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The role of melatonin biosynthetic enzymes has been well studied. However, the transcriptional regulation of melatonin biosynthetic enzymes and their integrative crosstalk with other signaling pathways remain elusive. Here, we summarize recent progress in the functional analysis of melatonin biosynthetic enzymes and the major sites of melatonin synthesis in plants. We focus on the dual roles of melatonin biosynthetic enzymes in melatonin biosynthesis and in the crosstalk between melatonin and autophagy, antioxidant signaling, and stress r