Maria Immacolata Ferrante

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Finding a partner in the ocean: molecular and evolutionary bases of the response to sexual cues in a planktonic diatom

Summary Microalgae play a major role as primary producers in aquatic ecosystems. Cell signalling regulates their interactions with the environment and other organisms, yet this process in phytoplankton is poorly defined. Using the marine planktonic diatom Pseudo‐nitzschia multistriata, we investigated the cell response to cues released during sexual reproduction, an event that demands strong regulatory mechanisms and impacts on population dynamics. We sequenced the genome of P. multistriata and performed phylogenomic and transcriptomic analyses, which allowed the definition of gene gains and losses, horizontal gene transfers, conservation and evolutionary rate of sex‐related genes. We also identified a small number of conserved noncoding elements. Sexual reproduction impacted on cell cycle progression and induced an asymmetric response of the opposite mating types. G protein‐coupled receptors and cyclic guanosine monophosphate (cGMP) are implicated in the response to sexual cues, which overall entails a modulation of cell cycle, meiosis‐related and nutrient transporter genes, suggesting a fine control of nutrient uptake even under nutrient‐replete conditions. The controllable life cycle and the genome sequence of P. multistriata allow the reconstruction of changes occurring in diatoms in a key phase of their life cycle, providing hints on the evolution and putative function of their genes and empowering studies on sexual reproduction.

Transcriptome sequencing of three Pseudo-nitzschia species reveals comparable gene sets and the presence of Nitric Oxide Synthase genes in diatoms

Diatoms are among the most diverse eukaryotic microorganisms on Earth, they are responsible for a large fraction of primary production in the oceans and can be found in different habitats. Pseudo-nitzschia are marine planktonic diatoms responsible for blooms in coastal and oceanic waters. We analyzed the transcriptome of three species, Pseudo-nitzschia arenysensis, Pseudo-nitzschia delicatissima and Pseudo-nitzschia multistriata, with different levels of genetic relatedness. These species have a worldwide distribution and the last one produces the neurotoxin domoic acid. We were able to annotate about 80% of the sequences in each transcriptome and the analysis of the relative functional annotations allowed comparison of the main metabolic pathways, pathways involved in the biosynthesis of isoprenoids (MAV and MEP pathways), and pathways putatively involved in domoic acid synthesis. The search for homologous transcripts among the target species and other congeneric species resulted in the discovery of a sequence annotated as Nitric Oxide Synthase (NOS), found uniquely in Pseudo-nitzschia multistriata. The predicted protein product contained all the domains of the canonical metazoan sequence. Putative NOS sequences were found in other available diatom datasets, supporting a role for nitric oxide as signaling molecule in this group of microalgae.

Establishment of Genetic Transformation in the Sexually Reproducing Diatoms Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis and Inheritance of the Transgene

We report the genetic transformation of the planktonic diatoms Pseudo-nitzschia arenysensis and Pseudo-nitzschia multistriata, members of the widely distributed and ecologically important genus Pseudo-nitzschia. P. arenysensis and P. multistriata present the classical size reduction/restitution life cycle and can reproduce sexually. Genetic transformation was achieved with the biolistic method, using the H4 gene promoter from P. multistriata to drive expression of exogenous genes. The transformation was first optimized introducing the Sh ble gene to confer resistance to the antibiotic zeocin. Integration of the transgene was confirmed by PCR and Southern blot analyses. Subsequently, we simultaneously transformed in P. arenysensis two plasmids, one encoding the β-glucuronidase (GUS) gene together with the plasmid carrying the Sh ble resistance gene, demonstrating the possibility of co-transformation. By transforming a gene encoding a fusion between the histone H4 and the green fluorescent protein (GFP), we demonstrated that fluorescent tagging is possible and that studies for protein localization are feasible. Importantly, we crossed P. arenysensis- and P. multistriata-transformed strains with a wild-type strain of opposite mating type and demonstrated that the transgene can be inherited in the F1 generation. The possibility to transform two diatom species for which genetic crosses are possible opens the way to a number of new approaches, including classical loss of function screens and the possibility to obtain different combinations of double transformants.

