Physiological and Biochemical Properties of Cotton Seedlings in Response to Cu²⁺ Stress

Abstract

Copper(II) (Cu²⁺) is essential for plant growth and development. However, high concentrations are extremely toxic to plants. We investigated the tolerance mechanism of cotton under Cu²⁺ stress in a hybrid cotton variety (Zhongmian 63) and two parent lines with different Cu²⁺ concentrations (0, 0.2, 50, and 100 μM). The stem height, root length, and leaf area of cotton seedlings had decreased growth rates in response to increasing Cu²⁺ concentrations. Increasing Cu²⁺ concentration promoted Cu²⁺ accumulation in all three cotton genotypes’ roots, stems, and leaves. However, compared with the parent lines, the roots of Zhongmian 63 were richer in Cu²⁺ and had the least amount of Cu²⁺ transported to the shoots. Moreover, excess Cu²⁺ also induced changes in cellular redox homeostasis, causing accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). Conversely, antioxidant enzyme activity increased, while photosynthetic pigment content decreased. Our findings indicated that the hybrid cotton variety fared well under Cu²⁺ stress. This creates a theoretical foundation for the further analysis of the molecular mechanism of cotton resistance to copper and suggests the potential of the large-scale planting of Zhongmian 63 in copper-contaminated soils.

1. Introduction

Heavy metal contamination is a potential environmental issue worldwide due to increasing levels caused by both natural and manufactured activities [1]. Copper(II) (Cu²⁺) is an essential micronutrient for biological functions, as a constituent of the enzymes and proteins necessary for plant growth and development [2]. Research proved that 5–30 mg/kg Cu²⁺ is ideal for plant tissue growth [3,4]. When deficient, the visible symptoms include stunted growth, chlorosis of young leaves, losses in biomass and fruit yield, and ultimately the death of the plant [5,6]. However, Cu²⁺ in excess concentrations is also potentially harmful. Excess accumulation can destabilize membrane integrity, decrease photosynthesis, and alter enzyme activity, which result in growth inhibition and other detrimental effects on the primary production and survival of plants [7,8]. Moreover, Cu²⁺ disrupts key physiological processes and the metabolic functions of essential elements, thereby causing cellular redox imbalance and oxidative stress [9,10]. In addition, Cu²⁺ at higher concentrations causes the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are cytotoxic and impair important cell compounds [6,11]. In response to Cu²⁺ toxicity, plants use homeostatic mechanisms to circumvent heavy metal toxicity through metal exclusion, immobilization in the cell wall, metal compartmentation, and binding of heavy metal by strong ligands [12,13]. In response to ROS production, both enzymatic and non-enzymatic antioxidant scavengers such as peroxidase (POD), superoxide dismutase (SOD), catalases (CAT), glutathione (GHS), ascorbate (ASA), and proline are highly activated to combat the oxidative injuries caused by heavy metal toxicity [14]. GSH, for example, is thought to play multiple anti-metal toxicity roles by reducing metal uptake and chelating metal ions [15,16]. Proline was found to reduce harmful heavy metal toxicity by acting as a hydroxyl radical scavenger in the cytoplasm [17]. Furthermore, POD, SOD, and CAT were found in high concentrations in response to heavy metal scavenging [17,18,19]. Similarly, in response to heavy metal toxicity, sugars can function as ROS eliminators or cell signals in response to metal toxicity in plants [19].

In China, a recent report showed that heavy metal contamination in cultivated farmlands exceeds 20 million hm², and Cu²⁺ is a vital metal pollutant [20]. Cotton (Gossypium hirsutum L.) is a well-known economic crop that is grown all over the world, which is highly cultivated in farm areas in China [21]. Therefore, it faces a significant threat by Cu²⁺ toxicity [22]. Cu²⁺ toxicity and tolerance in plant species were extensively studied, including in Arabidopsis [23], fenugreek [24], and riparian plant species [25]. In cotton, foliar application of Cu²⁺ was found to significantly affect the lint percentage and fiber properties [26]. Although cotton is resistant to heavy metals and other abiotic stresses, the non-edible characteristics of cotton fiber give it the ability to restore soil quality when grown in heavily polluted areas [20]. However, no comprehensive study has been conducted on the specific toxic effects and responses to Cu²⁺ of cotton seedlings. As a result, the current study aimed to identify Cu²⁺ toxicity syndrome in two parental lines and their hybrid variety as well as further investigate the mechanisms of Cu²⁺ tolerance in cotton. Primarily, we observed the physiological and biochemical properties of cotton seedling responses to Cu²⁺ stress among two cotton parental lines (9053 and sGK9708) and their hybrid variety (Zhongmian 63). To achieve the purpose of this study, the cotton seedlings were exposed to CuSO₄ at different concentrations to find the physio-molecular changes that occurred in the different genotypes. In addition, biomolecules, including POD and GHS, and ROS accumulation were assessed in the cotton seedlings in response to Cu²⁺ toxicity.

