Study On The Metabolites During Seed Symbiotic Germination Of Endangered Medicinal Plant Gymnadenia Conopsea(Orchidaceae)

Terrestrial orchid Gymnadenia conopsea is widely used for Tibetan medicine and Mongolian medicine,having evident pharmacological activities.Due to their tiny seeds devoid of endosperm,most orchid species are dependent on mycorrhizal fungi to supply the necessary nutrients for seed germination during early stages of plant development as well as in some adult orchid species.The fungal strain Ceratobasidium GS2,initially isolated from roots of G.conopsea,was able to support seed germination under both artificial and natural conditions.In this research,secondary metabolites of Ceratobasidium GS2,seeds and protocorm of G.conopsea were investigated using various chromatographic columns and their structures were identified by multiple spectroscopic methods.As a result,23 compounds including 1 new compound were isolated.15 compounds(1-15)including seven sterols(1-7)and five furanoids(8-13)were isolated from the symbiotic fungus Ceratobasidium sp.GS2.6 known compounds(20-25)from seeds of G.conopsea and 2 known compounds(26-27)from protocorm of G.conopsea were identified.This was the first chemical investigation of the symbiotic fungus,seeds and protocorm of G.conopsea.The structure of new compound 8 was determined mainly based on HR-ESI-MS and NMR spectral data.Modified Mosher’s reactions were used to establish the stereochemistry of the hydroxyl group in 8,which was not stable in Mosher’s reagents and transformed into four analogs 16-19.These degraded products(16-19)were elucidated based on UPLC-Q-TOF-MS/MS analysis,and compound 18 was further isolated from the degraded mixture and its structure was characterized through NMR experiments.Therefore,the absolute configuration of compound 8 was determined by ECD combined with quantum-chemical calculations adopting time-dependent density functional theory(TDDFT).Compounds 1-6 can promote the protocorm of G.conopsea formation and development,but compounds 10-11 strongly showed inhibition on the seed germination.In addition,compounds 8-12,and 18 showed weak antioxidant activities,and compounds 9-11 displayed phytotoxicity on punctured detached green foxtail leaves.UPLC-Q-TOF-MS/MS technology was used to explore the effect of metabolites of Ceratobasidium GS2 on symbiosis with seeds.A total of 1281 metabolites were annotated and 958 differential metabolites were screened,including 514 up-regulated metabolites and 444 down-regulated metabolites.The results of KEGG function annotation in the Ceratobasidium GS2 symbiotic group showed that the differentially expressed metabolites were significantly enriched in biosynthesis of amino acids.26 compounds not found in the blank group were detected.The results will further lay a good foundation for symbiosis germination.