## 2 Results
### Gene Ontology and Pathway Analysis of DEGs  
GO enrichment analysis was used for a global analysis of DEGs. The DEGs were classified into three categories of ontologies including the biological process, the cellular component, and molecular function (Figure 4). Among the biological process, the DEGs in four groups were mainly involved in the polysaccharide catabolic process (GO:0000272), protein transport (GO:0015031), protein localization (GO:0008104), establishment of protein localization (GO:0045184), macromolecule modification (GO:0043412), and amide biosynthetic process (GO:0043604). Typically, the amide biosynthetic process (GO:0043604), the peptide biosynthetic process (GO:0043043), and the peptide metabolic process (GO:0006518) were only enriched in the L4 group while the nitrogen compound metabolic process (GO:0006807) was enriched the most in L1 and L4 groups. Furthermore, the highly enriched terms included in cellular component among these groups were the extracellular region (GO:0005576), the nuclear part (GO:0044428), the protein complex (GO:0043234), and the ribosome  
Figure 3: Clustering analysis of 7721 differentially expressed genes (DEGs) among different groups in _Myriophyllum aquaticum_. (**A**) Heat map of DEGs expression in different groups through K-means clustering analysis. (**B**) Expression patterns and numbers of the DEGs in the nine clusters. Color keys in (**A**) and vertical ordinate in (**B**) were presented as log10 (TPM + 1). TPM: transcripts per million reads. LNC: group treated with 0 mM ammonium. L1: group treated with 0.1 mM ammonium. L2: group treated with 1 mM ammonium. L3: group treated with 12 mM ammonium. L4: group treated with 36 mM ammonium. (GO:0005840). Photosystem (GO:0009521) and photosystem II (GO:0009523) were only enriched in the L4 group while the chloroplast part (GO:0044434) was only enriched in L3 and L4 groups. Lastly, the molecular function contained 35, 18, 28, and 50 significantly (\(p<0.05\)) enriched GO terms in L1, L2, L3, and L4 groups, respectively. Among these terms, monooxygenase activity (GO:0004497), heme binding (GO:0020037), and iron ion binding (GO:0005506) were highly enriched in all groups. These results indicated that a global and complex regulation network existed in _M. aquaticum_, especially in the L4 group. These 7721 uni-genes were, subsequently, assigned to 126 KEGG pathways of which top 30 KEGG pathways were shown in Figure 5. Pathways of nitrogen metabolism (ko00910), starch and sucrose metabolism (ko00500), and the plant hormone signal transduction (ko04075) were significantly enriched in four groups. Genes involved in amino acid pathways in eukaryotes were also among the top 30 KEGG category, such as phenylalanine metabolism, alanine, aspartate and glutamate metabolism, carotenoid biosynthesis, valine, leucine and isoleucine degradation, tyrosine metabolism, cysteine and methionine metabolism, beta-Alanine metabolism, cyanoamino acid metabolism, histidine metabolism, tryptophan metabolism, and arginine and proline metabolism. In addition, many genes related to  
Figure 4: Analysis of Gene Ontology (GO) enrichment for differentially expressed genes (DEGs) among different groups in _Myrophyllum aquaticum_. LNC: group treated with 0 mM ammonium. L1: group treated with 0.1 mM ammonium. L2: group treated with 1 mM ammonium. L3: group treated with 12 mM ammonium. L4: group treated with 36 mM ammonium. Figure 3: Analysis of Gene Ontology (GO) enrichment for differentially expressed genes (DEGs) among different groups in _Myrophyllum aquaticum_. LNC: group treated with 0 mM ammonium. L1: group treated with 0.1 mM ammonium. L2: group treated with 1 mM ammonium. L3: group treated with 12 mM ammonium. L4: group treated with 36 mM ammonium. carbohydrate metabolisms including fructose and mannose metabolism, glycolysis/gluconeogenesis, oxidative phosphorylation, pentose and glucuronate interconversions, and galactose metabolism were also significantly (_p_ < 0.05) enriched (Table S5).