Acutely induced cell mortality in the unicellular green alga Chlamydomonas reinhardtii (Chlorophyceae) following exposure to acrylic resin nanoparticles.

Nanoparticles have unique properties that make them attractive for use in industrial and medical technology industries but can also be harmful to living organisms, making an understanding of their molecular mechanisms of action essential. We examined the effect of three different sized poly(isobutyl-cyanoacrylate) nanoparticles (iBCA-NPs) on the unicellular green alga Chlamydomonas reinhardtii. We found that exposure to iBCA-NPs immediately caused C. reinhardtii to display abnormal swimming behaviors. Furthermore, after one hour, most of the cells had stopped swimming and 10%-30% of cells were stained with trypan blue, suggesting that these cells had severely impaired plasma membranes. Observation of the cyto-ultrastructure showed that the cell walls had been severely damaged and that many iBCA-NPs were located in the space between the cell wall and plasma membrane, as well as inside the cytosol in some cases. A comparison of three strains of C. reinhardtii with different cell wall conditions further showed that the cell mortality ratio increased more rapidly in the absence of a cell wall. Interestingly, cell mortality over time was essentially identical regardless of iBCA-NP size if the total surface area was the same. Furthermore, direct observation of the trails of iBCA-NPs indicated that the first trigger was their contact with the cell wall, which is most likely accompanied by the inactivation or removal of adsorbed proteins from the cell wall surface. Cell mortality was accompanied by the overproduction of reactive oxygen species, which was detected more readily in cells grown under constant light rather than in the dark.

UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems.

KEY MESSAGE: In this investigation, we succeeded to generate Chlamydomonas mutants that bear dramatically enhanced ability for transgene expression. To yield these mutants, we utilized DNA methyltransferase deficient strain. These mutants must be useful as a plant cell factory. Chlamydomonas reinhardtii (hereafter Chlamydomonas) is a green freshwater microalga. It is a promising cell factory for the production of recombinant proteins because it rapidly grows in simple salt-based media. However, expression of transgenes integrated into the nuclear genome of Chlamydomonas is very poor, probably because of severe transcriptional silencing irrespective of the genomic position. In this study, we generated Chlamydomonas mutants by ultraviolet (UV)-mediated mutagenesis of maintenance-type DNA methyltransferase gene (MET1)-null mutants to overcome this disadvantage. We obtained several mutants with an enhanced ability to overexpress various transgenes irrespective of their integrated genomic positions. In addition, transformation efficiencies were significantly elevated. Our findings indicate that in addition to mechanisms involving MET1, transgene expression is regulated by a DNA methylation-independent transgene silencing system in Chlamydomonas. This is in agreement with the fact that DNA methylation occurs rarely in this organism. The generated mutants may be useful for the low-cost production of therapeutic proteins and eukaryotic enzymes based on their rapid growth in simple salt-based media.