OsFPN1 functions as an essential metal transporter for Fe homeostasis and Co/Ni tolerance in rice.

Abstract

Iron (Fe) homeostasis in rice (Oryza sativa) requires coordinated uptake and root-to-shoot distribution and can be perturbed by competing transition metals such as cobalt (Co) and nickel (Ni). This study investigated the roles of the metal transporter OsFPN1 in Fe, Co, and Ni transport. OsFPN1–sGFP localized predominantly to the Golgi apparatus. In yeast, OsFPN1 conferred phenotypes consistent with Fe/Co/Ni transport activity. Seedlings carrying CRISPR/Cas9-induced knockout mutations in OsFPN1 were hypersensitive to iron deficiency (–Fe) and Co/Ni stress; under –Fe supplemented with 100 µM Ni, seedlings exhibited severe growth inhibition and loss of viability by day 10. Mutant plants accumulated less Fe in shoots but more in roots under –Fe, while Co and Ni treatments resulted in reduced root accumulation and increased shoot translocation. This implies impaired root retention and enhanced long-distance transport of Co and Ni in the mutants. Yeast two-hybrid (Y2H) screening identified 58 non-redundant candidates as OsFPN1-interacting proteins, with interactions of OsPAR1 (a PRA1-family vesicle trafficking factor) and Os12g0168900 (a vacuolar V-ATPase subunit) validated by Y2H and bimolecular fluorescence complementation (BiFC). Reverse transcription quantitative PCR (RT-qPCR) analysis revealed elevated expression of OsIMA1, OsIMA2, OsPAR1, and Os12g0168900 in mutant shoots, and stronger induction of OsIMA1, OsIMA2, OsNAS2, OsPAR1, and Os12g0168900 in roots specifically under –Fe. These results show that Golgi-localized OsFPN1 is required for proper root-to-shoot Fe allocation and contributes to limiting Co/Ni translocation under metal stress, while the identified protein associations provide candidate links to endomembrane/vacuolar pathways for future functional investigation.