phycologia 34:501–507CrossRefGoogle Scholar
  31. Pearson GA, Hoarau G, Lago-Leston A, Coyer JA, Kube M, Reinhardt R, Henckel K, Serrão ETA, Corre E, Olsen JA (2010) An expressed sequence tag analysis of the intertidal brown seaweeds Fucus serratus (L.) and F. vesiculosus (L.) (Heterokontophyta, Phaeophyceae) in response to abiotic stressors. Mar Biotech 12:195–213CrossRefGoogle Scholar
  32. Peters AF, Marie D, Scornet D, Kloareg B, Cock JM (2004) Proposal of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) as a model organism from brown algal genetics and genomics. J Phycol 40:1079–1088CrossRefGoogle Scholar
  33. Reed RH, Davison IR, Chudek JA, Foster R (1985) The osmotic role of mannitol in the Phaeophyta–an appraisal. Phycologia 24:35–47CrossRefGoogle Scholar
  34. Richter DFE, Kirst GO (1987) d-Mannitol dehydrogenase and d-mannitol-1-phosphate dehydrogenase in Platymonas subcordformis: some characteristics and their role in osmotic adaptation. Planta 170:528–534CrossRefGoogle Scholar
  35. Roeder V, Collén J, Rousvoal S, Corre E, Leblanc C, Boyen C (2005) Identification of stress genes from Laminaria digitata (Phaeophyceae) protoplast cultures by expressed sequence tag analysis. J Phycol 41:1227–1235CrossRefGoogle Scholar
  36. Schmatz D (1997) The mannitol cycle in Eimeria. Parasitology 114:S81–S89PubMedGoogle Scholar
  37. Schneider KH, Giffhorn F, Kaplan S (1993) Cloning, nucleotide-sequence, and characterization of the mannitol dehydrogenase gene from Rhodobacter-sphaeroides. J Gen Microbiol 139:2475–2484PubMedGoogle Scholar
  38. Solomon PS, Waters ODC, Oliver RP (2007) Decoding the mannitol enigma in filamentous fungi. Trends Microbiol 15:257–262CrossRefPubMedGoogle Scholar
  39. Stoop JMH, Williamson JD, Pharr DM (1996) Mannitol metabolism in plants: a method for coping with stress. Trends Plant Sci 1:139–144CrossRefGoogle Scholar
  40. Studier FW (2005) Protein production by auto-induction in high density shaking culture. Protein Expr Purif 41:207–234CrossRefPubMedGoogle Scholar
  41. Thomas DN, Kirst GO (1991a) Differences in osmoacclimatation between sporophytes and gametophytes of the brown alga Ectocarpus siliculosus. Physiol Plant 83:218–289CrossRefGoogle Scholar
  42. Thomas DN, Kirst GO (1991b) Salt tolerance of Ectocarpus siliculosus (Dillw.) lyngb.: comparison of gametophytes, sporophytes and isolates of different geographic origin. Bot Acta 104:26–36Google Scholar
  43. Velez H, Glassbrook NJ, Daub ME (2007) Mannitol metabolism in the phytopathogenic fungus Alternaria alternuata. Fungal Genet Biol 44:258–268CrossRefPubMedGoogle Scholar
  44. Wisselink HW, Weusthuis RA, Eggink G, Hugenholtz J, Grobben GJ (2002) Mannitol production by lactic acid bacteria: a review. Int Dairy J 12:151–161CrossRefGoogle Scholar
  45. Wong TKM, Ho CL, Lee WW, Rahim RA, Phang SM (2007) Analyses of expressed sequence tags from Sargassum binderi (Phaeophyta). J Phycol 43:528–534CrossRefGoogle Scholar
  46. Wordern AZ, Lee JH, Mock T, Rouzé P, Simmons MP, Aerts A, Allen AE, Cuvelier ML, Derelle E, Everett MV et al (2009) Green evolution and dynamic adaptations revealed by genomes of the marine picoeukaryotes Micromonas. Science 324:268–274CrossRefGoogle Scholar
  47. Wright PJ, Chudeck JA, Foster R, Reed RH (1989) Turnover of the intracellular mannitol pool of Fucus spiralis L. (Fucales, Phaeophyta) during osmotic shock. J Exp Bot 40:1347–1353CrossRefGoogle Scholar
  48. Yamaguchi T, Ikawa T, Nisizawa K (1966) Incorporation of radioactive carbon from H14CO3 into sugar constituents by a brown alga, Eisenia bicyclis, during photosynthesis and its fate in the dark. Plant Cell Physiol 7:217–229Google Scholar
  49. Yamaguchi T, Ikawa T, Nisizawa K (1969) Pathway of mannitol formation during photosynthesis in brown algae. Plant Cell Physiol 10:425–440Google Scholar
  50. Zubia M, Payri C, Deslandes E (2008) Alginate, mannitol, phenolic compounds and biological activities of two range-extending brown algae, Sargassum mangarevense and Turbinaria ornate (Phaeophyta: Fucales), from Tahiti (French Polynesia). J Appl Phycol 20:1033–1043CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Sylvie Rousvoal
    • 1
    • 2
  • Agnès Groisillier
    • 1
    • 2
  • Simon M. Dittami
    • 1
    • 2
  • Gurvan Michel
    • 1
    • 2
  • Catherine Boyen
    • 1
    • 2
  • Thierry Tonon
    • 1
    • 2
    • 3
  1. 1.UPMC Univ Paris 6, UMR 7139 Marine Plants and BiomoleculesRoscoffFrance
  2. 2.CNRS, UMR 7139 Marine Plants and BiomoleculesRoscoffFrance
  3. 3.UMR 7139 CNRS-UPMCRoscoffFrance

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