Effects of Ni and Cu Stresses on Morphological and Physiological Characteristics of Euphorbia marginata Pursh Seedlings

Abstract

Increasing soil contamination with nickel (Ni) and copper (Cu) is a growing environmental concern, adversely affecting ecosystems and the survival of both plants and animals. This study investigated the morphological and physiological responses of Euphorbia marginata Pursh seedlings to varying concentrations of Ni and Cu over a 45-day period. The findings revealed that low concentrations of Ni and Cu enhanced morphological indexes, root indexes, biomass, and photosynthetic pigment content of E. marginata, while high concentrations inhibited these parameters. Compared to the control, Ni and Cu stresses induced membrane peroxidation, increased cell membrane permeability, and inhibited the synthesis of soluble proteins and proline in the leaves. The seedlings demonstrated an ability to mitigate Ni and Cu toxicity by increasing soluble sugar content and enhancing the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). Notably, E. marginata exhibited a higher capacity for Cu²⁺ enrichment and translocation compared to Ni²⁺. Combined Ni and Cu treatments reduced the maximum enrichment and translocation levels of both metals in E. marginata. This study highlights the superior tolerance of E. marginata to Ni and Cu stresses and elucidates the mechanisms underlying its response, providing a theoretical basis for the use of landscape plants in the remediation of heavy-metal-contaminated soils.

1. Introduction

The simultaneous advancement of urbanization and industrialization has led to both direct and indirect releases of heavy metals into the soil, significantly increasing soil heavy-metal activity and exacerbating existing pollution levels [1,2]. Natural sources of heavy metals include volcanic activity and soil matrices [3,4]. However, human activities such as mineral resource extraction, fossil fuel combustion, urban infrastructure development, and the improper use of fertilizers and pesticides in agriculture have substantially increased the composite contamination of soils with nickel (Ni), copper (Cu), and other heavy metals [5,6]. Currently, the most significant sources of contamination are mining sites for Ni, Cu, and other metals [7,8,9], where heavy metal ions infiltrate nearby farmland and urban areas through both natural and anthropogenic pathways [7,10,11]. This contamination further intensifies pollution levels in these environments. Moreover, these contaminants enter ecosystems through the food chain, causing detrimental effects on living organisms [12,13].

Trace amounts of Ni are integral to plant urease, influencing nitrogen metabolism and promoting plant growth and development [14]. Additionally, Ni can enhance seed germination and delay leaf senescence [15]. Cu is also crucial for plants, contributing to leaf senescence and maintaining high chlorophyll levels [16]. Cu is a vital component of chloroplast pigments and facilitates the transfer of photosynthetic electrons. It also acts as a catalyst for enzymes involved in chlorophyll synthesis [17]. However, excessive amounts of Ni and Cu can cause irreversible damage to plants and even lead to plant death. For instance, high levels of Ni can damage cell membranes, cause leaf greening, and result in the accumulation of reactive oxygen species (ROS) [18,19]. Similarly, excessive Cu can inhibit chlorophyll synthesis and damage photosynthetic organs, thus reducing the photosynthetic rate [20]. When plants absorb excess Ni and Cu, physiological indexes are affected [21]. Numerous studies have shown that Ni and Cu stress disrupts photosynthesis, osmoregulation, antioxidant enzyme activities, and root growth. These metals may inhibit enzyme activities by binding with proteins or disrupt normal metabolism by altering plasma membrane permeability [22]. Consequently, plants may exhibit chlorosis, root deformities, and stunted growth. The accumulation of Ni and Cu in plants leads to peroxidative damage and ROS accumulation [23,24], which in turn elevates the activity of antioxidant enzymes such as peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), thereby reducing cellular damage through catalytic reduction and other mechanisms. However, when heavy metal concentrations exceed regulatory thresholds, the structure and function of these enzymes are compromised [13,25,26,27]. Research also indicates that osmotic regulators, including soluble sugars, proteins, proline, and other substances, help mitigate the impact of heavy metal stress on plants [28,29,30,31].

Euphorbia marginata is a widely cultivated ornamental plant known for its resistance to heavy metal pollution. Its significant biomass makes it useful for remediation or soil rehabilitation in areas with low to moderate heavy metal contamination. Additionally, it is an ecological and economic plant capable of enhancing the environment while providing economic benefits [32,33,34]. However, studies on the effects of heavy metal stress on E. marginata are limited. Most research on heavy metal stress in plants focuses on combinations like Cu–Zn, Cu–Cd, and Cd–Pb [35,36,37], with few studies addressing the combined effects of Ni and Cu stress on plant growth and physiology. This study employed pot experiments to investigate the growth and physiological response mechanisms of E. marginata under Ni and Cu stress. The objectives were to simulate the effects of varying concentrations of Ni and Cu on the growth and physiology of E. marginata seedlings, specifically to (1) examine the morphology, biomass, photosynthetic pigment content, antioxidant enzyme activity, osmoregulatory substances, and cell membrane permeability of E. marginata under different Ni and Cu concentrations; (2) investigate the enrichment and translocation capacities of Ni²⁺ and Cu²⁺ in E. marginata under these conditions; and (3) deepen our understanding of the tolerance and response mechanisms of E. marginata to Ni and Cu stress, providing a theoretical basis for using ornamental plants in heavy metal pollution remediation.

2. Materials and Methods

2.1. Culture Conditions and Ni and Cu Treatment

E. marginata seeds were evenly distributed in seedling pots and allowed to germinate for 13 days. Seedlings with fully expanded cotyledons were first transplanted into trays, arranged in rows with 120 plants per tray, and grown for 20 days. Once the seedlings developed 2–3 true leaves, they were transplanted into pots with a diameter of 18 cm and a height of 15 cm, each placed on trays. The soil weight in the pots was standardized to 2.65 (±0.05) kg based on pot volume. Approximately 600 mL of distilled water was used for irrigation. After a period of 6–7 days, during which the transplanted seedlings acclimatized, heavy metal solutions were introduced to the pots, with distilled water serving as the control (A₀B₀) (Figure 1). To eliminate extraneous soil variables, the soil used in the experiment was sourced from the nursery of Jilin Agricultural University. The pots were incubated for 45 days at temperatures between 25–35 °C, under natural light, with a daily light period of 12–15 h, and humidity levels of 50–70%. The experiment was repeated five times. Ni²⁺ and Cu²⁺ solutions were prepared from analytically pure NiCl₂·6H₂O and CuSO₄·5H₂O, respectively. The concentration settings are shown in

Table 1. Design of Ni and Cu combination treatments.

