Delphine Scornet

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.

The cell wall polysaccharide metabolism of the brown alga Ectocarpus siliculosus. Insights into the evolution of extracellular matrix polysaccharides in Eukaryotes

• Brown algal cell walls share some components with plants (cellulose) and animals (sulfated fucans), but they also contain some unique polysaccharides (alginates). Analysis of the Ectocarpus genome provides a unique opportunity to decipher the molecular bases of these crucial metabolisms. • An extensive bioinformatic census of the enzymes potentially involved in the biogenesis and remodeling of cellulose, alginate and fucans was performed, and completed by phylogenetic analyses of key enzymes. • The routes for the biosynthesis of cellulose, alginates and sulfated fucans were reconstructed. Surprisingly, known families of cellulases, expansins and alginate lyases are absent in Ectocarpus, suggesting the existence of novel mechanisms and/or proteins for cell wall expansion in brown algae. • Altogether, our data depict a complex evolutionary history for the main components of brown algal cell walls. Cellulose synthesis was inherited from the ancestral red algal endosymbiont, whereas the terminal steps for alginate biosynthesis were acquired by horizontal gene transfer from an Actinobacterium. This horizontal gene transfer event also contributed genes for hemicellulose biosynthesis. By contrast, the biosynthetic route for sulfated fucans is an ancestral pathway, conserved with animals. These findings shine a new light on the origin and evolution of cell wall polysaccharides in other Eukaryotes.

Global expression analysis of the brown alga Ectocarpus siliculosus (Phaeophyceae) reveals large-scale reprogramming of the transcriptome in response to abiotic stress

BackgroundBrown algae (Phaeophyceae) are phylogenetically distant from red and green algae and an important component of the coastal ecosystem. They have developed unique mechanisms that allow them to inhabit the intertidal zone, an environment with high levels of abiotic stress. Ectocarpus siliculosus is being established as a genetic and genomic model for the brown algal lineage, but little is known about its response to abiotic stress.ResultsHere we examine the transcriptomic changes that occur during the short-term acclimation of E. siliculosus to three different abiotic stress conditions (hyposaline, hypersaline and oxidative stress). Our results show that almost 70% of the expressed genes are regulated in response to at least one of these stressors. Although there are several common elements with terrestrial plants, such as repression of growth-related genes, switching from primary production to protein and nutrient recycling processes, and induction of genes involved in vesicular trafficking, many of the stress-regulated genes are either not known to respond to stress in other organisms or are have been found exclusively in E. siliculosus.ConclusionsThis first large-scale transcriptomic study of a brown alga demonstrates that, unlike terrestrial plants, E. siliculosus undergoes extensive reprogramming of its transcriptome during the acclimation to mild abiotic stress. We identify several new genes and pathways with a putative function in the stress response and thus pave the way for more detailed investigations of the mechanisms underlying the stress tolerance ofbrown algae.

Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus

Development of the sporophyte and gametophyte generations of the brown alga E. siliculosus involves two different patterns of early development, which begin with either a symmetric or an asymmetric division of the initial cell, respectively. A mutant, immediate upright (imm), was isolated that exhibited several characteristics typical of the gametophyte during the early development of the sporophyte generation. Genetic analyses showed that imm is a recessive, single-locus Mendelian factor and analysis of gene expression in this mutant indicated that the regulation of a number of life-cycle-regulated genes is specifically modified in imm mutant sporophytes. Thus, IMM appears to be a regulatory locus that controls part of the sporophyte-specific developmental programme, the mutant exhibiting partial homeotic conversion of the sporophyte into the gametophyte, a phenomenon that has not been described previously.

Auxin Metabolism and Function in the Multicellular Brown Alga Ectocarpus siliculosus

Ectocarpus siliculosus is a small brown alga that has recently been developed as a genetic model. Its thallus is filamentous, initially organized as a main primary filament composed of elongated cells and round cells, from which branches differentiate. Modeling of its early development suggests the involvement of very local positional information mediated by cell-cell recognition. However, this model also indicates that an additional mechanism is required to ensure proper organization of the branching pattern. In this paper, we show that auxin indole-3-acetic acid (IAA) is detectable in mature E. siliculosus organisms and that it is present mainly at the apices of the filaments in the early stages of development. An in silico survey of auxin biosynthesis, conjugation, response, and transport genes showed that mainly IAA biosynthesis genes from land plants have homologs in the E. siliculosus genome. In addition, application of exogenous auxins and 2,3,5-triiodobenzoic acid had different effects depending on the developmental stage of the organism, and we propose a model in which auxin is involved in the negative control of progression in the developmental program. Furthermore, we identified an auxin-inducible gene called EsGRP1 from a small-scale microarray experiment and showed that its expression in a series of morphogenetic mutants was positively correlated with both their elongated-to-round cell ratio and their progression in the developmental program. Altogether, these data suggest that IAA is used by the brown alga Ectocarpus to relay cell-cell positional information and induces a signaling pathway different from that known in land plants.

