*3.4. Effects of Exogenous Leucine on Biomass, Leucine Content, Cell Viability, and ROS Accumulation in Leaves of Peach Seedlings under Cu Stress*

The fresh weights of the aboveground and belowground plant parts of seedlings under Cu stress were significantly lower than those of the control seedlings after 20 days. However, the fresh weights of the aboveground and belowground parts of seedlings under Cu stress with exogenous leucine were higher than those without exogenous leucine (Figure 4a). These findings indicate that Cu stress inhibited plant growth and reduced the fresh and dry weights of peach seedlings; exogenous leucine alleviated these effects and helped plants retain growth under Cu stress. The endogenous leucine contents in the leaves, stems, and roots of peach seedlings under Cu stress were significantly increased compared to those in the control seedlings. The exogenous leucine further significantly increased the endogenous leucine contents in the leaves, shoots, and roots (Figure 4b).

**Figure 3.** Effects of different treatments on antioxidant protective enzyme gene expression and enzyme activities in peach seedlings. (**a**) The expression of antioxidant enzyme genes; (**b**) CAT activity; (**c**) SOD activity; (**d**) GPx activity; (**e**) APX activity; (**f**) POD activity, (**g**) DHAR activity). Error bars represent standard deviations of the means (*n* = 3). Different lowercase letters indicate significant differences among different treatments (Duncan test, *p* < 0.05).

Evans blue staining was used to analyze the extent of damage in leaf cells after 6 days under Cu stress. The leaves of peach seedlings under Cu stress were darker, with a large percentage of the area stained. Meanwhile, the leaves of peach seedlings under Cu stress with exogenous leucine were lighter, with a smaller percentage of the area stained compared with that of the leaves under Cu stress alone (Figure 4c). These findings indicate that cell viability decreased under Cu stress and exogenous leucine mitigated Cu-stress-induced cell death (Figure 4d). High levels of ROS induced under heavy metal stress disrupt membrane stability and hinder plant growth. Cu stress significantly increased the production rate of O2 •− and the content of H2O2. However, exogenous leucine reduced the Cu-stress-induced O2 •− production rate and the H2O2 content (Figure 4e,f).

**Figure 4.** Effects of different treatments on biomass, leucine content, cell viability, and ROS accumulation in leaves of peach seedlings. (**a**) Fresh weight and dry weight of peach seedlings thirty days after the treatment; (**b**) leucine content; (**c**) Evans blue staining of leaves; (**d**) cell viability of leaves; (**e**) leaf O2 •− production rate; (**f**) H2O2 content. Error bars represent standard deviations of the means (*n* = 3). Different lowercase letters indicate significant differences among different treatments (Duncan test, *p* < 0.05).
