The SmMYB36-SmERF6/SmERF115 module regulates the biosynthesis of tanshinones and phenolic acids in salvia miltiorrhiza hairy roots

Abstract

Tanshinone and phenolic acids are the most important active substances of Salvia miltiorrhiza, and the insight into their transcriptional regulatory mechanisms is an essential process to increase their content in vivo. SmMYB36 has been found to have important regulatory functions in the synthesis of tanshinone and phenolic acid; paradoxically, its mechanism of action in S. miltiorrhiza is not clear. Here, we demonstrated that SmMYB36 functions as a promoter of tanshinones accumulation and a suppressor of phenolic acids through the generation of SmMYB36 overexpressed and chimeric SmMYB36-SRDX (EAR repressive domain) repressor hairy roots in combination with transcriptomic-metabolomic analysis. SmMYB36 directly down-regulate the key enzyme gene of primary metabolism, SmGAPC, up-regulate the tanshinones biosynthesis branch genes SmDXS2, SmGGPPS1, SmCPS1 and down-regulate the phenolic acids biosynthesis branch enzyme gene, SmRAS. Meanwhile, SmERF6, a positive regulator of tanshinone synthesis activating SmCPS1, was up-regulated and SmERF115, a positive regulator of phenolic acid biosynthesis activating SmRAS, was down-regulated. Furthermore, the seven acidic amino acids at the C-terminus of SmMYB36 are required for both self-activating domain and activation of target gene expression. As a consequence, this study contributes to reveal the potential relevance of transcription factors synergistically regulating the biosynthesis of tanshinone and phenolic acid.

Figure 1

SmMYB36 promotes the accumulation of tanshinone and inhibits the biosynthesis of salvianoli in transgenic hairy roots. a, b The contents of DTI, CT, TAI, TAIIA, and TTA (a), the major components of tanshinone, and RA, SalB, and TPA (b), the salvianoli principal concentrations, in SmMYB36 and SmMYB36-SRDX transgenic hairy roots. c, d Expression levels of SmCPS1, SmKSL1, and SmCYP76AH1, the key enzyme genes of tanshinone synthesis branch, and SmPAL1, SmRAS, and SmCYP98A14, the key enzyme genes of salvianoli synthesis branch. Empty ATCC15834 of transgenic hairy roots was used as control and error bars indicate standard deviation (SD) of three biological replicates. As determined by analysis of one-way and Duncan’s multiple range test (P < 0.05), different letters indicate significant differences.

Figure 2

Transcriptome combined with metabolome analysis in SmMYB36 overexpressed hairy roots. The wide heat map scale represents the relative level of metabolites and the narrow heat map scale represents the relative level of gene expression.

Figure 3

SmMYB36 binds directly to specific motifs in the promoters of SmGAPC, SmGGPPS1, SmCPS1, SmRAS, SmDXS2, SmERF6, and SmERF115 and promotes the expression of SmGGPPS1, SmCPS1, SmDXS2, SmERF6 and represses the expression of SmRAS, SmERF115, and SmGAPC expression. a Results of EMSA experiments. b–c Schematic diagram of dual-LUC vector construction (b) and experimental results (c). The LUC/REN activities of the experimental plasmid combinations (SmGAPCpro::LUC + GFP-SmMYB36, SmGGPPS1pro::LUC + GFP-SmMYB36, SmCPS1pro::LUC + SmMYB36-GFP, SmRASpro::LUC+ GFP-SmMYB36, SmDXS2pro::LUC + SmMYB36-GFP, SmERF6pro::LUC+ GFP-SmMYB36, and SmERF115pro:: LUC + SmMYB36-GFP) and the corresponding control plasmid combinations (SmGAPCpro::LUC + GFP, SmGGPPS1pro::LUC + GFP SmCPS1pro::LUC + GFP, SmRASpro::LUC + GFP, SmDXS2pro::LUC + GFP, SmERF6pro::LUC + GFP, and SmERF115pro::LUC + GFP) were measured and analysed, respectively. Error bars represent the SD (Student’s t-test, ^*P < 0.05, ^**P < 0.01).

Figure 4

SmMYB36 and SmERF6 cooperatively and positively regulate SmCPS1 expression, and SmMYB36 and SmERF115 antagonistically regulate SmRAS expression. a Schematic diagram of reporter vector containing SmRAS and SmCPS1 promoter and effector vector containing GFP-SmMYB36, SmERF115, and SmERF6 (b). LUC/REN activity was measured and comparatively analysed for co-transformation of different combinations of effector and reporter plasmids SmCPS1pro::LUC + GFP, SmCPS1pro::LUC + GFP-SmMYB36 + GFP, SmCPS1pro::LUC+ SmERF6 + GFP, SmCPS1pro::LUC + GFP-SmMYB36 + SmERF6, SmRASpro::LUC + GFP, SmRASpro::LUC + GFP-SmMYB36 + GFP, SmRASpro::LUC + SmERF115 + GFP, and SmRASpro::LUC + GFP-SmMYB36 + SmERF115. Error bars represent the SD (Student’s t-test, ^*P < 0.05, ^**P < 0.01).

Figure 5

SmMYB36^154–160 is both a self-activating structural domain and a region that activates the expression of SmGGPPS1, SmCPS1, SmDXS2, and SmERF6. a Analysis of the SmMYB36 self-activating structural domain in yeast. b Schematic diagram of the construction of the reporter plasmid and the effector plasmid. c Dual-LUC analysis of the effect of SmMYB36^1–153 on the expression of SmGAPC, SmRAS, SmDXS2, SmGGPPS1, SmCPS1, SmERF6, and SmERF115 genes. Error bars indicate the standard error of the mean of three experimental replicates (P < 0.05, P < 0.01).

Figure 6

SmMYB36^1–153 decreased the promoting effect of SmMYB36 on the accumulation of total tanshinone in S. miltiorrhiza hairy root. a, b The contents of DTI, CT, TAI, TAIIA and TTA, the major components of tanshinone, and RA, SaIB and TPA, the phenolic acids principal concentrations, in SmMYB36 and SmMYB36^1–153 transgenic hairy roots. c Expression levels of SmMYB36 target, genes SmGAPC, SmRAS, SmGGPPS1, SmCPS1, SmRAS, SmDXS2, SmERF6, and SmERF115. Empty ATCC15834 (ATCC) and empty vector (EV) of transgenic hairy roots was used as control and error bars indicate standard deviation (SD) of three biological replicates. A different letter indicates a significant difference between the groups, according to the analysis of one-way ANOVA and Duncan’s multiple range test (P < 0.05).

Figure 7

Schematic representation of SmMYB36 regulation of tanshinone and phenolic acid biosynthesis. The narrow solid arrows indicate single-step or proven reactions, while the wide dashed arrows represent multi-step or speculative reaction processes. Red font and arrows represent metabolites or genes that are positively regulated, blue arrows and font represent metabolites or genes that are negatively regulated.