Ostreococcus tauri is an easily cultured representative of unicellular algae (class Mamiellophyceae) that abound in oceans worldwide. Eight complete 13–22 Mb genomes of phylogenetically divergent species within this class are available, and their DNA sequences are nearly always present in metagenomic data produced from marine samples. Here we describe a simplified and robust transformation protocol for the smallest of these algae (O. tauri). Polyethylene glycol (PEG) treatment was much more efficient than the previously described electroporation protocol. Short (2 min or less) incubation times in PEG gave >10⁴ transformants per microgram DNA. The time of cell recovery after transformation could be reduced to a few hours, permitting the experiment to be done in a day rather than overnight as used in previous protocols. DNA was randomly inserted in the O. tauri genome. In our hands PEG was 20–40-fold more efficient than electroporation for the transformation of O. tauri, and this improvement will facilitate mutagenesis of all of the dispensable genes present in the tiny O. tauri genome.

Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration occurred randomly at a single locus in the genome and mainly as a single copy. Thus, we confirmed the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci. Furthermore, we often observed variations in the vector sequence itself. From these observations, we speculate that a nonhomologous end-joining-like mechanism is employed during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in this ecologically important marine alga.

Post-translational regulations of Shaker-like voltage-gated K⁺ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K⁺ fluorescence imaging in planta suggests that K⁺ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K⁺ distribution in leaves.