Overexpression of SmMYC2 enhances salt resistance in Arabidopsis thaliana and Salvia miltiorrhiza hairy roots.

Soil salinity significantly affects both Salvia miltiorrhiza growth and development as well as seed germination throughout field cultivation and production. The basic helix-loop-helix (bHLH) transcription factor (TF) MYC2 contributes significantly to plant stress resistance as a key regulator of the jasmonic acid signaling pathway. In transgenic S. miltiorrhiza hairy roots, SmMYC2 has been shown to promote the accumulation of tanshinone and salvianolic acid, but its role in S. miltiorrhiza of resistance to abiotic stress is unclear. Herein, we found methyl jasmonate (MeJA), NaCl, and PEG treatment all significantly increased SmMYC2 expression. In response to salt stress, SmMYC2 overexpression in yeast increased its rate of growth. Additionally, overexpression of SmMYC2 transgenic Arabidopsis thaliana and S. miltiorrhiza hairy root showed that it might improve salt resistance in transgenic plant. In particular, compared to WT, overexpression of SmMYC2 transgenic Arabidopsis had higher levels of three antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), proline (Pro) content, and ABA-dependent and ABA-independent genes expression. They also had lower levels of malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation. What's more, overexpression of SmMYC2 increases the expression of flavonoid synthesis genes and the accumulation of related components in Arabidopsis. These findings imply that SmMYC2 functions as a positive regulator that regulates plant tolerance to salt through ABA-dependent and independent signaling pathways.

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.

SmMYC2b Enhances Tanshinone Accumulation in Salvia miltiorrhiza by Activating Pathway Genes and Promoting Lateral Root Development.

Salvia miltiorrhiza Bunge (Lamiaceae) is an economically important medicinal plant as well as an emerging model plant. Our previous studies indicate that SmMYC2b is a positive transcription factor that can affect the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza. Moreover, MYC2s are well known to induce the development of lateral roots. As tanshinones are mainly distributed in the periderm, the promotion of lateral root development probably leads to increased accumulation of tanshinones. In this paper, we firstly discovered that SmMYC2b played a dual regulatory role in effectively enhancing the tanshinone accumulation by activating tanshinone biosynthetic pathway and promoting lateral root development. The expression levels of the previously studied pathway genes SmCPS1, SmKSL1, SmCYP76AH1, SmCYP76AH3, and SmCYP76AK1 dramatically increased. In addition, SmMYC2b was proved to exhibit a similar function as other homologs in promoting lateral root development, which increased the tanshinone produced tissue and further enhanced the biosynthesis of tanshinones. RNA-seq assays revealed that SmMYC2b-regulated genes comprised 30.6% (1,901 of 6,210) of JA-responsive genes, confirming that SmMYC2b played a crucial role in transcriptional regulation of JA-regulated genes. Overall, we concluded that SmMYC2b could enhance tanshinone accumulation by activating the tanshinone biosynthetic pathway and promoting lateral root development. Our study provides an effective approach to enhance the production of desired tanshinones and enriches our knowledge of the related regulatory network.

Overexpression of SmMYC2 Increases the Production of Phenolic Acids in Salvia miltiorrhiza.

MYC2 is a core transcription factor in the plant response to jasmonates. It also functions in secondary metabolism and various processes for growth and development. However, the knowledge about its role in Salvia miltiorrhiza is still very limited. We determined that the biosynthesis of salvianolic acid B (Sal B) was strongly induced in 2-month-old transgenic plants that over-expressed SmMYC2. In the roots of transgenic line 12 that over-expressed SmMYC2 (OEM-12), the Sal B concentration was as high as 5.95 ± 0.07 mg g-1, a level that was 1.88-fold higher than that in control plants that had been transformed with an empty vector. Neither tanshinone IIA nor cryptotanshinone was detected by high-performance liquid chromatography in any of the genotypes. Global transcriptomic analysis using RNA sequencing revealed that most enzyme-encoding genes for the phenylpropanoid biosynthesis pathway were up-regulated in the overexpression lines. Furthermore, both the phenylalanine and tyrosine biosynthesis pathways were activated in those transgenics. Our data demonstrate that overexpression of SmMYC2 promotes the production of phenolic acids by simultaneously activating both primary and secondary pathways for metabolism in S. miltiorrhiza.