Sylvie Rousvoal

The Ectocarpus genome and the independent evolution of multicellularity in brown algae

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.

Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.

IDENTIFICATION OF STRESS GENE TRANSCRIPTS IN LAMINARIA DIGITATA (PHAEOPHYCEAE) PROTOPLAST CULTURES BY EXPRESSED SEQUENCE TAG ANALYSIS1

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. Comparison of the ESTs with public databases allowed putative functions to be assigned to 45% of the sequences. Comparison with ESTs from L. digitata sporophytes showed that protoplasts expressed more stress genes than intact thalli. Several transcripts in the stress gene class coded for proteins involved in cell protection against oxygen radicals, including thioredoxins (six ESTs), thioredoxin peroxidases (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 identified 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 sporophytic‐thalli phase of L. digitata. The ESTs coding for 22 different mannuronan‐C5‐epimerases were identified among the cell wall biosynthesis genes, and several ESTs showed similarity with the genome of the Ectocarpus siliculosus virus.

Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus

BackgroundBrown algae are plant multi-cellular organisms occupying most of the world coasts and are essential actors in the constitution of ecological niches at the shoreline. Ectocarpus siliculosus is an emerging model for brown algal research. Its genome has been sequenced, and several tools are being developed to perform analyses at different levels of cell organization, including transcriptomic expression analyses. Several topics, including physiological responses to osmotic stress and to exposure to contaminants and solvents are being studied in order to better understand the adaptive capacity of brown algae to pollution and environmental changes. A series of genes that can be used to normalise expression analyses is required for these studies.ResultsWe monitored the expression of 13 genes under 21 different culture conditions. These included genes encoding proteins and factors involved in protein translation (ribosomal protein 26S, EF1alpha, IF2A, IF4E) and protein degradation (ubiquitin, ubiquitin conjugating enzyme) or folding (cyclophilin), and proteins involved in both the structure of the cytoskeleton (tubulin alpha, actin, actin-related proteins) and its trafficking function (dynein), as well as a protein implicated in carbon metabolism (glucose 6-phosphate dehydrogenase). The stability of their expression level was assessed using the Ct range, and by applying both the geNorm and the Normfinder principles of calculation.ConclusionComparisons of the data obtained with the three methods of calculation indicated that EF1alpha (EF1a) was the best reference gene for normalisation. The normalisation factor should be calculated with at least two genes, alpha tubulin, ubiquitin-conjugating enzyme or actin-related proteins being good partners of EF1a. Our results exclude actin as a good normalisation gene, and, in this, are in agreement with previous studies in other organisms.

The complete sequence of a brown algal mitochondrial genome, the ectocarpale Pylaiella littoralis (L.) Kjellm.

We describe here the complete sequence (58,507 bp) of the mitochondrial genome of the brown alga Pylaiella littoralis (Ectocarpales). This molecule displays an AT content of 62.0% and contains seventy-nine genes, most of them (73) encoded on one strand. They include the usual mitochondrial set of protist genes and a number of rarer genes. Among these, several ribosomal protein genes and the rn5 were identified. Twenty-four tRNA genes are present in this genome, insufficient to decode all genes. The other conspicuous features of this molecule are: a large (3018 nucleotides) in-frame insertion of unknown function in the cox2 gene; the presence of two different lineages of group II introns, including complete reverse transcriptase-like genes, one in the cox1 and the other in the rnl gene; the concomitant occurrence of a T7-like RNA polymerase and of several well-conserved α-proteobacterial-type promoters; and a small nad11 gene, coding for the first domain only of this NADH dehydrogenase subunit. Altogether, the mitochondrial genome of P. littoralis exhibits both α-proteobacterial characteristics and evidences of the independent integration of several exogenous DNA fragments.

