Abstracts

Bioluminescent fungi emit green light peaking at 530 nm. The biochemical mechanism and the genes involved in fungal bioluminescence have been recently described. The emission of light is one of the products of the so-called Caffeic Acid Cycle (CAC), wherein caffeic acid is firstly converted into hispidin in a reaction catalyzed by hispidin synthase (HispS), followed by its hydroxylation by hispidin-3-hydroxylase (H3H), giving rise to the fungal luciferin (3-hydroxyhispidin). Then, a luciferase (Luz) catalyzes the addition of molecular oxygen from the luciferin, yielding an endoperoxide as high-energy intermediate, whose decomposition leads to the formation of oxyluciferin (caffeylpyruvate) and light emission. In the last step, oxyluciferin is recycled to caffeic acid by caffeylpyruvate hydrolase (CPH), restarting the cycle. Despite metabolites and genes of CAC having been characterized, the molecular regulation and ecological functions of the fungal bioluminescence for the mycelium remain poorly understood. Previous work from our group has demonstrated that the bioluminescence of Neonothopanus gardneri, a fungus that can be found in Babaçu Forest in Brazil, is controlled by a temperature-compensated circadian clock. Based on the transcriptome and genome obtained by our group from N. gardneri mycelium, we have identified candidate genes for the biological clock and other ones acting as light sensors. We have developed reference genes for RT-qPCR assays that allow us to explore the regulation of light emission at the level of transcription. In this context, we investigate whether the expression of transcripts of CAC genes and other candidates to the biological clock and light sensors is responsive to light in N. gardneri’s mycelium. With this in mind, the mycelium was cultivated in liquid medium at 25ºC for i) 5 days in constant darkness (DD) and ii) subjected to 1-hour of light after constant darkness. After 5 days, the mycelium was macerated in liquid N₂, and submitted to RNA extraction, and the expression of target transcripts was assessed by RT-qPCR. Preliminary results indicate that some genes involved in fungal bioluminescence show a decrease in transcripts production after 1-hour of light exposure. We also incubated N. gardneri mycelium in a 12h-light/dark cycle at 25ºC and sampled it every 4h over 48h. HPLC-MS was used to quantify intermediates, and RT-qPCR was used to analyze the time-dependent expression of CAC genes at the transcript level. Results showed that CAC intermediates production peaked during subjective night periods, indicating a circadian rhythm. This study can contribute with new cues and insights on the regulation of transcripts related to the bioluminescence in fungi as well as the molecular aspects of the circadian regulation of the fungal bioluminescence.