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 dwarf1 (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 crosstalk 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.