## 3 Results
### Joint Metabolomic and Transcriptomic Analysis of Lotus Tissues  
The joint analysis of metabolism and transcription can unravel the relationship pattern of metabolites and genes and reveal the regulatory mechanism of metabolic pathways in organisms. Based on the number of differential molecules obtained from metabolome and transcriptome assays, the most DAMs were found between Rec and Plu, with 324 DAMs up-regulated and 311 DAMs down-regulated. This was followed by Sta with Rec, with 421 DAMs up-regulated and 212 DAMs down-regulated. The fewest DAMs were found between Sem and Plu, with 206 DAMs up-regulated and 305 DAMs down-regulated. The DEGs between Plu and Rec were the most, with 6793 DEGs up-regulated and 4118 DEGs down-regulated. The next largest number of DEGs was between Plu and Sem, with 5803 DEGs up-regulated and 4020 DEGs down-regulated. Rec and Flo had the least DEGs with 3050 up-regulated and 4019 down-regulated. This shows that the extent of difference in metabolites and genes between the two tissues is not strongly correlated with the distance of growth from each other (Figure 3B). From the KEGG pathway enrichment results of DAMs and DEGs, the top 20 pathways with the strongest degree of enrichment were selected to draw KEGG pathway bubble maps (Figure 4). In the nine sets of comparison combinations (Flo vs. Rec, Flo vs. Pet, Rec vs. Pet, Flo vs. Sem, Rec vs. Sem, Rec vs. Plu, Sem vs. Plu, Sta vs. Rec, Sta vs. Sem), the flavonoid biosynthesis pathway was enriched in both of the two omics approaches in six of these groups (Flo vs. Rec, Flo vs. Pet, Rec vs. Pet, Flo vs. Sem, Rec vs. Sem, Sta vs. Rec). The pathway was enriched to 25 DAMs and 31 DEGs in Flo vs. Rec, 26 DAMs and 35 DEGs in Flo vs. Pet, 22 DAMs and 37 DEGs in Rec vs. Pet, 25 DAMs and 35 DEGs in Flo vs. Sem, 23 DAMs and 29 DEGs in Rec vs. Sem, and 24 DAMs and 28 DEGs in Sta vs. Rec. KEGG pathway bar graphs were plotted based on the enrichment results (Figure 5), with the red horizontal line indicating a \(p\)-value of 0.01 and the blue horizontal line indicating a \(p\)-value of 0.05. The flavonoid biosynthesis pathway had a \(p\)-value <0.05 in both two omics approaches of the six comparative combinations, suggesting that this pathway is the key regulatory pathway; hence, DAMs and DEGs on this pathway were selected for further analysis. Figure 4: Cont. Figure 4: KEGG enrichment bubble map. (**A**) Flo vs. Rec; (**B**) Flo vs. Pet; (**C**) Rec vs. Pet; (**D**) Flo vs. Sem; (**E**) Rec vs. Sem; (**F**) Sta vs. Rec; (**G**) Rec vs. Plu; (**H**) Sem vs. Plu; (**I**) Sta vs. Sem. Figure 5: _Cont._  
To gain deeper insights into the accumulation mechanism of flavonoid compounds, the levels of differential metabolites and the expression patterns of differential genes within the pathway were visualized using heatmaps. These heatmaps were further integrated with the KEGG pathway to construct comprehensive metabolites and genes heatmaps for the flavonoid biosynthesis pathway (Figure 6). A total of 34 differential flavonoid components, 64 key-acting genes, and 17 key synthases were annotated in the flavonoid biosynthesis pathway. These included phenylalanine ammonia-lysae (_PAL_), 4-coumarate-CoA ligase (_4 CL_), chalclone synthase (_CHS_), transcinnamate 4-monooxygenase (_CYP3A_), shikimate O-hydroxycinnamoyltransferase (_HCT_), 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase (_C3'H_), cafeoyl-CoA O-methyltransferaseansferase (_CCoAOMT_), chalclone isomerase (_CHI_), phlorizin synthase (_PGT_), naringenin 3-dioxygenase (_F3H_), flavonoid 3'-monooxygenase (_F3'H_), flavonoid 3',5'-hydroxyl hydroxylase (_F3'5'H_), bifunctional dihydro flavonol 4-reductase (_DFR_), flavanol synthase (_FLS_), anthocyanin synthase (_ANS_), leucoanthocyanidin reductase (_LAR_), and anthocyanin reductase (_ANR_). Notably, _CHS_ catalyzes the production of various types of chalclone, acting as the first rate-limiting enzyme of the flavonoid biosynthesis pathway. Their expression levels can directly indicate the amount of flavonoids synthesized by the plant body [42; 43].