Temporal Dynamics and Regulatory Control of Ginsenoside Accumulation During Leaf Senescence in Panax ginseng

Ginsenosides, the principal bioactive triterpenoid saponins of Panax ginseng C.A. Meyer, accumulate abundantly in leaves. They form a large family of secondary metabolites comprising more than 300 structurally distinct compounds, yet how leaf senescence reshapes their accumulation remains unclear. We profiled 376 saponin metabolite features across four sampling stages spanning the natural senescence progression (T1–T3) and a frost-terminated stage (T4) by ultra-performance liquid chromatography–high-resolution mass spectrometry (UPLC-HRMS), modeled temporal effects across the natural senescence gradient (T1–T3) with generalized estimating equations (GEE), analyzed the frost-terminated stage T4 as a distinct endpoint, verified senescence histologically, and analyzed stage T1–T3 transcriptomes with replicate-based statistical testing. Leaf senescence globally reshaped the saponin metabolome, with 326 of the 376 saponin features (86.7%) showing significant temporal effects across the natural senescence gradient (T1–T3). The four classical aglycone classes—protopanaxadiol (PPD), protopanaxatriol (PPT), ocotillol (OT) and oleanolic acid (OA)—followed divergent accumulation programs across the natural senescence progression (T1–T3): sustained accumulation (PPT, OT and PPD) versus a U-shaped curve (OA). At the frost-terminated stage, T4, the classes diverged sharply—PPD-type abundance fell back to the T1 level (abundance-weighted class-level T4/T3 = 0.63), whereas the other classes rose further—a frost-associated remodeling pattern rather than a continuation of the senescence program. The frost-associated PPD decline was glycosylation-dependent—low-glycosylation compounds fell far more sharply than highly glycosylated ones (median of per-compound ratios T4/T3 0.51 vs. 1.04; one-sided permutation p = 1.2 × 10−4)—and coincided with T3 induction of β-glucosidase genes (exploratory and hypothesis-level, based on two qualified T3 replicates, and requiring validation with larger replicate numbers) and histologically verified vacuolar collapse, consistent with a deglycosylation-associated catabolic mechanism. Senescence signaling was consistent with differential hormone responses (salicylic acid activation, jasmonate and ethylene attenuation), broad suppression of Ca2+-signaling components and WRKY/NAC-centered transcriptional control, and joint analysis suggested a dichotomous saponin–gene correlation pattern. Collectively, these results establish a temporal atlas of ginsenoside accumulation during ginseng leaf senescence, delineate the class-specific coupling of saponin secondary metabolism to the senescence program, and offer a new perspective on ginsenosides as metabolites embedded in the physiological activities of the ginseng life cycle.