Effects of excess ammoniacal nitrogen (NH-N) on pigments, photosynthetic rates, chloroplast ultrastructure, proteomics, formation of reactive oxygen species and enzymatic activity in submerged plant Hydrilla verticillata (L.f.) Royle.

Although excess ammoniacal-nitrogen (NH-N) results in the disturbance of various important biochemical and physiological processes, a detailed study on the effects of NH-N stress on the photosynthesis and global changes in protein levels in submerged macrophytes is still lacking. Here, the changes of excess NH-N on physiological parameters in Hydrilla verticillata (L.f.) Royle, a submerged macrophyte were investigated, including the contents of photosynthetic pigments, soluble sugars, net photosynthesis and respiration, glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, chloroplast ultrastructure, chloroplast reactive oxygen species (ROS) accumulation and protein levels. Our results showed that the net photosynthetic rate and pigment content reached maximum values when the plants were treated with 1 and 2 mg L NH-N, respectively, and decreased at NH-N concentrations at 5, 10, 15 and 20 mg L. This decrease might be caused by ROS accumulation. Compared that in 0.02 mg L NH-N as a control, ROS generation in chloroplasts significantly increased in the presence of more than 2 mg L NH-N. Consistently, the damages caused by over-accumulated ROS were observed in chloroplast ultrastructure, showing a loose thylakoid membranes and swollen grana/stroma lamellae. Furthermore, through proteomic analysis, we identified 91 differentially expressed protein spots. Among them, six proteins involved in photosynthesis decreased in abundance in response to excess NH-N. Surprisingly, the abundance of all the identified proteins that were involved in nitrogen assimilation and amino acid metabolism tended to increase under excess NH-N compared with the control, suggestive of the imbalanced carbon and nitrogen (C-N) metabolisms. In support, activated GS and GOGAT cycle was observed, evidenced by higher activities of GS and GOGAT enzymes. To our knowledge, this work is the first description that excess NH-N results in chloroplast ultrastructural damages and the first proteomic evidence to support that excess NH-N can lead to a decline in photosynthesis and imbalance of C-N metabolism in submerged macrophytes.