Cross-stage single-cell and spatial metabolome analyses reveal periderm specialization and tanshinone biosynthesis in Salvia miltiorrhiza roots.

Perennial herbs develop long-lived roots that undergo repeated cycles of secondary growth, during which the periderm functions as a protective barrier and a major site of specialized metabolite accumulation. However, the cellular programs that coordinate periderm differentiation and secondary metabolism in perennial roots remain poorly understood. Single-cell RNA sequencing, together with spatial metabolome analyses, was employed to resolve cell-type composition and transcriptional dynamics in Salvia miltiorrhiza roots at different developmental stages. Based on Batch Integration via Precision Annotation and Consistency Testing-integrated single-cell data, the developmental trajectory from epidermal to peridermal lineages was reconstructed, revealing distinct transcriptional programs associated with diterpenoid biosynthesis. Spatial metabolome analyses confirmed that tanshinones were predominantly accumulated in cork cells of mature roots. Integrative analysis further indicated that SmCYP76AK5 participates in tanshinone biosynthesis. This cellular framework enables the prediction of metabolic activities in specific cell types and facilitates the inference of developmental differences between young and mature roots. It also provides a basis for integrating single-cell data into models of secondary metabolism in perennial plants.