DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus.

Brown algae are one of the most developmentally complex groups within the eukaryotes. As in many land plants and animals, their main body axis is established early in development, when the initial cell gives rise to two daughter cells that have apical and basal identities, equivalent to shoot and root identities in land plants, respectively. We show here that mutations in the Ectocarpus DISTAG (DIS) gene lead to loss of basal structures during both the gametophyte and the sporophyte generations. Several abnormalities were observed in the germinating initial cell in dis mutants, including increased cell size, disorganization of the Golgi apparatus, disruption of the microtubule network, and aberrant positioning of the nucleus. DIS encodes a TBCCd1 protein, which has a role in internal cell organization in animals, Chlamydomonas reinhardtii, and trypanosomes. Our study highlights the key role of subcellular events within the germinating initial cell in the determination of apical/basal cell identities in a brown alga and emphasizes the remarkable functional conservation of TBCCd1 in regulating internal cell organization across extremely distant eukaryotic groups.

Emission of volatile halogenated compounds, speciation and localization of bromine and iodine in the brown algal genome model Ectocarpus siliculosus.

This study explores key features of bromine and iodine metabolism in the filamentous brown alga and genomics model Ectocarpus siliculosus. Both elements are accumulated in Ectocarpus, albeit at much lower concentration factors (2-3 orders of magnitude for iodine, and < 1 order of magnitude for bromine) than e.g. in the kelp Laminaria digitata. Iodide competitively reduces the accumulation of bromide. Both iodide and bromide are accumulated in the cell wall (apoplast) of Ectocarpus, with minor amounts of bromine also detectable in the cytosol. Ectocarpus emits a range of volatile halogenated compounds, the most prominent of which by far is methyl iodide. Interestingly, biosynthesis of this compound cannot be accounted for by vanadium haloperoxidase since the latter have not been found to catalyze direct halogenation of an unactivated methyl group or hydrocarbon so a methyl halide transferase-type production mechanism is proposed.

Phototaxis and chemotaxis of brown algal swarmers.

Brown algae exhibit three patterns of sexual reproduction: isogamy, anisogamy, and oogamy. Unicellular swarmers including gametes and zoospores bear two heterogenous flagella, an anterior flagellum with mastigonemes (fine tripartite hairs) and a posterior one. In seawater, these flagellates usually receive physico-chemical signals for finding partners and good habitats. It is well known that brown algal swarmers change their swimming direction depending on blue light (phototaxis), and male gametes do so, based on the sex pheromones from female gametes (chemotaxis). In recent years, the comparative analysis of chemotaxis in isogamy, anisogamy, and oogamy has been conducted. In this paper, we focused on the phototaxis and chemotaxis of brown algal gametes comparing the current knowledge with our recent studies.

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.

Molecular phylogeny of Zeacarpa (Ralfsiales, Phaeophyceae) proposing a new family Zeacarpaceae and its transfer to Nemodermatales.

Zeacarpa leiomorpha is a crustose brown alga endemic to South Africa. The species has been tentatively placed in Ralfsiaceae, but its ordinal assignment has been uncertain. The molecular phylogeny of brown algae based on concatenated DNA sequences of seven chloroplast and mitochondrial gene sequences (atpB, psaA, psaB, psbA, psbC, rbcL, and cox1) of taxa covering most of the orders revealed the most related phylogenetic relationship of Z. leiomorpha to Nemoderma tingitanum (Nemodermatales) rather than Ralfsiaceae (Ralfsiales). Morphologically, Zeacarpa and Nemoderma share crustose thallus structure and multiple discoidal chloroplasts without pyrenoids in each cell, however, the formation of lateral unilocular zoidangia in tufts in loose upright filaments in Zeacarpa is distinctive in brown algae. Considering the relatively distant genetic divergence between the two taxa, comparable to that among families or orders in representative brown algae, in addition to the above-mentioned unique morphological features, we propose the classification of Zeacarpa in a new family Zeacarpaceae in the order Nemodermatales.

Optimization of volatile fatty acids and hydrogen production from Saccharina japonica: acidogenesis and molecular analysis of the resulting microbial communities.

