ORIGINAL ARTICLE Mannitol-1-phosphate dehydrogenase activity in Ectocarpus siliculosus, a key role for mannitol synthesis in brown algae Sylvie Rousvoal • Agne `s Groisillier • Simon M. Dittami • Gurvan Michel • Catherine Boyen • Thierry Tonon Received: 4 August 2010 / Accepted: 26 September 2010 / Published online: 28 October 2010 Ó Springer-Verlag 2010 Abstract Mannitol represents a major end product of photosynthesis in brown algae (Phaeophyceae), and is, with the b-1,3-glucan laminarin, the main form of carbon stor- age 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 dehy- drogenase (M1PDH). This activity was measured in algal extracts, and was shown to be regulated by NaCl concen- tration 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 pre- pared 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. Keywords Brown algae  Ectocarpus  Enzymatic activity  Mannitol  Primary metabolism Abbreviations ANOVA Analysis of variance ASW Artificial seawater CCAP Culture collection of algae and protozoa dbEST Database of expressed sequence tags DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EGTA Ethylene glycol tetraacetic acid F6P Fructose-6-phosphate HCA Hydrophobic cluster analysis Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HK Hexokinase H 2 O 2 Hydrogen peroxide LB Luria–Bertani medium M1P Mannitol-1-phosphate M1Pase Mannitol-1-phosphatase M1PDH Mannitol-1-phosphate dehydrogenase S. Rousvoal and A. Groisillier contributed equally to this work. S. Rousvoal  A. Groisillier  S. M. Dittami  G. Michel  C. Boyen  T. Tonon UPMC Univ Paris 6, UMR 7139 Marine Plants and Biomolecules, Station Biologique, 29682 Roscoff, France S. Rousvoal  A. Groisillier  S. M. Dittami  G. Michel  C. Boyen  T. Tonon CNRS, UMR 7139 Marine Plants and Biomolecules, Station Biologique, 29682 Roscoff, France T. Tonon (&) UMR 7139 CNRS-UPMC, Station Biologique, Place Georges Teissier, 29682 Roscoff, France e-mail: tonon@sb-roscoff.fr 123 Planta (2011) 233:261–273 DOI 10.1007/s00425-010-1295-6