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.

Inheritance of organelles in artificial hybrids of the isogamous multicellular chromist alga Ectocarpus siliculosus (Phaeophyceae)

Our knowledge about the sexual transmission of mitochondria and plastids (hereafter organelles) in isogamous eukaryotes comes mostly from studies of the yeast Saccharomyces cerevisiae and the green alga Chlamydomonas reinhardtii which are both unicellular species. To investigate organelle inheritance in a multicellular organism with morphological isogamy, we studied the filamentous brown alga Ectocarpus siliculosus, in which each gamete contributes one plastid and at least one mitochondrion to the zygote. We crossed strains whose organelle genotypes were distinguishable by PCR. Hybrids contained only maternal mitochondrial genotypes, indicating uniparental inheritance of this organelle. In contrast, hybrids were chimerical for the plastid genome. In 64 to 75% of cases examined, the two zygotic plastids were partitioned into the two halves of the sporophyte, which developed from the zygote. A smaller number of hybrids deviated from this pattern, suggesting more complex mechanisms such as irregular division, segregation, or exchange of genetic material between plastids. E. siliculosus is the first isogamous heterokont eukaryote in which inheritance of organelles has been determined with molecular markers. The deterministic pseudo-Mendelian mechanism of plastid inheritance in E. siliculosus is unusual among eukaryotes.

Reinstatement of Ectocarpus crouaniorum Thuret in Le Jolis as a third common species of Ectocarpus (Ectocarpales, Phaeophyceae) in Western Europe, and its phenology at Roscoff, Brittany

Based on morphological characters, cross‐fertility and molecular systematics, two species are currently recognized in the ubiquitous temperate brown algal genus Ectocarpus: the type species E. siliculosus (Dillwyn) Lyngbye and E. fasciculatus Harvey. We studied diversity, cross‐fertility and ecology of Ectocarpus in megatidal areas in northwest France (Western Europe) and propose to reinstate a third species, E. crouaniorum Thuret in Le Jolis. Genotyping of 67 individuals from five localities, including the type locality of E. crouaniorum, using internal transcribed spacer 1 (ITS1) length as a marker, showed that the three species co‐occurred whenever the habitat was suitable. Our survey also revealed a single putative field hybrid between E. crouaniorum and E. siliculosus, and a single individual of a further Ectocarpus genotype. In laboratory experiments, E. crouaniorum was crossed with E. siliculosus and E. fasciculatus. In 12 of 13 crosses, the zygotes did not develop (postzygotic sterility); in one experiment a viable hybrid was produced after crossing a female E. crouaniorum with a male E. siliculosus, but this hybrid was unable to form meiospores. Phylogenetic analysis of five molecular markers from the nuclear, mitochondrial and plastid genomes (in total 1818 bp) confirmed genetic separation of the three species. Ecologically, E. crouaniorum was confined to high intertidal pools and run‐offs, where the gametophyte was common from spring to summer. Another characteristic was that it usually occurred as an epiphyte of up to 12 cm in length on erect thalli of Scytosiphon lomentaria. Sporophytes of E. crouaniorum were found all year long; they were <3 cm in size or microscopic and were epilithic in the same habitat. The presence of a third species of Ectocarpus in Western Europe suggests that species diversity in this genus is larger than recognized during the last 40 years.

Ectocarpus: A Model Organism for the Brown Algae

The brown algae are an interesting group of organisms from several points of view. They are the dominant organisms in many coastal ecosystems, where they often form large, underwater forests. They also have an unusual evolutionary history, being members of the stramenopiles, which are very distantly related to well-studied animal and green plant models. As a consequence of this history, brown algae have evolved many novel features, for example in terms of their cell biology and metabolic pathways. They are also one of only a small number of eukaryotic groups to have independently evolved complex multicellularity. Despite these interesting features, the brown algae have remained a relatively poorly studied group. This situation has started to change over the last few years, however, with the emergence of the filamentous brown alga Ectocarpus as a model system that is amenable to the genomic and genetic approaches that have proved to be so powerful in more classical model organisms such as Drosophila and Arabidopsis.

How to Cultivate Ectocarpus

This article describes the standard procedure for growing Ectocarpus in the laboratory. The culture is started with partheno-sporophyte (or sporophyte) filaments because this is the stage that is usually maintained in strain collections. The standard medium is Provasoli-enriched natural seawater (PES), but Ectocarpus can also be grown in artificial seawater, which allows more precise control over the culture conditions. The algae can be cultivated either in plastic Petri dishes or in 10-L bottles with bubbling, if large amounts of biomass are required. Standard growth conditions are 13°C with a 12h/12h d/night cycle and 20 µmol photons m(-2) s(-1) irradiance using daylight-type fluorescent tubes. All manipulations of Ectocarpus cultures should be performed in a clean environment (if possible, under a laminar flow hood). Forceps should be dipped in ethanol and allowed to dry under the hood.