2. Materials and Methods

2.1. Plant Materials, Growth Condition, and Treatments

The glasshouse experiment was conducted using a conventional upland cotton line “9053” as the maternal parent, a new insect-resistant cotton variety improved line “sGK970” as the male parent, and their hybrid variety, Zhongmian 63, which is a bivalent transgenic insect-resistant cotton hybrid F1 generation and is planted in a large area of Yangtze River Basin, especially in Anhui Province and Hubei Province. Uniformly sized seeds were surface sterilized with a 1% (v/v) sodium hypochlorite solution for 20 min, washed with distilled water three times, and left for water imbibition for 24 h before growth in a sand culture medium. Seed germination occurred for 5 days at 28 °C in the dark. On the 6th day, uniform seedlings were transferred to hydroponic media and allowed to acclimatize for 10 days. The nutrient solution composition was 20 µM H₃BO₃, 1 µM ZnSO₄•⸳7H₂O, 0.2 µM CuSO₄⸳5H₂0, 1 µM MnSO₄⸳H₂O, 0.005 µM (NH₄)₆MO₇O₂₄⸳4H₂O, 2498.41 µM Ca(NO₃)₂⸳4H₂O, 499.7 µM KH₂PO₄, 1000.12 µM MgSO₄⸳7H₂O, 2504.70 µM KNO₃, and 100 µM Na Fe⸳EDTA at 22 ℃/27 ℃ during 8/16 h dark/light periods, respectively, with a light intensity of 180 μmol⸳m⁻²⸳s⁻¹ and 60% relative humidity. After 15 days, equally sized cotton seedlings were transferred into the nutrient solution with different Cu²⁺ concentrations. The treatment concentrations were composed of 0 (without addition of Cu²⁺), 0.2 (control group), 50, 100, and 200 μM Cu²⁺, supplied as CuSO₄⸳5H₂O. The nutrient solutions were continuously renewed every 4 days, and then the pH value was modified to 5.6–5.7 with 0.1 M HCl or 0.1 M NaOH. Plants were harvested after 10 days of Cu²⁺ treatment for future analysis.

2.2. Determination of Growth Parameters

Growth parameters of the cotton seedlings were evaluated by determining leaf area, root length, and shoot length. The leaf area of seedlings was determined by the area-weighing method, and the lengths of roots and shoots were recorded using a meter ruler.

2.3. Measurement of Photosynthetic Pigment Contents

Chlorophyll and carotenoid concentrations were determined according to Lichtenthhaler et al., 1983 [27], with a few modifications. Briefly, the top-most expanded leaves were randomly cut and soaked in 80% acetone in a ratio of 1:10 w/v until the pigments were completely extracted and the leaf became colorless. The process was performed in darkness. The extracts were centrifuged for 15 min at 4000× g to remove any residues. The supernatant was measured at 663, 645, and 470 nm using a visible spectrophotometer v-1000 (AoYi Instrument Co., Ltd., Shanghai, China) for chlorophyll a, chlorophyll b, and carotenoids, respectively, and 80% acetone was used as a blank control.

2.4. Determination of Copper Content

For Cu²⁺ content analysis, the root and leaf samples were harvested separately, rinsed with tap water, and immersed in 20 mM Na₂-EDTA for 15 min to remove any trace elements adhering to the tissue. The root and leaf samples were oven-dried at 75 °C for 48 h. Dried samples (0.1 g) were ground and acid-digested with an HNO₃ mixture for 24 h at 80 °C, followed by Cu²⁺ estimation using an atomic absorption spectrophotometer (Perkin Elmer-model 2380, C.R.G.R.) [28].

