*3.1. Effects of Exogenous Leucine on Growth, Chlorophyll Fluorescence Imaging, Net Photosynthetic Rate, SPAD Value, and Maximum Photochemical Efficiency of Peach Seedlings under Cu Stress*

It can be seen from Figure 1a that exogenous Leu can enhance the tolerance of peach seedlings under copper stress. Chlorophyll fluorescence can reflect the photosynthetic efficiency and the degree of stress of plants. We measured the changes in chlorophyll fluorescence (Figure 1b), the net photosynthetic rate (Figure 1c), the SPAD value (Figure 1d), and the maximum photochemical efficiency (Fv/Fm) (Figure 1e) of peach seedlings under Cu stress after leucine treatment. Figure 1a shows that the photosynthetic efficiency of leaves decreased under Cu stress, while the application of Leu improved the photosynthetic efficiency under Cu stress. The net photosynthetic rate, SPAD value, and maximum photochemical efficiency of peach seedlings decreased with an increase in time under Cu stress. However, Leu could alleviate the damage of photosynthetic system under Cu stress. Cu stress reduced the net photosynthetic rate, SPAD value, and maximum photochemical efficiency by 53.8%, 28.2%, and 14.5% compared with control, respectively, in peach seedlings within 6 days. Exogenous leucine increased the net photosynthetic rate, SPAD value, and maximum photochemical efficiency of seedlings under Cu stress by 83.35%, 28.6%, and 8.5% compared with the Cu treatment, respectively, within 6 days. The difference in the effect of Leu on the photosynthetic system was not significant under normal conditions compared to the control.

#### *3.2. Effects of Exogenous Leucine on Leaf Ultrastructure of Peach Seedlings under Cu Stress*

The ultrastructural changes in the cells of peach leaves under Cu stress and with exogenous leucine are shown in Figure 2a–c. In the control seedlings, mesophyll cells and organelles were visible, and cell walls were intact and smooth. The chloroplasts appeared full and fusiform, and the inner grana lamellae were stacked closely and arranged in order. Few starch granules and plastid globules were observed. Under Cu stress, compared with

the control, the starch granule volume increased significantly; starch grains occupied almost half of the chloroplast space. In addition, the number of plastid globules increased, and the chloroplast structure was deformed. With exogenous leucine, the volume of starch granules and the number of plastid globules decreased, and the chloroplast structure was similar to control, even under Cu stress.

**Figure 1.** Effects of different treatments on growth, chlorophyll fluorescence imaging, net photosynthetic rate, SPAD value, and maximum photochemical efficiency of peach seedlings. (**a**) Peach seedlings under different treatments; (**b**) chlorophyll fluorescence imaging; (**c**) net photosynthetic rate; (**d**) SPAD value; (**e**) maximum photochemical efficiency. Each data point represents the mean (±SD) of three replicates. Error bars represent standard deviations of the means (*n* = 3). Different lowercase letters indicate significant differences among different treatments (Duncan test, *p* < 0.05).

#### *3.3. Effects of Exogenous Leucine on Antioxidant Enzyme Gene Expression and Enzyme Activities in Peach Seedlings under Cu Stress*

We took the logarithm of the relative expression of antioxidant protective enzyme genes based on log2, so that the final value > 0 was positive regulation and otherwise it was negative regulation. We found that the relative gene expression of antioxidant protective enzymes in the control and Leu treatments was negative, indicating that the control and Leu treatments did not activate the antioxidant protective enzyme system. However, the Cu treatment and the Cu+Leu treatment could activate the antioxidant protective enzyme system in order to remove ROS in the plant. The relative expression of antioxidant protective enzymes in the Cu treatment with Leu addition was higher than that in the Cu treatment, indicating that Leu could reduce the production of ROS in the plant under Cu stress (Figure 3a). The activities of CAT, SOD, GPx, APX, POD, and DHAR

increased by 67.6%, 148.9%, 50.3%, 152.1%, 187.1%, and 181.4% compared with control, respectively, under the Cu+Leu treatment. However, the activities of CAT, SOD, GPx, APX, POD, and DHAR decreased by 29.5%, 28.9%, 14.7%, 6.8%, 41.3%, and 51.3%, respectively, under Cu stress compared with the Cu+Leu treatment, indicating that the production of ROS could be effectively reduced by Leu (Figures 3b–g and 4e,f).

**Figure 2.** Electron micrographs of leaf mesophyll cells of peach seedlings under different treatments. (**a**) Overall structure of cells with different treatments (bars correspond to 5 μm); (**b**,**c**) Organelle architecture with different treatments (bars correspond to 2 μm). CW: Cell wall; Chl: Chloroplast; SG: Starch granule; Pb: Plastid globule.
