Saponins are the main triterpenoid ingredients from Panax notoginseng, a well-known Chinese medicine, and are important sources for producing drugs to prevent and treat cerebrovascular and cardiovascular diseases. However, the transcriptional regulatory network of saponin biosynthesis in P. notoginseng is largely unknown. In the present study we demonstrated that one R2R3-MYB transcription factor, designated PnMYB4, acts as a repressor of saponin accumulation. Suppression of PnMYB4 in P. notoginseng calli significantly increased the saponin content and the expression level of saponin biosynthetic genes. PnMYB4 directly bound to the promoters of key saponin biosynthetic genes, including PnSS, PnSE, and PnDS, to repress saponin accumulation. PnMYB4 and the activator PnMYB1 could interacted with PnbHLH, which is a positive regulator of saponin biosynthesis, to modulate the biosynthesis of saponin. PnMYB4 competed with PnMYB1 for binding to PnbHLH, repressing activation of the promoters of saponin structural genes induced by the PnMYB1-PnbHLH complex. Our study reveals that a complex regulatory module of saponin biosynthesis is associated with positive and negative MYB transcriptional regulators and provides a theoretical basis for improving the content of saponins and efficacy of P. notoginseng.

Rhamnosides usually possess better bioavailabilities and improved solubilities compared with their aglycons and are a major source of bioactive natural products. However, biosynthesis of rhamnosides is hindered by the commercially expensive UDP-rhamnose (UDP-Rha) donor and a lack of universal rhamnosyltransferases. In the present study, an efficient UDP-Rha production system via a two-step enzymatic reactions using UDP-glucose (UDP-Glc) as a substrate was constructed. Extensive in vitro enzymatic assays and preparative reactions using the obtained UDP-Rha/UDP-Glc highlighted the robust glycosylation promiscuity of the reported rhamnosyltransferase AtUGT78D1. Based on HPLC-UV and HR-MS analyses, 30 of the tested aromatic compounds belonging to 7 structural types, including flavonoids, flavonoid glycosides, phenylethyl chromones, benzophenones, coumarins, lignanoids, and anthraquinones, were accepted by AtUGT78D1 to conduct the corresponding rhamnosylation and/or glucosylation with one or more glycosyl substitutions at different positions. Further preparative reactions expanded the catalytic characteristic of AtUGT78D1 since it can catalyse the rhamnosylation at the 3-OH position of the flavonols, glucosylation at the 7-OH position of the flavone baicalein, and multiple hydroxyl substitutions for diverse types of aromatics. Interestingly, a unique reversible catalysis activity of AtUGT78D1 was observed, and it has been effectively used in one-pot rhamnosylation of the desired rhamnoside. The enzymatic rhamnosylations of diverse “drug-like” scaffolds as well as bidirectional catalysis for one-pot rhamnosylations by plant rhamnosyltransferase were rarely reported before, which indicated that AtUGT78D1 was expected to be a universal and effective tool for chemo-enzymatic synthesis of diverse bioactive rhamnosylated derivatives for drug discovery.

Introduction: Salvia miltiorrhiza radix et rhizoma (Danshen) is a crucial medicinal material for treating cardiovascular and cerebrovascular diseases. However, the presence of adulterants and intraspecific variability poses challenges to its clinical safety.Methods: This study collected samples of S. miltiorrhiza from various regions and commonly encountered adulterants. The composition differences of S. miltiorrhiza radix and its adulterants were analyzed by fingerprint and broad-target metabolomics. Chloroplast genome was used to distinguish intra-genus species and DNA barcoding was used to identify germplasm sources.Results: The fingerprinting analysis proved that there is no chemical composition consistency between S. miltiorrhiza radix and its adulterants. Broad-targeted metabolomics can distinguish S. miltiorrhiza radix from Salvia yunnanensis radix, Dipsacus asperoides radix, and Arctium lappa radix. Additionally, comparative chloroplast genome analysis indicated that atpF and rps4-trnT-UGU were the potential DNA barcodes for S. miltiorrhiza. 259 samples from 13 provinces and 21 origins were amplified and sequenced, resulting in the identification of 62 haplotypes. The unique haplotypes found in Shanxi Luoyang, Shandong Qingdao and other places can be used as molecular geographic markers for the identification of the germplasm source of S. miltiorrhiza.Discussion: This study systematically differentiates S. miltiorrhiza from its adulterants and highlights the potential of unique haplotypes as markers for sourcing. The findings provide strong scientific evidence for the clinical safety of S. miltiorrhiza, emphasizing the importance of proper cultivation, selection, and breeding of varieties.