Polarization of brown algal zygotes.

Brown algae are a group of multicellular, heterokont algae that have convergently evolved developmental complexity that rivals that of embryophytes, animals or fungi. Early in development, brown algal zygotes establish a basal and an apical pole, which will become respectively the basal system (holdfast) and the apical system (thallus) of the adult alga. Brown algae are interesting models for understanding the establishment of cell polarity in a broad evolutionary context, because they exhibit a large diversity of life cycles, reproductive strategies and, importantly, their zygotes are produced in large quantities free of parental tissue, with symmetry breaking and asymmetric division taking place in a highly synchronous manner. This review describes the current knowledge about the establishment of the apical-basal axis in the model brown seaweeds Ectocarpus, Dictyota, Fucus and Saccharina, highlighting the advantages and specific interests of each system. Ectocarpus is a genetic model system that allows access to the molecular basis of early development and life-cycle control over apical-basal polarity. The oogamous brown alga Fucus, together with emerging comparative models Dictyota and Saccharina, emphasize the diversity of strategies of symmetry breaking in determining a cell polarity vector in brown algae. A comparison with symmetry-breaking mechanisms in land plants, animals and fungi, reveals that the one-step zygote polarisation of Fucus compares well to Saccharomyces budding and Arabidopsis stomata development, while the two-phased symmetry breaking in the Dictyota zygote compares to Schizosaccharomyces fission, the Caenorhabditis anterior-posterior zygote polarisation and Arabidopsis prolate pollen polarisation. The apical-basal patterning in Saccharina zygotes on the other hand, may be seen as analogous to that of land plants. Overall, brown algae have the potential to bring exciting new information on how a single cell gives rise to an entire complex body plan.

Transcriptional dynamics of gametogenesis in the green seaweed Ulva mutabilis identifies an RWP-RK transcription factor linked to reproduction.

BACKGROUND: The molecular mechanism underlying sexual reproduction in land plants is well understood in model plants and is a target for crop improvement. However, unlike land plants, the genetic basis involved in triggering reproduction and gamete formation remains elusive in most seaweeds, which are increasingly viewed as an alternative source of functional food and feedstock for energy applications. RESULTS: Gametogenesis of Ulva mutabilis, a model organism for green seaweeds, was studied. We analyzed transcriptome dynamics at different time points during gametogenesis following induction of reproduction by fragmentation and removal of sporulation inhibitors. Analyses demonstrated that 45% of the genes in the genome were differentially expressed during gametogenesis. We identified several transcription factors that potentially play a key role in the early gametogenesis of Ulva given the function of their homologs in higher plants and microalgae. In particular, the detailed expression pattern of an evolutionarily conserved transcription factor containing an RWP-RK domain suggested a key role during Ulva gametogenesis. CONCLUSIONS: Transcriptomic analyses of gametogenesis in the green seaweed Ulva highlight the importance of a conserved RWP-RK transcription factor in the induction of sexual reproduction. The identification of putative master regulators of gametogenesis provides a starting point for further functional characterization.

Genomic adaptations to an endolithic lifestyle in the coral-associated alga Ostreobium.

The green alga Ostreobium is an important coral holobiont member, playing key roles in skeletal decalcification and providing photosynthate to bleached corals that have lost their dinoflagellate endosymbionts. Ostreobium lives in the coral's skeleton, a low-light environment with variable pH and O2 availability. We present the Ostreobium nuclear genome and a metatranscriptomic analysis of healthy and bleached corals to improve our understanding of Ostreobium's adaptations to its extreme environment and its roles as a coral holobiont member. The Ostreobium genome has 10,663 predicted protein-coding genes and shows adaptations for life in low and variable light conditions and other stressors in the endolithic environment. This alga presents a rich repertoire of light-harvesting complex proteins but lacks many genes for photoprotection and photoreceptors. It also has a large arsenal of genes for oxidative stress response. An expansion of extracellular peptidases suggests that Ostreobium may supplement its energy needs by feeding on the organic skeletal matrix, and a diverse set of fermentation pathways allows it to live in the anoxic skeleton at night. Ostreobium depends on other holobiont members for vitamin B12, and our metatranscriptomes identify potential bacterial sources. Metatranscriptomes showed Ostreobium becoming a dominant agent of photosynthesis in bleached corals and provided evidence for variable responses among coral samples and different Ostreobium genotypes. Our work provides a comprehensive understanding of the adaptations of Ostreobium to its extreme environment and an important genomic resource to improve our comprehension of coral holobiont resilience, bleaching, and recovery.

Metabolic, Physiological, and Transcriptomics Analysis of Batch Cultures of the Green Microalga Chlamydomonas Grown on Different Acetate Concentrations.

