**1. Introduction**

Heavy metal pollution of farmland soil is one of the most severe environmental problems in the world [1]. Globally, there are 5 million sites of soil pollution, covering 500 million ha of land in which the soils are contaminated by different heavy metals or metalloids [2]. Heavy metal pollution in soil has a combined worldwide economic impact that is estimated to be in excess of USD 10 billion per year [3]. Heavy metal pollution not only decreases crop yield and quality but also affects human health via the food chain [4]. However, certain heavy metals, such as Cu at lower concentrations, are critical for plant growth and development. Cu is an essential trace element involved in many physiological processes of plants. It acts as a cofactor of enzymes such as the Cu/Znsuperoxide dismutase (Cu/Zn SOD) enzyme and polyphenol oxidase. It is involved in physiological processes such as photosynthesis, cell wall metabolism, and ethylene perception [5]. Cu is a component of the plastocyanin in plant chloroplasts, which is involved in the photosynthetic electron transport process and is also an activator of certain enzymes during chlorophyll formation [6]. Cu deficiency in plants can hinder normal growth, while excess Cu can be toxic. At higher concentrations, Cu ions (Cu2+) combine with oxygen to produce free radicals and other reactive oxygen species (ROS), which induce

**Citation:** Sun, M.; Li, S.; Gong, Q.; Xiao, Y.; Peng, F. Leucine Contributes to Copper Stress Tolerance in Peach (*Prunus persica*) Seedlings by Enhancing Photosynthesis and the Antioxidant Defense System. *Antioxidants* **2022**, *11*, 2455. https:// doi.org/10.3390/antiox11122455

Academic Editor: Nafees A. Khan

Received: 14 November 2022 Accepted: 9 December 2022 Published: 13 December 2022

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oxidative stress and lead to cell death. Moreover, the accumulation of Cu in the food chain can lead to poisoning in humans due to excessive consumption [7]. In recent years, Cu has become a major environmental pollutant due to overuse in the manufacturing and agricultural industries [8]. Therefore, in order to reduce plant damage due to Cu stress in soil, it is necessary to improve plant tolerance to Cu stress and reduce Cu ion enrichment under Cu stress.

Under normal conditions, the production and removal of reactive oxygen species (ROS) in plants are in dynamic balance. Oxidative stress occurs when the accumulated concentration of ROS exceeds the threshold of plant defense mechanisms under heavy metal stress [9]. Cu stress can induce cells to produce a large amount of ROS, leading to lipid peroxidation of the cell membrane and resulting in an increase in malondialdehyde (MDA) content, a reduction in the selective permeability of the plasma membrane, the leaching of intracellular materials, and then damage to photosynthetic organelles, thereby affecting the normal operation of physiological metabolic processes such as material exchange and photosynthesis [10]. ROS can change the distribution of ions and initiate the expression of nuclear genes in the process of transmitting and amplifying signals so that plants can tolerate various stresses [11]. Amino acids enhance the adaptive responses in plants to various stresses by directly/indirectly influencing the physiological processes or regulating stress-related gene expression and enzyme activities in plants [12]. In plants, leucine is not only a nutrient matrix for cell metabolism but also a signal molecule that can mediate protein metabolism, lipid decomposition, and other biological reactions. Leucine spray at different stages increased biomass and nitrogen content, promoted nitrogen absorption, and improved grain yield in rice [13].

Peach (*Prunus persica* L. Batsch) is one of the most widely cultivated fruit trees in China [14]. Previous studies have found that excessive copper in peach orchard soil can reduce the dry matter content of peach trees, reduce the photosynthetic rate of peach leaves, and reduce the chlorophyll content of leaves, which are not conducive to fruit yield and quality [15]. Therefore, it is crucial to reduce the Cu content in plant parts and alleviate the impact of Cu stress to improve the yield and quality of peach. Several studies have reported the beneficial effects of amino acids in plants exposed to metal stress [16]. In general, amino acid molecules have three major functions in response to heavy metal stress, namely metal binding, antioxidant defense, and signaling [17]. Due to their ability to bind metals, amino acids and their by-products can be used to respond to metal toxicity. Amino acids facilitate the chelation of heavy metal ions in cells and xylem sap, thereby resulting in the detoxification of heavy metals and an increase in plant resistance to toxic metal ions by activating antioxidant systems [18]. However, the role of leucine in the regulation of heavy metal stress is currently unclear. Therefore, in this study, peach seedlings were used as experimental materials to explore the mechanism of leucine alleviating Cu stress in peach seedlings.