Response surface methodology (RSM) was used to optimize the production of volatile fatty acids (VFAs) and hydrogen from mixed anaerobic cultures of Saccharina japonica with respect to two independent variables: methanogenic inhibitor concentration and temperature. The effects of four methanogenic inhibitors on acidogenic processes were tested, and qualitative microbial analyses were carried out. Escherichia, Acinetobacter, and Clostridium were the most predominant genera in samples treated with chloroform (CHCl3), iodoform (CHI3), 2-bromoethanesulfonate (BES), or ß-cyclodextrin (ß-CD), respectively. RSM showed that the production of VFAs reached a peak of 12.5 g/L at 38.6 °C in the presence of 7.4 g/L ß-CD; these were the conditions under which hydrogen production was also nearly maximal. The quantitative polymerase chain reaction (qPCR) showed that shifts in the bacterial community population correlated with the concentrations of ß-CD indicating that this compound effectively inhibited methanogens.

Plasmodesmata of brown algae.

Plasmodesmata (PD) are intercellular connections in plants which play roles in various developmental processes. They are also found in brown algae, a group of eukaryotes possessing complex multicellularity, as well as green plants. Recently, we conducted an ultrastructural study of PD in several species of brown algae. PD in brown algae are commonly straight plasma membrane-lined channels with a diameter of 10-20 nm and they lack desmotubule in contrast to green plants. Moreover, branched PD could not be observed in brown algae. In the brown alga, Dictyota dichotoma, PD are produced during cytokinesis through the formation of their precursor structures (pre-plasmodesmata, PPD). Clustering of PD in a structure termed "pit field" was recognized in several species having a complex multicellular thallus structure but not in those having uniseriate filamentous or multiseriate one. The pit fields might control cell-to-cell communication and contribute to the establishment of the complex multicellular thallus. In this review, we discuss fundamental morphological aspects of brown algal PD and present questions that remain open.

Identification of three proteins involved in fertilization and parthenogenetic development of a brown alga, Scytosiphon lomentaria.

Metabolic pathways of cell organelles may influence the expression of nuclear genes involved in fertilization and subsequent zygote development through a retrograde regulation. In Scytosiphon lomentaria, inheritance of chloroplast is biparental but mitochondria are maternally inherited. Male and female gametes underwent different parthenogenetic outcomes. Most (>99%) male gametes did not differentiate rhizoid cells or survived beyond four-cell stage, while over 95% of female gametes grew into mature asexual plants. Proteomic analysis showed that the protein contents of male and female gametes differed by approximately 1.7%, 12 sex-specific proteins out of 700 detected proteins. Three sex-specific proteins were isolated and identified using CAF-MALDI mass spectrometry and RACE-PCR. Among them, a male gamete-specific homoaconitate hydratase (HACN) and a female gamete-specific succinate semialdehyde dehydrogenase (SSADH) were predicted to be the genes involved in mitochondrial metabolic pathways. The expression level of both mitochondrial genes was dramatically changed at the fertilization event. During parthenogenetic development the male-specific HACN and GTP-binding protein were gradually down-regulated but SSADH stayed up-regulated up to 48h. To observe the effect of chemicals on the expression of these genes, male and female gametes were treated with γ-aminobutyric acid (GABA), hydrogen peroxide and L-ascorbic acid. Among them GABA treatment significantly reduced SSADH gene expression in female gamete but the same treatment induced high upregulation of the gene in male gamete. GABA treatment affected the behavior of gametes and their parthenogenetic development. Both gametes showed prolonged motile stage, retarded settlement and subsequent parthenogenetic development. Our results suggest that male and female gametes regulate mitochondrial metabolic pathways differentially during fertilization, which may be the reason for their physiological and behavioral differences.

Surface-bound iron: a metal ion buffer in the marine brown alga Ectocarpus siliculosus?

Although the iron uptake and storage mechanisms of terrestrial/higher plants have been well studied, the corresponding systems in marine algae have received far less attention. Studies have shown that while some species of unicellular algae utilize unique mechanisms of iron uptake, many acquire iron through the same general mechanisms as higher plants. In contrast, the iron acquisition strategies of the multicellular macroalgae remain largely unknown. This is especially surprising since many of these organisms represent important ecological and evolutionary niches in the coastal marine environment. It has been well established in both laboratory and environmentally derived samples, that a large amount of iron can be 'non-specifically' adsorbed to the surface of marine algae. While this phenomenon is widely recognized and has prompted the development of experimental protocols to eliminate its contribution to iron uptake studies, its potential biological significance as a concentrated iron source for marine algae is only now being recognized. This study used an interdisciplinary array of techniques to explore the nature of the extensive and powerful iron binding on the surface of both laboratory and environmental samples of the marine brown alga Ectocarpus siliculosus and shows that some of this surface-bound iron is eventually internalized. It is proposed that the surface-binding properties of E. siliculosus allow it to function as a quasibiological metal ion 'buffer', allowing iron uptake under the widely varying external iron concentrations found in coastal marine environments.

