The chromosome-scale assembly of the Salvia plebeia genome provides insight into the biosynthesis and regulation of rosmarinic acid

Abstract

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.

Keywords: CYP98A; Salvia plebeia; biosynthesis; genome assembly; rosmarinic acid.

Figure 1

Genomic features of S. plebeia. (a) Chromosome‐level landscape of the genome. (I, gene density; II, repeat density; III, GC content). (b) Phylogenetic tree of S. plebeia and 11 other species. (c) Distribution of Ks values of S. plebeia gene pairs in segmental duplications; from left, the two peaks reflect a WGD in Lamiales and an ancient WGT in core eudicots. (d) Collinearity between S. plebeia, S. officinalis, S. miltiorrhiza and S. splendens of Lamiales.

Figure 2

Functional verifications of S. plebeia RAS genes. (a) Proposed rosmarinic acid biosynthetic pathway. Abbreviation: RAS, rosmarinic acid synthase; CYP98A, cytochrome P450 98A. (b) LC–MS analyses of recombinant SpRAS enzyme assays using salvianic acid A and caffeoyl‐CoA as substrates. (c) LC–MS analyses of recombinant SpRAS enzyme assays using 4‐hydroxyphenyllactic acid and caffeoyl‐CoA as substrates. (d) LC–MS analyses of recombinant SpRAS enzyme assays using salvianic acid A and p‐coumaroyl‐CoA as substrates. (e) LC–MS analyses of recombinant SpRAS enzyme assays using 4‐hydroxyphenyllactic acid and p‐coumaroyl‐CoA as substrates. The products were detected using HPLC (330 nm) and LC–MS in negative ionization mode. Boiled enzymes are used as a control. The red boxes indicate molecular ion peaks.

Figure 3

Functional verifications of S. plebeia CYP98A genes. (a) HPLC analyses of recombinant SpCYP98A75, SpCYP98A77 and SpCYP98A78 enzymes assays using caffeoyl‐4′‐hydroxyphenyllactic acid as substrates. (b) HPLC analyses of recombinant SpCYP98A75, SpCYP98A77 and SpCYP98A78 enzymes assays using 4‐coumaroyl‐3′,4′‐hydroxyphenyllactic acid as substrates. (c) HPLC and EIC analyses of recombinant SpCYP98A75, SpCYP98A77 and SpCYP98A78 enzymes assays using 4‐coumaroyl‐4′‐hydroxyphenyllactic acid as substrates. The products were detected using HPLC (330 nm) and LC–MS in negative ionization mode. Microsomal proteins extracted from pESC‐WAT11 as negative control and rosmarinic acid standard as positive control.

Figure 4

Identification of TFs regulating rosmarinic acid biosynthesis. (a) Hairy root induction and cultures of S. plebeia using A. rhizogenes strain A4. I: Aseptic seedlings of S. plebeia. II: Infection of leaf explants by A. rhizogenes A4. III: Growth status of hairy root strains of S. plebeia on 1/2 MS solid culture medium. IV: Growth status of hairy roots of S. plebeia in 1/2 MS liquid culture medium. (b) Effects of MeJA induction days on the content of rosmarinic acid and homoplantaginin in hairy roots of S. plebeia. (c) The transcription expression levels of biosynthetic genes related to rosmarinic acid and homoplantaginin in hairy root culture system of S. plebeia treated with MeJA different induction times (0, 3, 6, 9 and 12 h). (c) The phylogenetic tree of the bHLH gene family in S. plebeia and A. thaliana, see Figure S15 for the detailed phylogeny. (e) Heatmap of differentially expressed bHLH transcription factor treated with MeJA in hairy root culture system of S. plebeia. Bars are means ± standard deviation (Student's t‐test, *P < 0.05, **P < 0.01).

Figure 5

SpbHLH54 positively regulates rosmarinic acid biosynthesis. (a) The content of rosmarinic acid and homoplantaginin in SpbHLH54 transgenic and wild‐type hairy root lines. (b) The transcription expression levels of biosynthetic genes related to rosmarinic acid homoplantaginin and in SpbHLH54 transgenic and wild‐type hairy root lines. (c, d) Yeast‐one‐hybrid (Y1H) assay showing the interaction of SpbHLH54 with SpRAS and SpFNS promoter. (e–g) Dual‐luciferase assay for SpbHLH54 and pSpRAS and pSpFNS in tobacco. Bars are means ± standard deviation (Student's t‐test, *P < 0.05, **P < 0.01, ***P < 0.001).