*2.4. Screening Di*ff*erential Metabolites in the Process of Leaf Color Change*

The differential metabolites were screened by combining the VIP values of fold change and OPLS-DA model. Following was the screening criteria: (1) If the difference of metabolites content between the control group and the experimental group is more than 2 times or less than 0.5, the difference is considered to be significant; (2) On the basis of the above, the metabolites with VIP ≥ 1 are selected. Volcano Plot was used to show the difference of expression level between the two groups of samples, and the difference was statistically significant (Figure 4). Compared with red leaves, 85 metabolites in green leaves were significantly different (10 up-regulated and 75 down-regulated), and 61 metabolites in green leaves were significantly different from yellow leaves (16 up-regulated and 45 down-regulated) (Tables S2 and S3). Compared with red leaves, 86 metabolites showed significant change in yellow leaves (3 up-regulated and 83 down-regulated) (Table S4). Through the analysis of Vennn diagram, it was found that there were 42 metabolites with different contents in the three periods of leaf color change. Except for the content of Liquiritinapioside, the other 41 substances decreased significantly with the change of leaf color (Figures S4 and S5). After a comprehensive analysis of the contents of all the differential metabolites, we found that the contents of 28 metabolites turned to zero in the process of leaf color change, as shown in Figure 5. Among these metabolites with drastic changes, the contents of Naringenin chalcone, an important intermediate in anthocyanin synthesis, and two important anthocyanin metabolites, Pelargonidin O-acetylhexoside and Anthocyanin 3-O-beta-D-glucoside, all decreased to zero in the green leaf stage.

**Figure 4.** (**a**) Volcano plot of differential metabolites for RL vs. GL, RL vs. YL and YL vs. GL. The colors of the scatter points in Figure 4a indicate the final screening results: red indicates metabolites that were significantly up-regulated; green indicates metabolites that were significantly down-regulated; grey indicates metabolites with no significant difference. (**b**) Venn diagram analysis of differential metabolites. RL, red leaves; YL, yellow leaves; GL, green leaves.

**Figure 5.** Differences in the content of 28 metabolites in the process of leaf color change. Y-scale represent the integral value of chromatographic peak area.
