Identification of circadian rhythms inNannochloropsisspecies using bioluminescence reporter lines

Abstract

32Circadian clocks allow organisms to predict environmental changes caused by the rotation of the 33 Earth. Although circadian rhythms are widespread among different taxa, the core components of 34 circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. 35Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly 36 characterized and no clock components have been identified. We have investigated cell division 37 and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division 38 occurred principally during the night period under diel conditions, however, rhythms dampened 39 within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for 40 long-term monitoring of in vivo transcriptional rhythms in two Nannochlropsis species, N. 41 oceanica CCMP1779 and N. salina CCMP537. The reporter lines express free-running 42 bioluminescence rhythms with periods of ~21-31 h that dampen within ~3-4 days under constant 43 light. Using different entrainment regimes, we demonstrate that these rhythms are regulated by a 44 circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be 45 modulated pharmacologically using a CK1 e/d inhibitor, suggesting a role of this kinase in the 46 Nannochloropsis clock. Together with the molecular and genomic tools available for 47 Nannochloropsis species, these reporter lines represent an excellent system for future studies on 48 the molecular mechanisms of stramenopile circadian oscillators. 49 50 Significance statement 51Stramenopiles are a large and diverse line of eukaryotes in which circadian rhythms have been 52 only poorly characterized and no clock components have been identified. We have developed 53 bioluminescence reporter lines in Nannochloropsis species and provide evidence for the presence 54 of a circadian oscillator in stramenopiles; these lines will serve as tools for future studies to 55 uncover the molecular mechanisms of circadian oscillations in these species. 56 57