2.5. Detection of H₂O₂ and MDA Levels

The accumulation of H₂O₂ in leaves was measured by monitoring the A415 of the titanium–peroxide complex, as previously described by Liu et al., 2018 [29], with some modifications. Absorbance values were calibrated to a standard curve generated with known concentrations of H₂O₂. Recovery was checked by adding various amounts of H₂O₂ to the leaf extracts as an internal standard. The level of lipid peroxidation was determined according to Thounaojam et al., 2012 [2]. Fresh leaves (0.2 g) were homogenized with 5 mL of 0.25% TBA. The homogenate was boiled for 30 min at 95 °C and centrifuged at 10,000× g for 10 min. The absorbance of the supernatant was recorded at 532 nm and 600 nm using a visible spectrophotometer 722N (Yidan Analytical Instrument Co., Ltd., Shanhai, China) with an extinction coefficient of 155 mM⁻¹·cm⁻¹.

2.6. Measurement of POD Activity and GSH Content

Frozen leaves were homogenized and centrifuged, and then the supernatant was immediately used for the antioxidant enzyme assays. The POD activity was measured by guaiacol oxidation prior to carrying out the method described by Zhang et al., 2016 [30]. To determine the GSH content, samples (0.5 g) were crashed with 5 mL of 10% (w/v) TCA, and the homogenate was centrifuged at 15,000× g for 15 min at 4 °C. The GSH content was determined according to the method previously described by Ates et al., 2009 [31].

2.7. Determination of Soluble Sugar Content

Soluble sugar content was conducted similarly to the method previously explained by Anjorin et al., 2016 [32], with a few modifications. Fresh leaves (0.5 g) were homogenized in 80% ethanol and then incubated at 75 °C for 10 min. Next, 40 mL of the supernatant were mixed with 80% carbolic acid and 4 mL of concentrated sulfuric acid, and then the absorbance was recorded at 490 nm using a visible spectrophotometer 722N (Yidan Analytical Instrument Co., Ltd., Shanhai, China). The concentration of soluble sugar was determined by a calibration curve prepared from a sucrose solution and was expressed as mg·g⁻¹ FW.

2.8. Statistical Analysis

All statistical analyses were performed using the SPSS 21.0 computer software package. Data for each treatment were expressed as mean values ± S.D., with three replicates. Differences among the groups were examined by one-way ANOVA followed by LSD. p < 0.05 was considered statistically significant.

3. Results

3.1. Plant Growth Conditions under Cu²⁺ Stress

The effect of the different Cu²⁺ concentrations on the growth of cotton seedlings is presented in Figure 1. The average data based on plant growth showed that the parental line sGK9708 and the hybrid variety Zhongmian 63 were more tolerant to Cu²⁺ than the maternal genotype 9053. Although the toxicity of Cu²⁺ greatly inhibited the growth of the cotton seedlings, the plant height and leaf area of the three cotton genotypes showed strong tolerance when the Cu²⁺ concentration was 50 μM, compared with the control group (Figure 1A,B), but the growth of the roots was inhibited (Figure 1C). This suggested that the roots are more vulnerable to Cu²⁺ stress than the shoots.

3.2. Content of Photosynthetic Pigments

The toxicity of Cu²⁺ severely reduced the content of chlorophyll a and chlorophyll b in the cotton seedling leaves (

Table 1. Effect of Cu²⁺ treatments on the contents of chlorophyll and carotenoid in cotton seedlings.

