Alberto Amato

Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

Diatoms, a major group of photosynthetic microalgae, have a high biotechnological potential that has not been fully exploited because of the paucity of available genetic tools. Here we demonstrate targeted and stable modifications of the genome of the marine diatom Phaeodactylum tricornutum, using both meganucleases and TALE nucleases. When nuclease-encoding constructs are co-transformed with a selectable marker, high frequencies of genome modifications are readily attained with 56 and 27% of the colonies exhibiting targeted mutagenesis or targeted gene insertion, respectively. The generation of an enhanced lipid-producing strain (45-fold increase in triacylglycerol accumulation) through the disruption of the UDP-glucose pyrophosphorylase gene exemplifies the power of genome engineering to harness diatoms for biofuel production.

Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy)

Twenty-six Pseudo-nitzschia multistriata cultures were tested for intracellular domoic acid production and fourteen were found to be toxic. Four suboptimal growth conditions were compared with conditions observed to be optimal to explore possible triggers for intracellular domoic acid production. Silica- and phosphate-limitation and low light treatment induced elevated toxin concentrations whereas high temperature appeared to suppress it. Inheritance of the toxin-production ability was investigated by measuring intracellular toxin content in a total of thirty-nine F(1) strains from two different crosses. Results showed radical differences in domoic acid levels among the F(1) offspring from the same parents.

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.

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.

Nutrient consumption and chain tuning in diatoms exposed to storm-like turbulence.

Current information on the response of phytoplankton to turbulence is linked to cell size and nutrient availability. Diatoms are considered to be favored by mixing as dissolved nutrients are more easily accessible for non-motile cells. We investigated how diatoms exploit microscale turbulence under nutrient repletion and depletion conditions. Here, we show that the chain-forming diatom Chaetoceros decipiens, continues to take up phosphorus and carbon even when silicon is depleted during turbulence. Our findings indicate that upon silica depletion, during turbulence, chain spectra of C. decipiens remained unchanged. We show here that longer chains are maintained during turbulence upon silica depletion whereas under still conditions, shorter chains are enriched. We interpret this as a sign of good physiological state leading to a delay of culture senescence. Our results show that C. decipiens senses and responds to turbulence both in nutrient repletion and depletion. This response is noteworthy due to the small size of the species. The coupling between turbulence and biological response that we depict here may have significant ecological implications. Considering the predicted increase of storms in Northern latitudes this response might modify community structure and succession. Our results partly corroborate Margalef’s mandala and provide additional explanations for that conceptualization.

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.

TURBOGEN: Computer-controlled vertically oscillating grid system for small-scale turbulence studies on plankton

In recent years, there has been a renewed interest in the impact of turbulence on aquatic organisms. In response to this interest, a novel instrument has been constructed, TURBOGEN, that generates turbulence in water volumes up to 13 l. TURBOGEN is fully computer controlled, thus, allowing for a high level of reproducibility and for variations of the intensity and characteristics of turbulence during the experiment. The calibration tests, carried out by particle image velocimetry, showed TURBOGEN to be successful in generating isotropic turbulence at the typical relatively low levels of the marine environment. TURBOGEN and its sizing have been devised with the long-term scope of analyzing in detail the molecular responses of plankton to different mixing regimes, which is of great importance in both environmental and biotechnological processes.

Regulation of chain length in two diatoms as a growth-fragmentation process

Chain formation in diatoms is relevant because of several aspects of their adaptation to the ecosystem. However, the tools to quantify the regulation of their assemblage and infer specific mechanisms in a laboratory setting are scarce. To address this problem, we define an approach based on a statistical physics model of chain growth and separation in combination with experimental evaluation of chain-length distributions. Applying this combined analysis to data from Chaetoceros decipiens and Phaeodactylum tricornutum, we find that cells of the first species control chain separation, likely through a cell-to-cell communication process, while the second species only modulates the separation rate. These results promote quantitative methods for characterizing chain formation in several chain-forming species and in diatoms in particular.

Animal-like prostaglandins in marine microalgae

Diatoms are among the most successful primary producers in ocean and freshwater environments. Deriving from a secondary endosymbiotic event, diatoms have a mixed genome containing bacterial, animal and plant genes encoding for metabolic pathways that may account for their evolutionary success. Studying the transcriptomes of two strains of the diatom Skeletonema marinoi, we report, for the first time in microalgae, an active animal-like prostaglandin pathway that is differentially expressed in the two strains. Prostaglandins are hormone-like mediators in many physiological and pathological processes in mammals, playing a pivotal role in inflammatory responses. They are also present in macroalgae and invertebrates, where they act as defense and communication mediators. The occurrence of animal-like prostaglandins in unicellular photosynthetic eukaryotes opens up new intriguing perspectives on the evolution and role of these molecules in the marine environment as possible mediators in cell-to-cell signaling, eventually influencing population dynamics in the plankton.

Grazer-induced transcriptomic and metabolomic response of the chain-forming diatom Skeletonema marinoi

Diatoms and copepods are main actors in marine food webs. The prey–predator interactions between them affect bloom dynamics, shape marine ecosystems and impact the energy transfer to higher trophic levels. Recently it has been demonstrated that the presence of grazers may affect the diatom prey beyond the direct effect of grazing. Here, we investigated the response of the chain-forming centric diatom Skeletonema marinoi to grazer cues, including changes in morphology, gene expression and metabolic profile. S. marinoi cells were incubated with Calanus finmarchicus or with Centropages typicus and in both cases responded by reducing the chain length, whereas changes in gene expression indicated an activation of stress response, changes in the lipid and nitrogen metabolism, in cell cycle regulation and in frustule formation. Transcripts linked to G protein-coupled receptors and to nitric oxide synthesis were differentially expressed suggesting involvement of these signalling transduction pathways in the response. Downregulation of a lipoxygenase in the transcriptomic data and of its products in the metabolomic data also indicate an involvement of oxylipins. Our data contribute to a better understanding of the gene function in diatoms, providing information on the nature of genes implicated in the interaction with grazers, a crucial process in marine ecosystems.