Functional characterization of AtAAS as a 5-hydroxytryptophan decarboxylase linking serotonin formation to melatonin metabolism in

Introduction: Serotonin and melatonin are plant signaling molecules involved in growth, stress responses, and redox balance. While animals produce serotonin via 5‑hydroxytryptophan (5‑HTP), plants are thought to use the tryptamine pathway. Although 5‑HTP exists in plants, its conversion to serotonin lacks a genetically defined enzyme. Methods: We performed phylogenetic and structural analyses of Arabidopsis aromatic aldehyde synthase (AtAAS), expressed recombinant AtAAS, and examined its 5-HTP decarboxylase activity in vitro. Enzyme kinetic parameters were determined, and endogenous 5-HTP levels in Arabidopsis were quantified. A T-DNA insertion mutant of AtAAS was further used to assess serotonin accumulation following 5-HTP feeding and to determine endogenous levels of serotonin, N-acetylserotonin, and melatonin under normal growth conditions. Results: AtAAS showed phylogenetic and structural similarities to animal aromatic amino acid decarboxylases and efficiently converted 5-HTP to serotonin in vitro (optimal pH 7.5, 55°C; Km = 1.806 mM, Vmax = 3.089 nmol/min/mg). Endogenous 5-HTP was detectable in Arabidopsis. Compared with wild-type plants, the AtAAS T-DNA insertion mutant exhibited reduced serotonin accumulation following 5-HTP feeding and decreased endogenous levels of downstream N-acetylserotonin and melatonin under normal growth conditions. Discussion: his discovery unveils enzymatic plasticity in plant serotonin biosynthesis and provides evidence for a previously unrecognized 5-HTP-dependent route in Arabidopsis.