Evolutionary origin of neohesperidoside, a bitter metabolite, and its potential role in biotic defense and citrus dissemination

Fruit secondary metabolites play pivotal roles in plant evolution by deterring herbivores and attracting seed dispersers. However, the mechanisms by which these compounds evolve and drive diversification in citrus remain poorly understood. In this study, we demonstrate that the emergence of the bitter compound neohesperidoside (Neo) has contributed to citrus dissemination by enhancing defense against biotic stresses. Targeted metabolomic analyses revealed that Neo accumulation emerged in early-diverging citrus lineages, whereas its non-bitter counterpart rutinoside (Rut) can be traced back to Citrus-related species. Comparative genomic analyses and enzyme functional assays further revealed that Neo biosynthesis originated from the duplication of two di-glucosyltransferase genes, CmdGlcT-1 and UGT79B203, in early-diverging citrus, followed by neofunctionalization into Cm1,2RhaT and UGT79B202, enzymes capable of synthesizing Neo. A structurally conserved amino acid residue-corresponding to Phe195 in Cm1,2RhaT and Leu201 in UGT79B203-was identified as critical for this functional transition. Compared with Rut, Neo exhibits stronger antifungal and anti-feeding activities, suggesting a role in enhanced biotic defense that may have contributed to the broader geographical distribution of early-diverging citrus species. Together, these findings provide new insights into the evolutionary origin of citrus bitterness and highlight the adaptive role of specialized metabolites in shaping plant-environment interactions.