Rhizosphere metabolomics reveals benzoxazinoid-mediated interspecific root interactions that promote N and Fe uptake in intercropped plants

Abstract

Interspecific root interactions are common in intercropping systems. However, limited information exists on whether specialized metabolites, such as benzoxazinoids (BXs) secreted by cereals in intercropping, mediate rhizosphere metabolite interactions between crops to enhance nitrogen (N) and iron (Fe) uptake. Compared with monoculture wheat (MW), intercropping wheat (IW) significantly increased the total BXs concentrations in the roots and rhizosphere. Additionally, BXs secreted by IW were detected in the rhizosphere of intercropped fava bean (IF), indicating their absorption by its roots. To analyze these interactions, three sets of rhizosphere differential metabolite profiles (IW_vs_MW, IF_vs_MF, and IF_vs_IW) were established, identfitying 251, 257, and 643 significant differential metabolites, respectively. These metabolites were primarily amino acids, phospholipids, monoterpenoids, and flavonoids. KEGG enrichment analysis revealed that the differential metabolites were significantly enriched in pathways such as BX biosynthesis and isoflavonoid biosynthesis, both of which belong to the biosynthesis of other secondary metabolites. Notably, BX biosynthesis was a shared pathway between IW_vs_MW and IF_vs_MF. Intergroup differences and correlation analyses of all compounds in the three differential metabolite sets highlighted that pyroglutamic acid, formononetin, and other flavonoids, along with perillic acid and other monoterpenes, were significantly affected by interspecific root interactions and positively correlated with 6-methoxy-2-benzoxazolinone (MBOA). These differential metabolites enhanced the activation and preference of intercropped plants for available N and Fe, thereby improving their uptake. In summary, our findings offer new insights into boosting N and Fe absorption through interspecific root DMs mediated by specialized metabolites (such as BX) in cereal-legume intercropping systems.