Although excess ammoniacal-nitrogen (NH₄⁺-N) results in the disturbance of various important biochemical and physiological processes, a detailed study on the effects of NH₄⁺-N stress on the photosynthesis and global changes in protein levels in submerged macrophytes is still lacking. Here, the changes of excess NH₄⁺-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^-1 NH₄⁺-N, respectively, and decreased at NH₄⁺-N concentrations at 5, 10, 15 and 20 mg L^-1. This decrease might be caused by ROS accumulation. Compared that in 0.02 mg L^-1 NH₄⁺-N as a control, ROS generation in chloroplasts significantly increased in the presence of more than 2 mg L^-1 NH₄⁺-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 NH₄⁺-N. Surprisingly, the abundance of all the identified proteins that were involved in nitrogen assimilation and amino acid metabolism tended to increase under excess NH₄⁺-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 NH₄⁺-N results in chloroplast ultrastructural damages and the first proteomic evidence to support that excess NH₄⁺-N can lead to a decline in photosynthesis and imbalance of C-N metabolism in submerged macrophytes.

尽管过量的氨氮（NH ₄ ⁺ -N）会导致各种重要的生化和生理过程受到干扰，但对NH ₄ ⁺ -N胁迫对淹没大型植物光合作用和蛋白质水平总体变化的影响的详细研究仍是仍然缺乏。在这里，过量的NH ₄ ⁺ -N对褐藻的生理参数的变化（Lf）研究了沉水植物Royle的含量，包括光合色素，可溶性糖，净光合作用和呼吸作用，谷氨酰胺合成酶（GS）和谷氨酸合成酶（GOGAT）的活性，叶绿体超微结构，叶绿体活性氧（ROS）积累和蛋白质水平。我们的结果表明，当分别用1和2 mg L ^-1 NH ₄ ⁺ -N处理植物时，净光合速率和色素含量达到最大值，而在NH ₄ ⁺ -N浓度分别为5、10、15时降低和20 mg L ^-1。这种下降可能是由于ROS积累引起的。相比于0.02 mg L ^-1 NH ₄ ⁺-N作为对照，在超过2 mg L ^-1 NH ₄ ⁺ -N的存在下，叶绿体中的ROS生成显着增加。一致地，在叶绿体超微结构中观察到由过度积累的ROS引起的损害，显示出类囊体膜松动和格兰纳/基质层膨胀。此外，通过蛋白质组学分析，我们确定了91个差异表达的蛋白质斑点。其中，参与光合作用的6种蛋白质因过量的NH ₄ ⁺ -N而大量减少。出乎意料的是，在过量的NH ₄ ⁺下，参与氮同化和氨基酸代谢的所有已鉴定蛋白的丰度趋于增加。-N与对照组相比，提示碳和氮（CN）代谢不平衡。在支持下，观察到活化的GS和GOGAT循环，这由GS和GOGAT酶的较高活性证明。据我们所知，这项工作是对过量的NH ₄ ⁺ -N导致叶绿体超微结构破坏的第一个描述，也是第一个支持蛋白质组学的证据，证明过量的NH ₄ ⁺ -N可能导致淹没后的光合作用下降和CN代谢失衡大型植物。