Acetate can be efficiently metabolized by the green microalga Chlamydomonasreinhardtii. The regular concentration is 17 mM, although higher concentrations are reported to increase starch and fatty acid content. To understand the responses to higher acetate concentrations, Chlamydomonas cells were cultivated in batch mode in the light at 17, 31, 44, and 57 mM acetate. Metabolic analyses show that cells grown at 57 mM acetate possess increased contents of all components analyzed (starch, chlorophylls, fatty acids, and proteins), with a three-fold increased volumetric biomass yield compared to cells cultivated at 17 mM acetate at the entry of stationary phase. Physiological analyses highlight the importance of photosynthesis for the low-acetate and exponential-phase samples. The stationary phase is reached when acetate is depleted, except for the cells grown at 57 mM acetate, which still divide until ammonium exhaustion. Surprisal analysis of the transcriptomics data supports the biological significance of our experiments. This allows the establishment of a model for acetate assimilation, its transcriptional regulation and the identification of candidates for genetic engineering of this metabolic pathway. Altogether, our analyses suggest that growing at high-acetate concentrations could increase biomass productivities in low-light and CO2-limiting air-bubbled medium for biotechnology.

Auxin Function in the Brown Alga Dictyota dichotoma.

Auxin controls body plan patterning in land plants and has been proposed to play a similar role in the development of brown algae (Phaeophyta) despite their distant evolutionary relationship with land plants. The mechanism of auxin action in brown algae remains controversial because of contradicting conclusions derived from pharmacological studies on Fucus In this study, we used Dictyota dichotoma as a model system to show that auxin plays a role during the apical-basal patterning of the embryo of brown algae. Indole-3-acetic acid was detectable in D. dichotoma germlings and mature tissue. Although two-celled D. dichotoma zygotes normally develop a rhizoid from one pole and a thallus meristem from the other, addition of exogenous auxins to one-celled embryos affected polarization, and both poles of the spheroidal embryo developed into rhizoids instead. The effect was strongest at lower pH and when variable extrinsic informational cues were applied. 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid, an inhibitor of the ABC-B/multidrug resistance/P-glycoprotein subfamily of transporters in land plants, affected rhizoid formation by increasing rhizoid branching and inducing ectopic rhizoids. An in silico survey of auxin genes suggested that a diverse range of biosynthesis genes and transport genes, such as PIN-LIKES, and the ATP-binding cassette subfamily (ABC-B/multidrug resistance/P-glycoprotein) transporters from land plants have homologs in D. dichotoma and Ectocarpus siliculosus Together with reports on auxin function in basal lineages of green algae, these results suggest that auxin function predates the divergence between the green and brown lineage and the transition toward land plants.

Insights into the Evolution of Multicellularity from the Sea Lettuce Genome.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.

Surprisal analysis of genome-wide transcript profiling identifies differentially expressed genes and pathways associated with four growth conditions in the microalga Chlamydomonas.

The usual cultivation mode of the green microalga Chlamydomonas is liquid medium and light. However, the microalga can also be grown on agar plates and in darkness. Our aim is to analyze and compare gene expression of cells cultivated in these different conditions. For that purpose, RNA-seq data are obtained from Chlamydomonas samples of two different labs grown in four environmental conditions (agar@light, agar@dark, liquid@light, liquid@dark). The RNA seq data are analyzed by surprisal analysis, which allows the simultaneous meta-analysis of all the samples. First we identify a balance state, which defines a state where the expression levels are similar in all the samples irrespectively of their growth conditions, or lab origin. In addition our analysis identifies additional constraints needed to quantify the deviation with respect to the balance state. The first constraint differentiates the agar samples versus the liquid ones; the second constraint the dark samples versus the light ones. The two constraints are almost of equal importance. Pathways involved in stress responses are found in the agar phenotype while the liquid phenotype comprises ATP and NADH production pathways. Remodeling of membrane is suggested in the dark phenotype while photosynthetic pathways characterize the light phenotype. The same trends are also present when performing purely statistical analysis such as K-means clustering and differentially expressed genes.

The Plastid Genome in Cladophorales Green Algae Is Encoded by Hairpin Chromosomes.

Virtually all plastid (chloroplast) genomes are circular double-stranded DNA molecules, typically between 100 and 200 kb in size and encoding circa 80-250 genes. Exceptions to this universal plastid genome architecture are very few and include the dinoflagellates, where genes are located on DNA minicircles. Here we report on the highly deviant chloroplast genome of Cladophorales green algae, which is entirely fragmented into hairpin chromosomes. Short- and long-read high-throughput sequencing of DNA and RNA demonstrated that the chloroplast genes of Boodlea composita are encoded on 1- to 7-kb DNA contigs with an exceptionally high GC content, each containing a long inverted repeat with one or two protein-coding genes and conserved non-coding regions putatively involved in replication and/or expression. We propose that these contigs correspond to linear single-stranded DNA molecules that fold onto themselves to form hairpin chromosomes. The Boodlea chloroplast genes are highly divergent from their corresponding orthologs, and display an alternative genetic code. The origin of this highly deviant chloroplast genome most likely occurred before the emergence of the Cladophorales, and coincided with an elevated transfer of chloroplast genes to the nucleus. A chloroplast genome that is composed only of linear DNA molecules is unprecedented among eukaryotes, and highlights unexpected variation in plastid genome architecture.

