Identification and Regulation of Melatonin Biosynthetic Genes in Sweet Pepper During Ripening and Melatonin Treatment

Since its discovery in higher plants, melatonin has attracted considerable attention for its antioxidant properties and its diverse roles in plant physiology and stress responses. However, its biosynthetic pathway remains only partially elucidated, particularly in horticultural crops of economic and nutritional importance, such as pepper (Capsicum annuum L.) fruits. In our previous work, we identified five genes encoding tryptophan decarboxylase (TDC), the first enzyme in the melatonin biosynthetic pathway in pepper. The present study expands on this by identifying and characterizing additional genes encoding enzymes involved in subsequent steps of the pathway, including four tryptamine 5-hydroxylase (T5H) genes, two serotonin N-acetyltransferase (SNAT) genes, three N-acetylserotonin O-methyltransferase (ASMT) genes, two caffeic acid O-methyltransferase (COMT) genes, and one N-acetylserotonin deacetylase (ASDAC) gene, representing a total of twelve newly identified genes. We further examined their expression in sweet pepper fruits and found that only nine of the identified genes are expressed in the fruit, with generally higher transcript levels during the unripe stages. Melatonin quantification in the California-type 'Masami' cultivar using UPLC with fluorescence detection (FD) revealed concentrations of 623 ng melatonin·g^-1 dry weight (DW) in green fruits and 431 ng melatonin·g^-1 DW in red fruits, consistent with the higher expression of melatonin biosynthetic genes in unripe fruit. Expression analysis of these genes by means of RNA-seq revealed differential modulation in response to exogenous melatonin treatments (20, 50, and 100 µM). To our knowledge, this is the first report demonstrating that exogenous melatonin regulates the expression of genes involved in its own biosynthetic pathway in sweet pepper fruits. Notably, treatment with 100 µM melatonin delayed ripening in these non-climacteric fruits, highlighting its potential biotechnological application for controlling fruit ripening and improving postharvest management.