The chromosome‐scale assembly of the <i>Salvia plebeia</i> genome provides insight into the biosynthesis and regulation of rosmarinic acid

Salvia plebeia is an important traditional Chinese medicinal herb, with flavonoids and phenolic acids as its primary bioactive components. However, the absence of a reference genome hinders our understanding of genetic basis underlying the synthesis of these components. Here, we present a high-quality, chromosome-scale genome assembly of S. plebeia, spanning 1.22 Gb, with a contig N50 of 91.72 Mb and 36 861 annotated protein-coding genes. Leveraging the genome data, we identified four catalytic enzymes-one rosmarinic acid synthase (RAS) and three cytochrome P450 monooxygenases (CYP450s) -in S. plebeia, which are involved in rosmarinic acid biosynthesis. We demonstrate that SpRAS catalyses the conjugation of various acyl donors and acceptors, resulting in the formation of rosmarinic acid and its precursor compounds. SpCYP98A75, SpCYP98A77 and SpCYP98A78 catalyse the formation of rosmarinic acid from its precursors at either the C-3 or the C-3' position. Notably, SpCYP98A75 exhibited a stronger hydroxylation capacity at the C-3' position, whereas SpCYP98A77 and SpCYP98A78 demonstrate greater hydroxylation efficiency at the C-3 position. Furthermore, SpCYP98A75 hydroxylated both the C-3 and C-3' positions simultaneously, promoting the conversion of 4-coumaroyl-4'-hydroxyphenyllactic acid to rosmarinic acid. Next, using a hairy root genetic transformation system for S. plebeia, we identified a basic helix-loop-helix protein type transcription factor, SpbHLH54, which positively regulates the biosynthesis of rosmarinic acid and homoplantaginin in S. plebeia. These findings provide a valuable genomic resource for elucidating the mechanisms of rosmarinic acid biosynthesis and its regulation and improve the understanding of evolutionary patterns within the Lamiaceae family.