Identification and functional characterization of SmABCG24 regulating tanshinone transport in Salvia miltiorrhiza

Salvia miltiorrhiza is a perennial herb of the Lamiaceae family, in which tanshinones, a group of diterpenoid compounds, mainly accumulate in roots. While the biosynthesis and regulation of tanshinone have been extensively investigated, their transmembrane transport remains poorly understood. In this study, we identified SmABCG24, which encodes a half-size ATP-binding cassette (ABC) transporter localized to the plasma membrane. Heterologous expression in Saccharomyces cerevisiae demonstrated that SmABCG24-expressing yeast cells efficiently exported tanshinone IIA and alleviated its cytotoxicity, a process dependent on both ATP hydrolysis and the proton gradient, as evidenced by inhibition with Na₃VO₄ and NH₄Cl. Overexpression of SmABCG24 in S. miltiorrhiza hairy roots resulted in root enlargement and darkening, accompanied by decreased intracellular tanshinone levels but enhanced secretion into the culture medium, whereas CRISPR/Cas9-mediated knockout caused severe root browning and a marked reduction of tanshinone contents in both roots and medium. Consistently, stable transgenic plants exhibited pronounced alterations in root morphology and epidermal structure following SmABCG24 manipulation. Tissue-specific metabolite analyses revealed that tanshinones predominantly accumulated in the root epidermis, with SmABCG24 loss leading to increased intracellular retention, whereas overexpression promoted their redistribution toward the epidermal region. These findings demonstrate that SmABCG24 mediates tanshinones efflux from the periderm tissue of S. miltiorrhiza roots. This study provides the first functional characterization of a plant ABCG24 transporter, providing direct evidence of its involvement in specialized metabolite transport. Our results not only advance understanding of the distribution and accumulation of tanshinones but also identify SmABCG24 as a promising target for metabolic engineering.