## 3 Discussion
### Involvement of TF Families and Protein Kinases in the Ammonium Mediated Signalling Response  
A number of TFs and protein kinases, including MYB, WRKY, NAC, bHLH, bZIP, and AP2, were involved in nitrogen mediated signalling and regulatory networks [12; 22; 42; 43] including some that were even involved in long-distant signalling between the leaf and the root [42]. In this study, comparative analysis on transcription factors that were significantly regulated responding to different concentrations of ammonium demonstrated that MYB, AP2, and WRKY TFs were the most abundant TF families (Figure 6). Previous studies proved the involvement of MYB TFs in the expression of nitrogen transporters and assimilatory genes [43], as well as their important roles in GS isogenes regulation in _Arabidopsis_ and rice [34]. Although the exact role of AP2 in nitrogen-related signaling mediation is not known, involvement of AP2 adapting to nitrogen deficiency was reported in a previous study [37]. Dof1 TFs can increase nitrogen assimilation in many plants such as maize and tobacco [37]. Two Dof1 genes in _M. aquaticum_ were identified including one that (DN552745_c4_g1) was significantly upregulated (_p_ < 0.05) at low ammonium concentrations (L1 and L2 group). The other (DN545277_c1_g1) was upregulated at high ammonium concentrations (L4 group), which indicated a universal stimulation of nitrogen assimilation in _M. aquaticum_. In general, the differential expression of different transcription factors in response to different concentrations of ammonium may also be one of the factors responsible for high NUE in _M. aquaticum_.  
Protein kinases also played important roles in nitrogen signalling [31; 44; 45]. In our transcriptome data, histidine kinase 1 (DN569908_c5_g2, DN569908_c5_g1 and DN569944_c1_g1) was upregulated at a low ammonium concentration. Histidine kinases took part in a variety of plant signalling responses to abiotic stress [22; 46]. Therefore, upregulation of histidine kinase 1 in response to low ammonium treatment may also reveal its involvement in ammonium-mediated signalling in _M. aquaticum_. In plants, regulation of nitrogen sensing and transportation by calcium signalling requires Ca\({}^{2+}\) dependent protein kinases (CDPKs) and CBL-interacting protein kinases (CBL/CIPKs) [1; 31; 44; 45]. In this study, eight CDPKs (CDPK 3; 5; 8; 11; 16; 28; 29; 32) and four CIPKs (CIPK 1, 5, 11, and 12) were differentially regulated in response to ammonium treatment. The CDPK11, CDPK16, and CDPK28 were commonly induced at low concentrations of ammonium, whereas CDPK8 and CDPK32 were specifically induced at high concentrations of ammonium. In _Arabidopsis_, CDPK was involved in the activation of anion channels such as SLAC1 [47]. Therefore, the induction of CDPK in _M. aquaticum_ responding to ammonium treatment indicated its role in nitrogen sensing and transportation. As for CIPKs, CIPK 11 and CIPK 12 were significantly induced (_p_ < 0.05) at low concentrations of ammonium while CIPK 1 and CIPK 11 were significantly induced (_p_ < 0.05) at high concentrations of ammonium. However, CIPK 5 was significantly repressed at high concentrations of ammonium, which indicated a strategy to ammonium stress [44; 45]. Previous studies emphasized the involvement of CIPKs in regulating the nitrogen-mediated response in plants [31; 44]. Therefore, it could be hypothesized that CIPKs identified in this study might be important components of nitrogen signalling in _M. aquaticum_.