Efficient nitrogen assimilation is important for sustainable agriculture1, yet its subcellular organization remains unknown. Here we show that plastoglobules (PGs) in the chloroplasts of mesophyll cells function as a metabolic hub that orchestrates nitrogen utilization in maize. Nitrogen-responsive dynamics of PGs represent a conserved feature across plant species. We identify two key enzymes, nitrite reductase 2 (ZmNIR2) and glutamine synthetase 1 (ZmGLN1), specifically targeted to PGs by a chloroplast transit peptide and hydrophobic region. Cryogenic electron microscopy analysis of recombinant ZmGLN1 shows a decameric complex, enabling a metabolon with ZmNIR2 for enhanced efficiency. Among two NIR and six GLN enzymes, ZmNIR2 and ZmGLN1 are the primary PG-localized components that orchestrate sub-organellar nitrogen assimilation and dictate nitrogen use efficiency. Genetic variation in ZmNIR2 splicing in cultivated germplasm generates a PG-targeted isoform (ZmNIR2T1) that boosts NUE. Our work establishes PGs as a central compartment for primary nitrogen assimilation, providing a promising strategy to develop high-NUE crops for global food security.