Cotton Variety	Cu2+ Concentration (μM)	Chlorophyll a (mg/g FW)	Chlorophyll b (mg/g FW)	Chlorophyll (a + b) (mg/g FW)	Chlorophyll a/b	Carotenoid (mg/g FW)
9053	0	0.28 ± 0.01 bcd	0.08 ± 0.01 b	0.36 ± 0.02 bcd	3.36 ± 0.14 c	0.051 ± 0.003 bc
	0.2	0.33 ± 0.05 cd	0.09 ± 0.02 bc	0.43 ± 0.06 cd	3.52 ± 0.09 c	0.048 ± 0.006 ab
	50	0.30 ± 0.04 cd	0.09 ± 0.02 bc	0.39 ± 0.06 cd	3.12 ± 0.09 b	0.045 ± 0.004 ab
	100	0.27 ± 0.03 bcd	0.08 ± 0.01 b	0.35 ± 0.04 bcd	3.26 ± 0.06 bc	0.049 ± 0.007 abc
	200	0.11 ± 0.01 a	0.04 ± 0.01 a	0.16 ± 0.01 a	2.64 ± 0.21 a	0.049 ± 0.004 abc
Zhongmian 63	0	0.24 ± 0.02 bcd	0.08 ± 0.01 b	0.31 ± 0.02 bcd	3.06 ± 0.20 c	0.044 ± 0.003 ab
	0.2	0.25 ± 0.04 bc	0.08 ± 0.02 b	0.32 ± 0.05 bc	3.06 ± 0.11 b	0.038 ± 0.004 a
	50	0.35 ± 0.06 d	0.11 ± 0.02 c	0.46 ± 0.08 d	3.15 ± 0.03 b	0.050 ± 0.005 bc
	100	0.28 ± 0.10 cd	0.09 ± 0.03 bc	0.37 ± 0.13 bcd	3.11 ± 0.10 b	0.048 ± 0.016 abc
	200	0.19 ± 0.02 ab	0.07 ± 0.01 b	0.26 ± 0.03 ab	2.67 ± 0.07 a	0.055 ± 0.006 bc
sGK9708
	0	0.30 ± 0.03 cd	0.10 ± 0.02 bc	0.40 ± 0.04 cd	3.09 ± 0.17 bc	0.050 ± 0.003 bc
	0.2	0.32 ± 0.08 cd	0.09 ± 0.03 bc	0.42 ± 0.11 cd	3.37 ± 0.03 bc	0.044 ± 0.005 ab
	50	0.34 ± 0.06 cd	0.10 ± 0.02 bc	0.43 ± 0.08 cd	3.39 ± 0.13 bc	0.068 ± 0.004 abc
	100	0.29 ± 0.03 cd	0.09 ± 0.01 bc	0.38 ± 0.04 bcd	3.13 ± 0.09 b	0.049 ± 0.003 abc
	200	0.17 ± 0.08 a	0.07 ± 0.02 b	0.24 ± 0.1 ab	2.34 ± 0.4 a	0.058 ± 0.007 c

Each column represents the mean ± SD. Different lowercase letters in the same column represent the significant differences at (p < 0.05, Tukey’s multiple comparison).

). In the absence of Cu²⁺, the chlorophyll content of the three genotypes of cotton seedlings decreased compared with that of the control group. When Cu²⁺ was in excess, the chlorophyll content of the three genotypes of cotton seedlings also decreased compared to that of the control group. On the contrary, the chlorophyll contents of sGK9708 and Zhongmian 63 temporarily increased when the Cu²⁺ concentration was 50 μM, significantly so for Zhongmian 63, but subsequently decreased as the Cu²⁺ concentration increased. The ratio of chlorophyll-a content to chlorophyll-b content tended to reduce with increasing Cu²⁺ concentrations in the nutrient solution. This indicated that the content of chlorophyll-a decrease was higher than that of chlorophyll-b.

3.3. Determination of Cu²⁺ Content

Under Cu²⁺ stress, different cotton varieties exhibited differences in Cu²⁺ uptake and transport (

Table 2. The Cu²⁺ contents in the leaf, stem, and root of cotton under different Cu²⁺ concentrations.

