Mutational studies of transcription factors in Salvia miltiorrhiza

Transcription factors play a critical regulation function in secondary metabolic pathways in Salvia miltiorrhiza. Herein, we reported a bHLH transcription factor, SmbHLH125, cloned from S. miltiorrhiza. Overexpression of SmbHLH125 severely inhibited the hairy root elongation. CRISPR/Cas9 mediated knockout of SmbHLH125 did not result in a significant difference in growth. Notably, knockout of SmbHLH125 led to a notable decrease in rosmarinic acid content and a marked increase in salvianolic acid B and four tanshinones. Integrated transcriptomic and metabolomic studies revealed alterations in the expression levels of several enzyme genes, including Sm4CL family genes in the phenolic acid biosynthesis and SmDXR in tanshinone biosynthesis, upon SmbHLH125 knockout. In addition, association analyses showed that knockout of SmbHLH125 caused changes in the gene expression levels of enzymes involved in the biosynthetic pathways of lignin, anthocyanin, flavonoids, terpenoids, and gibberellin, as well as changes in the accumulation of related metabolites. Transcription factors associated with SmbHLH125 were also further analyzed. Correlation analysis indicated that SmLAC7 and SmLAC27 may be potential enzyme genes for phenolic acid synthesis, and CYP71AU50, CYP736A12, CYP71A6, and CYP92B28 may be catalase genes for phenolic acid synthesis and tanshinone synthesis. Yeast single heterozygote(Y1H) assays showed that SmbHLH125 binds to Sm4CL family genes in the phenolic acid pathway, as well as the SmRAS and SmHPPR3 promoter, and to the promoter regions of 13 enzyme genes in the tanshinone synthesis pathway, including SmDXR, SmAACT2 and so on. In conclusion, we added a new member to the regulation of the secondary metabolic network of S. miltiorrhiza.