ETOILE regulates developmental patterning in the filamentous brown alga Ectocarpus siliculosus.

Brown algae are multicellular marine organisms evolutionarily distant from both metazoans and land plants. The molecular or cellular mechanisms that govern the developmental patterning in brown algae are poorly characterized. Here, we report the first morphogenetic mutant, étoile (etl), produced in the brown algal model Ectocarpus siliculosus. Genetic, cellular, and morphometric analyses showed that a single recessive locus, ETL, regulates cell differentiation: etl cells display thickening of the extracellular matrix (ECM), and the elongated, apical, and actively dividing E cells are underrepresented. As a result of this defect, the overrepresentation of round, branch-initiating R cells in the etl mutant leads to the rapid induction of the branching process at the expense of the uniaxial growth in the primary filament. Computational modeling allowed the simulation of the etl mutant phenotype by including a modified response to the neighborhood information in the division rules used to specify wild-type development. Microarray experiments supported the hypothesis of a defect in cell-cell communication, as primarily Lin-Notch-domain transmembrane proteins, which share similarities with metazoan Notch proteins involved in binary cell differentiation were repressed in etl. Thus, our study highlights the role of the ECM and of novel transmembrane proteins in cell-cell communication during the establishment of the developmental pattern in this brown alga.

Non-cell autonomous regulation of life cycle transitions in the model brown alga Ectocarpus.

The model brown alga Ectocarpus has a haploid-diploid life cycle, involving alternation between two independent multicellular generations, the gametophyte and the sporophyte. Recent work has shown that alternation of generations is not determined by ploidy but is rather under genetic control, involving at least one master regulatory locus, OUROBOROS (ORO). Using cell biology approaches combined with measurements of generation-specific transcript abundance we provide evidence that alternation of generations can also be regulated by non-cell autonomous mechanisms. The Ectocarpus sporophyte produces a diffusible factor that causes major developmental reprogramming in gametophyte cells. Cells become resistant to reprogramming when the cell wall is synthetized, suggesting that the cell wall may play a role in locking an individual into the developmental program that has been engaged. A functional ORO gene is necessary for the induction of the developmental switch. Our results highlight the role of the cell wall in maintaining the differentiated generation stage once the appropriate developmental program has been engaged and also indicate that ORO is a key member of the developmental pathway triggered by the sporophyte factor. Alternation between gametophyte and sporophyte generations in Ectocarpus is surprisingly labile, perhaps reflecting an adaptation to the variable seashore environment inhabited by this alga.

Cryopreservation of the model alga Ectocarpus (Phaeophyceae).

The brown alga Ectocarpus has recently become the first fully sequenced multicellular alga and is an important biological model. Due to the large and growing number of Ectocarpus strains isolated and maintained by the research community, including increasing numbers of mutants, there is an urgent need for developing reliable, cost-effective long-term maintenance techniques. We report here that cryopreservation constitutes an attractive option in this respect, using a simple two-step protocol employing combined DMSO 10 percent (v/v) and sorbitol 9 percent (w/v) as cryoprotectants. This model organism appears to be remarkably robust and post-cryo recovery has been observed in all strains tested in this study. Cultures can be regenerated by the germination of cryopreserved zooids (spores), or the recovery of vegetative cells. In the latter case, dividing surviving cells may grow into the cell lumen of a neighbouring dead cell, eventually regenerating a phenotypically normal thalloidal structure.

Discovery of a novel brown algal genus and species Setoutiphycus delamareoides (Phaeophyceae, Ectocarpales) from the Seto Inland Sea, Japan.

We describe a new genus and species of brown algae from the Seto Inland Sea, Japan. This species is similar to Delamarea in gross morphology and anatomy, but distinctive in having longer thalli with rare branching and shorter cortical cells. In culture, pluri-zoids derived from plurilocular zoidangia on the erect thalli developed into filamentous gametophytes bearing ectocarpoid plurilocular zoidangia, but also formed parenchymatous erect thalli of sub-sympodial growth similar to Trachynema often having branches, and formed lateral and terminal plurilocular zoidangia. Molecular phylogenies using concatenated chloroplast and mitochondrial gene sequences showed the new alga nested in the clade composed of ectocarpalean genera with diffuse growth, parenchymatous thalli, and multiple chloroplasts, but this species is distinctive. Therefore, we propose Setoutiphycus delamareoides gen. & sp. nov. for this new alga, and provisionally place it in Chordariaceae, Ectocarpales. The Seto Inland Sea repeatedly dried during sea level regressions during glacial periods, and the present sea level recovered after the last glacial maximums (LGM), ca. 10,000 years ago. Therefore, it is unlikely that the species evolved within this area. Its distribution in the area may be explained as a remnant population that survived in refugia in southern Japan during the LGM.

