Arbuscular mycorrhizal fungi alleviate manganese toxicity in trifoliate orange through dual mechanisms of external barrier and internal detoxification.

Manganese (Mn) toxicity, exacerbated by soil acidification and anthropogenic pollution, poses a significant threat to plant growth and ecosystem health, particularly in economically important citrus plants. This study tried to uncover the mechanisms by which the arbuscular mycorrhizal fungus Funneliformis mosseae enhances Mn tolerance in trifoliate orange (Poncirus trifoliata) seedlings under excess Mn stress (20 mmol/L MnSO4). Although Mn stress inhibited root colonization by F. mosseae, inoculation with F. mosseae significantly alleviated the Mn-induced suppression of plant growth, root system architecture, photosynthetic efficiency, and photochemical activity of photosystem II. Crucially, AMF colonization reduced Mn accumulation in leaves, stems, and roots, alongside decreased the Mn bioconcentration and translocation factors. Mechanistically, F. mosseae enhanced Mn tolerance in trifoliate orange through two complementary strategies: external exclusion and internal detoxification. Externally, F. mosseae increased levels of difficultly extractable glomalin-related soil protein to sequestrate Mn in the rhizosphere, and significantly upregulated fungal suppressor of mitochondrial fission genes (FmSMF1 and FmSMF2) to enhance hyphal sequestration, thereby blocking Mn entry into roots. Internally, F. mosseae modulated the subcellular distribution and chemical forms of Mn in trifoliate orange: they promoted Mn compartmentalization into the cell wall and soluble (vacuolar) fractions, while reducing its accumulation and proportion in sensitive organelles such as chloroplasts and mitochondria. Furthermore, Mn was transformed from highly active, toxic forms (ethanol- and water-extractable) into more stable, inert forms (e.g., pectate- and phosphate-bound). Consistent with the reduced Mn uptake and toxicity, the expression of host metal tolerance protein (PtMTP8 and PtMTP11) and superoxide dismutase (PtMnSOD) genes was significantly downregulated in mycorrhizal roots under Mn toxicity. In conclusion, F. mosseae enhances Mn tolerance in trifoliate orange through a synergistic dual mechanism, establishing an external barrier via fungal-mediated immobilization and building an internal defense line by reprogramming host Mn compartmentalization and detoxification pathways, thereby minimizing Mn phytotoxicity.