*3.5. Effects of Exogenous Leucine on Root Biomass, Root Cell Viability, Root Viability, Proline, Malondialdehyde Contents, and Electrolyte Leakage of Peach Seedlings under Cu Stress*

Figure 5a and Supplementary Table S2 show that Leu had little effect on the roots of peach seedlings under normal conditions; however, the root biomass was significantly reduced under Cu stress, and Leu could effectively reduce the reduction in root biomass of peach seedlings. The plant root system is the organ that is directly exposed to heavy metals in soil. Evans blue staining was used to analyze the viability of root cells under Cu

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stress. The roots were darker under Cu stress, and the color became lighter with exogenous leucine (Figure 5b). In addition, the Cu+Leu treatment significantly increased root cell viability and root viability relative to the Cu treatment (Figure 5c,d). The contents of proline in the peach seedlings under Cu stress were significantly higher than those in the control seedlings (1.3 times higher, Figure 5e). Exogenous leucine significantly reduced the Custress-induced increase in proline contents in peach seedlings. The results indicate that the leucine treatment significantly alleviated the osmotic stress caused by high concentrations of Cu in peach seedlings. The malondialdehyde (MDA) content of seedlings under Cu stress was significantly higher than that of the control seedlings. Exogenous leucine reduced the stress-induced increase in the MDA content; however, the content was significantly higher than that of the control. The results indicate that leucine reduced the MDA content and that electrolyte leakage improved membrane stability in peach seedlings under Cu stress (Figure 5f,g). As can be seen from Supplementary Table S3, Cu ions were mainly enriched in the roots of peach seedlings, and the Cu+Leu treatment could significantly reduce the Cu ion content in the roots compared with the Cu treatment.

**Figure 5.** Effects of different treatments on root biomass, root cell viability, root viability, proline, malondialdehyde content, and electrolyte leakage of peach seedlings. (**a**) Root biomass; (**b**) Evans blue staining of roots; (**c**) root cell viability; (**d**) root viability; (**e**) proline content; (**f**) MDA content; (**g**) electrolyte leakage. Error bars represent standard deviations of the means (*n* = 3). Different lowercase letters indicate significant differences among different treatments (Duncan test, *p* < 0.05).

#### *3.6. Effects of Exogenous Leucine on Nitrogen Metabolism in Peach Seedlings under Cu Stress*

Under normal conditions, the activities of NR, GS, and GOGAT treated with control and Leu were at high levels from 0 to 6 days. The activities of NR, GS, and GOGAT decreased significantly with time under Cu stress. Although the enzyme activity of leucinetreated seedlings under Cu stress was significantly lower than that of control seedlings, the enzyme activity decreased slightly after 4 days, which was significantly higher than that of pure Cu stress seedlings (Figure 6a–c). Cu stress decreased the nitrate–nitrogen content and increased the ammonium–nitrogen and free amino acid contents in the peach seedlings (Figure 6d–f). The application of leucine to the seedlings under Cu stress increased the nitrate–nitrogen content; however, it was lower than that in the control group. Meanwhile, with exogenous leucine, the ammonium–nitrogen content was the same as that in the control group, while the total free amino acid content was significantly higher than that in control group.

**Figure 6.** Effects of different treatments on nitrogen metabolism in peach seedlings. (**a**) Nitrate reductase (NR) activity; (**b**) glutamine synthase (GS) activity; (**c**) glutamic acid synthetase (GOGAT) activity; (**d**) nitrate–nitrogen (NO3-N) content; (**e**) ammonium–nitrogen (NH4+-N) content; (**f**) free amino acid 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).