The diatom molecular toolkit to handle nitrogen uptake

Nutrient concentrations in the oceans display significant temporal and spatial variability, which strongly affects growth, distribution and survival of phytoplankton. Nitrogen (N) in particular is often considered a limiting resource for prominent marine microalgae, such as diatoms. Diatoms possess a suite of N-related transporters and enzymes and utilize a variety of inorganic (e.g., nitrate, NO3(-); ammonium, NH4(+)) and organic (e.g., urea; amino acids) N sources for growth. However, the molecular mechanisms allowing diatoms to cope efficiently with N oscillations by controlling uptake capacities and signaling pathways involved in the perception of external and internal clues remain largely unknown. Data reported in the literature suggest that the regulation and the characteristic of the genes, and their products, involved in N metabolism are often diatom-specific, which correlates with the peculiar physiology of these organisms for what N utilization concerns. Our study reveals that diatoms host a larger suite of N transporters than one would expected for a unicellular organism, which may warrant flexible responses to variable conditions, possibly also correlated to the phases of life cycle of the cells. All this makes N transporters a crucial key to reveal the balance between proximate and ultimate factors in diatom life.

Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta.

BackgroundSexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase.ResultsThe exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestral loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase.ConclusionsOur results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.

The dynamics of sexual phase in the marine diatom Pseudo‐nitzschia multistriata (Bacillariophyceae)

Sexual reproduction represents a fundamental phase in the life cycle of diatoms, linked to both the production of genotypic diversity and the formation of large‐sized initial cells. Only cells below a certain size threshold can be sexualized, but various environmental factors can modulate the success of sexual reproduction. We investigated the role of cell density and physiological conditions of parental strains in affecting the success and timing of sexual reproduction in the marine heterothallic diatom Pseudo‐nitzschia multistriata. We also studied the dynamics of the sexual phase in still conditions allowing cell sedimentation and in gently mixed conditions that keep cells in suspension. Our results showed that successful sexual reproduction can only be achieved when crossing parental strains in the exponential growth phase. Evidence was provided for the fact that sexual reproduction is a density‐dependent event and requires a threshold cell concentration to start, although this might vary considerably amongst strains. Moreover, the onset of the sexual phase was coupled to a marked reduction in growth of the vegetative parental cells. The crosses carried out in physically mixed conditions produced a significantly reduced number of sexual stages as compared to crosses in still conditions, showing that mixing impairs sexualization. The results of our experiments suggest that the signaling that triggers the sexual phase is favored when cells can accumulate, reducing the distance between them and facilitating contacts and/or the perception of chemical cues. Information on the progression of the sexual phase in laboratory conditions help understanding the conditions at which sex occurs in the natural environment.

The sexual phase of the diatom Pseudo-nitzschia multistriata: cytological and time-lapse cinematography characterization

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.

Clonal expansion behind a marine diatom bloom

Genetic diversity is what selection acts on, thus shaping the adaptive potential of populations. We studied micro-evolutionary patterns of the key planktonic diatom Pseudo-nitzschia multistriata at a long-term sampling site over 2 consecutive years by genotyping isolates with 22 microsatellite markers. We show that both sex and vegetative growth interplay in shaping intraspecific diversity. We document a brief but massive demographic and clonal expansion driven by strains of the same mating type. The analysis of an extended data set (6 years) indicates that the genetic fingerprint of P. multistriata changed over time with a nonlinear pattern, with intermittent periods of weak and strong diversification related to the temporary predominance of clonal expansions over sexual recombination. These dynamics, rarely documented for phytoplankton, contribute to the understanding of bloom formation and of the mechanisms that drive microevolution in diatoms.

Marine diatoms change their gene expression profile when exposed to microscale turbulence under nutrient replete conditions

Diatoms are a fundamental microalgal phylum that thrives in turbulent environments. Despite several experimental and numerical studies, if and how diatoms may profit from turbulence is still an open question. One of the leading arguments is that turbulence favours nutrient uptake. Morphological features, such as the absence of flagella, the presence of a rigid exoskeleton and the micrometre size would support the possible passive but beneficial role of turbulence on diatoms. We demonstrate that in fact diatoms actively respond to turbulence in non-limiting nutrient conditions. TURBOGEN, a prototypic instrument to generate natural levels of microscale turbulence, was used to expose diatoms to the mechanical stimulus. Differential expression analyses, coupled with microscopy inspections, enabled us to study the morphological and transcriptional response of Chaetoceros decipiens to turbulence. Our target species responds to turbulence by activating energy storage pathways like fatty acid biosynthesis and by modifying its cell chain spectrum. Two other ecologically important species were examined and the occurrence of a morphological response was confirmed. These results challenge the view of phytoplankton as unsophisticated passive organisms.