Wheat growth and nitrogen (N) uptake gradually decrease in response to high NH₄ ⁺/NO₃ ^- ratio. However, the mechanisms underlying the response of wheat seedling roots to changes in NH₄ ⁺/NO₃ ^- ratio remain unclear. In this study, we investigated wheat growth, transcriptome, and proteome profiles of roots in response to increasing NH₄ ⁺/NO₃ ^- ratios (N _a : 100/0; N _r1: 75/25, N _r2: 50/50, N _r3: 25/75, and N _n : 0/100). High NH₄ ⁺/NO₃ ^- ratio significantly reduced leaf relative chlorophyll content, Fv/Fm, and ΦII values. Both total root length and specific root length decreased with increasing NH₄ ⁺/NO₃ ^- ratios. Moreover, the rise in NH₄ ⁺/NO₃ ^- ratio significantly promoted O₂ ^- production. Furthermore, transcriptome sequencing and tandem mass tag-based quantitative proteome analyses identified 14,376 differentially expressed genes (DEGs) and 1,819 differentially expressed proteins (DEPs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that glutathione metabolism and phenylpropanoid biosynthesis were the main two shared enriched pathways across ratio comparisons. Upregulated DEGs and DEPs involving glutathione S-transferases may contribute to the prevention of oxidative stress. An increment in the NH₄ ⁺/NO₃ ^- ratio induced the expression of genes and proteins involved in lignin biosynthesis, which increased root lignin content. Additionally, phylogenetic tree analysis showed that both A0A3B6NPP6 and A0A3B6LM09 belong to the cinnamyl-alcohol dehydrogenase subfamily. Fifteen downregulated DEGs were identified as high-affinity nitrate transporters or nitrate transporters. Upregulated TraesCS3D02G344800 and TraesCS3A02G350800 were involved in ammonium transport. Downregulated A0A3B6Q9B3 is involved in nitrate transport, whereas A0A3B6PQS3 is a ferredoxin-nitrite reductase. This may explain why an increase in the NH₄ ⁺/NO₃ ^- ratio significantly reduced root NO₃ ^--N content but increased NH₄ ⁺-N content. Overall, these results demonstrated that increasing the NH₄ ⁺/NO₃ ^- ratio at the seedling stage induced the accumulation of reactive oxygen species, which in turn enhanced root glutathione metabolism and lignification, thereby resulting in increased root oxidative tolerance at the cost of reducing nitrate transport and utilization, which reduced leaf photosynthetic capacity and, ultimately, plant biomass accumulation.

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