Jeroen Gillard

Metabolomics Enables the Structure Elucidation of a Diatom Sex Pheromone

Diatoms are unicellular photosynthetic organisms that often dominate primary production in pelagic and benthic aquatic ecosystems. Despite their central role in the biosphere, little is known about their pheromone chemistry and their lifecycle, which is characterized by asexual population growth alternating with short bursts of sexual reproduction. Diatoms are unique among microalgae in that sexual reproduction is only possible below a species-specific sexual size threshold (SST). This SST is intimately linked to the characteristic cell division of diatoms. Due to their rigid biomineralized cell wall, mitotic division results in a reduction in size (Figure 1). Size is restored typically by sexual reproduction. Upon germination, a zygote generates a large initial cell, which begins a new round of vegetative proliferation. In the ancestral group of predominantly planktonic centric diatoms, environmental cues induce meiosis in cells below the SST, resulting in the formation of eggs and flagellated sperm. However, in the youngest and most species-rich pennate raphid diatoms, which have adopted a primarily benthic lifestyle, it is the pairing of diploid cells that triggers the production of isogametes. Although the SST is known to control the mating capacity of pennate diatoms and indirect evidence suggests the involvement of pheromones, the regulatory principles underlying the differentiation of mating cells and the identity of the pheromones remain unknown. We studied the physiological and metabolic changes associated with sexual reproduction in Seminavis robusta, a model for studies of pennate diatom lifecycles, with the goal to elucidate its pheromone chemistry. Mating is strictly size-dependent with a narrowly defined SST of (51.6 0.5) mm (Figure 2a). Like most pennate diatoms, S. robusta is a heterothallic species, that is, a species with morphologically identical but physiologically distinct cell types between which fertilization can take place. Mixing G1 phase-synchronized cells below the SST revealed the physiological differences between cells of different mating types (Figure 2b). Depending on the density of the partners, pairs or clusters of a migrating mating type (designated MT) around an attracting cell (designated MT ) were observed. The increased motility of MT in the presence of medium from a mating culture suggested the involvement of regulatory pheromones. We developed a bioassay to unambiguously prove and quantify the effect of such pheromones. Therefore hydrophilic/lipophilic-balanced solid-phase extraction cartridges (HLB-SPE) were loaded with media of S. robusta cultures. The SPE absorbent beads were then removed from the cartridges and served as pheromone sources. Behavioral responses towards such beads were monitored using light microscopy. Beads loaded with medium from amating culture attracted MT cells below the SST proving that the pheromone can be extracted (Figure 2b, movie in the Supporting Information). Attraction by MT cells was dependent on cell size and on prior perception of sexually mature (i.e. below the SST) mating partners. Extracts of MT cultures below the SST were only active when the MT cells were previously conditioned with medium from MT below the SST (Figure 2c). Extracts of MT above the SSTwere not active, even after conditioning with medium from MT below the SST (Figure S1 in the Supporting Information). Likewise, theMT motility response is primed by MT signals. Attraction to pheromone-loaded beads only occurred if MT cells were

Genome-wide analysis of the diatom cell cycle unveils a novel type of cyclins involved in environmental signaling

BackgroundDespite the enormous importance of diatoms in aquatic ecosystems and their broad industrial potential, little is known about their life cycle control. Diatoms typically inhabit rapidly changing and unstable environments, suggesting that cell cycle regulation in diatoms must have evolved to adequately integrate various environmental signals. The recent genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum allows us to explore the molecular conservation of cell cycle regulation in diatoms.ResultsBy profile-based annotation of cell cycle genes, counterparts of conserved as well as new regulators were identified in T. pseudonana and P. tricornutum. In particular, the cyclin gene family was found to be expanded extensively compared to that of other eukaryotes and a novel type of cyclins was discovered, the diatom-specific cyclins. We established a synchronization method for P. tricornutum that enabled assignment of the different annotated genes to specific cell cycle phase transitions. The diatom-specific cyclins are predominantly expressed at the G1-to-S transition and some respond to phosphate availability, hinting at a role in connecting cell division to environmental stimuli.ConclusionThe discovery of highly conserved and new cell cycle regulators suggests the evolution of unique control mechanisms for diatom cell division, probably contributing to their ability to adapt and survive under highly fluctuating environmental conditions.

