IDENTIFICATION OF STRESS GENE TRANSCRIPTS IN LAMINARIA DIGITATA (PHAEOPHYCEAE) PROTOPLAST CULTURES BY EXPRESSED SEQUENCE TAG ANALYSIS 1 Vincent Roeder, Jonas Colle´n, Sylvie Rousvoal, Erwan Corre, Catherine Leblanc, and Catherine Boyen 2 Centre National de la Recherche Scientifique and Universite´ Pierre et MarieCurie, Station Biologique, BP 74, 29682 Roscoff cedex, France To characterize stress and defense-induced genes in the brown alga Laminaria digitata (Hudson) J.V. Lamouroux, 1985 expressed sequence tags (ESTs) were generated from L. digitata protoplasts. Com- parison of the ESTs with public databases allowed putative functions to be assigned to 45% of the se- quences. Comparison with ESTs from L. digitata sporophytes showed that protoplasts expressed more stress genes than intact thalli. Several tran- scripts in the stress gene class coded for proteins involved in cell protection against oxygen radicals, including thioredoxins (six ESTs), thioredoxin per- oxidases (two ESTs), and glutathione-S-transferase (GST) (41 ESTs). The GSTs appear to be part of the sigma class, making them the first GST sigma iden- tified in a photosynthetic organism. Other stress genes included a new type of vanadium-dependent bromoperoxidases (vBPO) showing 71% similarity with vBPOs previously identified in the sporophy- tic-thalli phase of L. digitata. The ESTs coding for 22 different mannuronan-C5-epimerases were iden- tified among the cell wall biosynthesis genes, and several ESTs showed similarity with the genome of the Ectocarpus siliculosus virus. Key index words: defense; expressed sequences tags; glutathione-S-transferase; Laminaria digitata; man- nuronan-C5-epimerase; protoplast; stress; vana- dium-dependent bromoperoxidase Abbreviations: EST, expressed sequence tag; EsV, Ectocarpus siliculosus virus; GST, glutathione-S- transferase; HSP, heat shock protein; Man-C5-E, mannuronan-C5-epimerase; ROS, reactive oxygen species; vBPO, vanadium-dependent bromoper- oxidase Laminaria digitata (Hudson) J.V. Lamouroux is a brown alga, and as such it belongs to the phylum Het- erokonta, a eukaryotic lineage that also includes dia- toms and oomycetes (Baldauf et al. 2003). The life cycle is heteromorphic and consists of a microscopic haploid filamentous phase, the gametophyte, and a macroscopic diploid phase, the sporophyte, which can attain a length of up to 2 m. The family Laminariales includes several economically important species and is thus an important marine resource. Some of its mem- bers can concentrate iodine to 30,000 times the con- centration in the surrounding seawater and are used as an iodine source in China (Saenko et al. 1978). Lamin- aria digitata has therefore been used as a model organ- ism to investigate halogen metabolism (Ku ¨pper et al. 1998). The Laminariales are also the primary source of alginates, anionic polysaccharides, which are the main component of the cell wall (30%–45% of dry weight), consisting of heteropolysaccharidic chains of mann- uronic and guluronic acid (Kloareg and Quatrano 1988). Despite their ecological, economic, and evolutionary importance, few studies have focused on brown algal genomes or genes. The haploid genome of L. digitata has been estimated at 640 Mbp (Le Gall et al. 1993) divided into 31 chromosomes (Lewis 1996). In parti- cular, the molecular biology of the metabolic pathways of iodine and alginate remains poorly understood. Similarly, the molecular bases of innate immunity have been barely explored. In a previous study, we described the generation of two expressed sequence tag (EST) catalogs from gametophytes and sporo- phytes of L. digitata (Cre ´pineau et al. 2000). Other studies have focused on genes and groups of genes, such as the light-harvesting complex multigenic family (De Martino et al. 2000), the mannuronan-C5-epi- merases (Man-C5-Es; Nyvall et al. 2003), and the va- nadium-dependent haloperoxidases (Colin et al. 2003, 2005). More recently, over 12,000 ESTs have been generated from another heterokont, the diatom Phaeo- dactylum tricornutum (Scala et al. 2002, Maheswari et al. 2005). Our previous EST work on L. digitata (Cre´pineau et al. 2000) revealed several genes of interest, such as vanadium-dependent bromoperoxidases (vBPOs), and genes involved in carbon metabolism (Moulin et al. 1999). However, one limitation of the approach used, which analyzed ESTs from plants growing under nor- mal conditions, was that most of the ESTs identified corresponded to house-keeping proteins, especially ribosomal proteins. The present study identified genes involved in stress responses and cell wall biosynthesis. Protoplasts were considered as a favorable experimen- tal tool for this type of molecular investigation. 1 Received 21 January 2005. Accepted 29 August 2005. 2 Author for correspondence: boyen@sb-roscoff.fr. 1227 J. Phycol. 41, 1227–1235 (2005) r 2005 Phycological Society of America DOI: 10.1111/j.1529-8817.2005.00150.x