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

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.