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SmSPL6 Induces Phenolic Acid Biosynthesis and Affects Root Development in Salvia miltiorrhiza.

ABSTRACT: Salvia miltiorrhiza is a renowned model medicinal plant species for which 15 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family genes have been identified; however, the specific functions of SmSPLs have not been well characterized as of yet. For this study, the expression patterns of SmSPL6 were determined through its responses to treatments of exogenous hormones, including indole acetic acid (IAA), gibberellic acid (GA₃), methyl jasmonic acid (MeJA), and abscisic acid (ABA). To characterize its functionality, we obtained SmSPL6-ovexpressed transgenic S. miltiorrhiza plants and found that overexpressed SmSPL6 promoted the accumulation of phenolic acids and repressed the biosynthesis of anthocyanin. Meanwhile, the root lengths of the SmSPL6-overexpressed lines were significantly longer than the control; however, both the fresh weights and lateral root numbers decreased. Further investigations indicated that SmSPL6 regulated the biosynthesis of phenolic acid by directly binding to the promoter regions of the enzyme genes Sm4CL9 and SmCYP98A14 and activated their expression. We concluded that SmSPL6 regulates not only the biosynthesis of phenolic acids, but also the development of roots in S. miltiorrhiza.

SUBMITTER: Cao Y

PROVIDER: S-EPMC8348295 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionSalvia 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.ObjectivesOur 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.MethodsHairy 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.ResultsBeing 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.ConclusionOur 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.

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SmSPL6 Induces Phenolic Acid Biosynthesis and Affects Root Development in Salvia miltiorrhiza.

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