Objectives Brassica juncea (B. juncea) is characterized by strong tolerance to infertile soils and abundant genetic variation. In this study, two B. juncea accessions, H39 and L25, which differ markedly in their responses to low-N stress, were used to systematically compared their response differences in phenotypic, physiological, and genomic mechanisms underlying their contrasting nitrogen utilization under different nitrogen levels.

Methods In the hydroponic experiment, two treatments, normal nitrogen and low nitrogen, were set up. At the seedling stage, phenotypes related to plant nitrogen utilization and changes in physiological indicators for low-nitrogen tolerance were measured. Meanwhile, whole-genome resequencing was used to detect SNP and InDel variations between H39, L25 and the reference genome. GO and KEGG enrichment analyses were carried out to explore the categories of candidate functional genes and pathways related to nitrogen use efficiency. In the field experiment, low-nitrogen and normal-nitrogen treatments were set up, and yield and indicators related to nitrogen utilization were measured at the maturity stage.

Results Under the same N supply, H39 consistently exhibited significantly greater total dry weight, total N accumulation, and NUE than L25 at both the hydroponic seedling stage and field maturity stage. Under low-N stress, reductions in biomass and yield were markedly smaller in H39, indicating stronger adaptation to nitrogen deficiency. N partitioning analysis further showed that H39 allocated a greater proportion of absorbed N to the shoot at the seedling stage, and a higher proportion to the grain at maturity, together with a significantly higher NHI than L25, suggesting superior N transport and remobilization capacity. Physiological and biochemical analyses demonstrated that, under low N treatment, H39 accumulated anthocyanins more rapidly, showed significantly higher SOD, POD, and CAT activities, and had lower MDA content, indicating a stronger antioxidant defense system. In addition, H39 displayed significantly higher NR and GS activities, lower shoot nitrate and free amino acid contents, and higher soluble protein content than L25, reflecting greater nitrogen assimilation efficiency and a stronger capacity for conversion into organic nitrogen. Whole-genome resequencing revealed abundant SNP and InDel variation between H39 and L25. Among these, 6,932 NUE-related polymorphic genes were identified, which were mainly enriched in catalytic activity, transport activity, plant hormone signal transduction, and metabolic pathways.

Conclusions The high nitrogen-use efficiency trait of H39 mainly relies on its strong antioxidant defense capacity, more efficient nitrogen assimilation metabolism, and superior nitrogen translocation and redistribution ability under low-nitrogen stress. The occurrence of a large number of differentially expressed genes related to catalysis, transport, hormone signaling and metabolic regulation is the genetic basis of H39 for its high nitrogen-use efficiency.