Untargeted metabolomics reveals novel metabolites in Lotus japonicus roots during arbuscular mycorrhiza symbiosis

Arbuscular mycorrhiza (AM) improves mineral nutrient supply, stress tolerance, and growth of host plants through re-programing of plant physiology. We investigated the effect of AM on the root secondary metabolome of the model legume Lotus japonicus using untargeted metabolomics. Acetonitrile extracts of AM and control roots were analysed using ultra-high-performance liquid chromatography-electrospray ionization-ion mobility-time-of-flight-mass spectrometry (UPLC-ESI-IM-ToF-MS). We characterized AM-regulated metabolites using co-chromatography with authentic standards or isolation and structure identification from L. japonicus roots using preparative high-performance liquid chromatography and nuclear magnetic resonance spectroscopy. Arbuscular mycorrhiza triggered major changes in the root metabolome, with most features representing unknown compounds. We identified three novel polyphenols: 5,7-dihydroxy-4′-methoxycoumaronochromone (lotuschromone), 4-hydroxy-2-(2′-hydroxy-4′-methoxyphenyl)-6-methoxybenzofuran-3-carbaldehyde (lotusaldehyde), and 7-hydroxy-3,9-dimethoxypterocarp-6a-ene (lotuscarpene). Further AM-enhanced secondary metabolites included the previously known lupinalbin A and B, ayamenin D, biochanin A, vestitol, acacetin, coumestrol, and betulinic acid. Lupinalbin A, biochanin A, ayamenin D, liquiritigenin, isoliquiritigenin, lotuscarpene, medicarpin, daidzein, genistein, and 2′-hydroxygenistein inhibited Rhizophagus irregularis spore germination upon direct application. Our results show that AM enhances the production of polyphenols in L. japonicus roots and highlights a treasure trove of numerous unknown plant secondary metabolites awaiting structural identification and functional characterization.