Phenolic acids and tanshinones are two major bioactive components in Salvia miltiorrhiza Bunge. A novel endogenous R2R3-MYB transcription factor, SmMYB36, was identified in this research. This transcript factor can simultaneously influence the content of two types of components in SmMYB36 overexpression hairy roots. SmMYB36 was mainly localized in the nucleus of onion epidermis and it has transactivation activity. The overexpression of SmMYB36 promoted tanshinone accumulation but inhibited phenolic acid and flavonoid biosynthesis in Salvia miltiorrhiza hairy roots. The altered metabolite content was due to changed metabolic flow which was regulated by transcript expression of metabolic pathway genes. The gene transcription levels of the phenylpropanoid general pathway, tyrosine derived pathway, methylerythritol phosphate pathway and downstream tanshinone biosynthetic pathway changed significantly due to the overexpression of SmMYB36. The wide distribution of MYB binding elements (MBS, MRE, MBSI and MBSII) and electrophoretic mobility shift assay results indicated that SmMYB36 may be an effective tool to regulate metabolic flux shifts.

Jasmonates (JAs) are important plant hormones that regulate a variety of plant development and defense processes, including biosynthesis of secondary metabolites. The JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators in the JA signaling pathways of plants. We first verified that methyl jasmonate (MeJA) enhanced the accumulation of both salvianolic acids and tanshinones in Salvia miltiorrhiza (Danshen) hairy roots by inducing the expression of their biosynthetic pathway genes. Nine JAZ genes were cloned from Danshen and their expression levels in hairy roots were all increased by treatment with MeJA. When analyzed in detail, however, SmJAZ8 showed the strongest expression in the induced hairy roots. Overexpression or RNAi of SmJAZ8 deregulated or up-regulated the yields of salvianolic acids and tanshinones in the MeJA-induced transgenic hairy roots, respectively, and transcription factors and biosynthetic pathway genes showed an expression pattern that mirrored the production of the compounds. Genetic transformation of SmJAZ8 altered the expression of other SmJAZ genes, suggesting evidence of crosstalk occurring in JAZ-regulated secondary metabolism. Furthermore, the transcriptome analysis revealed a primary-secondary metabolism balance regulated by SmJAZ8. Altogether, we propose a novel role for SmJAZ8 as a negative feedback loop controller in the JA-induced biosynthesis of salvianolic acids and tanshinones.

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.

Salvia miltiorrhiza is one of the most widely used traditional Chinese medicinal plants because of its excellent performance in treating heart diseases. Tanshinones and phenolic acids are two important classes of effective metabolites, and their biosynthesis has attracted widespread interest. Here, we functionally characterized SmGRAS1 and SmGRAS2, two GRAS family transcription factors from S. miltiorrhiza. SmGRAS1/2 were highly expressed in the root periderm, where tanshinones mainly accumulated in S. miltiorrhiza. Overexpression of SmGRAS1/2 upregulated tanshinones accumulation and downregulated GA, phenolic acids contents, and root biomass. However, antisense expression of SmGRAS1/2 reduced the tanshinones accumulation and increased the GA, phenolic acids contents, and root biomass. The expression patterns of biosynthesis genes were consistent with the changes in compounds accumulation. GA treatment increased tanshinones, phenolic acids, and GA contents in the overexpression lines, and restored the root growth inhibited by overexpressing SmGRAS1/2. Subsequently, yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays (EMSA) showed SmGRAS1 promoted tanshinones biosynthesis by directly binding to the GARE motif in the SmKSL1 promoter and activating its expression. Yeast two-hybrid assays showed SmGRAS1 interacted physically with SmGRAS2. Taken together, the results revealed that SmGRAS1/2 acted as repressors in root growth and phenolic acids biosynthesis but as positive regulators in tanshinones biosynthesis. Overall, our findings revealed the potential value of SmGRAS1/2 in genetically engineering changes in secondary metabolism.

Summary The medicinal plant Scutellaria baicalensis Georgi is rich in specialized 4′‐deoxyflavones, which are reported to have many health‐promoting properties. We assayed Scutellaria flavones with different methoxyl groups on human cancer cell lines and found that polymethoxylated 4′‐deoxyflavones, like skullcapflavone I and tenaxin I have stronger ability to induce apoptosis compared to unmethylated baicalein, showing that methoxylation enhances bioactivity as well as the physical properties of specialized flavones, while having no side‐effects on healthy cells. We investigated the formation of methoxylated flavones and found that two O‐methyltransferase (OMT) families are active in the roots of S. baicalensis. The Type II OMTs, SbPFOMT2 and SbPFOMT5, decorate one of two adjacent hydroxyl groups on flavones and are responsible for methylation on the C6, 8 and 3′‐hydroxyl positions, to form oroxylin A, tenaxin II and chrysoeriol respectively. The Type I OMTs, SbFOMT3, SbFOMT5 and SbFOMT6 account mainly for C7‐methoxylation of flavones, but SbFOMT5 can also methylate baicalein on its C5 and C6‐hydroxyl positions. The dimethoxylated flavone, skullcapflavone I (found naturally in roots of S. baicalensis) can be produced in yeast by co‐expressing SbPFOMT5 plus SbFOMT6 when the appropriately hydroxylated 4′‐deoxyflavone substrates are supplied in the medium. Co‐expression of SbPFOMT5 plus SbFOMT5 in yeast produced tenaxin I, also found in Scutellaria roots. This work showed that both type I and type II OMT enzymes are involved in biosynthesis of methoxylated flavones in S. baicalensis.

