Metabolic adaptations to ammonia-induced oxidative stress in leaves of the submerged macrophyte Vallisneria natans (Lour.) Hara

Ammonia (i.e. the total of NH3 and NH4+) has been one of the main causes of the decline of macrophytes in fresh water. In order to study the effects of ammonia toxicity, plants of the submersed macrophyte Vallisneria natans (Lour.) Hara were treated with various concentrations of NH4Cl (0.1, 0.4, 1.2, 2 and 2.8 mM) for 4 days or with 2 mM NH4Cl for different lengths of time (12 h, 1, 2, 4 and 8 days). The toxic effect and oxidative stress caused by NH4Cl resulted in a reduction of total chlorophyll (chlorophyll a and b) and an increase in the levels of reactive oxygen species (ROS) O2− and H2O2, with an increased concentration of NH4Cl and duration of exposure. Meanwhile, weak chlorosis and water-soaked symptoms were observed in older leaves exposed to 2.8 mM NH4Cl for 4 days. The activity of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR) was up-regulated in leaves treated with 1.2, 2 and 2.8 mM NH4Cl for 4 days or with 2 mM NH4Cl for 1, 2 and 4 days, when compared with controls. Among these enzymes, the activity of superoxide dismutase, ascorbate peroxidase and glutathione reductase was significantly up-regulated in plants treated with 0.4 mM NH4Cl for 4 days, while they were down-regulated at 4 and 8 days from their peak values in leaves treated with 2 mM NH4Cl. The content of ascorbic acid decreased significantly in leaves treated with 0.4-2.8 mM NH4Cl for 4 days or with 2 mM NH4Cl for 2-8 days. The content of total glutathione (tGSH; reduced and oxidized glutathione) increased in leaves treated with NH4Cl at 0.4, 1.2 and 2 mM for 4 days or with 2 mM NH4Cl at 1, 2 and 4 days, while tGSH was decreased below the level of controls by treatment with 2.8 mM NH4Cl for 4 days or to the level of controls by treatment with 2 mM NH4Cl for 8 days. However, the content of malondialdehyde (MDA) decreased with increased concentration of NH4Cl and duration of exposure. Results from staining with 3,3′-dimethoxybenzidine (DAB) further indicated that the level of H2O2 and the activity of guaiacol peroxidase increased significantly in plants exposed to 2 mM NH4Cl for 4 days. These results suggested that ammonia exerted its toxic effect on the growth of V. natans (Lour.) Hara, at least in part, by induction of oxidative stress and inhibition of photosynthesis. The decrease in the content of malondialdehyde is discussed.