Functional Characterization Of SmMYC2a And SmMYC2b Which Regulate Multiple Jasmonate-inducible Steps In The Biosynthesis Of Active Compounds In Salvia Miltiorrhiza

Danshen is recorded in Shen Nong’s herbal classic as an effective medicine. It is an ancient Chinese herbal medicine which comes from the root and rhizome of Salvia miltiorrhiza Bunge. Danshen is clinically used for the treatment of coronary heart disease, angina pectoris, ischemic stroke and other cardiac symptoms. Medicinal research suggested that phenolic acids including Salvianolic acid B (Sal B) and terpenoids including tanshinone IIA are important material basis of pharmacological activities of S. miltiorrhiza. Previous study suggested that the accumulation of tanshinones and Sal B in S. miltiorrhiza hairy root cultures dramatically enhanced by improving JA level, with the former enhanced 4.5 folds and the latter enhanced 6 folds. However, the specific role of methyl jasmonate (MeJA) in the activation of transcription factors (TFs) which regulates various pathway genes was still unknown in S. miltiorrhiza. Rational engineering to boost the yield of plant bioactive compounds has been greatly impeded owing to our poor understanding of comprehensive plant metabolic network. Here we capitalized on MYC2 TFs which are proposed to be involved in JA pathway.Based on assembled transcription profiling database, we successfully amplified SmMYC2a and SmMYC2b. SmMYC2a contains an 1803 bp ORF which encodes 600 amino acid residues, while SmMYC2b contains an 1824 bp ORF which encodes 607 amino acid residues. Domain analysis showed that both SmMYC2a and SmMYC2b contained basic helix-loop-helix (bHLH) domain, which defined bHLH protein family. Meanwhile,SmMYC2a and SmMYC2b contain a conserved JID motif, suggesting the probabale binding of Jasmonate ZIM-Domain (JAZ) proteins.Methyl jasmonate (MeJA) indicated that both of SmMYC2a and SmMYC2b were induced by treatment with MeJA, reaching a maximal level at 0.5 h after treatment. Afterwards, the variation trends of transcript levels of SmMYC2a and SmMYC2b slightly differed. Transcript level of SmMYC2a declined slowly to basic value till 3 h. However, transcript level of SmMYC2b decreased sharply to basic value at 1 h after MeJA treatment. The different reduction velocity of SmMYC2a and SmMYC2b transcripts indicated that compared to SmMYC2b, SmMYC2a could maintain high expression longer and might serve as a regulator with more extensive modulatory effects.SmMYC2a and SmMYC2b were detected to be localized in nuleus. The nucleus localized characteristic confirmed the expectation of SmMYC2a and SmMYC2b as TFs.To further explore whether the biosynthesis of tanshinones and LAB were regulated by SmMYC2s, RNAi-mediated expression knockdown experiments were carried out in S. miltiorrhiza. QRT-PCR analysis was performed to seek for evidence of the relationships between the two TFs and critical pathway genes. Not only the phenolic acid biosynthetic genes were affected, but also the tanshinone pathway was repressed in SmMYC2a/b knockdown lines. In addition to common pathway genes (SmPAL, SmRAS6, SmCYP98A14, SmCMK, SmTPS and SmCPS) both of the MYC2s affected, SmMYC2a was discovered to affect SmHMGR, SmMCS, SmGPPS, SmGGPPS and SmKSL, and SmMYC2b could influence the expression of SmDXR and SmMK, indicating SmMYC2a and SmMYC2b strongly and specifically regulated pathway genes in the biosynthesis of tanshinones and phenolic acids.A significant decrease was observed in the biosynthesis of intermediate product (L-phenylalanine, homogentisic acid, rosmarinic acid) and end products (Sal A, Sal B) in SmMYC2a or SmMYC2b knockdown lines, showing consistent repression effect of phenolic acid biosynthesis pathway. The biosynthesis of tanshinones (tanshinone I, dihydrotanshinone I, tanshinone IIA, cryptotanshinone) were remarkably depressed in SmMYC2a knockdown transgenic lines, and knockdown of SmMYC2b caused resemble changes in the production of tanshinones. All the results of compound determination indicated that SmMYC2a and SmMYC2b were positive regulators which exerted resemble but also irreplaceable functions in the regulation of JA-responsive genes, leading to the biosynthetic regulation of tanshinones and phenolic acids.To further explore the genes that MYC2 could transcriptionally activated, EMS assays were performed using purified target proteins and biotin-modified promoter fragments containing the E-box. Both of SmMYC2a and SmMYC2b protein were discovered to bind to G-box motif-containing SmCYP98A14 promoter. In addition, SmMYC2a could bind to E-box motif in SmRAS6 promoter. The specific binding revealed that SmMYC2a might regulate the biosynthesis pathway of phenolic acids by activating the transcription of SmRAS6 and SmCYP98A14 while the regulation of SmMYC2b might via the binding to SmCYP98A14.Y2H assay was implemented to investigate the interactive ability of SmMYC2 with SmJAZ repressors. Both of SmMYC2 exhibited strong interactions with SmJAZ1 and SmJAZ2, implying that the two MYC2 TFs might function as direct targets of JAZ proteins. The binding effect of MYC2-JAZ might result in the repression of MYG2 proteins that could bind to the promoters of critical pathway genes (SmRAS6 and SmCYP98A14) and activate their transcription.In a word, this study explored the regulation effect of SmMYC2a/b on the biosynthesis of tanshinones and phenolic acids. This research will undoubtedly contribute to the characterization of JAs effect on secondary metabolites and construction of JA messenger-TFs-genes-second metabolites network to ultimately achieve high yield of target compounds in S. miltiorrhiza (tanshinones and Sal B).