*3.7. Correlation Analysis of Leucine Content with Osmotic System and Antioxidant System in Peach Seedlings*

Figure 7 exhibits a heatmap correlation matrix among different physiological and molecular traits. For the correlation study, the leucine content of the peach seedlings as well as osmotic regulatory system and antioxidant system activity (SOD, POD, CAT, APX, GPx, and DHAR) were evaluated. As can be seen from Figure 7, the leucine content in peach seedlings was positively correlated with plant antioxidant enzyme activities, and the correlation was significantly different and was negatively correlated with the hydrogen peroxide content and the MDA content. The MDA content was negatively correlated with the proline content, the SPAD value, the cell viability of roots, the cell viability of leaves, the APX activity, and the net photosynthetic rate. The hydrogen peroxide content was negatively correlated with the SOD activity, POD activity, DHAR activity, CAT activity, and GPx activity.

**Figure 7.** Correlation analysis of leucine content with the osmotic system and the antioxidant system in peach seedlings (Duncan test, \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, \*\*\* *p* ≤ 0.001).

#### **4. Discussion**

Plants are inevitably challenged by various environmental stresses, in particular salt, heat, intense irradiance, and heavy metal stress. Abiotic stress can reduce crop growth, plant leaf area, and photosynthesis rates [29]. Leaves, the central organs of photosynthesis in plants, respond to changes in the external environment or internal metabolism via changes in the ultrastructure, opening angle, aspect ratio, or photosynthesis [30]. It has been reported that a low concentration of Cu2+ (<300 mg·kg−1) could increase the chlorophyll content of '*Hanfu*' apple seedlings and keep the leaves in a healthy state. However, when the Cu2+ concentration exceeded 300 mg·kg<sup>−</sup>1, the plants were stressed, and the chlorophyll content and photosynthetic efficiency of seedlings decreased [31]. Under Cu stress, the net photosynthetic rate of peach seedlings decreased, probably due to the change in chloroplast composition [32]. Chlorophyll a/chlorophyll b were significantly decreased after the application of 600 mg·kg−<sup>1</sup> Cu2+, indicating that a high Cu concentration seriously inhibited the synthesis process of chlorophyll and thus reduced plant photosynthetic efficiency [33]. The maximum photochemical efficiency is a useful indicator of photosystem function and efficiency. The maximum photochemical efficiency is inversely proportional to stress, and any significant change in this value reflects the influence of stress on plants [34]. In this study, the SPAD values of leaves decreased significantly under Cu stress. However, exogenous leucine significantly increased the chlorophyll fluorescence, net photosynthetic rate, SPAD value, and maximum photochemical efficiency (Figure 1b–e).

The morphology and ultrastructure of chloroplasts also directly affect the photosynthetic performance of plants. Several studies have investigated the effect of Cu on the chlorophyll ultrastructure [35,36]. Lin et al. (2008) found that higher concentrations of Cu ruptured the chloroplasts completely with an expanded thylakoid matrix [37]. Ji et al. (2007) also found expanded chloroplasts, distorted grana lamella, and ruptured membranes in *Potamogeton malaianus* under Cu stress [38]. An appropriate concentration of Cu2+ can promote plant photosynthesis, but an excessive concentration of Cu2+ will inactivate chlorophyll proteins, change the chloroplast ultrastructure, destroy the structure and function of thylakoids, ultimately inhibit photosynthesis, and seriously affect nutrient accumulation [39]. In this study, under Cu stress the cell ultrastructure changed, the chloroplasts were distorted, and the number and volume of starch granules and plastid globules increased. In addition, the cell walls were ruptured, and starch granules occupied almost half of the chloroplasts. After the application of leucine, the number of starch granules and plastid globules decreased significantly, and the chloroplast morphology returned to normal (Figure 2). The possible reason is that exogenous leucine inhibited pigment oxidative decomposition, increased protochlorophyllide reductase activity, promoted chlorophyll synthesis, increased the SPAD value, and maintained the shape of chloroplasts, which in turn improved the photosynthetic system of leaves.

