## 3 Results  
### Differential Analysis of Secondary Metabolism in Lotus Tissues  
The total ion chromatogram (TIC) of six tissues of the lotus plant in positive and negative ion modes are shown in Figure 1A,B, respectively. After screening differential metabolites from the confidently identified metabolite list, 8638 and 8125 differential metabolites were identified in positive and negative ion modes, respectively. Principal component analysis (Figure 1C,D) of metabolites in positive and negative ion modes showed differences in metabolites between groups of different tissues; differences within groups were not apparent, which could explain the unique metabolic characteristics of different tissues to a certain extent. The Nelumbinis Stamen (Sta) was clearly distinguished from other tissues with unique metabolic features. The Nelumbinis Semen (Sem) and Nelumbinis Plumula (Flu) had similar metabolic characteristics. Notably, the Nelumbinis Petiolus (Pet), Nelumbinis Receptaculum (Rec), and Nelumbinis Flos (Flo) are the light-seeing tissues of the lotus growing on the water surface. Flo, Rec, and Sem are the flower, the fruit, and the seed of the lotus. The lotus pollen comes from Sta, and after successful pollination, the pistil develops into Rec; inside Rec, the Sem develops, and the embryonic root, Flu, develops in Sem. Therefore, we set up the following comparison combinations: Flo vs. Rec, Flo vs. Pet, Rec vs. Pet, Flo vs. Sem, Rec vs. Sem, Sta vs. Rec, Rec vs. Flu, Sem vs. Flu, and Sta vs. Sem. Figure 1: _Cont._The Z-score plot can visualize the overall trend and degree of difference in the quantitative values of metabolites among different groupings and samples. Figure 1E shows the top 30 differential metabolites, which exhibited the most significant degree of variation among the six tissues of the lotus. All the metabolites with a greater degree of variation were significantly enriched in Sta. Based on the results of the principal component analysis, the Z-score plot displayed a unique metabolic profile of Sta. Amygdalin, which has the highest variation among the six tissues and the highest relative content in Sta, is usually broadly distributed in Rosaceae [38], and more commonly found in almond seed kernels as well as apricot pollen [39,40]. Pharmacological effects include cough suppressant, anticancer, anti-inflammatory, analgesic, and hypoglycemic effects [38,41]. In addition, metabolite contents including butin, galactaric acid, liquidambaric, geranial, and echinatin in Sta were significantly higher than that in the other five tissues. MS/MS secondary analysis identified 1094 secondary metabolites in six tissues of the lotus plant (Figure 2A, Table S1), including 122 flavonoids, 95 preenol lipids, 92 organooxygen compounds, 86 fatty acyls, 57 carboxylic acids and derivatives, 54 benzene and substituted derivatives, 50 phenols, 40 terpenoids, and 32 alkaloids and other compounds. A total of 92 differential metabolites were common to different comparative combinations in different tissues of lotus, including 16 flavonoids, 7 phenols, 6 terpenoids, 6 organooxygen compounds, 4 alkaloids, 4 benzene and substituted derivatives, and 4 preenol lipids and other compounds. Among them, flavonoids had the largest percentage, showing that they had the greatest effect on the differentiation of different tissues of the lotus (Figure 2B). To investigate the accumulation pattern of secondary metabolites in different tissues of the lotus, K-means cluster analysis was performed on 1094 secondary metabolites identified in six tissues of the lotus plant (Figure 2C, Table S2). The findings revealed that secondary metabolites accumulated in different tissues with different patterns. The secondary metabolites were classified into nine different clusters based on the distribution trend of their contents in different tissues. Clusters 1 and 7 contained 125 and 116 secondary metabolites, most of which were organooxygen compounds, with a trend of specific accumulation in Pet and Sem.