Spatial metabolomics reveals brassinosteroid-mediated compartmentalization of saikosaponin biosynthesis in Bupleurum chinense DC. under abiotic stress

Although the biosynthesis of active ingredients in medicinal plants is co-regulated by diverse environmental stimuli, the endocrine mechanism by which brassinosteroids (BRs) coordinate physiological resilience with the in situ spatial distribution of saikosaponins in Bupleurum chinense DC. under combined drought and salinity remains largely elusive. We hypothesized that exogenous brassinosteroids (BRs) application alleviates oxidative impairment provoked by single and combined osmotic/ionic stresses while orchestrating the tissue-level topological routing of saikosaponin biosynthesis. Here, B. chinense seedlings were exposed to drought (15% PEG6000), salinity (150 mM NaCl), and combined stress with or without root-applied BRs (0.2 mg/L). Plant growth, foliar cytomembrane integrity, antioxidant enzyme kinetics, and key transcriptional profiles were evaluated and integrated with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)-based spatial metabolomics. Stress-induced morphological inhibition and membrane lesion ranked salinity > combined stress > drought. Exogenous BRs markedly counteracted stress-evoked membrane lipid peroxidation by sustaining CAT, SOD, and POD activities and curtailing H 2 O 2 and superoxide accumulation, as corroborated by in situ DAB and NBT cytochemical visualization. Under drought and salinity, BRs further amplified the stress-elicited elevation of SS-a, SS-c, SS-d, and total saikosaponins through coordinated transcriptional activation of MVA/MEP pathway genes and pentacyclic triterpene synthases (HMGR, FPS, SS, SE, and β-AS). In sharp contrast, under combined drought and salinity, BRs application failed to upregulate these biosynthetic genes, revealing an antagonistic metabolic arrest driven by acute carbon allocation trade-offs. Spatial metabolomics demonstrated that common precursors (IPP, FPP, and 2,3-oxidosqualene) displayed an ubiquitous distribution across the whole root cross-section (cork, cortex, phloem, and xylem), whereas bioactive saikosaponins (SS-a, SS-c, SS-e, SS-f, and acetyl-SS-a) were preferentially compartmentalized in the secondary phloem and periderm. Exogenous BRs dynamically intensified precursor flux toward the outer cortical/phloem sink under single stress regimes. In summary, this work systematically characterizes BRs-regulated spatial patterns of saikosaponins under combined stress. It offers theoretical evidence for decoding stress adaptation mechanisms of B. chinense and provides practical support for optimizing its ecological cultivation techniques.