	CuSO4·5H2O Treatment	0 mg·kg−1	300 mg·kg−1	600 mg·kg−1	900 mg·kg−1
NiCl2·6H2O Treatment
0 mg·kg−1		A0B0	B1	B2	B3
100 mg·kg−1		A1	A1B1	A1B2	A1B3
300 mg·kg−1		A2	A2B1	A2B2	A2B3
500 mg·kg−1		A3	A3B1	A3B2	A3B3

.

2.2. Measurement Items and Methods

2.2.1. Growth Indicators

Plant height: The heights of 15 randomly selected plants were measured from the base of the stem to the growth point using a straightedge, and the resulting values were averaged.

Stem diameter: A vernier caliper was used to measure the diameter of the base of the stems of 15 randomly selected plants, and the resulting values were averaged.

Leaf length and width: The length and maximum width of the seventh expanded leaf of E. marginata were measured.

Root scanning: An Epson Expression 12000XL scanner, EPSON, Los Alamitos, CA, USA was employed for morphological scanning, while WinRHIZO 2022 Root Measurement and Analysis Software was used to determine the root length, root surface area, and root volume of the plants.

Biomass: The plants were first washed with distilled water and dried with filter paper. The fresh weights of the aboveground and belowground parts were then determined using a balance. The plants were subsequently air-dried at room temperature until a constant weight was achieved, after which the dry weights of the aboveground and belowground parts were measured.

2.2.2. Measurement of Physiological Indicators

Photosynthetic pigment content: Using the ethanol–acetone extraction method [38], approximately 0.1 g of fresh leaves were cut into pieces and placed in 10 mL of an ethanol–acetone mixture (1:1 v/v) at room temperature, while ensuring the leaves were not exposed to light. The leaves were allowed to completely fade, turning white over approximately 36 h. The mixture was used as a blank to adjust the zero at wavelengths of 665 nm, 649 nm, and 470 nm to determine the absorbance.

POD activity: Using the Guaiacol method [39], approximately 0.1 g of liquid-nitrogen-frozen leaves were mixed with 10 mL of 0.01 mol L⁻¹ phosphate buffer solution (pH 6.0). The mixture was homogenized in an ice bath and centrifuged at 4000 rpm for 15 min. A 1 mL volume of the supernatant was taken and mixed with 1 mL of phosphate buffer for control. A 3 mL volume of the reaction mixture was added, and the absorbance at 470 nm was measured at 1 min intervals for a total of five readings.

SOD activity: Using the nitro blue tetrazolium photochemical method [40], approximately 0.1 g of liquid-nitrogen-frozen leaves were mixed with 0.05 mol L⁻¹ phosphate buffer (pH 7.8). Five milliliters of an ice bath were added to the mixture, which was then homogenized. The mixture was centrifuged at 10,000 rpm for 20 min. In a transparent test tube, 0.05 mL of the supernatant was mixed with 1.5 mL of phosphate buffer (50 mmol L⁻¹), 0.3 mL of EDTA-Na₂ solution (0.1 mmol L⁻¹), 0.3 mL of riboflavin solution (0.02 mmol L⁻¹), 0.3 mL of nitroblue tetrazolium solution (0.75 mmol L⁻¹), 0.3 mL of methionine solution (130 mmol L⁻¹), and 0.25 mL of distilled water for the assay tube. For the light control tube, 1.5 mL of phosphate buffer (50 mmol L⁻¹), 0.3 mL of EDTA-Na2 solution (0.1 mmol L⁻¹), 0.3 mL of riboflavin solution (0.02 mmol L⁻¹), 0.3 mL of nitroblue tetrazolium solution (0.75 mmol L⁻¹), 0.3 mL of methionine solution (130 mmol L⁻¹), and 0.3 mL of distilled water were mixed. The dark control tube, with the same composition as the light control tube, was placed in the dark to protect it from light, while the remaining test tubes were placed under 4000 Lux fluorescent lamps for 20 min to develop the color reaction. After the reaction, the dark control tube was used as a blank to adjust to zero, and the absorbance of the assay tubes and the light control tube was measured at 560 nm.

CAT activity: using the UV absorption method [39], approximately 0.1 g of liquid-nitrogen-frozen leaves were collected and homogenized in 5 mL of 0.1 mol L⁻¹ phosphate buffer (pH 7.5) containing 5 mmol L⁻¹ DTT and 5% PVP, in an ice bath. The homogenate was centrifuged at 12,000 rpm for 10 min at 4 °C. Then, 100 μL of the supernatant was mixed with 2.9 mL of 0.02 mol L⁻¹ hydrogen peroxide, and the absorbance was measured at 240 nm at 1 min intervals for a total of five readings. CAT activity was determined by referencing the standard linear equation and the change in absorbance value.

Soluble sugar content: using the anthrone method [41], approximately 0.1 g of liquid-nitrogen-frozen leaves were homogenized in 1.5 mL of 80% ethanol in an ice bath. The mixture was transferred to a 50 °C water bath and incubated for 20 min, with mixing every 2 min. After cooling, the mixture was centrifuged at 12,000 rpm for 10 min at room temperature. A 25 μL sample of the supernatant was combined with 75 μL of distilled water, 30 μL of anthrone reagent, and 250 μL of concentrated sulfuric acid. The mixtures were then heated in a water bath at 95 °C for 10 min. The absorbance was measured at 620 nm, and the soluble sugar content was calculated using the standard linear equation.