#### **2. Materials and Methods**

#### *2.1. Experimental Design*

Experiments were performed in the experiment center of Shandong Agricultural University (Tai'an, China) in April 2020 (117◦13 E and 36◦16 N). First, 60-day-old '*lu xing*' peach plants, *Prunus persica* (L.) Batsch., were planted in pots. Seedlings (10 cm tall) grown from peach seeds with the same growth trend and without diseases or insect pests were selected and planted. The pots were cylindrical with an inner diameter of 20 cm and a height of 30 cm. Approximately 2.5 kg of garden soil was placed in each pot, and each treatment was repeated in 60 pots (replicates). The basic physical and chemical properties of the tested soil were as follows: the pH value was 6.68, the alkaline hydrolyzable nitrogen content was 45.65 mg·kg−1, the organic matter content was 12.57 g·kg<sup>−</sup>1, the available phosphorus content was 35.33 mg·kg−1, and the available potassium content was 83.72 mg·kg<sup>−</sup>1. The growing conditions for the peach trees were day and night temperatures of 29 ◦C/23 ◦C, a natural photoperiod of around 12.5 h, and a constant relative

humidity of 30%. The seedlings were maintained following conventional management practices. In the preliminary Cu stress screening test (Supplementary File, Figure S1), we found that Cu-induced damage to the peach seedlings began to appear after their treatment with 5 mmol·L−<sup>1</sup> CuCl2·2H2O for 6 days, and leucine (10 mmol·L−1) significantly alleviated Cu stress for 6 days, which met the test requirements. Therefore, in this experiment, the concentrations of 5 mmol·L−<sup>1</sup> for CuCl2 ·2H2O and 10 mmol·L−<sup>1</sup> for leucine were chosen. The seedlings were treated as follows: water (control), 10 mmol·L−<sup>1</sup> leucine (Leu, Shanghai Yuan ye Biotechnology Co., Ltd., Shanghai, China), 5 mmol·L−<sup>1</sup> CuCl2·2H2O (Cu), and 5 mmol·L−<sup>1</sup> CuCl2·2H2O + 10 mmol·L−<sup>1</sup> leucine (Cu+Leu). In this experiment, CuCl2·2H2O and leucine were uniformly applied to the soil one time at a reagent dosage of 200 mL per tree. All treatments were applied in soil pots at 9 am to avoid excessively high temperatures. On the sixth day after treatment, image indicators were tested with fresh samples, and for the other indicators samples were frozen in liquid nitrogen and placed in an ultra low temperature refrigerator at −80 ◦C for further testing. Three biological repetitions were included per treatment.

## *2.2. Determination of Endogenous Leucine Content*

On the sixth day after treatment, the leaves, stems, and roots were dried and sifted through a 100-mesh sieve. Approximately 0.5 g of the powdered sample was accurately weighed, placed in a 10 mL centrifuge tube, and mixed with 10 mL of ultrapure water. This mixture was ultrasonicated for 30 min and centrifuged at 10,000 rpm for 2 min. The pH of the supernatant was adjusted from 5.3 to 2.2 with 1 mol/L hydrochloric acid. The resultant sample was used to determine the leucine content with an automatic amino acid analyzer (Biochrom, Cambridge, UK) [19].

#### *2.3. Determination of SPAD Value, Photosynthetic Rate, and Maximum Photochemical Efficiency*

In order to observe the dynamic changes in the physiological indexes of peach seedlings after the treatments, we chose to measure them on the 2nd, 4th, and 6th days after treatment. Chlorophyll fluorescence was measured using an Imaging-PAM chlorophyll fluorometer (HeinzWalz GmbH, Effeltrich, Germany). Before the determination of Fo, the plants were dark-adapted for 30 min. Then, the leaves were adapted to actinic light (250 mmol/m2/s), and the maximum fluorescence (Fm) and steady-state fluorescence (Fs) under actinic light were measured with a saturated pulse (3000 mmol/m2/s). The variable fluorescence/fluorescence maximum (Fv/Fm), the quantum efficiency of PSII (φPSII), the photochemical quenching coefficient (qP), and the non-photochemical quenching coefficient (NPQ) were automatically calculated by the software.

The fifth and sixth uppermost leaves from the top of the plant were selected and fully unfolded. The SPAD values were measured using a chlorophyll tester (SPAD-502 plus, Tokyo, Japan), and the photosynthetic rate was measured using a ciras-3 portable photosynthetic instrument (PP Systems, Hitchin, UK). On the sixth day after treatment, the maximum photochemical efficiency (Fv/Fm) was measured using a portable pulse adjustment fluorometer (Handy PEA, Hansatech, UK). Two leaves per plant and three plants per treatment were used.