Membrane fusion process and assembly of cell wall during cytokinesis in the brown alga, Silvetia babingtonii (Fucales, Phaeophyceae).

During cytokinesis in brown algal cells, Golgi-derived vesicles (GVs) and flat cisternae (FCs) are involved in building the new cell partition membrane. In this study, we followed the membrane fusion process in Silvetia babingtonii zygotes using electron microscopy together with rapid freezing and freeze substitution. After mitosis, many FCs were formed around endoplasmic reticulum clusters and these then spread toward the future cytokinetic plane. Actin depolymerization using latrunculin B prevented the appearance of the FCs. Fusion of GVs to FCs resulted in structures that were thicker and more elongated (EFCs; expanded flat cisternae). Some complicated membranous structures (MN; membranous network) were formed by interconnection of EFCs and following the arrival of additional GVs. The MN grew into membranous sacs (MSs) as gaps between the MNs disappeared. The MSs were observed in patches along the cytokinetic plane. Neighboring MSs were united to form the new cell partition membrane. An immunocytochemical analysis indicated that fucoidan was synthesized in Golgi bodies and transported by vesicles to the future cytokinetic plane, where the vesicles fused with the FCs. Alginate was not detected until the MS phase. Incubation of sections with cellulase-gold showed that the cellulose content of the new cross wall was not comparable to that of the parent cell wall.

Role of endoreduplication and apomeiosis during parthenogenetic reproduction in the model brown alga Ectocarpus.

• The filamentous brown alga Ectocarpus has a complex life cycle, involving alternation between independent and morphologically distinct sporophyte and gametophyte generations. In addition to this basic haploid-diploid life cycle, gametes can germinate parthenogenetically to produce parthenosporophytes. This article addresses the question of how parthenosporophytes, which are derived from a haploid progenitor cell, are able to produce meiospores in unilocular sporangia, a process that normally involves a reductive meiotic division. • We used flow cytometry, multiphoton imaging, culture studies and a bioinformatics survey of the recently sequenced Ectocarpus genome to describe its life cycle under laboratory conditions and the nuclear DNA changes which accompany key developmental transitions. • Endoreduplication occurs during the first cell cycle in about one-third of parthenosporophytes. The production of meiospores by these diploid parthenosporophytes involves a meiotic division similar to that observed in zygote-derived sporophytes. By contrast, meiospore production in parthenosporophytes that fail to endoreduplicate occurs via a nonreductive apomeiotic event. • Our results highlight Ectocarpus's reproductive and developmental plasticity and are consistent with previous work showing that its life cycle transitions are controlled by genetic mechanisms and are independent of ploidy.

Microtubule biogenesis and cell shape in Ochromonas. I. The distribution of cytoplasmic and mitotic microtubules.

In the first of two companion papers which attempt to correlate microtubules and their nucleating sites with developmental and cell division patterns in the unicellular flagellate, Ochromonas, the distribution of cytoplasmic and mitotic microtubules and various kinetosome-related fibers are detailed. Of the five kinetosome-related fibers, which have been found in Ochromonas, two, the kineto-beak fibers and the rhizoplast fibers are utilized as attachment sites for distinct groups of microtubules. The set of microtubules attached to the kineto-beak fibers apparently shape the anterior beak region of the cell whereas the rhizoplast microtubules appear to extend into and shape the tail in vegetative cells. In mitotic cells a rhizoplast is found at each spindle pole apparently serving as foci for the spindle microtubules. These findings are discussed in relation to the less well defined attachment sites for vegetative and mitotic microtubules in other kinds of cells. It is noted that the effects of depolymerizing microtubules in vivo might be easily quantitated in whole populations since no external wall or pellicle contributes to the maintenance or the biogenesis of the characteristic cell form of Ochromonas.

Microtubule biogenesis and cell shape in Ochromonas. II. The role of nucleating sites in shape development.

