ELONGATED HYPOCOTYL 5-mediated suppression of melatonin biosynthesis is alleviated by darkness and promotes cotyledon opening

Inhibition of melatonin biosynthesis by the transcription factor HY5 occurs by direct suppression of its major synthetase gene SNAT6, which can be relieved by darkness, resulting in inhibition of cotyledon opening in Arabidopsis. Abstract Melatonin (N-acetyl-5-methoxytryptamine) biosynthesis in plants is induced by darkness and high-intensity light; however, the underlying transcriptional mechanisms and upstream signalling pathways are unknown. We identified a dark-induced and highly expressed melatonin synthetase in Arabidopsis thaliana, AtSNAT6, encoding serotonin N-acetyltransferase. We assessed melatonin content and AtSNAT6 expression in mutants lacking key regulators of light/dark signalling. AtCOP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and AtHY5 (ELONGATED HYPOCOTYL 5), which control light/dark transition and photomorphogenesis, promoted and suppressed melatonin biosynthesis, respectively. Using EMSA and ChIP-qPCR analysis, we showed that AtHY5 inhibits AtSNAT6 expression directly. An analysis of melatonin content in snat6 hy5 double mutant and AtHY5+AtSNAT6-overexpressing plants confirmed the regulatory function of AtHY5 and AtSNAT6 in melatonin biosynthesis. Exogenous melatonin further inhibited cotyledon opening in hy5 mutant and AtSNAT6-overexpressing seedlings, but partially reversed the promotion of cotyledon opening in AtHY5-overexpressing seedlings and snat6. Additionally, CRISPR/Cas9-mediated mutation of AtSNAT6 increased cotyledon opening in hy5 mutant, and overexpression of AtSNAT6 decreased cotyledon opening in AtHY5-overexpressing seedlings via changing melatonin biosynthesis, confirming that AtHY5 decreased melatonin-mediated inhibition of cotyledon opening. Our data provide new insights into the regulation of melatonin biosynthesis and its function in cotyledon opening.