Coptis species are rich in protoberberine-type benzylisoquinoline alkaloids (BIAs). However, the differential BIA accumulation between Coptis chinensis and C. teeta, two primary botanical sources of traditional Chinese medicine ‘Huanglian’, remains mechanistically poorly understood. Here, we combined widely targeted metabolomics, matrix-assisted laser desorption/ionization mass spectrometry imaging, histological characterization, and transcriptomic analyses to investigate the mechanisms underlying the specialized BIA accumulation in C. chinensis versus C. teeta. Clearly, we observed significantly elevated BIA accumulation in C. chinensis rhizomes compared to C. teeta, in particular, the preferential BIA localization within the cortical tissues of C. chinensis rhizomes, consistent with the anatomically expanded cortical and xylem regions. This structural specialization facilitates BIA compartmental distribution patterns. Integrated transcriptomic–metabolomic analysis further constructed a BIA biosynthetic regulatory network, identifying key transcription factors that synergistically promote BIA accumulation in C. chinensis rhizomes, establishing their roles as speciation-associated regulators of medicinal quality divergence between C. chinensis and C. teeta. Overall, this study provides the first integrated anatomical and transcriptional framework explaining interspecies differences in BIA accumulation, enabling the development of quality improvement strategies for medicinal plants.

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