Soluble protein content: Using the Coomassie brilliant blue staining method [42], approximately 0.1 g of liquid-nitrogen-frozen leaves were homogenized with 5 mL of distilled water in an ice bath. The mixture was centrifuged at 3000 rpm for 10 min, and 1 mL of the supernatant was aspirated. The supernatant was then added to 5 mL of Coomassie brilliant blue reagent. After standing for 2 min, the absorbance was measured at 595 nm. The soluble protein content was determined using a prepared standard curve.

Proline content: Using the ninhydrin color development method [43], approximately 0.1 g of liquid-nitrogen-frozen leaves were homogenized in 1 mL of 3% sulfosalicylic acid solution in an ice bath. The extracts were heated at 90 °C with shaking for 10 min, followed by centrifugation at 12,000 rpm for 10 min. The supernatants were then cooled. A 150 μL aliquot of the cooled supernatant was combined with 150 μL of distilled water, 150 μL of glacial acetic acid, and 300 μL of 3% sulfosalicylic acid. The mixtures were heated at 95 °C for 30 min. After cooling, the absorbance was measured at 520 nm, and the proline content was calculated using the standard linear equation.

Malondialdehyde (MDA) content: Using the thiobarbituric acid method [44], approximately 0.1 g of liquid-nitrogen-frozen leaves were ground in an ice bath with 5 mL of 5% TCA. The mixture was centrifuged at 4000 rpm for 10 min, and 2 mL of the supernatant was collected. For the control, 2 mL of distilled water was used. Subsequently, 2 mL of 0.6% TBA was added to the solutions. The mixtures were boiled in a water bath for 10 min, then cooled and centrifuged at 3000 rpm for 15 min. The absorbance of the supernatant was measured at wavelengths of 523 nm and 600 nm to determine the MDA content.

Relative conductivity: Using the soaking method [45], approximately 0.1 g of fresh leaves were collected, cut into pieces, and added to 20 mL of distilled water. The mixtures were allowed to soak at room temperature for 24 h. The conductivity of the extract was measured using a conductivity meter (R1). The extract was then heated in a boiling water bath for 15 min, and the conductivity was measured again after cooling (R2). The relative conductivity values were then calculated.

2.2.3. Determination of Ni and Cu Content under Different Treatments

After a 45-day treatment period, the plants were washed with distilled water, dried with filter paper, and weighed to 0.2 g each for roots, stems, and leaves. The nickel and copper contents were subsequently determined through microwave digestion using inductively coupled plasma mass spectrometry [46]. Post-treatment, the soil was separated from the remaining plant tissues, dried in a constant temperature oven at 37 °C until reaching a stable weight, ground with a mortar, and sieved through a 60-mesh sieve. The nickel and copper contents were then analyzed as previously described.

The bioconcentration factor was calculated as the ratio of the concentration of Ni or Cu in the plant to the concentration of Ni or Cu in the soil.

The transfer factor was calculated as the ratio of the Ni or Cu content in the aboveground parts of the plant to the Ni or Cu content in the roots of the plant.

2.3. Statistical Analysis

Data were processed and analyzed using Microsoft Excel 2019 and IBM SPSS Statistics 25.0. Statistical analysis was performed using one-way analysis of variance followed by Duncan’s multiple comparison test (p < 0.05). Results are presented as mean values ± standard errors or standard deviations.

3. Results

3.1. Effect of Ni and Cu Stresses on Morphology Traits

The E. marginata plants exhibited an initial increase followed by a decrease in heights, stem diameters, leaf widths, and leaf lengths under both single and combined stresses of Ni and Cu (

Table 2. Morphology traits of E. marginata under Ni and Cu stresses.

Treatment	Plant Height (cm)	Stem Diameter (cm)	Leaves Length (cm)	Leaves Width (cm)
A0B0	51.25 ± 0.77 g	0.49 ± 0.02 ab	7.87 ± 0.19 d	4.13 ± 0.02 abcde
A1	52.67 ± 1.39 ef	0.49 ± 0.04 ab	8.17 ± 0.06 c	4.59 ± 0.05 a
A2	51.90 ± 0.36 fg	0.47 ± 0.03 ab	7.65 ± 0.02 e	4.21 ± 0.04 abcde
A3	48.86 ± 0.79 i	0.44 ± 0.10 b	7.25 ± 0.08 f	4.07 ± 0.11 abcde
B1	53.08 ± 0.81 ef	0.50 ± 0.04 ab	7.90 ± 0.04 d	4.19 ± 0.03 abcde
B2	53.32 ± 0.63 de	0.51 ± 0.05 ab	7.65 ± 0.07 e	4.09 ± 0.08 abcde
B3	51.07 ± 0.65 g	0.48 ± 0.01 ab	7.33 ± 0.12 f	3.82 ± 0.19 e
A1B1	55.31 ± 1.24 bc	0.51 ± 0.12 ab	8.36 ± 0.10 ab	4.39 ± 0.74 abcd
A1B2	56.09 ± 0.81 b	0.51 ± 0.08 ab	8.38 ± 0.05 ab	4.41 ± 0.11 abc
A1B3	59.00 ± 0.36 a	0.56 ± 0.03 a	8.51 ± 0.07 a	4.56 ± 0.13 ab
A2B1	55.60 ± 0.39 bc	0.49 ± 0.06 ab	8.23 ± 0.20 bc	4.32 ± 0.03 abcde
A2B2	54.47 ± 0.33 cd	0.49 ± 0.01 ab	8.16 ± 0.15 c	4.16 ± 0.05 abcde
A2B3	51.32 ± 0.51 g	0.49 ± 0.05 ab	7.94 ± 0.08 d	4.04 ± 0.09 bcde
A3B1	50.54 ± 0.53 gh	0.48 ± 0.02 ab	7.92 ± 0.14 d	3.93 ± 0.71 cde
A3B2	49.41 ± 1.12 hi	0.47 ± 0.08 ab	7.89 ± 0.07 d	3.98 ± 0.04 cde
A3B3	48.81 ± 0.29 i	0.46 ± 0.02 ab	7.67 ± 0.03 e	3.88 ± 0.01 de

Note: Different lowercase letters indicate significant differences between treatments (p ≤ 0.05).

