Overexpression of pxAlaAT3 in Populus × xiaohei alleviates root growth inhibition under ammonium nitrogen stress

Overexpressed AlaAT3 in Populus enhances ammonium tolerance by modulating carbohydrate metabolism, nitrogen metabolism, and antioxidant system-related metabolic processes. Alanine aminotransferase (AlaAT) is a critical enzyme involved in the nitrogen assimilation process in plant cells, catalyzing the reversible transfer of an amino group from alanine to α-ketoglutarate. This reaction is essential for maintaining metabolic homeostasis. Previous studies have suggested that AlaAT plays a role in alleviating ammonium toxicity in plants. To investigate this hypothesis, transgenic Populus × xiaohei plants overexpressing AlaAT3 were generated, and their phenotypic, physiological, and transcriptional traits were compared with those of wild-type (WT) plants. Under treatment with 3 mM NH₄⁺ ammonium nitrogen, the transgenic plants exhibited significantly enhanced root biomass. Compared with WT plants, the transgenic lines demonstrated higher activities of GS, SOD, and CAT enzymes, while POD activity was notably reduced. Levels of soluble protein, free amino acids, sucrose, starch, soluble sugars, and proline were significantly elevated, whereas concentrations of O₂^-, and NH₄⁺ were markedly reduced. Transcriptomic analysis revealed significant enrichment in glutathione metabolism, peroxisome, nitrogen metabolism, and starch and sucrose metabolism pathway in the transgenic plants, with corresponding genes displaying notable transcriptional changes. Regulatory network analysis identified key transcription factors, including WRKY53, DOF3.4, and DOF1.5, as potential regulators of ammonium toxicity resistance in these transgenic lines. These findings demonstrate that AlaAT3 overexpression enhances Populus × xiaohei tolerance to NH₄⁺ by modulating glutathione metabolism, peroxisome, nitrogen metabolism, and starch and sucrose metabolism pathway. This study provides candidate genes and lays a strong foundation for future research into the mechanisms underlying NH₄⁺ tolerance in Populus plants overexpressing AlaAT3.