Metabolic and transcriptomic analysis reveals single seed mutants of ephedra saxatilis as dominant variants in high altitude Xizang

Ephedra species, important Tibetan medicinal plants, are widely distributed across the Qinghai-Tibet Plateau at altitudes of 2700-5000 meters. Their adaptation to high-altitude environments, such as low temperatures, strong UV radiation and low oxygen, is still poorly understood. This study investigated the morphological, metabolic, and genetic mechanisms underlying the reproductive advantage of a unique single-seed variant observed in high-germination-rate Ephedra species. Seeds from six Ephedra species were collected for germination assays and electron microscopic analysis. Results showed that E. saxatilis, E. intermedia, and E. monosperma exhibited significantly higher germination rates (Germination rates > 65%) and predominantly produced single-seed variants, while others mainly produced double seeds. Analysis of burr and fold numbers of phenotypic traits showed a significant positive correlation with germination rates. Time-course metabolomics analysis identified 762 KEGG annotated metabolites, and revealed E. saxatilis as the dominant species due to its faster metabolic rate, particularly simulated high-altitude conditions. Absolute hormone quantification highlighted the mutant single-seed variant of E. saxatilis as the dominant type, with ABA content peaking in the shed seed coat. ABA exhibited antagonistic interactions with 2MeScZR, SA, IAA, GA7, IPR, and t-CA, suggesting a complex hormonal regulation network. Co-expression network analysis integrating transcriptome and hormone data predicted 26 key genes regulating seed germination adaptation. This study provides novel insights into the ecological and evolutionary significance of single-seed variation in high-altitude adaptation. The findings have potential applications in high-altitude plant breeding, conservation, and sustainable utilization of Ephedra species. Future research should focus on the genetic basis of single-seed variation and its role in other high-altitude plant species.