Integrative omics reveals GCCGCC-mediated DREB regulation of serotonin biosynthesis in the southwestern China endemic walnut Juglans sigillata

Juglans sigillata is an economically important endemic walnut species in southwest China, renowned for its unique stress adaptation abilities and significantly higher serotonin content than the widely cultivated common walnut (J. regia). Serotonin, a neurotransmitter and a precursor of melatonin, is a key functional ingredient in nutraceuticals and functional foods, significantly contributing to the industrial value of walnut products. However, the molecular mechanism underlying serotonin biosynthesis and regulation in this species remains completely unexplored. In this study, we systematically characterized serotonin accumulation patterns and dissected its biosynthesis regulatory network in J. sigillata via an integrated workflow of genome mining, transcriptome profiling, and molecular functional validation. We found that serotonin levels peaked in late-stage embryos during seed development and were 10-fold higher in bud tissues than in mature embryos. JsTDC1 and JsT5H were identified as core biosynthetic genes, with JsTDC1 expression showing a strong positive correlation with serotonin accumulation. Transient expression assays in Nicotiana benthamiana confirmed their catalytic functions, and the meristem-specific CAT-box and seed-specific RY-element in the JsTDC1 promoter likely account for distinct spatiotemporal accumulation of serotonin. Crucially, using luciferase assays, yeast one-hybrid, and electrophoretic mobility shift assays, we demonstrated that two DREB transcription factors, JsDREB2D1 and JsDREB2D2, directly activate JsTDC1 by binding to a non-classical GCCGCC motif. This study provides the first mechanistic insight into walnut serotonin biosynthesis, reveals a novel DREB-mediated regulatory module, and delivers actionable molecular targets for breeding high-serotonin walnut varieties to support the nutraceutical industry.