In search of new tractable diatoms for experimental biology

Diatoms are a species-rich group of photosynthetic eukaryotes, with enormous ecological significance and great potential for biotechnology. During the last decade, diatoms have begun to be studied intensively using modern molecular techniques and the genomes of four diatoms have been wholly or partially sequenced. Although new insights into the biology and evolution of diatoms are accumulating rapidly due to the availability of reverse genetic tools, the full potential of these molecular biological approaches can only be fully realized if experimental control of sexual crosses becomes firmly established and widely accessible to experimental biologists. Here we discuss the issue of choosing new models for diatom research, by taking into account the broader context of diatom mating systems and the place of sex in relation to the intricate cycle of cell size reduction and restitution that is characteristic of most diatoms. We illustrate the results of our efforts to select and develop experimental systems in diatoms, using species with typical life cycle attributes, which could be used as future model organisms to complement existing ones.

Physiological and Transcriptomic Evidence for a Close Coupling between Chloroplast Ontogeny and Cell Cycle Progression in the Pennate Diatom Seminavis robusta

Despite the growing interest in diatom genomics, detailed time series of gene expression in relation to key cellular processes are still lacking. Here, we investigated the relationships between the cell cycle and chloroplast development in the pennate diatom Seminavis robusta. This diatom possesses two chloroplasts with a well-orchestrated developmental cycle, common to many pennate diatoms. By assessing the effects of induced cell cycle arrest with microscopy and flow cytometry, we found that division and reorganization of the chloroplasts are initiated only after S-phase progression. Next, we quantified the expression of the S. robusta FtsZ homolog to address the division status of chloroplasts during synchronized growth and monitored microscopically their dynamics in relation to nuclear division and silicon deposition. We show that chloroplasts divide and relocate during the S/G2 phase, after which a girdle band is deposited to accommodate cell growth. Synchronized cultures of two genotypes were subsequently used for a cDNA-amplified fragment length polymorphism-based genome-wide transcript profiling, in which 917 reproducibly modulated transcripts were identified. We observed that genes involved in pigment biosynthesis and coding for light-harvesting proteins were up-regulated during G2/M phase and cell separation. Light and cell cycle progression were both found to affect fucoxanthin-chlorophyll a/c-binding protein expression and accumulation of fucoxanthin cell content. Because chloroplasts elongate at the stage of cytokinesis, cell cycle-modulated photosynthetic gene expression and synthesis of pigments in concert with cell division might balance chloroplast growth, which confirms that chloroplast biogenesis in S. robusta is tightly regulated.

Daily bursts of biogenic cyanogen bromide (BrCN) control biofilm formation around a marine benthic diatom

The spatial organization of biofilms is strongly regulated by chemical cues released by settling organisms. However, the exact nature of these interactions and the repertoire of chemical cues and signals that micro-organisms produce and exude in response to the presence of competitors remain largely unexplored. Biofilms dominated by microalgae often show remarkable, yet unexplained fine-scale patchy variation in species composition. Because this occurs even in absence of abiotic heterogeneity, antagonistic interactions might play a key role. Here we show that a marine benthic diatom produces chemical cues that cause chloroplast bleaching, a reduced photosynthetic efficiency, growth inhibition and massive cell death in naturally co-occurring competing microalgae. Using headspace solid phase microextraction (HS-SPME)-GC-MS, we demonstrate that this diatom exudes a diverse mixture of volatile iodinated and brominated metabolites including the natural product cyanogen bromide (BrCN), which exhibits pronounced allelopathic activity. Toxin production is light-dependent with a short BrCN burst after sunrise. BrCN acts as a short-term signal, leading to daily “cleaning” events around the algae. We show that allelopathic effects are H2O2 dependent and link BrCN production to haloperoxidase activity. This strategy is a highly effective means of biofilm control and may provide an explanation for the poorly understood role of volatile halocarbons from marine algae, which contribute significantly to the atmospheric halocarbon budget.

Selective silicate-directed motility in diatoms.

