Salvia miltiorrhiza, commonly known as Danshen, is a traditional medicinal plant from the Lamiaceae family. According to the Chinese Pharmacopoeia (2020 edition), Danshen has a bitter flavor and cold nature. Its dried rhizomes possess pharmacological activities such as activating blood circulation, removing blood stasis, relieving pain, and clearing the heart[1], attributed to hydrophilic phenolic acids and lipophilic tanshinones. The biosynthesis pathways of these compounds specified in the pharmacopoeia are still hot research[2], but more medicinal compounds biosynthesis remains unclear.

Phenolic acid compounds often exist as multimers, and the conversion of rosmarinic acid (RA) to salvianolic acid B (SAB) involves the polymerization of a dimer into a tetramer. Recent study indicated that that RA and SAB levels inversely correlated with root hardness[3]. Rosmarinic acid synthase SmRAS and the cytochrome P450 gene family are key enzyme families for SA synthesis. Among the SmRAS gene family members, SmRAS1/2/4 exhibit predominant expression in root tissues. Functional studies involving overexpression have demonstrated that SmRAS1 and SmRAS4 significantly enhance the accumulation of RA and SAB, while simultaneously reducing danshensu (DSS) levels - a crucial precursor in phenolic acid biosynthesis[4,5]. These findings collectively indicate that SmRAS1 and SmRAS4 serve as critical regulatory components in the phenolic acid biosynthetic pathway. Functional studies of the SmCYP98A subfamily have enhanced knowledge of the SA synthesis pathway. SmRAS mediates the catalytic conversion of diverse acyl donors and acceptors to generate RA precursors. While two SmCYP98A subfamily SmCYP98A14 and SmCYP98A75 execute regiospecific meta-hydroxylation at distinct positions of these intermediates[6]. These mechanistic insights position CYP98A enzymes as strategic metabolic engineering targets for enhancing SA biosynthesis. Laccase genes family have been reported to participate in this polymerization process[7], but the pathway remained unclear. The relevant enzyme genes continue to be an area of active investigation. Tanshinone, another active diterpene compound in Danshen, is synthesized from GGPP. GGPP[8] would transform by type-II diterpene synthases CPS and KSL to produce miltiradiene, which will convert into various tanshinones through the action of cytochrome P450 oxidases(CYP450s)[9]. Despite these advancements, the enzymatic reaction mechanisms involved in the biosynthesis of tanshinones remain obscure and require further research.

The basic helix-loop-helix (bHLH) transcription factor is the second largest family in eukaryotes[10,11], characterized by a highly conserved HLH region. Numerous studies have highlighted the pivotal role of bHLH transcription factors in diverse physiological processes, including growth, development, and secondary metabolism[[12], [13], [14], [15], [16]]. In S. miltiorrhiza, SmbHLH37[17], SmbHLH53[18], SmbHLH92 and SmbHLH60 [19]have been reported to regulate phenolic acids and tanshinones based on overexpression or CRISPR/Cas 9. Thus, analyzing bHLH transcription factors in Danshen is crucial for understanding the regulatory mechanisms of metabolites.

In recent years, integrated transcriptomic and metabolomic methods have become a powerful approach to studying gene function[20]. This approach allows for the analysis of differentially expressed genes and the accumulation levels of downstream metabolites, providing a comprehensive understanding of the biological functions of transcription factors at the molecular level. However, most studies have focused on transcriptome profiling of TF regulation in S. miltiorrhiza, there have been no precedents for exploring gene functions through integrated omics approaches. Therefore, we tried combining the transcriptomic and metabolomic data to analyze gene functions at the molecular level and use co-expression analysis to predict potential synthetic genes in the secondary metabolic pathways. These findings offered new insights into the regulatory mechanisms of the secondary metabolic network.