, Figure 2). Plant heights were significantly suppressed under treatments A₃, B₃, A₃B₁, A₃B₂, and A₃B₃, with decreases ranging from 0.35% to 4.76% (p < 0.01) compared to the control (A₀B₀). Stem diameters were similarly suppressed under treatments A₂, A₃, B₃, A₃B₁, A₃B₂, and A₃B₃, showing decreases from 0.76% to 9.12% compared to the control. Leaf widths were suppressed under treatments A₂, A₃, B₂, B₃, and A₃B₃, with decreases from 2.51% to 7.90% (p < 0.05) compared to the control. Conversely, treatments A₁, A₂, B₁, A₁B₁, A₁B₂, A₁B₃, A₂B₁, and A₂B₂ resulted in increased leaf lengths, with observed increases ranging from 0.54% to 11.05% (p < 0.01) compared to the control. The promotion of plant morphological growth was enhanced by the application of low concentrations of Ni and Cu complexes.

3.2. Effect of Ni and Cu Stresses on Root Morphology Traits

The single addition of Ni and Cu inhibited the increase in both aboveground and belowground fresh weights of E. marginata compared to the control (A₀B₀) (

Table 3. Root system index of E. marginata under Ni and Cu stresses.

Treatment	Root Length (cm)	Root Surface Area (cm2)	Root Volume (cm3)
A0B0	301.84 ± 6.24 c	55.06 ± 0.47 g	0.80 ± 0.01 abcd
A1	339.58 ± 15.18 b	65.66 ± 0.28 c	1.01 ± 0.10 ab
A2	273.75 ± 5.71 de	54.84 ± 0.95 g	0.90 ± 0.25 abcd
A3	265.79 ± 12.30 def	49.57 ± 0.04 i	0.70 ± 0.21 d
B1	315.04 ± 4.84 c	61.45 ± 1.06 e	0.97 ± 0.23 abc
B2	299.37 ± 11.97 c	56.00 ± 0.50 fg	0.73 ± 0.19 cd
B3	232.15 ± 13.63 hi	47.50 ± 0.08 j	0.68 ± 0.05 d
A1B1	365.39 ± 8.23 a	69.06 ± 0.11 b	1.04 ± 0.10 a
A1B2	356.31 ± 10.34 ab	70.58 ± 0.54 a	1.04 ± 0.15 a
A1B3	263.73 ± 1.48 ef	68.30 ± 1.78 b	1.03 ± 0.03 ab
A2B1	345.45 ± 17.78 b	64.26 ± 1.42 d	1.00 ± 0.05 ab
A2B2	282.41 ± 9.52 d	56.89 ± 0.38 f	0.97 ± 0.15 abc
A2B3	250.66 ± 3.14 fg	55.93 ± 0.61 fg	0.97 ± 0.04 abc
A3B1	237.97 ± 10.02 gh	51.68 ± 0.44 h	0.92 ± 0.17 abcd
A3B2	220.44 ± 2.85 i	44.54 ± 0.23 k	0.76 ± 0.09 bcd
A3B3	184.64 ± 6.60 j	39.61 ± 1.22 l	0.66 ± 0.12 d

Note: Different lowercase letters indicate significant differences between treatments (p ≤ 0.05).

). In the low-concentration treatments (A₁, B₁), the changes in aboveground dry weights were similar for the single additions of the two metals. At these concentrations, Cu had a stronger inhibitory effect on the increase in belowground biomass, while the inhibitory effect of Ni increased significantly with higher stress concentrations. The biomass of E. marginata increased more in treatments combining the same concentrations of Ni and Cu compared to the single metal treatments.

3.3. Effect of Ni and Cu Stresses on Biomass

The individual addition of Ni and Cu resulted in a reduction in both aboveground and belowground fresh weights of E. marginata compared to the control (A₀B₀) (Figure 3). In the low-concentration treatments (A₁, B₁), the changes in aboveground dry weights were similar for the single additions of the two metals. However, Cu exhibited a stronger inhibitory effect on the increase in belowground weights, while the inhibitory effect of Ni significantly increased with higher stress concentrations. The biomass of E. marginata increased more in treatments combining the same concentrations of Ni and Cu compared to the single metal treatments.

3.4. Effect of Ni and Cu Stresses on Photosynthetic Pigment Content

The chlorophyll content of E. marginata leaves following single Ni treatments exhibited a significant increase at the A₁ treatment level (Figure 4), with a gradual decline observed as treatment concentrations increased. Carotenoid content was reduced by 0.12% to 14.86% in comparison to the control (A₀B₀). Low concentrations of Cu treatments (B₁) resulted in increased chlorophyll content, total chlorophyll, chlorophyll a/b ratio, and carotenoid content in the leaves. However, the increase in chlorophyll b content was inhibited, showing a decrease of 1.61% to 9.46% with increasing treatment concentration compared to the control. The photosynthetic pigment content of leaves after Ni and Cu combination treatments increased in the A₁B₁, A₁B₂, A₁B₃, and A₂B₁ treatments compared to the control but decreased in all other treatments. As the concentration of Ni and Cu treatments increased, only the chlorophyll a/b ratio remained higher than the control. The addition of low concentrations of Ni may reduce the adverse effect of Cu on the synthesis of photosynthetic pigments in E. marginata. However, the inhibitory effect on the photosynthetic pigments of the leaves was significantly enhanced by the combination of the two metals at high concentrations.