Egg activation-triggered shape change in the Dictyota dichotoma (Phaeophyceae) zygote is actin-myosin and secretion dependent.

Background and Aims: Cellular morphogenesis in land plants and brown algae is typically a slow process involving growth established by an interplay of turgor pressure and cell wall rigidity. However, a recent study showed that zygotes of the brown alga Dictyota dichotoma undergo a rapid shape change from a sphere to an elongated spheroid in about 90 s, establishing the first body axis. Methods: Using a combination of pharmacology, staining techniques, membrane depolarization and microscopy techniques (brightfield, transmission electron microscopy and confocal laser scanning microscopy), egg activation and the shape change of the egg cell of D. dichotoma was studied. Key Results: It was established that elongation of the zygote does not involve growth, i.e. a positive change in size. The elongation is dependent on F-actin and myosin but independent of microtubules. Secretion was also found to be necessary for elongation after addition of brefeldin A. Moreover, a temporal correlation between extracellular matrix secretion and elongation was observed. Ionomycin and high potassium seawater are capable of triggering the onset of elongation, suggesting a role for membrane depolarization and calcium influx in the signalling mechanism. The elongated cells are shorter in the presence of ionomycin, suggesting a role for calcium in elongation. Conclusions: A model is proposed in which the fast elongation of the fertilized egg in Dictyota is accomplished by a force generated by F-actin and myosin, regulated by cytoplasmic calcium concentrations and by secretion during elongation lowering the antagonistic force. The finding of early extracellular matrix secretion, membrane depolarization and ionophore-triggered egg activation suggest significant differences in the mechanism of egg activation signalling between D. dichotoma and the oogamous brown algal model system Fucus .

Two-step cell polarization in algal zygotes.

In most complex eukaryotes, development starts with the establishment of cell polarity determining the first axis of the body plan. This polarity axis is established by the asymmetrical distribution of intrinsic factors1-3, which breaks the symmetry in a single step. Zygotes of the brown alga Fucus, which unlike land plant and animal zygotes4,5 do not possess a maternally predetermined polarity axis, serve as models to study polarity establishment6,7. Here, we studied this process in Dictyota, and concluded that sense and direction of the cell polarization vector are established in two mechanistically and temporally distinct phases that are under control of different life cycle stages. On egg activation, the zygote elongates rapidly according to a maternally predetermined direction expressing the first phase of cell polarization. Which of the two poles of the resulting prolate spheroidal zygote will acquire the basal cell fate is subsequently environmentally determined. The second phase is accompanied by and dependent on zygotic transcription instead of relying uniquely on maternal factors8. Cell polarization, whereby determination of direction and sense of the polarization vector are temporally and mechanistically uncoupled, is unique and represents a favourable system to gain insight into the processes underlying cell polarity establishment in general.

Photopolarization of Fucus zygotes is determined by time sensitive vectorial addition of environmental cues during axis amplification.

Fucoid zygotes have been extensively used to study cell polarization and asymmetrical cell division. Fertilized eggs are responsive to different environmental cues (e.g., light, gravity) for a long period before the polarity is fixed and the cells germinate accordingly. First, it is commonly believed that the direction and sense of the polarization vector are established simultaneously as indicated by the formation of an F-actin patch. Secondly, upon reorientation of the zygote, a new polar gradient is formed and it is assumed that the position of the future rhizoid pole is only influenced by the latter. Here we tested these two hypotheses investigating photopolarization in Fucus zygotes by reorienting zygotes 90° relative to a unilateral light source at different time points during the first cell cycle. We conclude that fixation of direction and sense of the polarization vector is indeed established simultaneously. However, the experiments yielded a distribution of polarization axes that cannot be explained if only the last environmental cue is supposed to determine the polarization axis. We conclude that our observations, together with published findings, can only be explained by assuming imprinting of the different polarization vectors and their integration as a vectorial sum at the moment of axis fixation. This way cells will average different serially perceived cues resulting in a polarization vector representative of the dynamic intertidal environment, instead of betting exclusively on the perceived vector at the moment of axis fixation.

Brown algae as a model for plant organogenesis.

Brown algae are an extremely interesting, but surprisingly poorly explored, group of organisms. They are one of only five eukaryotic lineages to have independently evolved complex multicellularity, which they express through a wide variety of morphologies ranging from uniseriate branched filaments to complex parenchymatous thalli with multiple cell types. Despite their very distinct evolutionary history, brown algae and land plants share a striking amount of developmental features. This has led to an interest in several aspects of brown algal development, including embryogenesis, polarity, cell cycle, asymmetric cell division and a putative role for plant hormone signalling. This review describes how investigations using brown algal models have helped to increase our understanding of the processes controlling early embryo development, in particular polarization, axis formation and asymmetric cell division. Additionally, the diversity of life cycles in the brown lineage and the emergence of Ectocarpus as a powerful model organism, are affording interesting insights on the molecular mechanisms underlying haploid-diploid life cycles. The use of these and other emerging brown algal models will undoubtedly add to our knowledge on the mechanisms that regulate development in multicellular photosynthetic organisms.