Characterization of Mannuronan C-5-Epimerase Genes from the Brown Alga Laminaria digitata

Alginate is an industrially important polysaccharide obtained commercially by harvesting brown algae. The final step in alginate biosynthesis, the epimerization of β-1,4-d-mannuronic acid to α-1,4-l-guluronic acid, a structural change that controls the physicochemical properties of the alginate, is catalyzed by the enzyme mannuronan C-5-epimerase. Six different cDNAs with homology to bacterial mannuronan C-5-epimerases were isolated from the brown alga Laminaria digitata (Phaeophyceae). Hydrophobic cluster analysis indicated that the proteins encoded by the L. digitata sequences have important structural similarities to the bacterial mannuronan C-5-epimerases, including conservation of the catalytic site. The expression of the C-5-epimerase genes was examined by northern-blot analysis and reverse transcriptase-polymerase chain reaction in L. digitata throughout a year. Expression was also monitored in protoplast cultures by northern and western blot, reverse transcriptase-polymerase chain reaction, and activity measurements. From both the structural comparisons and the expression pattern, it appears that the cDNAs isolated from L. digitata encode functional mannuronan C-5-epimerases. The phylogenetic relationships of the bacterial and brown algal enzymes and the inferences on the origin of alginate biosynthetic machinery are discussed.

An expressed sequence tag analysis of thallus and regenerating protoplasts of Chondrus crispus (Gigartinales, Rhodophyceae)

In order to identify genes involved in cell wall regeneration and stress responses in red algae, expressed sequence tags (ESTs) from protoplasts (2002 ESTs) and thalli (2052 ESTs) from the seaweed Chondrus crispus (Stackh.) were studied. Clustering gave 2291 non‐redundant sequences; 50% of the ESTs showed similarity (e<10−4) to known sequences. The fraction of stress‐related ESTs was five‐times higher in the protoplast library than in the thallus library. The ESTs that were statistically over‐represented in protoplasts included: glutathione S‐transferases, heat shock proteins, vanadium bromoperoxidase, and several genes of unknown function; in all 32 transcripts. Over‐represented genes in thallus included: NADH dehydrogenase, a peroxidase, and several genes of unknown function; in all 12 transcripts. In general, the ESTs from the two libraries were very different; for example, only 38% of contigs had members of both catalogues. The approach allowed the identification of numerous stress genes; including 23 different heat shock proteins and molecular chaperones, antioxidative enzymes, and several genes potentially involved in detoxification. Genes potentially involved in the construction of the cell wall or the extracellular matrix included α‐galactosidase, pullulanase, sulfohydrolase, and several sequences with von Willebrand factor type A domains with similarities to cochlin, integrin, and vitrin.

Isolation and characterization of microsatellite markers in the nuclear genome of the brown alga Laminaria digitata (Phaeophyceae)

© 1998 Blackwell Science Ltd, Molecular Ecology, 7, 1771Ð1788 Table 2 Cross-specific amplification of Mustelid primers. Size range of PCR product for each locus amplified in each species is given in base pairs. Numbers in parentheses indicate the number of alleles observed in N = 3 individuals of each species. PCR reactions resulting in no product are indicated by Ð, while PCR reactions producing multibanding patterns are denoted by . Locus GG-14 amplifies two microsatellite loci in otter (Lontra canadensis) and marten (Martes americana)

Towards deciphering dynamic changes and evolutionary mechanisms involved in the adaptation to low salinities in Ectocarpus (brown algae)

Colonizations of freshwater by marine species are rare events, and little information is known about the underlying mechanisms. Brown algae are an independent lineage of photosynthetic and multicellular organisms from which few species inhabit freshwater. As a marine alga that is also found in freshwater, Ectocarpus is of particular interest for studying the transition between these habitats. To gain insights into mechanisms of the transition, we examined salinity tolerance and adaptations to low salinities in a freshwater strain of Ectocarpus on physiological and molecular levels. We show that this isolate belongs to a widely distributed and highly stress-resistant clade, and differed from the genome-sequenced marine strain in its tolerance of low salinities. It also exhibited profound, but reversible, morphological, physiological, and transcriptomic changes when transferred to seawater. Although gene expression profiles were similar in both strains under identical conditions, metabolite and ion profiles differed strongly, the freshwater strain exhibiting e.g. higher cellular contents of amino acids and nitrate, higher contents of n-3 fatty acids, and lower intracellular mannitol and sodium concentrations. Moreover, several stress markers were noted in the freshwater isolate in seawater. This finding suggests that, while high stress tolerance and plasticity may be prerequisites for the colonization of freshwater, genomic alterations have occurred that produced permanent changes in the metabolite profiles to stabilize the transition.

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

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.