Diatoms are highly abundant unicellular algae that often dominate pelagic as well as benthic primary production in the oceans and inland waters. Being strictly dependent on silica to build their biomineralized cell walls, marine diatoms precipitate 240 × 1012 mol Si per year, which makes them the major sink in the global Si cycle. Dissolved silicic acid (dSi) availability frequently limits diatom productivity and influences species composition of communities. We show that benthic diatoms selectively perceive and behaviourally react to gradients of dSi. Cell speed increases under dSi-limited conditions in a chemokinetic response and, if gradients of this resource are present, increased directionality of cell movement promotes chemotaxis. The ability to exploit local and short-lived dSi hotspots using a specific search behaviour likely contributes to micro-scale patch dynamics in biofilm communities. On a global scale this behaviour might affect sediment–water dSi fluxes and biogeochemical cycling.

Transcriptional analysis of cell growth and morphogenesis in the unicellular green alga Micrasterias (Streptophyta), with emphasis on the role of expansin

BackgroundStreptophyte green algae share several characteristics of cell growth and cell wall formation with their relatives, the embryophytic land plants. The multilobed cell wall of Micrasterias denticulata that rebuilds symmetrically after cell division and consists of pectin and cellulose, makes this unicellular streptophyte alga an interesting model system to study the molecular controls on cell shape and cell wall formation in green plants.ResultsGenome-wide transcript expression profiling of synchronously growing cells identified 107 genes of which the expression correlated with the growth phase. Four transcripts showed high similarity to expansins that had not been examined previously in green algae. Phylogenetic analysis suggests that these genes are most closely related to the plant EXPANSIN A family, although their domain organization is very divergent. A GFP-tagged version of the expansin-resembling protein MdEXP2 localized to the cell wall and in Golgi-derived vesicles. Overexpression phenotypes ranged from lobe elongation to loss of growth polarity and planarity. These results indicate that MdEXP2 can alter the cell wall structure and, thus, might have a function related to that of land plant expansins during cell morphogenesis.ConclusionsOur study demonstrates the potential of M. denticulata as a unicellular model system, in which cell growth mechanisms have been discovered similar to those in land plants. Additionally, evidence is provided that the evolutionary origins of many cell wall components and regulatory genes in embryophytes precede the colonization of land.

A sex-inducing pheromone triggers cell cycle arrest and mate attraction in the diatom Seminavis robusta

Although sexual reproduction is believed to play a major role in the high diversification rates and species richness of diatoms, a mechanistic understanding of diatom life cycle control is virtually lacking. Diatom sexual signalling is controlled by a complex, yet largely unknown, pheromone system. Here, a sex-inducing pheromone (SIP+) of the benthic pennate diatom Seminavis robusta was identified by comparative metabolomics, subsequently purified, and physicochemically characterized. Transcriptome analysis revealed that SIP+ triggers the switch from mitosis-to-meiosis in the opposing mating type, coupled with the transcriptional induction of proline biosynthesis genes, and the release of the proline-derived attraction pheromone. The induction of cell cycle arrest by a pheromone, chemically distinct from the one used to attract the opposite mating type, highlights the existence of a sophisticated mechanism to increase chances of mate finding, while keeping the metabolic losses associated with the release of an attraction pheromone to a minimum.

Heterothallic sexual reproduction in the model diatom Cylindrotheca

Cylindrotheca is one of the main model diatoms for ecophysiological and silicification research and is among the few diatoms for which a transformation protocol is available. Knowledge of its life cycle is not available, however, although sexual reproduction has been described for several related genera. In this study, 16 Cylindrotheca closterium strains from a single rbcL lineage were used to describe the life cycle of this marine diatom, including the sexual process and mating system. Similar to other Bacillariaceae, sexual reproduction was induced by the presence of a suitable mating partner, with two gametes produced per gametangium, resulting in two auxospores per gametangial pair. Differences with other Bacillariaceae include details of cell pairing, gamete behaviour, auxospore orientation and chloroplast configuration, and perizonium structure. The mating system is heterothallic, since strains fell into two mating type groups, with several strains of one mating type occasionally displaying intraclonal auxosporulation. Initial cell lengths were on average 95–100 µm, the sexual size threshold was at least 66 µm, and the minimal viable cell length c. 11 µm. Sexual reproduction could be synchronized by dark conditions, which allowed us to determine that the whole sexual process is completed in less than 24 h. Furthermore, large percentages of cells at defined sexual stages can easily be obtained and the possibility to experimentally manipulate the life cycle provides a valuable tool for future research on all aspects of the biology of this model diatom.