Multi-omics analysis reveals flavonoid metabolic changes and hormonal responses associated with seed dormancy release during cold stratification in Fritillaria unibracteata

Abstract

Fritillaria unibracteata, an endangered medicinal plant endemic to the Qinghai–Tibet Plateau, produces seeds characterized by prolonged dormancy and low germination rates, thereby severely limiting artificial propagation, germplasm conservation, and sustainable cultivation. This study optimized a two-stage low-temperature stratification treatment and integrated physiological, transcriptomic (RNA-seq), and non-targeted metabolomic (LC–MS) analyses to investigate the physiological and molecular changes during seed dormancy release. Seeds began to germinate after 180 days of stratification, reaching a germination rate of 92.63%, indicating that prolonged low-temperature stratification effectively promoted seed dormancy release under the experimental conditions used in this study. During stratification, gibberellic acid (GA₃) content increased, whereas abscisic acid (ABA) content and the ABA/GA₃ ratio decreased. Starch, soluble protein, and soluble sugar contents declined markedly. Key enzyme activities involved in glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway increased, whereas catalase (CAT) and peroxidase (POD) activities decreased. Transcriptome analysis and WGCNA revealed significant enrichment of pathways related to lipid storage, energy metabolism, and hormone signaling. The diterpenoid biosynthesis pathway and GA signaling were consistently enriched across stratification stages. WGCNA identified GA2ox as a hub gene, while multi-omics integration revealed significant correlations between flavonoid biosynthesis genes and metabolite accumulation. CCoAOMT expression was positively correlated with the accumulation of multiple flavonoids, including quercetin, epicatechin, and xanthohumol. Collectively, the results indicate that seed dormancy release during cold stratification is associated with coordinated changes in hormone balance, reserve mobilization, energy metabolism, and flavonoid metabolism. These findings provide an effective strategy for seed treatment and cultivation, thereby facilitating the large-scale propagation, sustainable cultivation, and industrial utilization of F. unibracteata.