Promotion of BR Biosynthesis by miR444 Is Required for Ammonium-Triggered Inhibition of Root Growth

Rice (Oryza sativa), the staple food for almost half of the world's population, prefers ammonium (NH₄ ⁺) as the major nitrogen resource, and while NH₄ ⁺ has profound effects on rice growth and yields, the underlying regulatory mechanisms remain largely unknown. Brassinosteroids (BRs) are a class of steroidal hormones playing key roles in plant growth and development. In this study, we show that NH₄ ⁺ promotes BR biosynthesis through miR444 to regulate rice root growth. miR444 targeted five homologous MADS-box transcription repressors potentially forming homologous or heterogeneous complexes in rice. miR444 positively regulated BR biosynthesis through its MADS-box targets, which directly repress the transcription of BR-deficient dwarf 1 (OsBRD1), a key BR biosynthetic gene. NH₄ ⁺ induced the miR444-OsBRD1 signaling cascade in roots, thereby increasing the amount of BRs, whose biosynthesis and signaling were required for NH₄ ⁺ -dependent root elongation inhibition. Consistently, miR444-overexpressing rice roots were hypersensitive to NH₄ ⁺ depending on BR biosynthesis, and overexpression of miR444's target, OsMADS57, resulted in rice hyposensitivity to NH₄ ⁺ in root elongation, which was associated with a reduction of BR content. In summary, our findings reveal a cross talk mechanism between NH₄ ⁺ and BR in which NH₄ ⁺ activates miR444-OsBRD1, an undescribed BR biosynthesis-promoting signaling cascade, to increase BR content, inhibiting root elongation in rice.