The proposal made in the preceding paper that the species-specific shape of Ochromonas is mediated by cytoplasmic microtubules which are related to two nucleating sites has been experimentally verified. Exposure of cells to colchicine or hydrostatic pressure causes microtubule disassembly and a correlative loss of cell shape in a posterior to anterior direction. Upon removal of colchicine or release of pressure, cell shape regenerates and microtubules reappear, first in association with the kineto-beak site concomitant with regeneration of the anterior asymmetry, and later at the rhizoplast site concomitant with formation of the posterior tail. It is concluded that two separate sets of cytoplasmic tubules function in formation and maintenance of specific portions of the total cell shape. On the basis of the following observations, we further suggest that the beak and rhizoplast sites could exert control over the position and timing of the appearance, the orientation, and the pattern of microtubule distribution in Ochromonas. (a) the two sites are accurately positioned in the cell relative to other cell organelles; (b) in regenerating cells microtubules reform first at these sites and appear to elongate to the cell posterior; (c) microtubules initially reappear in the orientation characteristic of the fully differentiated cell; (d) the two sets of tubules are polymerized at different times, in the same sequence, during reassembly or resynthesis of the microtubular system. Experiments using cycloheximide, after a treatment with colchicine, have demonstrated that Ochromonas cannot reassume its normal shape without new protein synthesis. This suggests that microtubule protein once exposed to colchicine cannot be reassembled into microtubules. Pressure-treated cells, on the other hand, reassemble tubules and regenerate the normal shape in the presence or absence of cycloheximide. The use of these two agents in analyzing nucleating site function and the independent processes of synthesis and assembly of microtubules is discussed.

Cytochalasin treatment disrupts the endogenous currents associated with cell polarization in fucoid zygotes: studies of the role of F-actin in embryogenesis.

We determined the distribution of F-actin in fucoid (Pelvetia, Fucus) embryos with nitrobenzoxadiazole-phallacidin, and studied the effect of cytochalasin upon the endogenous currents associated with cell polarization by using the vibrating probe. F-actin is not localized at the presumptive rhizoid immediately after experimental induction of the polar axis with a light gradient; however, a preferential distribution of F-actin develops at the presumptive rhizoid by the time the position of the polar axis is fixed. F-actin continues to be localized at the tip of the rhizoid after germination, except during cytokinesis, when the furrow is the only brightly staining region of the embryo. Incubation with cytochalasin can result in either an enhanced or a diminished pool of F-actin in the embryonic cortex (see Results). Cytochalasin D (100 micrograms/ml) significantly reduces the inward current at the rhizoid pole (n = 11) after a 2.5-h incubation. This drop is concentration dependent and occurs within approximately 30 min at 100 micrograms/ml and approximately 60 min at 10 micrograms/ml. Cytochalasin treatment eliminates the pulsatile component of the current. Preliminary results suggest that 100 micrograms/ml cytochalasin D prevents development of inward current at the presumptive rhizoid but does not completely delocalize this locus if added after photopolarization. We conclude that microfilaments are required for the establishment and maintenance of the pattern of endogenous currents observed during early embryogenesis. This suggests a new model for axis formation and fixation.

Cell wall is required for fixation of the embryonic axis in Fucus zygotes.

Establishment of a primary developmental axis generally is thought to involve rearrangements in the plasma membrane or cytoplasm of the egg. In this report the additional requirement for cell wall in polarization of Fucus zygotes was investigated. Protoplasts of fertilized eggs were tested for their ability to establish an axis in accordance with an orienting vector of unilateral light. The results demonstrate that cell wall is not required for axis formation. However, the orientation of the axis remains labile until new cell wall is synthesized. The presence of a cell wall is an absolute requirement for axis fixation.

Underpinning the Development of Seaweed Biotechnology: Cryopreservation of Brown Algae (Saccharina latissima) Gametophytes.

Sugar kelp (Saccharina latissima) is an economically important species, and natural populations provide diverse and productive habitats as well as important ecosystem services. For seaweed aquaculture to be successful in newly emerging industry in Europe and other Western countries, it will have to develop sustainable production management strategies. A key feature in this process is the capacity to conserve genetic diversity for breeding programs aimed at developing seed stock for onward cultivation, as well as in the management of wild populations, as potentially interesting genetic resources are predicted to disappear due to climate change. In this study, the cryopreservation of male and female gametophytes (haploid life stage) of S. latissima by different combinations of two-step cooling methods and cryoprotectants was explored. We report here that cryopreservation constitutes an attractive option for the long-term preservation of S. latissima gametophytes, with viable cells in all treatment combinations. The highest viabilities for both male and female gametophytes were found using controlled-rate cooling methods combined with dimethyl sulfoxide 10% (v/v). Morphological normal sporophytes were observed to develop from cryopreserved vegetative gametophytic cells, independent of treatment. This indicates that cryopreservation is a useful preservation method for male and female S. latissima gametophytes.