Mass Spectrometry Imaging Combined With Single‐Cell Transcriptional Profiling Reveals the Multidimensional Spatial Distributions and Biosynthetic Pathways of Medicinal Components in Andrographis paniculata

ABSTRACT The synthesis and accumulation of active ingredients in medicinal plants are distributed in specific organs, tissues, and cell types, which are important for the exploitation of medicinal plants. However, the fine distribution of active ingredients is difficult to know. Here, the system of mass spectrometry imaging (MSI) integrated with single‐cell RNA sequencing was established for the first time in Andrographis paniculata ( A. paniculata ), a medicinal plant widely utilised in China and Southeast Asia. MSI shows specific distribution of andrographolides in A. paniculata , with higher accumulation in non‐veinal leaf regions and outer stem cortex (leaf > stem; outer > inner cortex), as validated by LC‐QQQ‐MS/MS assays. Leaf scRNA‐seq demonstrates that ApCPS2 (the key terpene synthase for andrographolide biosynthesis) exhibits pronounced cell‐type‐specific expression in photosynthetic mesophyll subclusters, indicating mesophyll cells as the primary site for light‐modulated andrographolide production. Interestingly, light may enhance the accumulation of andrographolide biosynthesis, confirming the light sensitivity of metabolism in mesophyll cells. This study explores medicinal components' multidimensional spatial distributions and biosynthetic pathways in A. paniculata via MSI combined with single‐cell technology, providing a novel strategy for determining plant metabolites' fine synthesis and distribution.