Cotton Variety	Cu2+ Concentration (μM)	Leaf (μg/g DW)	Stem (μg/g DW)	Root (μg/g DW)
9053	0	8.67 ± 0.85 a	4.43 ± 0.70 a°	20.27 ± 1.46 a
	0.2	10.40 ± 1.20 a	5.33 ± 0.35 a	22.20 ± 2.00 a
	50	28.95 ± 3.13 b	8.70 ± 1.45 bc	121.67 ± 5.8 b
	100	46.27 ± 4.76 c	15.87 ± 2.06 e	262.87 ± 23.1 de
	200	75.54 ± 11.40 e	25.37 ± 2.80 g	386.30 ± 31.96 f
Zhongmian 63	0	7.60 ± 0.36 a	4.47 ± 0.70 a	20.47 ± 1.74 a
	0.2	8.83 ± 0.81 a	5.43 ± 0.59 a	24.77 ± 3.32 a
	50	23.79 ± 2.36 b	9.03 ± 0.83 c	158.63 ± 10.92 bc
	100	39.43 ± 5.80 c	13.60 ± 1.55 de	286.20 ± 17.76 de
	200	65.07 ± 4.73 d	20.57 ± 2.41 f	489.20 ± 32.03 g
sGK9708	0	8.50 ± 0.70 a	5.63 ± 0.45 ab	18.57 ± 1.12 a
	0.2	9.47 ± 1.20 a	6.30 ± 0.50 abc	20.07 ± 2.14 a
	50	30.27 ± 1.99 b	12.40 ± 2.05 d	139.83 ± 10.63 bc
	100	43.53 ± 3.56 c	20.27 ± 1.96 f	253.00 ± 10.50 d
	200	76.97 ± 8.06 e	33.90 ± 3.60 h	475.73 ± 24.83 g

Each column represents the mean ± SD. Different lowercase letters in the same column represent the significant differences at (p < 0.05, Tukey’s multiple comparison).

). Although the absorption of Cu²⁺ was different, the trends of the Cu²⁺ accumulation in different tissues of all three varieties were similar. Under different Cu²⁺ treatments, the Cu²⁺ concentrations were highest in the roots, then in the leaves, and finally in the stems. However, the highest accumulation of Cu²⁺ was found in Zhongmian 63, followed by that of sGK9708 and 9053 (Table 2). In comparison to the parent lines, the quantity of Cu²⁺ transport from the roots to the shoots was lower in Zhongmian 63. This indicated that Zhongmian 63 is more capable of enriching Cu²⁺ in the roots, effectively reducing the toxicity of Cu²⁺ to the cotton seedlings.

3.4. Determination of Morphological Characters and Soluble Sugar Content

Morphological changes, including stem height, root height, and leaf area, were measured in response to different Cu²⁺ concentrations. As shown in Figure 2, cotton stem and root height increased in all three genotypes at 0, 0.2, and 50 µM but decreased as Cu²⁺ concentration increased from 100 to 200 µM (Figure 2A,C). The cotton leaf area responses to Cu²⁺ showed a similar trend, and the 9053 variety showed an extreme decrease in leaf area at 100 and 200 µm (Figure 2B). Regarding the effects of different concentrations of Cu²⁺ on the soluble sugar content of all three cotton genotypes, the results showed that the soluble sugar content in leaves reached its maximum at a 50 μM Cu²⁺ concentration and then decreased with further increases in the Cu²⁺ concentration. At Cu²⁺ concentrations of 100 μM and 200 μM, the soluble sugar content of Zhongmian 63 was significantly higher than that of the parent lines (Figure 2D).

3.5. Determination of H₂O₂ and MDA Content and Antioxidant Scavenging Ability

The leaf H₂O₂ and MDA content of the three genotypes increased with an increasing Cu²⁺ concentration (Figure 3). At a Cu²⁺ concentration of 200 μM, the H₂O₂ and MDA in the three cotton genotypes reached the highest levels. However, compared with the parent lines, the H₂O₂ and MDA content in the Zhongmian 63 leaves increased slowly with increasing Cu²⁺ concentrations (Figure 3A,B). This indicated that Zhongmian 63 has a substantial ability to scavenge ROS compounds. The leaf GSH content of the three genotypes increased with increasing Cu²⁺ concentrations (Figure 4A). When Cu²⁺ was excessive, the GSH content increased with the increase in Cu²⁺ concentration in the three cotton genotypes. At the Cu²⁺ concentration of 200 μM, the GSH content in the leaves of the three cotton genotypes also reached the highest levels. However, under the conditions of different Cu²⁺ concentration treatments, the GSH content in the Zhongmian 63 leaves was significantly higher than that of the parent lines. Generally, regarding Cu²⁺, POD activity in the leaves of the cotton seedlings first increased and then decreased (Figure 4B). In 9053, POD activity in the leaves of seedlings peaked at a Cu²⁺ concentration of 50 μM and then declined significantly. Similarly, at Cu²⁺ concentrations of 100 μM, sGK9708 and Zhongmian 63 had extremely high POD scavenging activities, with higher expression in Zhongmian 63. Lastly, POD activity decreased significantly when the Cu²⁺ concentration reached 200 μM.

