Abstract

Carthamidin and isocarthamidin as well as their 7-O-glucuronides are abundant tetrahydroxyflavanones in Scutellaria barbata , but their biosynthesis remains poorly defined. Here, we combined metabolite profiling, transcriptome-guided gene discovery, heterologous expression, enzymatic assays, virus-induced gene silencing (VIGS), and transient pathway reconstruction to elucidate their biosynthetic pathway. SbarCHS1 and SbarCHI form the naringenin-producing module. Three CYP82D enzymes then catalyze regioselective A-ring hydroxylation: SbarCYP82D1 and SbarCYP82D8 function as naringenin 6-hydroxylases to generate carthamidin, whereas SbarCYP82D5 acts as an efficient naringenin 8-hydroxylase to produce isocarthamidin. A UGT88-family enzyme, SbarUGAT, catalyzes 7-O-glucuronidation of both hydroxylated flavanones. Non-enzymatic interconversion observed between carthamidin and isocarthamidin and between their corresponding 7-O-glucuronides further suggests that chemical equilibration contributes to this branched biosynthetic pathway. VIGS confirmed the in planta roles of SbarCHS1, SbarCYP82D8, and SbarUGAT, identifying SbarCYP82D8 as the dominant hydroxylation enzyme in leaves. Reconstitution in Nicotiana benthamiana enabled de novo production of both glucuronides, and co-expression of SbarUGDH further enhanced glucuronide accumulation. These findings define the S. barbata biosynthesis of bioactive flavanone glucuronides and provide enzymes for future metabolic engineering of such specialized flavonoids.