Flavonoids play multiple roles in plant coloration and stress resistance and are closely associated with human health. Flavonoids and non-flavonoids (such as phenolic acids) are produced via the phenylpropanoid-derived pathway. Anthocyanidin synthase (ANS) catalyzes the synthesis of anthocyanins from leucoanthocyanidin in the flavonoids branched pathway. In this study, SmANS from Salvia miltiorrhiza was cloned and mainly localized in the endoplasmic reticulum (ER), plastids, Golgi, plasma membrane, and nucleus of tobacco epidermal cells, and was most highly expressed in purple petals in S. miltiorrhiza, whereas it showed almost no expression in white petals, green calyxes, and pistils in S. miltiorrhiza Bge f. alba. Overexpressed SmANS enhanced anthocyanin accumulation but reduced salvianolic acid B (SAB) and rosmarinic acid (RA) biosynthesis in S. miltiorrhiza and S. miltiorrhiza Bge f. alba plantlets, meanwhile, it restored the purple-red phenotype in S. miltiorrhiza Bge f. alba. These changes were due to reallocation of the metabolic flow, which was influenced by the SmANS gene. These findings indicate that SmANS not only plays a key role in anthocyanin accumulation in S. miltiorrhiza, but also acts as a “switch” for the coloration of S. miltiorrhiza Bge f. alba. This study provides baseline information for further research on flavonoids metabolism and improvement of anthocyanin or phenolic acid production by genetic engineering.

The ethylene response factor family genes are involved in the regulation of secondary metabolism in Salvia miltiorrhiza, but the mechanism underlying this regulation remains elusive. In the present study, based on the cDNA library of S. miltiorrhiza, an AP2/ERF gene was cloned and named SmERF1b-like. This gene exhibited a significant response to exogenous ethylene supply, such that ethylene remarkably upregulated SmERF1b-like expression levels in the leaves of S. miltiorrhiza. Subcellular localization showed that SmERF1b-like is located in the nucleus. Furthermore, SmERF1b-like showed a binding affinity with a GCC-box motif in the promoter region of genes associated with tanshinone biosynthesis in S. miltiorrhiza. Overexpression of SmERF1b-like in hairy roots of S. miltiorrhiza substantially upregulated SmCPS1 and SmKSL1 expression levels, resulting in increased biosynthesis of tanshinone I and cryptotanshinone contents. This finding provides valuable theoretical support for the utilization of a plant genetic engineering strategy to enhance S. miltiorrhiza resources.

Background: Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants with high medicinal value. Gibberellins are growth-promoting phytohormones that regulate numerous growth and developmental processes in plants. However, their role on the secondary metabolism regulation has not been investigated. Results: In this study, we found that gibberellic acid (GA) can promote hairy roots growth and increase the contents of tanshinones and phenolic acids. Transcriptomic sequencing revealed that many genes involved in the secondary metabolism pathway were the GA-responsive. After further analysis of GA signaling pathway genes, which their expression profiles have significantly changed, it was found that the GRAS transcription factor family had a significant response to GA. We identified 35 SmGRAS genes in S. miltiorrhiza, which can be divided into 10 subfamilies. Thereafter, members of the same subfamily showed similar conserved motifs and gene structures, suggesting possible conserved functions. Conclusions: Most SmGRAS genes were significantly responsive to GA, indicating that they may play an important role in the GA signaling pathway, also participating in the GA regulation of root growth and secondary metabolism in S. miltiorrhiza.

Senna obtusifolia is a famous medicinal plant that is widely used in Asian countries. Its seed plays an important role in the treatment of many diseases because it contains various anthraquinones and flavonoids. Our previous studies have indicated that three space environment-induced S. obtusifolia lines (SP-lines) i.e., QC10, QC29, and QC46, have higher seed yield and aurantio-obtusin (AO) content. However, the underlying mechanism of higher AO content in SP-lines is still unknown. Herein, transcriptome sequencing and HPLC were employed to analyze the differences between SP-lines and ground control (GC3) and elucidate the regulatory mechanisms of AO accumulation in SP-lines. The results show that 4002 differentially expressed genes (DEGs) were identified in SP-lines versus (vs.) GC3. DEGs in the QC10 vs. GC3, QC29 vs. GC3, and QC46 vs. GC3 comparisons were classified into 28, 36, and 81 GO terms and involved in 63, 74, and 107 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG pathway and gene expression analysis revealed that DEGs involved in anthraquinone pathways were significantly elevated in QC10 and QC46. Integrating the results of GO annotation, KEGG enrichment, and gene expression analysis, we propose that the elevated genes such as DAHPS, DHQS, and MenB enhance the metabolic flux in the anthraquinone pathway and promote AO content in QC10 and QC46. Taken together, this study elucidated the mechanism of AO content in SP-lines and provides valuable genetic information for S. obtusifolia. In addition, to the best of our knowledge, this study presents the first transcriptome analysis of environment-induced medicinal plants and paves the way to select elite S. obtusifolia varieties in the future.

As the antibiotic resistance of pathogens becomes increasingly severe, it is becoming more feasible to use methods that suppress the virulence of pathogens rather than exerting selective pressure on their growth. Pseudomonas aeruginosa, a dangerous opportunistic pathogen, infects hosts by producing multiple virulence factors, which are regulated by quorum-sensing (QS) systems, including the las systems, rhl systems, and pqs systems. This study used the chromosome lacZ transcription fusion reporter model to screen the traditional Chinese medicine monomer library and found that bakuchiol can effectively inhibit the pqs system and related virulence phenotypes of P. aeruginosa, including the production of virulence factors (pyocyanin, hydrogen cyanide, elastase, and lectin) and motility (swarming, swimming, and twitching motility) without affecting its growth. Subsequently, through genetic complementation analysis, we found that bakuchiol inhibited the function of the transcriptional activation protein PqsR of the pqs system in P. aeruginosa in a concentration-dependent manner. Furthermore, molecular dynamics simulation study results indicated that bakuchiol can target PqsR of the pqs system, thereby inhibiting the pqs system. Among the amino acids in PqsR, ALA-168 may be a key amino acid residue in the hydrophobic interaction between PqsR protein and bakuchiol. Finally, in vivo experiments demonstrated that bakuchiol attenuated the pathogenicity of P. aeruginosa to Chinese cabbage (Brassica pekinensis) and Caenorhabditis elegans. In summary, this study suggests that bakuchiol is an effective inhibitor that targets the pqs system of P. aeruginosa, providing a new strategy for addressing P. aeruginosa infections.