4. Discussion

4.1. Morpho-Physiological Changes and Copper Absorption

In recent decades, increasing modern agricultural practices, anthropogenic activities, rapid industrialization, rapid urbanization, and burning of fossils fuels have amplified the threshold level of various heavy metals in the plant–soil complex, especially in aquatic environments, causing negative impacts on the living forms [33,34]. Much land under plant cultivation faces heavy metal contamination because of the unlimited and excessive usage of chemical agriculture products (chemical manures, pesticides, fertilizers, weedicides, and herbicides), mining, urban and rural municipal solid waste, metalliferous mines, energy and fuel production, mining, the mismanagement of agribusiness wastewater, power transmission, and industrial sources, resulting in heavy metal release [35,36,37]. Over the last 1000 years, the increased use of Cu²⁺ as a pesticide has become one of the world’s major environmental problems. According to previous studies, the management of Cu²⁺ as a fungicide has always been a part of agriculture [25]. However, Cu²⁺ does not degrade as quickly as it accumulates in soil, which causes elevated levels that can reach up to 3000 mg kg⁻¹in agricultural areas [38]. Yang et al., 2015 [39], proposed that Cu²⁺ toxicity largely affects root growth and morphology in rice and maize and tends to accumulate in the root tissue, which can be transferred to the shoots. Studies on the effects of heavy metal pollutants on the physiological and biochemical characteristics of plants and remedial techniques have been well-documented. For instance, Bouazizi et al. 2010 showed that CuSO₄ causes a reduction in biomass and leaf number in Phaseolus vulgaris. Ref. [28] showed that CuSO4 reduces the biomass and leaf number in Phaseolus vulgari. Moreover, existing research shows that CuSO4 toxicity reduces Cu²⁺ growth rates and necrosis as well as stunted growth in woody ornamentals [25,40]. Further studies proved that Cu²⁺ accumulation also affects plant morphology by lowering the stem height of rice seedlings and the chlorophyll content of cotton roots [2,20]. In the present study, a decrease in cotton morphological growth under an increasing level of Cu²⁺ indicated a Cu²⁺ toxicity effect at elevated concentrations on cotton roots, leaves, and stems (Figure 2A–C). The hybrid genotype, Zhongmian 63, was more tolerant to Cu²⁺ than the parent lines (9053 and sGK9708). We hypothesized that growth retardation might be due to a significant reduction in oxidative stress and probably the photosynthetic pigments in the leaves. Chlorophyll is considered an important biomarker of abiotic stress, including from heavy metals [41]. The existing research proved that excess Cu²⁺ is capable of disrupting the chloroplast and thylakoid membrane composition, triggers oxidative stress in plant cells, and reduces stomatal conductance and declining photosynthetic gas exchange and chlorophyll fluorescence parameters [42,43]. In addition, Cu²⁺ concentrations significantly affect the expression levels of the chlorophyll-a and carotenoid content of the phytoplankton Nitzschia sp. [44]. Furthermore, other previous studies demonstrated that elevated Cu²⁺ concentrations not only cause chlorosis, which reduces plant biomass, but also interfere with the electron transport chain system by saturating the protein pigment plastocyanin, which is involved in photosynthesis [34]. Similarly, in arable crops, such as rice and tomatoes, higher Cu²⁺ concentrations cause chlorophyll reduction [45,46]. In Urtica dioica and Fallopia japonica, the chlorophyll content slightly increased with increasing Cu²⁺ [25]. A recent report on lentil plants showed a significantly reduced chloroplast pigment composition, including chl a and chl b, along with carotenoids, at high Cu²⁺ concentrations (3.0 mM) [47]. Similarly, in the present investigation, the chlorophyll content in the leaves of cotton seedlings decreased with increasing Cu²⁺ concentration (Table 1). However, the carotenoid content increased under Cu²⁺ stress conditions compared to that of the control group (Table 1). From previous studies, carotenoids are known to increase under Cu²⁺ toxicity and act as biological indicators of heavy metal effects in cyanobacteria [48]. We speculate that the decrease in chlorophyll content and increase in carotenoid content in cotton genotypes may be a signal of Cu²⁺ toxicity. Heavy metal toxicity in plants may result from the increasing contents in the soil, which directly affects the quality and safety of agricultural products [49]. Cotton is well-known for alleviating poor soil conditions by adsorbing polluting heavy metals, such as Cu²⁺, Ag, Au and Pb, in farmland [20]. Nevertheless, reproductive processes are greatly inhibited at higher concentrations [50]. According to previous studies, high concentrations of Cu²⁺ lead to plant root toxicity, causing root stunting, which directly interferes with the root’s capacity to absorb the nutrients delivered by diffusion [51]. In the present study, cotton roots had the highest level of Cu²⁺ absorption, followed by leaves and stems (Table 2). In addition, at a CuSO₄ level of 200 µM, cotton roots were stunted in all three genotypes. We hypothesized that root stagnation development in cotton roots could be the result of a high accumulation of Cu²⁺ (Figure 1).

