## 3 Discussion
### Nitrogen Metabolism and Amino Acid Synthesis  
It is believed that the high NUE in _M. aquaticum_ is closely linked to highly efficient nitrogen metabolic systems, including ammonium transport and assimilation systems. Although studies had investigated the function of some key enzymes in ammonium assimilation of _M. aquaticum_[13], a global investigation on the gene regulation mechanism has not been carried out. In this study, the differential expression of genes closely related to ammonium transportation and assimilation at different concentrations of ammonium indicated that these functional genes would be one of the crucial factors contributing to differential responses to ammonium treatment. Excessive ammonium is toxic to _M. aquaticum_. Therefore, the ammonium transportation and assimilation systems should be synergistically regulated [1]. In _Arabidopsis_, three dominant AMTs (AMT1;1, AMT1;2, and AMT1;3) import 95% of the ammonium at low ammonium concentrations and AMT1;3 is critical for ammonium-triggered lateral root branching [1; 12; 28]. In _M. aquaticum_, genes (DN547206_c0_g1 and DN557461_c1_g1) encoding AMT1;2 and AMT3;1 were upregulated at low concentrations of ammonium and downregulated at high concentrations of ammonium. Such responsive regulations on gene expression may help _M. aquaticum_ to better cope with different surrounding ammonium concentrations, which is similar to the gene regulation in _Arabidopsis_[13; 20]. Genes encoding NRTs (DN565032_c0_g1 and DN565032_c0_g2) were downregulated at high concentrations of ammonium and upregulated at low concentrations of ammonium in _M. aquaticum_. Previous studies showed that NRTs played an important role in unfavorable environment adaptation of plants [29; 30]. Meanwhile, deletion of _NRT1;1_ could significantly increase the ammonium tolerance in _Arabidopsis_[30]. In this study, downregulation of genes encoding NRTs at high concentrations of ammonium might also assist _M. aquaticum_ to resist ammonium toxicity while upregulation of genes encoding NRTs at low concentrations of ammonium might produce more NRTs for nitrate uptake to alleviate nitrogen starvation. Ammonium transportation into _M. aquaticum_ tissues is much more likely to induce responses for ammonium assimilation into amino acids. Previous studies reported the increase in enzyme activity for ammonium assimilation, including GOGAT, AS, and GS [1; 13]. In this study, several uni-genes encoding GDH and GOGAT were induced at high concentrations of ammonium (Table S2). GDH is a catabolic enzyme mainly contributing to amino acid breakdown under C deficient conditions to provide 2-OG for the citric acid (TCA) cycle [17] and to ammonium assimilation [14; 15]. Even though it was reported that the primary role of GDH under non-stress conditions was catalyzing glutamate decomposition [31; 32], several studies demonstrated that GDH played an important assimilating role under stress conditions of high concentrations of ammonium [16; 18; 33]. Additionally, high concentrations of ammonium also substantially stimulated the expression of AS (DN549208_c0_g1), which indicates its close association with ammonium assimilation in _M. aquaticum_, as reported in a previous study [13; 24]. Although GS was one of the main contributors to primary nitrogen assimilation and protein storage in plant tissues [16; 17; 34], the expression of GS was dramatically downregulated at high concentrations of ammonium. Considering the significantly increased Gln content at high concentrations of ammonium, one explanation was that a post-transcriptional regulation of GS activity existed in _M. aquaticum_[12; 17; 34]. Another explanation was that GS activity could be upregulated and then downregulated during the long period (14 days) cultivation with high concentrations of ammonium, which needed to be further confirmed. Besides the increase in the nitrogen scavenging mechanism, upregulation of the amidase encoding gene (DN569606_c1_g2) indicated that _M.