Scutellaria baicalensis, is one of the most traditional medicinal plants in the Lamiaceae family, and has been widely used to treat liver and lung complaints and as a complementary cancer treatment in traditional Chinese medicine. The preparation from its roots, called “Huang Qin,” is rich in specialized flavones such as baicalein, wogonin, and their glycosides which lack a 4′-hydroxyl group on the B ring (4′-deoxyflavones), with anti-tumor, antioxidant, and antiviral activities. Baicalein has recently been reported to inhibit the replication of the COVID-19 virus. These 4′-deoxyflavones are found only in the order Lamiales and were discovered in the genus Scutellaria, suggesting that a new metabolic pathway synthesizing 4′-deoxyflavones evolved recently in this genus. In this review, we focus on the class of 4′-deoxyflavones in S. baicalensis and their pharmacological properties. We also describe the apparent evolutionary route taken by the genes encoding enzymes involved in the novel, root-specific, biosynthetic pathway for baicalein and wogonin, which provides insights into the evolution of specific flavone biosynthetic pathways in the mint family.

Subclass III members of the sucrose non-fermenting-1-related protein kinase 2 (SnRK2) play essential roles in both the abscisic acid signaling and abiotic stress responses of plants by phosphorylating the downstream ABA-responsive element (ABRE)-binding proteins (AREB/ABFs). This comprehensive study investigated the function of new candidate genes, namely SmSnRK2.3, SmSnRK2.6, and SmAREB1, with a view to breeding novel varieties of Salvia miltiorrhiza with improved stress tolerance stresses and more content of bioactive ingredients. Exogenous ABA strongly induced the expression of these genes. PlantCARE predicted several hormones and stress response cis-elements in their promoters. SmSnRK2.6 and SmAREB1 showed the highest expression levels in the leaves of S. miltiorrhiza seedlings, while SmSnRK2.3 exhibited a steady expression in their roots, stems, and leaves. A subcellular localization assay revealed that both SmSnRK2.3 and SmSnRK2.6 were located in the cell membrane, cytoplasm, and nucleus, whereas SmAREB1 was exclusive to the nucleus. Overexpressing SmSnRK2.3 did not significantly promote the accumulation of rosmarinic acid (RA) and salvianolic acid B (Sal B) in the transgenic S. miltiorrhiza hairy roots. However, overexpressing SmSnRK2.6 and SmAREB1 increased the contents of RA and Sal B, and regulated the expression levels of structural genes participating in the phenolic acid-branched and side-branched pathways, including SmPAL1, SmC4H, Sm4CL1, SmTAT, SmHPPR, SmRAS, SmCHS, SmCCR, SmCOMT, and SmHPPD. Furthermore, SmSnRK2.3 and SmSnRK2.6 interacted physically with SmAREB1. In summary, our results indicate that SmSnRK2.6 is involved in stress responses and can regulate structural gene transcripts to promote greater metabolic flux to the phenolic acid-branched pathway, via its interaction with SmAREB1, a transcription factor. In this way, SmSnRK2.6 contributes to the positive regulation of phenolic acids in S. miltiorrhiza hairy roots.

Scutellaria baicalensis Georgi (S. baicalensis ) produces abundant root-specific flavones (RSFs), which provide various benefits to human health. We have elucidated the complete biosynthetic pathways of baicalein and wogonin. However, the transcriptional regulation of flavone biosynthesis in S. baicalensis remains unclear. We show that the SbMYB3 transcription factor functions as a transcriptional activator involved in the biosynthesis of RSFs in S. baicalensis. Yeast one-hybrid and transcriptional activation assays showed that SbMYB3 binds to the promoter of flavone synthase II-2 (SbFNSII-2) and enhances its transcription. In S. baicalensis hairy roots, RNAi of SbMYB3 reduced the accumulation of baicalin and wogonoside, and SbMYB3 knockout decreased the biosynthesis of baicalein, baicalin, wogonin, and wogonoside, whereas SbMYB3 overexpression enhanced the contents of baicalein, baicalin, wogonin and wogonoside. Transcript profiling by qRT-PCR demonstrated that SbMYB3 activates SbFNSII-2 expression directly, thus leading to more abundant accumulation of RSFs. This study provides a potential target for metabolic engineering of RSFs.