CRISPR/Cas9 is a powerful tool for genome editing. We constructed an easy-to-handle expression vector for application in the model organism Phaeodactylum tricornutum and tested its capabilities in order to apply CRISPR/Cas9 technology for our purpose. In our experiments, we targeted two different genes, screened for mutations and analyzed mutated diatoms in a three-step process. In the end, we identified cells, showing either monoallelic or homo-biallelic targeted mutations. Thus, we confirm that application of the CRISPR/Cas9 system for P. tricornutum is very promising, although, as discussed, overlooked pitfalls have to be considered.

Unicellular organisms that live in marine environments must cope with considerable fluctuations in the availability of inorganic phosphate (P i). Here, we investigated the extracellular P i concentration-dependent expression, as well as the intracellular or extracellular localization, of phosphatases and phosphate transporters of the diatom Phaeodactylum tricornutum. We identified P i-regulated plasma membrane-localized, ER-localized, and secreted phosphatases, in addition to plasma membrane-localized, vacuolar membrane-localized, and plastid-surrounding membrane-localized phosphate transporters that were also regulated in a P i concentration-dependent manner. These studies not only add further knowledge to already existing transcriptomic data, but also highlight the capacity of the diatom to distribute P i intracellularly and to mobilize P i from extracellular and intracellular resources.

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.Oceanic plankton are characterised by a huge biodiversity spanning over many phyla. The different life strategies and behaviours displayed by such diverse organisms were selected also to cope with a patchy and variable 3D world driven by water motion 1 . Key factors for their survival, e.g., light availability, nutrient concentrations, prey abundance and water temperature, are all strongly tuned by the fluid motion at different scales. Fluid motion introduces kinetic energy in the system and turbulence is the way kinetic energy is transferred, through a dissipation cascade, over several eddy-like structures down to the smallest scale. Below this scale, energy is dissipated to heat via the friction of viscosity and water motion cannot prevail over molecular diffusion but can control it by changing local gradients 2, 3 . This is particularly important for unicellular phytoplankton which are surrounded by a fluid boundary layer where molecular diffusion is the dominant process and only solute (nutrient) chemical gradients assure cell provisioning. A distortion of the boundary layer would change these gradients, hence nutrients would diffuse more rapidly, enhancing the uptake rate 4 . For non-motile cells, like diatoms, the distortion of the boundary layer can be produced only by sinking or by the shear generated by the decay of turbulent kinetic energy. Therefore there is no more turbulence, as a random motion of water parcels, but the effects of turbulence which dissipates though sheared flow. Shear stress is what cells below Kolmogorov scale would ultimately perceive

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.