Abstract
Nitrogen (N) availability is a key determinant of plant growth and development. Here, we investigate how different N sources shape Arabidopsis thaliana root system architecture, metabolism and hormone dynamics. L-glutamine (L-GLN) significantly enhances root biomass compared to nitrate (KNO(3)) without compromising shoot growth. This effect emerges after 2 weeks and is independent of L-GLN's role as a carbon or ammonium source or of potential L-GLN-induced pH changes due to ammonium release, indicating a specific function of L-GLN as a N source and signaling molecule. A reverse genetic screen identified AMINO ACID PERMEASE 1 (AAP1)-mediated uptake and GLUTAMINE SYNTHETASE (GS)-dependent assimilation as essential for L-GLN-induced root biomass. In contrast, the N-sensing regulators NITRATE TRANSPORTER 1.1 (NRT1.1) and AMMONIUM TRANSPORTER (AMT) family members contribute to the differential root responses between KNO(3) and L-GLN. Metabolic profiling revealed distinct amino acid signatures under these N sources, irrespective of genotype. Hormonal analyses showed that L-GLN modulates auxin homeostasis, with auxin supplementation restoring primary root growth and lateral root symmetry under L-GLN conditions. L-GLN also reconfigures cytokinin balance by elevating cZ while reducing tZ, collectively shaping root system architecture through hormone-dependent regulation. Together, these findings establish L-GLN as an integrator of N metabolism and hormone signaling in root development, highlighting its signaling capacity beyond nutrient supply and offering new perspectives for improving N use efficiency.