Multi-omics analysis revealed adaptation mechanisms in roots of different nitrogen-efficiency peanut genotypes under low-nitrogen stress

1. N-efficient genotype maintained higher N metabolism ( via N uptake and assimilation), antioxidant capacity (via increased antioxidant activities), and flavonoid accumulation under N-deficiency. 2. The key genes encoding CCoAOMT, CHS, IFR and HCT affected epicatechin, kaempferol, calycosin, and biochanin A levels, with relatively higher levels in JH under low-N stress. 3. Under low-N treatment, WRKY40 and MYB30 , MYB4 , and bHLH35 might regulate flavonoid accumulation as positive and negative regulators, respectively. Overapplication of nitrogen (N) is an important limiting factor in sustainable agricultural development. Breeding N-efficient genotypes is an effective approach to reduce crop N input, increase N-efficiency, and improve crop productive. However, the molecular mechanisms underlying low-N adaptations in peanut ( Arachis hypogaea L.) roots are unknown. Herein, we compared root adaptation mechanisms to low-N stress between the N-efficient genotype JH15 (JH) and the N-inefficient genotype HY20 (HY), focusing on N metabolism and antioxidant capacity. Under N deficiency, JH exhibited a more developed root architecture, higher antioxidant activity, and higher N-metabolic enzyme levels under N deficiency. The expression of both high- and low-affinity nitrate transporter proteins (NRT2.5, NRT1.6), and the chloride channel protein CLC was upregulated in JH, with higher expression of genes encoding glutamine synthetase and asparagine synthase. However, only the low-affinity N transporters (NPF5.2, NPF7.3) were upregulated in HY. Flavonoid and isoflavonoid biosynthesis were the main metabolic pathways underlying the differences between the two genotypes under low-N treatment. The results of weighted gene co-expression network analysis and correlation network analysis revealed that differential expression of the key genes encoding caffeoyl-CoA O-methyltransferase, chalcone synthase, 2’-hydroxyisoflavone reductase, and shikimate hydroxycinnamoyl-CoA transferase affected key metabolites levels (epicatechin, kaempferol, calycosin, and biochanin A). We also found that WRKY40 and MYB30 , MYB4 , and bHLH35 may regulate flavonoids accumulation as positive and negative regulators, respectively. In summary, enhanced N uptake and assimilation and flavonoid accumulation in JH enhanced N metabolism and antioxidant capacity, improving N-efficiency.