## 3 Results
### Kyoto Encyclopedia of Genes and Genomes Metabolic Pathway Enrichment Analysis  
Pathway enrichment analysis of DEGs using the KEGG database ([[http://www.genome.jp/kegg/](http://www.genome.jp/kegg/)]([http://www.genome.jp/kegg/](http://www.genome.jp/kegg/))) identified 122, 123, 118, 110, 124, and 124 KEGG pathways for the six comparison groups CKvsNT (15), CKvsNT (814), CKvsAT (15), CKvsAT (814), NTvsAT (15), and NTvsAT (814), respectively. The top 15 enriched pathways for the two genotypes contained more upregulated DEGs than downregulated DEGs for CKvsNT (Figure S3) but more downregulated DEGs than upregulated DEGs for CKvsAT (Figure S4). DEGs involved in ribosome, biosynthesis of secondary metabolites and phenylalanine metabolism were more enriched in CKvsNT, whereas DEGs involved in biosynthesis of secondary metabolites, phenylalanine metabolism and phenylpropanol biosynthesis were more enriched in CKvsAT. In NTvsAT (Figure 5), there were more downregulated than upregulated DEGs, and DEGs involved in ribosome, phenylalanine metabolism and biosynthesis of secondary metabolites were highly enriched.  
Genes Involved in Starch and Sucrose Metabolism, Fatty Acid Biosynthesis, Ribosome and Cell Wall Biogenesis  
Carbohydrates, lipids and proteins are the main organic materials that compose plant cells and provide energy. In the pathway enrichment analysis in this study, we found three metabolic pathways, namely, starch and sucrose metabolism (SSM) (ko00500), fatty acid biosynthesis (FAB) (ko00061) and ribosome (RIB) (ko03010), which are the primary pathways of carbohydrate metabolism, lipid metabolism and translation, respectively, to be enriched in CKvsNT and NTvsAT. DEGs involved in SSM, FAB, and RIB showed the same trend. The numbers of DEGs involved in SSM, FAB, and RIB in CKvsNT (15 and 814) and NTvsAT (15 and 814) were greater than those in CKvsAT (15 and 814) (Table 2). Moreover, there were more upregulated DEGs in SSM, FAB, and RIB and fewer downregulated DEGs in the NT treatment group than in the CK treatment group, despite few differences in response to the  
Figure 5: Top fifteen enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for NTvsAT (15 and 814). The \(x\)-axis represents the number of DEGs involved in each pathway; the \(y\)-axis depicts pathways arranged from bottom to top based on the degree of enrichment.  
AT treatment. There were fewer upregulated DEGs in SSM, FAB, and RIB and more downregulated DEGs in the AT treatment group than in the NT treatment group. Information on the DEGs involved in SSM, FAB, and RIB in the six comparison groups is provided in Table S5. To further determine whether the molecular levels of carbohydrates, lipids and proteins were significantly altered in the different N treatments, we examined the numbers of up- and downregulated DEGs for each pathway in carbohydrate metabolism, lipid metabolism and translation in the six comparison groups (Table S6). The results indicate that carbohydrate metabolism, lipid metabolism and protein metabolism were inhibited in the AT treatment group, which led to decreased growth.  
The cell wall is the major component of plant roots. In the GO term analysis performed in this study, we found that the numbers of DEGs involved in primary cell wall biogenesis (CWB) (GO: 0009833) differed between the comparison groups (Table 2). There were 8 and 6 upregulated DEGs in CKvsNT (15 and 814), 9 and 5 downregulated DEGs in NTvsAT (15 and 814) and, interestingly, no DEGs in CKvsAT (15 and 814). Furthermore, DEGs associated with CWB are members of the cellulose synthase (_CESA_) gene superfamily, including _CESA1_, _CESA2_, _CESA3_, and _CESA6_ (Table S5). Therefore, NT treatment enhanced the transcriptional levels of genes related to CWB, whereas AT treatment had no effect on primary CWB.