To explore the mechanisms of NO₃^–-dependent alleviation of NH₄⁺ toxicity in the roots of wheat (Triticum aestivum), both transcriptomic and proteomic approaches were used to investigate the differential expressions of genes and proteins under different nitrogen treatments. Compared with 7.5 mmol·L^-1 NO₃^‑ (control), 7.5 mmol·L^-1 NH₄⁺ inhibited the root growth of wheat seedlings. Transcriptome analysis showed that sole NH₄⁺ treatment upregulated expressions of genes encoding glycolysis- and fermentation-related enzymes, including pyruvate decarboxylase, alcohol dehydrogenase and lactate dehydrogenase, while downregulating expressions of genes encoding TCA cycle enzymes and the ATP synthases in the roots compared with control. Expressions of genes encoding the respiratory burst oxidase homologs (Rbohs), alternative oxidase (AOX) and dioxygenases were significantly upregulated, and expression of the PIP-type aquaporin genes was downregulated. The addition of 1 mmol·L^-1 NO₃^– to the solution containing 7.5 mmol·L^-1 NH₄⁺ downregulated the expression of genes encoding glycolysis enzymes, fermentation enzymes, Rbohs, AOX and dioxygenases, and increased the expression of genes encoding the TCA cycle enzymes, the ATP synthases and PIP-type aquaporins. Proteomic analysis showed that expressions of glycolysis enzymes, fermentation enzymes and AOX were upregulated, while PIP-type aquaporins were downregulated under sole NH₄⁺ conditions compared with the control. The addition of NO₃^– downregulated expressions of glycolysis enzymes, fermentation enzymes, Rbohs and AOX and upregulated expressions of PIP-type aquaporins. In conclusion, sole NH₄⁺ treatment promotes glycolytic and fermentation pathways, inhibits the TCA cycle and energy generation, and ultimately inhibits root growth of wheat seedlings. The inhibition of root growth may be due to the sole NH₄⁺-induced hypoxic stress on the roots. The addition of NO₃^– inhibits the glycolysis and fermentation pathways, promotes the TCA cycle and energy production, significantly alleviates the hypoxia stress and thereby attenuates the inhibitory effect of NH₄⁺ on root growth.

Agropyron mongolicum is one of northern China’s most representative perennial forage grasses, showing strong tolerance to cold and drought. In plants, caffeic acid O-methyltransferase (COMT) is a key gene involved in the biosynthesis of lignin and melatonin, and plays an important role in regulating plant growth, biomass quality, and stress resistance. In this study, through the analysis of the full-length transcriptome data of Agropyron mongolicum, the COMT candidate gene AmCOMT1 was cloned. AmCOMT1 is highly expressed in tissues with high lignin content, such as stem and root, and its expression is induced by a variety of abiotic stresses, including drought and salt. Expression of AmCOMT1 in Arabidopsis wild type (Col-0) and mutant (omt1-2) significantly promoted the synthesis of lignin in transgenic Arabidopsis, restoring the lignin content and composition of the mutant to wildtype level and the lignin content in Col-0/35S:AmCOMT1 was increased by 11%. In addition, overexpression of AmCOMT1 increased the melatonin content in Col-0/35S:AmCOMT1 transgenic Arabidopsis. Under salt stress conditions, the average root length of this strain increased by 20.3% compared to the wildtype, showing higher stress resistance. In this study, we identified AmCOMT1 from Agropyron mongolicum as a key gene regulating both lignin biosynthesis and melatonin biosynthesis, improving the stress tolerance of transgenic Arabidopsis. Our results highlighted the application potential of AmCOMT1 in genetic improvement of forage grasses through molecular breeding.