Plants suffering from adverse heavy metal stress, including Cu stress, iron stress, and arsenic stress, produce excess internal reactive ROS, such as superoxide (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH-), which negatively elicit oxidative stress on cellular structures and metabolism [40–43]. The antioxidant system is divided into the enzymatic system and the non-enzymatic antioxidant system. These enzymes include SOD, POD, CAT, APX, GPx, and other antioxidant enzymes as well as non-enzymatic antioxidant substances such as GSH, ascorbic acid (ASA), MTs, and proline [44]. Previous studies found that the activities of POD, SOD, and CAT in grape roots increased first and then decreased under different Cu concentrations (0.5, 1, 1.5, and 2 mmol/L) [45]. In this study, we found that the expression levels of antioxidant genes were significantly upregulated when seedlings were exposed to Cu stress, among which the expression levels of *PpCAT*, *PpSOD,* and *PpGPx* were more upregulated. Under Cu stress, the expression of antioxidant genes and the activity of antioxidant enzymes were increased after adding Leu, indicating that Leu could reduce the peroxidation damage under Cu stress (Figure 3). According to Figure 4e,f, compared with the Cu treatment, Cu+Leu can significantly reduce the content of H2O2 and the production of O2- in plants. Therefore, it is speculated that Leu has no obvious effect on the antioxidation system under normal conditions, but under Cu stress Leu can activate the antioxidant system and reduce the oxidative damage caused by ROS.

The AsA-GSH cycle is an important way for plants to respond to stress, mainly through the joint action of multiple enzymatic reactions involving the reduced antioxidant AsA and GSH as well as APX and DHAR so as to realize the process of H2O2 removal and the regeneration of ASA and GSH and maintain the REDOX homeostasis of cells, improving the stress resistance of plants under stress conditions [46]. Previous studies have shown that the ASA and GSH contents in wheat roots were significantly higher under the high concentration of Cu2+ at 1 mmol/L than in the control treatment [47]. Wu et al. found that Taxus chinensis var. chinensis synthesized a large amount of ASA, which was used to remove a large amount of H2O2 accumulated in the root system to weaken high Cu toxicity [48]. In addition, plants can regulate the osmotic balance by increasing the contents of proline and other osmotic regulators, thus maintaining normal cell metabolism and improving plant stress resistance under heavy metal stress. Our results showed that the relative expression and enzyme activities of *PpAPX*, P*pGPx,* and *PpDHAR* were enhanced by Leu supplementation under Cu stress, which partially explained the enhanced ROS scavenging capacity and the decreased proline and MDA contents in Leu-treated peach seedlings under Cu stress (Figures 3–5). As can be seen from the Figure 7, the leucine content in peach seedlings was positively correlated with plant antioxidant enzyme activities, and the correlation was significantly different and was negatively correlated with the hydrogen peroxide content and the MDA content. These results indicate that exogenous leucine reduced the accumulation of ROS in peach seedlings under Cu stress, thus reducing the oxidative damage.