4.2. ROS Accumulation Levels and Peroxidation

The production of ROS is a common phenomenon of stress, and ROS attack polyunsaturated fatty acids and lead to lipid peroxidation [52]. Chronically overexposing plants to copper increases the production of reactive oxygen species, causing the accumulation of different ROS such as superoxide (O₂•−), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•HO), which disrupt redox homeostasis and cause oxidative damage to cells at the lipid, protein, and nucleic acid levels [51,53,54]. Many studies showed that heavy metal toxicity causes significant accumulation of H₂O₂ and MDA [45,55]. For instance, in wheat, long-term exposure to a high level of CuSO₄ (75 µM) influenced ROS generation [51]. Moreover, in M. sativa seedlings, excess Cu²⁺ increased ROS production by destroying the H₂O₂ scavenging system [29,56]. Similarly, in fenugreek, Cu²⁺ toxicity induced an increase in the rate of hydrogen peroxide production and lipid peroxidation, displaying oxidative stress [24]. MDA concentrations in Glycine max and Lupinus albus L. were reported to increase in root nodules when exposed to a high level of CuSO₄ (192 µM) [57]. In this study, increasing the CuSO₄ concentration in the culture medium led to an increase in H₂O₂ and MDA in the leaves of cotton seedlings at values of 100 and 200 µM in all the cotton genotypes, with higher levels in the 9053 and sGK9708 varieties (Figure 3). We hypothesized that high concentrations of ROS and MDA may be a signal of oxidative stress activation, enzymatic inactivation, and lipid peroxidation [58].