3.5. Effect of Ni and Cu Stresses on Antioxidant Activity

The antioxidant enzyme activities of E. marginata leaves were significantly affected by Ni and Cu stress. The activities of POD, SOD, and CAT were higher than those of the control (A₀B₀) under Ni and Cu mono-treatments (Figure 5). In the A₁ treatments, POD, SOD, and CAT activities increased by 67.01%, 24.17%, and 37.66%, respectively, compared to the control. The peak activities of POD and CAT were observed in the B₃ treatments. SOD activity increased by 41.14% and 21.86% compared to the control in the B₁ treatments. These increases indicate enhanced tolerance to Ni and Cu stresses in the seedlings, as evidenced by the elevated activities of POD, SOD, and CAT. The highest antioxidant enzyme activities were observed in the A₁B₂ treatments following Ni and Cu combination treatments. POD and CAT activities increased by 43.40% and 35.67%, respectively, compared to the control, while SOD activities showed the strongest increase of 28.38% in the A₁B₁ treatments. The inhibitory effect on antioxidant enzyme activity in E. marginata leaves was stronger with increasing concentrations of Ni and Cu treatments.

3.6. Effect of Ni and Cu Stresses on Osmoregulatory Substance Content

The soluble sugar content of E. marginata demonstrated an increasing trend in response to elevated stress concentrations in both single Ni and Cu treatments (Figure 6a). The maximum soluble sugar content was observed at 500 mg kg⁻¹ (A₃) for Ni treatments and 900 mg kg⁻¹ (B₃) for Cu treatments. This increase in soluble sugar content was beneficial in mitigating the stress effects of Ni and Cu on E. marginata. Similarly, the soluble sugar content exhibited an increasing trend with rising stress concentrations in combined Ni and Cu treatments. However, this trend was suppressed once the plants’ regulatory threshold was exceeded. As the concentration of Ni in the combination treatments increased, the addition of Cu was beneficial for increasing soluble sugar concentrations.

In the Ni single treatments, the soluble protein content in the leaves was highest at the A1 concentration, increasing by 0.62% compared to the control (A₀B₀) (Figure 6b). This increase helps alleviate the stress on E. marginata. In the Cu single-stress treatments, the soluble protein content increased with higher treatment concentrations. However, the soluble protein content in both the Cu single treatments and the Ni and Cu combination treatments was lower than in the control. During this time, protein synthesis was inhibited, weakening the alleviation of Ni–Cu stress. The inhibitory effect of Ni single treatments on the soluble protein content in the leaves was slightly less than that of the Cu single treatments and the Ni and Cu combination treatments.

The reduction in proline content reflected the discomfort of E. marginata under Ni and Cu stresses. The proline content in the leaves of E. marginata subjected to Ni and Cu treatments was lower than that of the control (A₀B₀), with a decrease ranging from 18.17% to 37.24% compared to the control (p < 0.01) (Figure 6c). High concentrations of Ni and Cu treatments had significant inhibitory effects on proline content in E. marginata leaves. Comparing the effects of Ni and Cu single and combined stresses revealed that the addition of Ni could, to some extent, alleviate the inhibitory effect of Cu stress on proline synthesis.

3.7. Effect of Ni and Cu Stresses on Cell Membrane Permeability

The MDA content in E. marginata leaves increased significantly under Ni and Cu treatments (p ≤ 0.01) (Figure 7a). Specifically, the MDA content increased by 7.43% to 14.86% after Ni single treatments compared to the control (A₀B₀). Similarly, the MDA content increased by 0.57% to 16.57% after Cu single treatments and by 12.00% to 23.43% after combined Ni and Cu treatments compared to the control. The relative conductivity of the leaves exhibited a similar trend (Figure 7b), increasing with the concentrations of the Ni and Cu treatments. The relative conductivity ranged from 2.73% to 16.92% compared to the control.

3.8. Ni and Cu Uptake and Transport in Different Treatments

Under single Ni stress, the uptake of Ni²⁺ by E. marginata reached its peak in the A₃ treatment (

Table 4. Ni²⁺ content of E. marginata under Ni and Cu stresses.

Treatment	Ni2+ Content in Roots (mg kg−1)	Ni2+ Content in Stems (mg kg−1)	Ni2+ Content in Leaves (mg kg−1)	BCF	Translocation Factor
A0B0	7.23 ± 0.33 g	1.85 ± 0.35 ef	3.66 ± 0.61 h	0.38 ± 0.03 d	0.77 ± 0.16 e
A1	8.55 ± 0.19 f	2.69 ± 0.35 bcd	7.94 ± 0.91 f	0.44 ± 0.01 c	1.24 ± 0.04 b
A2	11.94 ± 0.54 cd	3.94 ± 0.12 a	14.76 ± 0.76 b	0.51 ± 0.01 b	1.57 ± 0.13 a
A3	23.42 ± 0.60 a	2.85 ± 0.17 bc	16.86 ± 0.87 a	0.56 ± 0.00 a	0.84 ± 0.05 de
A1B1	10.56 ± 0.57 e	1.39 ± 0.42 f	4.36 ± 0.36 gh	0.39 ± 0.03 d	0.55 ± 0.04 f
A1B2	13.16 ± 0.70 b	1.52 ± 0.45 f	4.44 ± 0.25 gh	0.47 ± 0.01 bc	0.45 ± 0.01 f
A1B3	12.79 ± 0.20 b	1.53 ± 0.41 f	4.78 ± 0.70 g	0.48 ± 0.02 bc	0.49 ± 0.03 f
A2B1	11.38 ± 0.44 d	2.15 ± 0.25 de	7.81 ± 0.34 f	0.44 ± 0.02 c	0.88 ± 0.03 de
A2B2	12.94 ± 0.2 b	2.66 ± 0.23 bcd	9.48 ± 0.47 cd	0.57 ± 0.02 a	0.94 ± 0.07 cd
A2B3	12.34 ± 0.35 bc	2.15 ± 0.19 de	8.54 ± 0.49 def	0.50 ± 0.03 b	0.86 ± 0.03 de
A3B1	12.36 ± 0.44 bc	2.45 ± 0.40 cd	8.28 ± 0.24 ef	0.38 ± 0.02 d	0.87 ± 0.02 de
A3B2	12.83 ± 0.11 b	3.79 ± 0.25 a	9.20 ± 0.32 cde	0.45 ± 0.02 c	1.01 ± 0.05 c
A3B3	12.54 ± 0.57 bc	3.04 ± 0.30 b	9.89 ± 0.22 c	0.44 ± 0.02 c	1.03 ± 0.04 c

Note: Different lowercase letters indicate significant differences between treatments (p ≤ 0.05).

