Transcriptional signatures of nitrogen and amino acid metabolism under excess ammonium stress in Nicotiana tabacum leaves

High concentrations of soil ammonium are toxic to most plants, limiting their ability to use nitrogen efficiently. This study aims to identify candidate transcription factors that respond to excess ammonium stress in Nicotiana tabacum , a sensitive species. Seedlings at the seven-leaf stage were grown hydroponically for three weeks with 10 mM NH₄⁺, 10 mM NO₃⁻, or a mixture of 5 mM each as their sole nitrogen source. Growth parameters and physiological traits, including enzymatic activities, free amino acid pools, and the transcriptome, were determined. The high NH ₄ ⁺ treatment significantly reduced plant growth, as well as the accumulation of C, N, P and K, causing a premature senescence. The free amino acid concentrations increased by around 30%, which featuring the increased accumulation of free ammonium, hydroxyproline, threonine and valine, while the decrease in glutamate, aspartate, GABA and homoalanine. Ammonium stress was found to upregulate genes involved in signaling, transcriptional regulation, mitochondrial activity and transporters, while downregulating genes in photosynthesis, the thylakoid membrane, responses to chemicals and cellular homeostasis. Notably, four genes encoding glutamate dehydrogenase and three encoding aspartate amino transferases were exclusively upregulated in stressed leaves. An inferred gene regulatory network (iGRN) identified six transcription factors as candidate regulators that are highly associated with the response to excess ammonium stress conditions. High ammonium levels lead to intricate interactions between nitrogen nutrition and other cellular processes. Our results pave the way for further deciphering of the molecular mechanisms underlying ammonium sensitivity and tolerance.