ABSTRACT: Most land plants have evolved both a direct root uptake pathway and a symbiotic pathway, via association with arbuscular mycorrhizal (AM) fungi, to facilitate nutrient acquisition, particularly of phosphorus (P) and nitrogen (N), from soil. Recently, we revealed a highly efficient symbiotic pathway for nitrate uptake, mediated by an AM-specific NPF/NRT1 transporter, OsNPF4.5, in rice. However, the regulatory mechanism controlling the AM-specialized expression of OsNPF4.5 remains unclear. Here, we demonstrate that two cis-acting elements, the CArG and GCC-box, are essential for activating the expression of OsNPF4.5 in rice mycorrhizal roots. Deletion of either of the two motifs in its promoter caused almost complete abolition of the promoter activity of OsNPF4.5. An AM-responsive MADS (MCM1, AG, DEFA, and SRF) transcript factor, OsMADS61, could positively regulate OsNPF4.5 and another nitrate transporter gene, OsNRT2.2, involved in direct nitrate uptake. Knockout of OsMADS61 decreased root biomass, N accumulation and mycorrhization efficiency in its mutants. OsMADS61 could be directly regulated by another AM-upregulated OsMADS paralogue, OsMADS26, which itself can also activate OsNPF4.5, OsNRT2.2, and OsNAR2.1, encoding a nitrate transporter activating protein. Together, our results reveal a dual regulatory role for OsMADS61 and OsMADS26 in governing both direct and symbiotic nitrate uptake pathways.
