Circadian regulation of key physiological processes by the <scp>RITMO</scp>1 clock protein in the marine diatom <i>Phaeodactylum tricornutum</i>

Phasing biological and physiological processes to periodic light-dark cycles is crucial for the life of most organisms. Marine diatoms, as many phytoplanktonic species, exhibit biological rhythms, yet their molecular timekeepers remain largely uncharacterized. Recently, the bHLH-PAS protein RITMO1 has been proposed to act as a regulator of diatom circadian rhythms. In this study, we first determined the physiological conditions to monitor circadian clock activity and its perturbation in the diatom model species Phaeodactylum tricornutum by using cell fluorescence as a circadian output. Employing ectopic overexpression, targeted gene mutagenesis, and functional complementation, we then investigated the role of RITMO1 in various circadian processes. Our data reveal that RITMO1 significantly influences the P. tricornutum circadian rhythms not only of cellular fluorescence, but also of photosynthesis and of the expression of clock-controlled genes, including transcription factors and putative clock input/output components. RITMO1 effects on rhythmicity are unambiguously detectable under free-running conditions. By uncovering the complex regulation of biological rhythms in P. tricornutum, these findings advance our understanding of the endogenous factors controlling diatom physiological responses to environmental changes. They also offer initial insights into the mechanistic principles of oscillator functions in a major group of phytoplankton, which remain largely unexplored in chronobiology.