Integrative physiological and transcriptomic analysis provides insights into seed dormancy release induced by cold stratification in Pseudostellaria heterophylla

Pseudostellaria heterophylla is an important medicinal herb whose seed propagation is hindered by physiological dormancy. We investigated cold stratification-induced dormancy release via integrative physiological, anatomical, and transcriptomic analyses. Optimal dormancy release was achieved after 55 days of cold stratification at 0–1 °C with 16% moisture, yielding 86.67% germination. Anatomically, embryo volume progressively increased while endosperm decreased, accompanied by asynchronous cotyledon elongation and vascular tissue formation. Physiologically, three distinct phases were identified. Soluble protein peaked at 20 days. Starch showed transient increases at 20 and 45 days before declining after 45 days. Soluble sugars accumulated only after 45 days. Endogenous hormone analysis revealed that ABA decreased while GA₃ and IAA increased from 20 days onward. Respiratory metabolism shifted toward the TCA cycle (MDH increased) with no change in G6PDH. Transcriptomic analysis identified 52,582 unigenes and 11,425–15,212 differentially expressed genes across stratification stages. WGCNA revealed a blue module (5234 genes) strongly correlated with germination traits and enriched in starch/sucrose metabolism, glycolysis, phenylpropanoid biosynthesis, and photosynthesis-related pathways. p -coumaric acid, a phenylpropanoid intermediate, progressively declined during stratification, and exogenous application confirmed its dose-dependent germination inhibition, identifying it as a candidate endogenous germination inhibitor. These findings suggest that cold stratification is associated with a temporally structured, multi-stage reprogramming of embryo development, hormone signaling, energy metabolism, and secondary metabolite clearance, with the blue module integrating these processes. This study provides a theoretical foundation for developing evidence-based seed priming technologies for medicinal plants.