bHLH-PAS protein RITMO1 regulates diel biological rhythms in the marine diatom Phaeodactylum tricornutum

Periodic light–dark cycles govern the timing of basic biological processes in organisms inhabiting land as well as the sea, where life evolved. Although prominent marine phytoplanktonic organisms such as diatoms show robust diel rhythms, the mechanisms regulating these processes are still obscure. By characterizing a Phaeodactylum tricornutum bHLH-PAS nuclear protein, hereby named RITMO1, we shed light on the regulation of the daily life of diatoms. Alteration of RITMO1 expression levels and timing by ectopic overexpression results in lines with deregulated diurnal gene expression profiles compared with the wild-type cells. Reduced gene expression oscillations are also observed in these lines in continuous darkness, showing that the regulation of rhythmicity by RITMO1 is not directly dependent on light inputs. We also describe strong diurnal rhythms of cellular fluorescence in wild-type cells, which persist in continuous light conditions, indicating the existence of an endogenous circadian clock in diatoms. The altered rhythmicity observed in RITMO1 overexpression lines in continuous light supports the involvement of this protein in circadian rhythm regulation. Phylogenetic analysis reveals a wide distribution of RITMO1-like proteins in the genomes of diatoms as well as in other marine algae, which may indicate a common function in these phototrophs. This study adds elements to our understanding of diatom biology and offers perspectives to elucidate timekeeping mechanisms in marine organisms belonging to a major, but under-investigated, branch of the tree of life. Phaeodactylum tricornutum wild type (Wt) and bHLH1a overexpressing line (OE-1) cultures were entrained to 16 hours of light and 8 hours of dark (16L:8D) for 2 weeks. The preadapted Wt and OE-1 lines were transferred to DD and sampled every 3 hours for a period of 24 hours, starting one hour after the beginning of the presumptive light period (time point 1). Three biological replicates were sampled for each condition. The expression of 15 selected genes, identified by their expression profiles in a previous screening in LD and DD conditions, was analyzed over the time series in both the Wt and OE-1 lines.