NanoLuciferase Reporter Assays to Investigate Protein Dynamics and Circadian Clock Regulation in the Diatom Model Species Phaeodactylum tricornutum

ABSTRACT

Adaptation to environmental variations is crucial for organisms to optimize cellular and metabolic functions and ensure survival. A fundamental endogenous mechanism underlying this adaptation is the circadian clock, which coordinates the timing of diverse biological processes with the daily environmental cycle. The circadian system that regulates daily patterns of gene expression and protein dynamics has been extensively studied in terrestrial organisms, yet it remains poorly understood in prominent eukaryotic phytoplankton organisms such as diatoms.

Progress has been limited so far by the lack of automated, sensitive tools to monitor gene expression dynamics and circadian rhythms. To address this gap, we here leveraged the NanoLuciferase (NLuc) reporter to investigate circadian regulation in diatoms, focusing on the first identified diatom clock component RITMO1 as a molecular readout. We created transgenic lines in the model species Phaeodactylum tricornutum that express either transcriptional RITMO1p::NLuc or translational RITMO1p::RITMO1:NLuc fusions. Our results show that while NLuc is not suitable for transcriptional studies, it is highly effective for tracking diatom protein dynamics. In vivo assays revealed that RITMO1:NLuc faithfully recapitulates the diel rhythms of the endogenous protein and that the proteasome contributes to its degradation, during dark periods. Additionally, we established a protocol for continuous, automated in vitro luminescence monitoring and showed that RITMO1:NLuc expression profile can be used as a tool for studying circadian rhythms in diatoms. These methods can be adapted to other algae and cell lines, aiding the characterization of still-unexplored clock systems in organisms that shape aquatic ecosystems.