), with the aboveground content of Ni²⁺ exceeding the under-ground content in the A₁ and A₂ treatments. The Ni²⁺ enrichment coefficient of E. marginata under combined Ni and Cu stresses was 0.57 in the A₂B₂ treatment, and the transport coefficient was 1.57 in the A₂ treatment. Additionally, the presence of Cu increased the uptake and transport of Ni²⁺ by E. marginata to some extent under the same concentrations of Ni treatments. However, the enrichment and translocation capacities of E. marginata for Ni²⁺ were diminished following the combined treatments compared to the single treatments.

The enrichment and transport coefficients of Cu²⁺ in the roots, stems, and leaves of E. marginata under single Cu²⁺ stress showed a highly significant (p < 0.01) gradual increase (

Table 5. Cu²⁺ content of E. marginata under Ni and Cu stresses.

Treatment	Cu2+ Content in Roots (mg kg−1)	Cu 2+ Content in Stems (mg kg−1)	Cu 2+ Content in Leaves (mg kg−1)	BCF	Translocation Factor
A0B0	7.93 ± 0.33 j	3.85 ± 0.35 g	5.96 ± 0.52 f	0.55 ±0.01 h	1.24 ± 0.05 d
B1	15.54 ± 0.80 h	6.26 ± 1.79 def	13.43 ± 0.91 de	0.58 ± 0.03 gh	1.27 ± 0.22 d
B2	19.89 ± 0.89 g	9.52 ± 1.02 ab	26.34 ± 1.62 b	0.65 ± 0.01 f	1.81 ± 0.12 b
B3	23.87 ± 1.93 f	5.88 ± 1.86 ef	49.36 ± 0.62 a	0.72 ± 0.02 e	2.32 ± 0.14 a
A1B1	14.09 ± 0.21 hi	5.99 ± 0.97 ef	14.43 ± 0.58 d	0.63 ± 0.04 fg	1.45 ± 0.09 c
A1B2	24.16 ± 0.82 ef	5.40 ± 0.49 fg	12.26 ± 0.30 e	0.77 ± 0.01 de	0.73 ± 0.06 f
A1B3	34.74 ± 0.38 a	7.61 ± 0.25 cde	12.27 ± 0.44 e	0.84 ± 0.02 c	0.57 ± 0.01 g
A2B1	12.65 ± 0.58 i	7.80 ± 1.16 bcd	16.96 ± 0.85 c	0.72 ± 0.06 e	1.95 ± 0.07 b
A2B2	26.23 ± 0.41 cd	8.04 ± 0.25 bc	17.60 ± 0.54 c	1.15 ± 0.04 b	0.98 ± 0.02 ef
A2B3	30.37 ± 1.22 b	7.91 ± 0.77 bcd	17.55 ± 0.45 c	0.89 ± 0.04 c	0.84 ± 0.04 fg
A3B1	14.06 ± 0.23 hi	8.40 ± 0.35 bc	18.20 ± 1.03 c	0.83 ± 0.01 cd	1.89 ± 0.07 b
A3B2	26.99 ± 0.84 c	10.55 ± 0.61 a	18.32 ± 1.27 c	1.36 ± 0.09 a	1.07 ± 0.04 e
A3B3	25.47 ± 0.67 de	7.61 ± 0.37 cde	17.23 ± 0.25 c	0.73 ± 0.01 e	0.98 ± 0.05 ef

Note: Different lowercase letters indicate significant differences between treatments (p ≤ 0.05).

). The maximum enrichment coefficient of Cu²⁺ in E. marginata under combined Ni and Cu stresses was 1.36 in the A₃B₂ treatment, with the maximum transport coefficient reaching 1.95 in the A₂B₁ treatment. Compared to the single treatments, the combined treatments demonstrated an enhanced Cu²⁺ enrichment capacity but a diminished Cu²⁺ transport capacity in E. marginata.

4. Discussion

4.1. Ni and Cu Effect Morphology Traits of Seedings

Plant growth is primarily the result of coordinated physiological and biochemical processes [47], making it a straightforward and effective method for assessing plant response to external stimuli [48]. This assessment can be achieved by observing plant growth traits, as demonstrated in the case of E. marginata under Ni and Cu single and combined treatments. The results indicated that plant height, stem diameter, leaf width, and leaf length were promoted under low-concentration treatments. This promotion can be attributed to the fact that nickel is an essential element for numerous biomolecules in plants, which are required for maintaining their normal structure and function (e.g., SOD, urease, Ni-Fe hydrogenase, carbon monoxide dehydrogenase) [19]. Copper, being an essential trace element for plant growth, is involved in various physiological and biochemical processes, including the production of chlorophyll, photosynthesis, protection against oxidative stress, and the metabolism of proteins and carbohydrates [16,17]. This observation contrasts with the findings of Hossain et al., who reported that Cu treatments reduced seedling growth in plants [49]. Exposure to elevated levels of heavy metals can overwhelm the plant’s natural regulatory capacity, leading to cellular-level damage [23]. Consequently, as the treatment concentrations increased, the growth of E. marginata was inhibited at 500 mg kg⁻¹ for Ni treatments and 900 mg kg⁻¹ for Cu treatments. This finding is corroborated by the study on copper stress in maize by S. Reckova et al. [50].

