Screening of regulatory factors for tanshinone biosynthesis in response to drought stress

Salvia miltiorrhiza Bunge is a widely used medicinal herb whose roots and rhizomes are clinically applied to treat cardiovascular and cerebrovascular diseases. As one of its major bioactive components, tanshinone biosynthesis can be significantly induced by drought stress. Nevertheless, our knowledge of the regulatory factors for tanshinone biosynthesis in response to drought is still lacking. In the present study, the physiological and molecular mechanisms underlying drought response in S. miltiorrhiza were systematically investigated. It was revealed that drought stress significantly promoted tanshinone accumulation in S. miltiorrhiza . Osmotic balance was maintained primarily by proline accumulation, while increased malondialdehyde content indicated oxidative stress and activated the antioxidant enzyme system to mitigate damage. Analysis of the transcriptome indicated that genes with differential expression tended to be concentrated in metabolic processes, catalytic activity, and stress response pathways. Among them, the phenylpropanoid biosynthetic pathway and the jasmonic acid pathway were activated. The transcriptional levels of key enzyme genes involved in tanshinone synthesis exhibited a trend of initial up-regulation followed by down-regulation. A total of 20 candidate transcription factors associated with tanshinone biosynthesis were screened. Among them, eight representative factors ( SmERF2 , SmERF112 , SmERF1A , SmbZIP44 , SmbZIP53 , SmNAC2 , SmNAC83 , and SmWRKY22 ) were highly expressed in roots and localized in the nucleus. Dual-luciferase assays showed that these transcription factors modulated the promoter activities of key tanshinone biosynthetic genes. Y1H assays further detected direct binding of five transcription factors to specific cis-elements in target promoters, whereas no direct binding was detected for SmbZIP53 , SmNAC2 , and SmNAC83 under the same conditions. These findings identify candidate transcriptional regulators and promoter-level regulatory modules potentially associated with drought-responsive tanshinone biosynthesis, providing a foundation for future in planta functional validation.