Duchatelet, L.; Galeazzo, G. A.; Coubris, C.; Bridoux, L.; Rezsohazy, R.; Melo, M. R. S.; Marek, M.; Dupont, S.; Oliveira, A. G.; Delroisse, J.

Bioluminescence is the production of visible light by living organisms. This process occurs biochemically through the primary oxidation of specific luciferin substrates, facilitated by enzymes known as luciferases. Moreover, the bioluminescent reaction often involves additional elements like auxiliary proteins or cofactors. Auxiliary proteins, such as fluorescent proteins and coelenterazine-binding proteins, for example, may serve various roles, such as altering the wavelength of the emitted light or stabilizing the highly reactive and unstable luciferin, respectively. Calcium has been demonstrated to serve as an essential ion in luminescence processes across various species. Despite the existence of numerous bioluminescent species, only a fraction of bioluminescent systems and their associated molecular actors have been thoroughly studied. Within anthozoans, the bioluminescence system of the Renilla genus stands out as the sole extensively characterized system, involving a coelenterazine-dependent luciferase, a calcium-dependent coelenterazine-binding protein, and a green fluorescent protein. Through a multidisciplinary approach, we investigated the bioluminescent system of three sea pen species: Pennatula phosphorea, Funiculina quadrangularis, and Anthoptilum murrayi (Pennatuloidea, Anthozoa, Cnidaria). We first confirmed or characterized the emission spectra of these species, elucidated the coelenterazine-based reaction, and generated reference transcriptomes for the two first species. Our transcriptomic analysis revealed transcripts encoding luciferases, fluorescent proteins, and coelenterazine-binding proteins, also shedding light on their expression profiles. Immunodetection confirmed the presence of luciferase in P. phosphorea and F. quadrangularis, while autofluorescence patterns attributed to green fluorescent protein or the coelenterazine were observed in P. phosphorea. Furthermore, a recombinant luciferase was expressed for A. murrayi. Our findings also underscore the essential role of calcium ions in bioluminescence, possibly associated with the mechanism of coelenterazine binding and substrate release. Finally, we propose a mechanistic model of bioluminescence in anthozoans, laying the groundwork for further functional characterization and ecological exploration of these sessile and benthic luminous organisms.