Enhanced Activity of Glutamine Synthetase by Manipulating a GWAS‐Identified <i>BnaA02.GLN1;2</i> Gene Augments Nitrate Uptake and Yield in <i>Brassica napus</i>

ABSTRACT Exploiting natural variation to uncouple yield from nitrogen (N) input is essential for sustainable oilseed rape production. Here, we screened 505 accessions and discovered a 10‐fold gradient in chlorate (ClO₃⁻) sensitivity that mirrors genotypic differences in nitrogen use efficiency (NUE). Chlorate‐sensitive genotypes (CSG) exhibited 35% higher ¹⁵NO₃⁻ influx, 40%–62% greater root glutamine synthetase (GS) activity, and almost twice the seed yield of chlorate‐insensitive genotypes (CIG) under high‐N supply, whereas nitrate (NO₃⁻) reductase activity was unchanged. Chemical inhibition of GS using L ‐methionine‐S‐sulfoximine (MSX) resulted in the loss of ClO₃⁻ sensitivity, mechanistically linking assimilation flux to the observed phenotypic variation. Root proteomic analysis identified differentially abundant proteins between CSG and CIG and showed that BnaA02.GLN1;2 (a cytosolic GS1 homologue) was a hub protein with higher abundance in CSG. Overexpression of BnaA02.GLN1;2 enhanced rapeseed chlorate sensitivity under sufficient NO₃⁻ supply, promoted seedling growth, and enhanced both NO₃⁻ uptake and assimilation. Mature transgenic plants produced more pods and higher yields under normal N conditions while simultaneously reducing seed erucic acid and glucosinolate concentrations. Genome‐wide association study mapped a major quantitative trait locus on chromosome A02, with BnaA02.GLN1;2 as the causal gene. Eleven single‐nucleotide polymorphisms (SNPs) classified BnaA02.GLN1;2 into three haplotypes: BnaA02.GLN1;2 Hap1 showed higher gene expression level, GS activity and shoot N content than BnaA02.GLN1;2 Hap3 . Our findings reposition GS1 as a key regulator that coordinates NO₃⁻ uptake and assimilation and establish BnaA02.GLN1;2 Hap1 as a breeding‐ready target to develop high‐yield, low‐input rapeseed.