Systematic characterization of endogenous and heterologous promoters establishes a modular expression toolkit for Salvia miltiorrhiza

Promoters are central regulatory elements in plant synthetic biology, governing the magnitude, spatial pattern, and stability of transgene expression. However, the repertoire of promoters available for Salvia miltiorrhiza hairy roots remains limited, constraining rational pathway design in this cultivated medicinal crop and potential industrial production chassis. Here, we developed and characterized a promoter toolkit for S. miltiorrhiza by integrating transcriptome-guided endogenous promoter mining, benchmarking of commonly used constitutive promoters, promoter truncation analysis, and terminator pairing. Guided by the S. miltiorrhiza hairy root transcriptome, we selected eight endogenous genes and compared their candidate promoters with eight viral, plant-derived, and synthetic constitutive promoters using the visible RUBY betalain reporter. The selected endogenous genes exhibited distinct tissue-expression patterns, including hairy root-biased expression of SmUBQ , root and hairy root enrichment of SmKIR , SmMLP31 , and SmEXT , and stem- and leaf-biased expression of SmPR10 and SmCAT . In RUBY reporter assays, P MAS , P MinSyn110 , and P SmPR10 displayed strong activity in transgenic hairy roots. Truncation analysis identified candidate active fragments, notably P SmPR10–2 , which retained robust reporter activity. Terminator-pairing experiments further demonstrated that P SmPR10–2 was broadly compatible with the seven terminators tested, whereas P SmUBQ-1 exhibited greater sensitivity to specific terminators. Regenerated transgenic plants confirmed visible, promoter-dependent RUBY accumulation in roots, stems, and leaves. This work provides a modular promoter resource for S. miltiorrhiza hairy root engineering and supports spatially resolved pathway design for the production of plant-derived compounds.