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Melatonin is a bioactive, edible ingredient that promotes human health and exists widely in plants, but little is known about its biosynthetic routes and underlying molecular mechanisms in the herbaceous peony. In this contribution, we found that herbaceous peony flowers are rich in melatonin that is found in the greatest quantities in the white series, followed by the ink series, the red series and then the pink series. On this basis, the melatonin content fluctuates during flower development and peaks during the bloom stage. Moreover, it is apparent that sun exposure and blue light induce melatonin production whereas green light restrains it during a 24-h light/dark cycle of melatonin content, as there were ‘dual peaks’ at 2 p.m. and 2 a.m. Additionally, the corresponding expression pattern of the herbaceous peony tryptophan decarboxylase gene (TDC) was positively related with melatonin production. These results suggest that color series, development stage and light play an important role in melatonin accumulation, and that TDC is a rate-limiting gene in melatonin biosynthesis. [ABSTRACT FROM AUTHOR]