NRT1.1-Related NH₄ ⁺ Toxicity Is Associated with a Disturbed Balance between NH₄ ⁺ Uptake and Assimilation

A high concentration of ammonium (NH₄ ⁺) as the sole source of nitrogen in the growth medium often is toxic to plants. The nitrate transporter NRT1.1 is involved in mediating the effects of NH₄ ⁺ toxicity; however, the mechanism remains undefined. In this study, wild-type Arabidopsis (Arabidopsis thaliana Columbia-0 [Col-0]) and NRT1.1 mutants (chl1-1 and chl1-5) were grown hydroponically in NH₄NO₃ and (NH₄)₂SO₄ media to assess the function of NRT1.1 in NH₄ ⁺ stress responses. All the plants grew normally in medium containing mixed nitrogen sources, but Col-0 displayed more chlorosis and lower biomass and photosynthesis than the NRT1.1 mutants in (NH₄)₂SO₄ medium. Grafting experiments between Col-0 and chl1-5 further confirmed that NH₄ ⁺ toxicity is influenced by NRT1.1. In (NH₄)₂SO₄ medium, NRT1.1 induced the expression of NH₄ ⁺ transporters, increasing NH₄ ⁺ uptake. Additionally, the activities of glutamine synthetase and glutamate synthetase in roots of Col-0 plants decreased and soluble sugar accumulated significantly, whereas pyruvate kinase-mediated glycolysis was not affected, all of which contributed to NH₄ ⁺ accumulation. By contrast, the NRT1.1 mutants showed reduced NH₄ ⁺ accumulation and enhanced NH₄ ⁺ assimilation through glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase. Moreover, the up-regulation of genes involved in ethylene synthesis and senescence in Col-0 plants treated with (NH₄)₂SO₄ suggests that ethylene is involved in NH₄ ⁺ toxicity responses. This study showed that NH₄ ⁺ toxicity is related to a nitrate-independent signaling function of NRT1.1 in Arabidopsis, characterized by enhanced NH₄ ⁺ accumulation and altered NH₄ ⁺ metabolism, which stimulates ethylene synthesis, leading to plant senescence.