Impaired photoprotection in Phaeodactylum tricornutum KEA3 mutants reveals the proton regulatory circuit of diatoms light acclimation.

Diatoms are successful phytoplankton clades able to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity via the nonphotochemical quenching (NPQ) process. While the molecular effectors of NPQ as well as the involvement of the proton motive force (PMF) in its regulation are known, the regulators of the PET/PMF relationship remain unidentified in diatoms. We generated mutants of the H+ /K+ antiporter KEA3 in the model diatom Phaeodactylum tricornutum. Loss of KEA3 activity affects the PET/PMF coupling and NPQ responses at the onset of illumination, during transients and in steady-state conditions. Thus, this antiporter is a main regulator of the PET/PMF coupling. Consistent with this conclusion, a parsimonious model including only two free components, KEA3 and the diadinoxanthin de-epoxidase, describes most of the feedback loops between PET and NPQ. This simple regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory calcium ion (Ca2+ )-binding domain in KEA3 modulating its activity. This circuit is likely tuned by the NPQ-effector proteins, LHCXs, providing diatoms with the required flexibility to thrive in different ocean provinces.

Mixotrophic growth of the extremophile Galdieria sulphuraria reveals the flexibility of its carbon assimilation metabolism.

Galdieria sulphuraria is a cosmopolitan microalga found in volcanic hot springs and calderas. It grows at low pH in photoautotrophic (use of light as a source of energy) or heterotrophic (respiration as a source of energy) conditions, using an unusually broad range of organic carbon sources. Previous data suggested that G. sulphuraria cannot grow mixotrophically (simultaneously exploiting light and organic carbon as energy sources), its photosynthetic machinery being repressed by organic carbon. Here, we show that G. sulphuraria SAG21.92 thrives in photoautotrophy, heterotrophy and mixotrophy. By comparing growth, biomass production, photosynthetic and respiratory performances in these three trophic modes, we show that addition of organic carbon to cultures (mixotrophy) relieves inorganic carbon limitation of photosynthesis thanks to increased CO2 supply through respiration. This synergistic effect is lost when inorganic carbon limitation is artificially overcome by saturating photosynthesis with added external CO2 . Proteomic and metabolic profiling corroborates this conclusion suggesting that mixotrophy is an opportunistic mechanism to increase intracellular CO2 concentration under physiological conditions, boosting photosynthesis by enhancing the carboxylation activity of Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and decreasing photorespiration. We discuss possible implications of these findings for the ecological success of Galdieria in extreme environments and for biotechnological applications.

Generation of Mutants of Nuclear-Encoded Plastid Proteins Using CRISPR/Cas9 in the Diatom Phaeodactylum tricornutum.

Genome modifications in microalgae are becoming a widespread and mandatory tool for research in both fundamental and applied biology. Among genome editing methods in these photosynthetic organisms, CRISPR/Cas9 offers a specific, powerful and efficient tool for genome engineering by inducing mutations in targeted regions of the genome. Here we described a protocol that allows the generation of knockout mutants by CRISPR/Cas9 in the diatom Phaeodactylum tricornutum using biolistic transformation.