4.2. Ni and Cu Effect Root Morphology Traits of Seedings

The study of root system changes in Arabidopsis thaliana revealed that these changes may be related to mechanisms for avoiding harmful metals [51]. Similarly, the responses of E. marginata to Ni, Cu, and their combined stresses can be visualized through root system indexes. This experiment indicated that Ni and Cu stresses, specifically the A₁, B₁, A₁B₁, A₁B₂, and A₂B₁ treatments, promote the plant root system. These increases are related to the properties of the two metals in influencing the plant’s growth physiology. A similar phenomenon was observed in the study by Benakova et al. on Brassica napus [52], where root changes under Zn stress were documented. The root indexes of E. marginata demonstrated varying degrees of inhibition with increasing concentrations of both metals in single and combined treatments. This is consistent with the response of Solanum lycopersicum L. under different concentrations of Ni stress [53]. The disruption of the plasma membrane of root cells by Ni and Cu ions leads to the production of ROS, which attacks the cells and inhibits mitosis. Consequently, this results in reduced uptake of nutrients and water, decreased root system vigor, and impeded root growth [54,55].

4.3. Ni and Cu Effect Biomass of Seedings

When heavy metal elements in the soil are excessively absorbed by the plant root system, it hinders the growth and development of the roots, thereby affecting overall plant growth and reducing plant biomass [56,57,58]. In the presence of Ni, the fresh weight of both the aboveground and belowground parts of E. marginata decreased with increasing treatment concentrations, while the dry weight increased at low concentrations. Under Cu single stress, the biomass of E. marginata generally exhibited a downward trend with increasing treatment concentrations. Ni and Cu have dual roles in plants: at low concentrations, they can participate in various normal physiological activities, but at higher concentrations, they inhibit plant growth. When these metals are beneficial to plant growth, their combined effect on heavy metal uptake may either alleviate the stress of a single metal or induce a stress response, resulting in increased plant biomass [59]. The positive effects on growth observed in this study under the A₁B₁, A₁B₂, A₁B₃, and A₂B₁ treatments can be attributed to the excitatory phenomenon, where low doses of metals activate the plant’s stress defense system, resulting in beneficial effects [60,61]. The observed reduction in biomass with increasing stress concentrations in this experiment aligns with the findings of Junren Chen et al. [62].

4.4. Ni and Cu Effect Photosynthetic Pigment Content of Seedings

A reduction in photosynthetic pigment content is a significant consequence of Cu toxicity [63], while excessive Ni inhibits plant photosynthesis. Chlorophylls a and b are the primary light-absorbing pigments and are crucial for the stability of light-harvesting complex II (LHCII). The content of chlorophyll b is a key factor for the accumulation of LHCII. Furthermore, the degradation of chlorophyll b triggers the degradation of LHCII, leading to a decrease in the light-harvesting capacity of the plant, which subsequently causes photodamage [64]. In addition to their structural function in photosynthetic reaction centers, carotenoids play an important role in the plant antioxidant system by scavenging ROS. This protects the plant photosynthetic system from oxidative damage [65] The effects of single and combined Ni and Cu stress treatments on the photosynthetic pigment content of E. marginata were generally promotive at low concentrations and inhibitory at high concentrations. Alterations in pigment content are a common plant response to metal stress. In the A₁B₁, A₂B₁, and A₃B₁ treatments, the proline content of E. marginata exhibited a positive correlation with increasing Ni concentrations when the Cu concentration was 300 mg kg⁻¹. However, when the Cu concentration exceeded 300 mg kg⁻¹, the combination of Ni and Cu exhibited a pronounced inhibitory effect on proline. This response differs from the proline changes observed in Ocimum basilicum L. under 100 ppm, 210 ppm, 500 ppm Ni, and 500 ppm Cu treatments [66].

4.5. Ni and Cu Effect Antioxidant Activity of Seedings

Heavy metal ions, upon uptake by plants, stimulate the production of a large number of oxygen-containing free radicals (e.g., superoxide anion radicals, hydrogen peroxide), leading to oxidative stress [67]. The increase in antioxidant enzymes can mitigate the damage caused by oxidative stress in plants [68]. SOD can disproportionate superoxide anion radicals to produce H₂O₂, while CAT and POD can disproportionately decompose H₂O₂ into non-toxic H₂O and O₂ [69,70]. In this study, the antioxidant system of E. marginata was significantly altered under the single-stress treatments of Ni and Cu. The activities of SOD, POD, and CAT in E. marginata were higher than those of the control under single-stress treatments. Under compound stress, SOD, POD, and CAT activities exhibited an initial increase followed by a decrease. Additionally, the antioxidant enzyme activities under compound stress were significantly lower than those under single treatments of Ni and Cu. This suggests that the ROS generated by compound stress might exceed the capacity of the antioxidant enzymes. Rehman et al. found that in Boehmeria nivea L. under Cu stress, the activities of SOD and POD were higher than those of the control at low Cu concentration (200 mg kg⁻¹). However, when the concentrations were increased to 300 mg kg⁻¹ and 400 mg kg⁻¹, the activities of SOD and POD were significantly reduced [71].

4.6. Ni and Cu Effect Osmoregulatory Substance Content of Seedings

The accumulation of heavy metal ions in plants can lead to water deficiency, which can be compensated for by the use of soluble proteins, soluble sugars, and proline as osmoregulatory substances [72]. These substances help regulate osmotic pressure balance, enabling the plant to resist adverse environmental conditions [73]. Additionally, osmoregulatory substances play a crucial role in regulating plant metabolism under stress [74].

Soluble sugars and soluble proteins typically exist as small molecules within cells, whereas proline is often distributed in the free state in plants [75]. In this study, under single Ni stress, the soluble sugar content of E. marginata exhibited an increasing trend with rising stress concentration, while the soluble protein content initially increased and then decreased with increasing Ni stress concentration. In the context of single Cu treatment, the soluble sugar content of E. marginata increased with higher stress concentration, while the soluble protein content showed a similar trend. However, both values remained below those observed in the control. Under combined Ni and Cu treatment, the soluble sugar and soluble protein contents of E. marginata gradually declined with increasing stress concentration. Heavy metal stress caused disturbances in carbohydrate metabolism in seedlings, with soluble sugars playing a role in osmotic potential regulation by reducing intracellular osmotic potential and ensuring normal cellular water supply [76,77]. As the concentrations of Ni and Cu increased, the protein content of E. marginata decreased. This may be attributed to Ni and Cu ions altering protein structure and inactivating them, or oxidative stress producing a large amount of ROS that attacked protein molecules, leading to reduced soluble protein synthesis and accelerated hydrolysis. These findings are consistent with those of Haque et al., who studied changes in the soluble protein content of sugar beet under Cd stress at the cellular level [78].

