Subcellular accumulation and source of O2[rad]− and H2O2 in submerged plant Hydrilla verticillata (L.f.) Royle under NH4+-N stress condition

Abstract

In this study, the effects of excess NH4+-N on the subcellular accumulation of O2− and H2O2 in submerged plant Hydrilla verticillata (L.f.) Royle were investigated using both histochemical and cytochemical methods. Treatments with ≥ 2.00 and ≥ 5.00 mg L−1 NH4+-N for 5 d significantly increased production of O2− and H2O2, respectively. The activities of plasma membrane-bound NADPH (nicotinamide adenine dinucleotide phosphate) oxidases and antioxidant enzymes (superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, dehydroascorbate reductase and glutathione reductase) were also increased correspondingly. This study also provides the first cytochemical evidence of subcellular accumulation of O2− and H2O2 in the submerged plants. In the leaves of H. verticillata treated with 20.0 mg L−1 NH4+-N, O2− dependent DAB precipitates were found primarily on the inner side of the plasma membrane, extracellular space and chloroplasts. H2O2-CeCl3 precipitates were mainly localized on the inner side of the plasma membrane and extracellular space of the mesophyll cells. Treatments with the inhibitors of NADPH oxidase (diphenylene iodonium and imidazole) indicate that NH4+-N-induced production of O2− and H2O2 in H. verticillata leaves may involve plasma membrane-bound NADPH oxidase. Moreover, low-light treatment decreased NH4+-induced O2− production, suggesting that alterations in the photosynthetic electron transfer chain due to NH4+ toxicity could lead to O2− production.