SmbHLH60 and SmMYC2 antagonistically regulate phenolic acids and anthocyanins biosynthesis in Salvia miltiorrhiza.

INTRODUCTION: Salvia miltiorrhiza is a renowned traditional Chinese medicinal plant with extremely high medicinal value, especially for cardiovascular and cerebrovascular diseases. The jasmonic acid (JA) signaling pathway plays an important role in the improved biosynthesis of secondary metabolites, which is mediated by a major transcriptional regulator, MYC2. However, the JA regulatory mechanism of secondary metabolites biosynthesis in S. miltiorrhiza is still largely unknown. OBJECTIVES: Our work focuses on the dissection of the molecular mechanism of transcriptional regulation in MeJA-mediated biosynthesis of medicinal components of S. miltiorrhiza. We examined the role of MeJA-responsive bHLH transcription factors (TFs) in improving bioactive secondary metabolites accumulation in S. miltiorrhiza. METHODS: Hairy root transformation based on CRISPR/Cas9 technique was used to decipher gene function(s). Changes in the content of phenolic acids were evaluated by HPLC. Y1H, EMSA and dual-LUC assays were employed to analyze the molecular mechanism of SmbHLH60 in the regulation on the biosynthesis of phenolic acids and anthocyanins. Y2H, BiFC and pull-down affinity assays were used to corroborate the interaction between SmbHLH60 and SmMYC2. RESULTS: Being one of the most significantly negatively regulated bHLH genes by MeJA, a new transcription factor SmbHLH60 was discovered and characterized. Over-expression of SmbHLH60 resulted in significant inhibition of phenolic acid and anthocyanin biosynthesis in S. miltiorrhiza by transcriptionally repressing of target genes such as SmTAT1 and SmDFR, whereas CRISPR/Cas9-generated knockout of SmbHLH60 resulted in the opposite effect. In addition, SmbHLH60 and SmMYC2 formed a heterodimer to antagonistically regulate phenolic acid and anthocyanin biosynthesis. CONCLUSION: Our results clarified that SmbHLH60 is a negativeregulator on the biosynthesis of phenolic acids and anthocyanins. SmbHLH60 competed with SmMYC2 in an antagonistic manner, providing new insights for the molecular mechanism of MeJA-mediated regulation on the biosynthesis of secondary metabolites in S. miltiorrhiza.

Soybean Processing Wastes: Novel Insights on Their Production, Extraction of Isoflavones, and Their Therapeutic Properties.

Soybean processing waste (SPW) has potential as a sustainable source of phytochemicals and functional foods. A variety of phytochemicals, nutrients, and minerals have been characterized from SPW using various analytical methods. SPW utilization strategies may provide a new way to increase production of bioactive compounds, nutritional supplements, and cosmetic ingredients. SPW has the potential for value-added processing, to improve commercial use, and to lower environmental pollution through proper use. Okara, a byproduct generated during soybean processing of tofu and soy milk, is rich in dietary fiber, isoflavones, and saponins. Isoflavones, an important class of biologically active compounds owing to their multifunctional and therapeutic effects, are extracted from SPW. Further, studies have shown that okara has potential prebiotic and therapeutic value in lowering the risk of noncommunicable diseases. Therefore, in this review, we focus on several extraction methods and pharmacotherapeutic effects of different SPWs. Their effective uses in functional foods, nutraceuticals, and health applications, as biocatalysts, and as value-added resources have been discussed.

The biosynthesis of phenolic acids is positively regulated by the JA-responsive transcription factor ERF115 in Salvia miltiorrhiza.

Phenolic acids are important secondary metabolites produced in the Chinese medicinal plant Salvia miltiorrhiza, but little is known about the transcription factors involved in the regulation of tanshinone and phenolic acid biosynthesis. Here, a novel AP2/ERF transcription factor SmERF115 was isolated and functionally characterized. SmERF115 was most responsive to methyl jasmonate (MeJA) treatment and was localized in the nucleus. The phenolic acid production was increased in SmERF115-overexpressing hairy roots, but with a decrease in tanshinone content. In contrast, silencing of SmERF115 reduced the phenolic acid level, but increased tanshinone content. The expression of the key biosynthetic gene SmRAS1 was up-regulated in SmERF115 overexpression lines but was down-regulated in SmERF115-RNAi lines. Yeast one-hybrid (Y1H) assay and EMSA showed that SmERF115 directly binds to the promoter of SmRAS1, while dual-luciferase assays showed that SmERF115 could activate expression of SmRAS1 in vivo. Furthermore, global transcriptomic analysis by RNA sequencing revealed that expression of other genes such as PAL3, 4CL5, TAT3, and RAS4 was also increased in the overexpression line, implying that they were potentially involved in the SmERF115-mediated pathway. Our data show that SmERF115 is a positive regulator of phenolic acid biosynthesis, and may be a potential target for further metabolic engineering of phenolic acid biosynthesis in S. miltiorrhiza.

The AP2/ERF transcription factor SmERF1L1 regulates the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza.

Tanshinones and phenolic acids are two important metabolites synthesized by the traditional Chinese medicinal plant Salvia miltiorrhiza. There is increasing market demand for these compounds. Here, we isolated and functionally characterized SmERF1L1, a novel JA (Jasmonic acid)-responsive gene encoding AP2/ERF transcription factor, from Salvia miltiorrhiza. SmERF1L1 was responsive to methyl jasmonate (MJ), yeast extraction (YE), salicylic acid (SA) and ethylene treatments. Subcellular localization assay indicated that SmERF1L1 located in the nucleus. Overexpression of SmERF1L1 significantly increased tanshinones production in transgenic S. miltiorrhiza hairy roots by comprehensively upregulating tanshinone biosynthetic pathway genes, especially SmDXR. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that SmERF1L1 binds to the GCC-box of SmDXR promoter while dual luciferase (Dual-LUC) assay showed that SmERF1L1 positively regulated the expression of SmDXR. Our study suggested that the SmERF1L1 may be a good potential target for further metabolic engineering of bioactive component biosynthesis in S. miltiorrhiza.