Metabolite and Transcriptomic Changes Reveal the Cold Stratification Process in Sinopodophyllum hexandrum Seeds

(Royle) Ying, an endangered perennial medicinal herb, exhibits morpho-physiological dormancy in its seeds, requiring cold stratification for germination. However, the precise molecular mechanisms underlying this transition from dormancy to germination remain unclear. This study integrates transcriptome and plant hormone-targeted metabolomics techniques to unravel these intricate molecular regulatory mechanisms during cold stratification in seeds. Significant alterations in the physicochemical properties (starch, soluble sugars, soluble proteins) and enzyme activities (PK, SDH, G-6-PDH) within the seeds occur during stratification. To characterize and monitor the formation and transformation of plant hormones throughout this process, extracts from seeds at five stratification stages of 0 days (S0), 30 days (S1), 60 days (S2), 90 days (S3), and 120 days (S4) were analyzed using UPLC-MS/MS, revealing a total of 37 differential metabolites belonging to seven major classes of plant hormones. To investigate the biosynthetic and conversion processes of plant hormones related to seed dormancy and germination, the transcriptome of seeds was monitored via RNA-seq, revealing 65,372 differentially expressed genes associated with plant hormone synthesis and signaling. Notably, cytokinins (CKs) and gibberellins (GAs) exhibited synergistic effects, while abscisic acid (ABA) displayed antagonistic effects. Furthermore, key hub genes were identified through integrated network analysis. In this rigorous scientific study, we systematically elucidate the intricate dynamic molecular regulatory mechanisms that govern the transition from dormancy to germination in seeds during stratification. By meticulously examining these mechanisms, we establish a solid foundation of knowledge that serves as a scientific basis for facilitating large-scale breeding programs and advancing the artificial cultivation of this highly valued medicinal plant.