Excessive Cu2+ is preferentially accumulated in plant roots, and root length will decrease with an increase in the Cu2+ concentration, which will affect the absorption of water and mineral elements by roots and inhibit the growth of the aboveground parts [49]. Michaud et al. found that the root length of wheat decreased by 10%, 25%, and 50%, respectively, when the Cu ion concentration in the root reached 100, 150, and 250–300 mg/kg, indicating that, in a certain range, the higher the Cu ion concentration, the more obvious the inhibitory effect on root growth [50]. In this study, the Cu content was the highest in the roots and the lowest in the stems (Supplementary Table S3). In the seedlings treated with leucine under Cu stress, the Cu contents in the roots, stems, and leaves were significantly lower than those in the seedlings under Cu stress alone. In addition, the Cu content in the roots decreased to a greater extent with exogenous leucine. The decrease may be due to the chelating function of amino acids such as leucine and their derivatives [51]. As a result, Cu ions may not be transported through the roots to the aboveground parts. Thus, exogenous leucine also increased the root viability under Cu stress by reducing the accumulation of Cu in the roots and leaves, which in turn improved the root absorption ability, nutrient transportation, and photosynthetic performance, which finally promoted plant growth. Root damage can directly affect the growth and development of plant aboveground parts, in which biomass is usually used as a physiological index to detect the degree of the metal toxicity of plants. Huang et al. found that the biomass of white pomelo did not change significantly under the Cu concentration of 0.5–300 μmol/L, but when the Cu concentration reached 400 μmol/L, the biomass decreased significantly. Studies have shown that Cu stress inhibits plant growth [52]. In Abutilon theophrasti, Cu inhibited root tip cells [53]. Meanwhile, sublethal levels of Cu resulted in lipid peroxidation, which destroyed the membrane structure and affected the root physiological function of beans [54]. In this study, Cu stress significantly reduced the length, area, and volume of roots, the number of root tips, and the number of bifurcations and fibrous roots (Figure 5a, Supplementary Table S2). In addition, the fresh and dry weights of the aboveground and belowground plant parts were significantly reduced (Figure 4a). These changes are consistent with a reduction in root viability under Cu stress (Figure 5b–d).

Nitrogen metabolism is the primary source of protein and amino acids in plants [55]. Plants use a series of enzymes, such as NR, GS, and GOGAT, that are involved in nitrogen metabolism to absorb and efficiently utilize nitrogen [56]. NR is a rate-limiting enzyme in the nitrate assimilation pathway, and its activity is sensitive to H2O2 [57]. In this study, Cu stress significantly increased the H2O2 content of plants, which led to a decrease in NR enzyme activity. GS and GOGAT are key enzymes that convert inorganic nitrogen to organic nitrogen. The higher content of ammonium–nitrogen in peach seedlings under Cu stress may be related to the decrease in GS/GOGAT activities. The inhibition of the GS/GOGAT pathway hindered the assimilation of inorganic nitrogen into soluble protein [58]. However, a high ammonium–nitrogen content was harmful to peach growth. Cu stress also induced an increase in free amino acids, which may be related to autophagy and accelerated protein degradation [59]. In seedlings with exogenous leucine under Cu stress, the free leucine content increased significantly, probably from exogenous leucine. To maintain an amino acid balance, plants absorb and synthesize more of other amino acids, which further increases the total amount of free amino acids in the peach seedlings. Thus, in this study, leucine retained nitrogen metabolism efficiency in peach seedlings under Cu stress (Figure 6).

#### **5. Conclusions**

Exogenous leucine improved the leaf ultrastructure, ionic balance, and photosynthetic parameters. Furthermore, it improved the nitrogen metabolism efficiency of plants and attenuated Cu-stress-induced oxidative damage via a decrease in reactive oxygen species (ROS) and the regulation of the antioxidant and osmotic systems. These effects in turn ameliorated cell viability and biomass accumulation with improved resistance of peach seedlings to Cu stress. This study illustrates that leucine alleviates the damage caused by Cu stress in peach seedlings, which may provide more reference data for environmental risk assessments of Cu and make it possible to reuse soils with excessive copper contents caused by heavy metals.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/antiox11122455/s1, Table S1, Primer sequences, Figure S1, growth of 66-day-old peach seedlings under different amino acid treatments, Table S2, Effect of exogenous leucine on root architecture of peach seedlings under copper stress, Table S3, Effect of exogenous leucine on the copper ions content of peach seedlings under copper stress.

**Author Contributions:** M.S.: Conceptualization, Methodology, Software, Data curation, and Writing—original draft; S.L.: Visualization, Software, Validation, and Investigation; Q.G.: Software and Investigation; Y.X.: Methodology, Supervision, and Funding acquisition; F.P.: Conceptualization, Methodology, Software, Supervision, and Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Shandong Province Rural Revitalization Science and Technology Innovation Boost Action Plan Project (2021TZXD013) and the National Modern Agroindustry Technology Research System Fund (No. CARS-30-2-02).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data are contained within the article.

**Conflicts of Interest:** The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

#### **References**