The proline content in E. marginata leaves was lower than that of the control under both single and combined treatments of Ni and Cu, exhibiting a trend of initially increasing and then decreasing. It was postulated that the addition of Ni and Cu might inhibit the production and synthesis of free proline in the plants. Shahzad K. et al. demonstrated that exogenous application of proline to Vigna radiata plants could stabilize the structure of biomacromolecules and regulate cellular redox, thereby reducing stress induced by Ni [79]. This further suggests that proline plays an important role in alleviating heavy metal stress. During combined Ni and Cu stresses, proline content in E. marginata increased with rising Ni concentrations at 300 mg kg⁻¹ Cu concentrations in the A₁B₁, A₂B₁, and A₃B₁ treatments. When copper concentration exceeded 300 mg kg⁻¹, the Ni and Cu complex significantly inhibited proline. This differs from the proline changes observed in Ocimum basilicum L. under 100 ppm, 210 ppm, 500 ppm Ni, and 500 ppm Cu treatments [80].

4.7. Ni and Cu Effect Cell Membrane Permeability of Seedings

Plants can mitigate the detrimental effects of excess heavy metal ions on growth and physiology by increasing the activity of antioxidant enzymes and the production of osmoregulatory substances. However, the balance between scavenging and generation of ROS can be gradually disrupted with prolonged stress, leading to further damage to biomolecules, including lipids, proteins, and nucleic acids [81]. Among these, membrane lipid peroxidation is the most common, resulting in the disruption of cell membrane structure and function, increased permeability, and the leakage of cytosol. This allows external substances to freely enter and exit the cells, causing cellular damage [82,83]. In this experiment, the MDA content and relative conductivity of E. marginata exhibited a continuous increase with the concentration of stresses applied, indicating that Ni and Cu treatments damage the cell membranes of E. marginata and affect its functionality. A similar study on membrane peroxidation in wheat seedlings under 75 μM copper, cadmium, and nickel treatments was conducted by Ewa Gajewska et al. [84].

4.8. Ni and Cu Accumulation and Transport

The magnitude of ion absorption, enrichment, and translocation capacity varies considerably among different plants. Furthermore, the absorption, enrichment, and translocation capacity of the same plant can differ for various metals. In these experiments, under single Ni treatments, the enrichment and translocation ability of E. marginata increased in the A₁ and A₂ treatments. However, their translocation ability decreased at higher concentrations, with Ni²⁺ beginning to accumulate in the roots. This may be attributed to the rise in heavy metal ion concentration within their regulatory ranges, where the underground portion continuously accumulates ions and then transports them to the aboveground parts, resulting in increased transport coefficients [85]. Jun Ge et al. also found that Sedum alfredii under high Ni stress accumulated more than 3000 mg kg⁻¹ of Ni in the roots, and its ability to tolerate high Ni stress was mainly attributed to metal homeostasis in the root cells [86]. When stress concentrations exceed the tolerance threshold, plant growth is inhibited, and the absorbed heavy metals are retained within the plant body. In the context of single Cu treatments, E. marginata exhibited increasing Cu²⁺ enrichment, with a trans-location capacity of 2.32 observed after 900 mg kg⁻¹ treatments. This indicates that E. marginata has a relatively robust absorption and enrichment capacity for Cu²⁺ and the potential for Cu pollution remediation. Hamada AbdElgawad et al. demonstrated that maize exhibited significantly higher Cu²⁺ accumulation levels in all plant parts under Cu stress [58]. A comparable phenomenon was observed in the case of Cu²⁺ uptake in E. marginata. In the presence of both metals, the enrichment capacity of E. marginata for Ni and Cu increased, but their transport capacity decreased compared to the single treatments. Plants transport metal ions through the cationic form [85], resulting in a more complex mechanism of uptake and transport of the two metals than that observed in single treatments. Additionally, the transfer of the two metals to the aboveground parts may be inhibited by each other [87].

5. Conclusions

This study investigated the growth, physiology, and changes in Ni²⁺ and Cu²⁺ accumulation and transport capacity in E. marginata under different Ni and Cu stress treatments through potting experiments. Low concentrations of Ni and Cu treatments promoted the growth indices of E. marginata, including plant height, leaf length, leaf width, biomass, root indices, and the synthesis of photosynthetic pigments. However, higher concentrations of Ni and Cu inhibited the growth of E. marginata. Ni and Cu stresses caused significant peroxide damage to E. marginata, increasing the membrane permeability of leaf cells and inhibiting proline synthesis. It has been demonstrated that E. marginata can be made more tolerant to Ni and Cu stresses by increasing the activities of SOD, POD, and CAT, as well as increasing soluble sugars and soluble proteins. E. marginata showed a higher accumulation and transport capacity for Cu²⁺ than for Ni²⁺. Under combined treatments, the addition of a suitable amount of Cu²⁺ improved the uptake and translocation capacity of Ni²⁺, while the addition of Ni²⁺ promoted Cu²⁺ uptake but inhibited its translocation. The results demonstrated that the stress levels of Ni and Cu on E. marginata increased with higher treatment concentrations. Additionally, combined Ni and Cu treatments exhibited significantly greater damage to E. marginata compared to individual treatments. These findings provide a theoretical basis for studying gene expression differences in response to different heavy metals and help investigate the gene regulation mechanism of E. marginata’s Ni and Cu tolerance, enhancing its application in the field of contaminated soils.