Multi-omics profiling 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 m. 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 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 23 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.