Metabolomic and transcriptomic analyses reveal the mechanisms underlying Smilax glabra seed dormancy release.

Smilax glabra, a perennial vine with high pharmaceutical value, exhibits seed dormancy under natural conditions that severely restricts artificial propagation and industrial-scale cultivation. This study aims to systematically investigate the physiological and molecular mechanisms underlying temperature-stratification-induced (25 °C/4 °C) dormancy release in S. glabra seeds, providing a scientific basis for optimizing seed propagation protocols. In this study, ultraviolet spectrophotometry (UV) was employed to monitor the dynamic variations in soluble sugars, starch, soluble protein content, and enzymatic activities (including phosphoglucose isomerase (PGI), malate dehydrogenase (MDH), and glucose-6-phosphate dehydrogenase (G6PDH)) during temperature stratification (25 °C/4 °C) of S. glabra seeds. Additionally, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and RNA-seq were integrated to systematically characterize the phytohormone metabolic profiles and molecular biological mechanisms underlying seed stratification. The results demonstrated that alternating temperatures of 25 °C/4 °C effectively broke seed dormancy. During the dormancy release process, seeds consumed soluble proteins and starch, while starch-degrading genes (AMY and BAM) were significantly upregulated to drive starch conversion into soluble sugars. Meanwhile, PGI activity exhibited a marked decrease, while MDH and G6PDH activities showed overall upward trends. At the hormone metabolism level, downregulation of NCED and upregulation of CYP707A synergistically reduced ABA levels, while upregulation of GA20ox and KAO promoted GA biosynthesis; downregulation of DELLA protein-encoding genes relieved GA signal suppression, collectively forming the "ABA decline-GA rise" hormonal balance shift that drives seed germination. Simultaneously, JA biosynthesis genes LOX2 and AOS were upregulated, leading to OPDA accumulation, but OPDA was not converted into JA. Furthermore, the upregulation of the negative regulator JAZ blocked JA signal transduction, thereby relieving JA-mediated inhibition of germination. This study reveals the molecular mechanisms underlying 25 °C/4 °C temperature-stratification-mediated dormancy release in S. glabra seeds through integrated physiological, metabolic, and transcriptional analyses.