4.3. Antioxidant Alleviation and Soluble Sugar Defensive Mechanisms

At the cellular level, plants protect their cells from oxidative stress through a range of biochemical mechanisms (i.e., enzymatic and non-enzymatic antioxidants) [59]. Plant enzymatic defenses comprise antioxidant enzymes (catalase, ascorbate peroxidase (APX), phenol peroxidase (POX), glutathione peroxidase (GPX), and superoxide dismutase), together with non-enzymatic substances (proline, GHS, and ascorbate (ASA)), which promote the scavenging of ROS [20,34,60]. According to previous reports, high Cu²⁺ levels induce the activities of antioxidant enzymes (SOD, CAT, and POD) as well as non-enzymatic antioxidants (GSH) [51]. In the present study, both enzymatic and non-enzymatic defense mechanisms were stimulated in response to increased Cu²⁺ levels (Figure 4). POD plays a major role in the H₂O₂ scavenging enzymes that remove H₂O₂ from chloroplasts and the cytosol of plant cells [61]. The role of POD scavenging in plants against heavy metal toxicity was proven to increase and decrease as metal toxicity increases [56,62]. The enhancement of peroxidase (POD) activities was reported in the roots and leaves of cotton genotypes exposed to Cu²⁺ (100 mg kg⁻¹) [22]. Application of Cu²⁺ in Linum usitatissimum resulted into SOD and POD activities’ enhancement in relation to increasing Cu²⁺ concentrations [17]. Similarly, in this study, POD antioxidant activity first increased at 0, 0.2, 50, and 100 µM but then significantly declined at 200 µM. We speculate that the POD fluctuations caused by Cu²⁺ concentrations might be a biomarker for heavy metals or lipid peroxidation [63]. GSH acts as an antioxidant and detoxifies H₂O₂ via the ascorbate–glutathione cycle. Most importantly, it acts as a precursor of phytochelatins (PCs) by chelating Cu²⁺ and participating in detoxification and tolerance [20]. Wang et al., 2023 [23], proposed that Cu2⁺-induced chlorosis in Arabidopsis seedlings could be mitigated by the exogenous application of glutathione (GSH). Cu²⁺-induced elevation in GSH content was observed in rice [2]. In addition, the toxicity of cadmium (Cd) and other heavy metal ions is primarily reduced by PC complexation activity [19,64]. In this work, Cu²⁺ levels of 50, 100, and 200 µM dramatically increased the GSH content in Zhongmian 63 (Figure 4A). We hypothesized that GHS may play a key role in metal chelation and removal under the condition of excess Cu²⁺ content in cotton seedlings [13]. Soluble sugars were declared to take part in the control of photosynthetic activity, sensing, and the control of ROS balance [65]. Li et al., 2020 [20], explained that plant cells usually accumulate soluble sugars to diminish intracellular osmotic potential and guarantee the normal supply of water under heavy metal stress, in order to maintain normal physiological functioning. In the present work, the content of soluble sugar in the leaves of cotton seedling first increased and then decreased with the increase in the Cu²⁺ concentration (Figure 2D). We speculated that a lower Cu²⁺ concentration accelerates the decomposition of high-density carbohydrates in plants and inhibits their synthesis. Therefore, photosynthetic products directly form low molecular mass substances, such as sucrose, resulting in soluble sugar accumulation. On the contrary, at high Cu²⁺ concentrations, the anabolism and growth of plants were repressed, and the photosynthetic capacity of the plants reduced, resulting in a decrease in soluble sugar content. Hence, Cu²⁺ toxicity significantly inhibits soluble sugar content, affecting cotton tolerance.

Heavy metals arise from many sources, such as industry, mining, and agriculture. However, sources in the agricultural sector can be categorized into fertilization, pesticides, livestock manure, and wastewater [66]. Heavy metal pollution in the environment has been rapidly expanding and causing havoc, particularly in the agricultural sector, by accumulating in soil and plant uptake [67]. Cu²⁺ accumulation can be useful for plant growth and development but is extremely toxic to plant seedlings. Previous studies showed that Cu²⁺ toxicity has a less significant effect on maize [68]. Nonetheless, Cu²⁺ in higher contents had a damaging impact on the growth as well as root morphology of plants [11]. Cotton can withstand adverse environmental conditions in several phases and through phytoremediation [69]. However, seedlings may have a different response to heavy metal toxicity. As observed in this study, the morphological effect of Cu²⁺ shows no significant changes in plant leaves; however, at extreme concentrations, the cotton root morphology exhibits a declination in growth. We argue that plants may have specific threshold points for metal toxicity, but levels above the desired threshold may be detrimental to growth and development. This depicts the notion that the agricultural usage of heavy metals such as Cu²⁺ may be useful for plants, but excessive usage resulting in high soil accumulation may cause toxicity thresholds above normal, which may affect plants at tender growth stages. Hence, we recommend that a deficiency of Cu²⁺ in cotton seedlings may be solved by applying Cu²⁺-encompassing fertilizers to the soil, but high Cu²⁺ levels can be managed by reducing Cu²⁺-related fertilizer application during the seedling’s growth stages [70]. We further suggest cotton seedlings be cultivated in a controlled environment (hydroponic culture), which may also be effective for cotton growth, since varying soil copper concentrations may be monitored and adjusted until fully mature to withstand adverse environmental conditions before being exposed to the outside environment.

In summary, this study demonstrated that an excessive Cu²⁺ concentration severely restricts the root, shoot, leaf, and photosynthetic parameters of cotton seedlings. However, the cotton seedlings showed strong tolerance to a range of Cu²⁺ concentrations. Higher levels of Cu²⁺-induced oxidative stress increased the H₂O₂ production in cotton seedlings. On the other hand, when compared with the parent line, the hybrid lines effectively increased their tolerance to Cu²⁺ by increasing GSH content, POD activity, and soluble sugar content and reducing the transfer of Cu²⁺ to the ground.