Pb Pollution Stress in Alnus cremastogyne Monitored by Antioxidant Enzymes

Abstract

Lead (Pb) is a common toxic heavy metal element that can be absorbed by plant roots and enter the food chain, damaging human health. Alnus cremastogyne has a wide native range, is fast growing, has a wide range of timber uses, and has rhizomatous roots that can improve planted soils. In this study, we evaluated whether Alnus cremastogyne has the potential to remediate Pb-contaminated soils through a 6-month pot experiment in soils with different Pb concentrations (0, 50, 100, 200, and 400 mg/kg). Our results indicate that Alnus cremastogyne plant height, basal diameter, and organ biomass decreased, H₂O₂ and MDA content increased, and the activities of antioxidant enzymes and osmotic regulators increased and then decreased with increasing lead concentrations. The Pb bioconcentration factor of Alnus cremastogyne was less than 1 at all Pb concentrations, and Pb accumulated mainly in the root system. This indicates that Alnus cremastogyne is not a Pb-enriched plant and does not have outstanding Pb transport capacity. The growth of Alnus cremastogyne was not significantly affected at low Pb concentrations, and its plant height, basal diameter, and biomass were significantly suppressed under high Pb stress. Therefore, Alnus cremastogyne is not a suitable species for the remediation of lead-contaminated soils but can be used as a silvicultural species in environments with low lead levels.

1. Introduction

The prevalence of heavy metal contamination in soils is increasing due to industrialization and urbanization. [1,2]. Lead (Pb) is highly toxic and can persist in soil for 5000 to 150,000 years and maintain high concentrations for up to 150 years [3]. Pb in soil is directly taken up by crops and enriched through the food chain, posing a threat to food safety and human health [4]. Pb is not involved in the growth and development of animals and plants, and it only takes very low concentrations to exhibit toxic hazards to various tissue systems in the human body [5]. When the concentration of Pb in the human body exceeds the critical value, it will seriously damage central nervous system activity and damage the lungs, liver, kidneys, and other organs [6]. Pb pollution has become a worldwide problem affecting the health of human beings.

Phytoremediation represents a method of environmental remediation that employs plant life to extract, sequester, and detoxify heavy metal contaminants. This process is cost-effective and environmentally sustainable, and a widely applicable solution for remediating soils polluted with heavy metals [7,8]. This approach encompasses two primary strategies: phytoextraction and phytostabilization. Phytoextraction involves plants absorbing heavy metals from the soil through their roots and accumulating them in aboveground tissues, which can then be managed as hazardous waste or incinerated to recover metals [9]. The aim of phytostabilization is to fix heavy metals on plant roots through a chelating process, thereby reducing the fluidity and bioavailability of heavy metals in the soil, and thus stabilizing the contaminants and minimizing risks to human health and the environment [10]. Plants utilized in phytoextraction that can accumulate high concentrations of heavy metals in their aboveground parts are termed hyperaccumulators [11]. Over the years, numerous hyperaccumulators of heavy metals have been identified, with herbaceous plants comprising the majority [12,13]. Despite their ability to accumulate heavy metals in significant concentrations, herbaceous plants are constrained by their limited biomass capacity, which restricts the amount of heavy metals they can enrich [14]. In addition, their relatively low economic value and susceptibility to entering the human food chain via livestock and poultry have limited their extensive use [15,16]. On the contrary, woody plants have favorable characteristics, such as rapid growth, high biomass, and well-developed root systems, and the ability to uptake various heavy metals without posing risks to human food chains [17]. These attributes make them promising candidates for the phytostabilization of contaminated soils.

Currently, most of the hyper-enriched plants found are herbaceous. Although herbaceous hyper-enriched plants have outstanding potential for soil heavy metal remediation, some inherent characteristics, such as low biomass, small plant size, and small total enriched amount, have largely limited their application in practice. Therefore, the search and development of woody plants with a large biomass, rapid growth, and high heavy metal enrichment capacity is the key to moving phytoremediation technology from the laboratory to practical application. Alnus cremastogyne is a native tree species in Southwest China, widely distributed in Sichuan, Guizhou, Shaanxi, and Gansu provinces. Alnus cremastogyne has a large biomass, fast growth, and a wide range of timber uses; so, in addition to its timber production and ecological importance, it is also a high-priority intercropping species widely used to improve soil quality [18]. Alnus cremastogyne is widely distributed in Pb-polluted areas, with a fast growth rate, having light and soft material, being easy to process, with a special aroma and corrosion-resistant properties, and is a good material for construction, furniture, and shipbuilding [19]. Currently, research on Alnus cremastogyne mainly focuses on its impact on soil microbial communities, but little has been reported on its research under Pb stress. The objectives of this study include (1) assessing the tolerance of Alnus cremastogyne to Pb; (2) quantifying the uptake of Pb in various organs of Alnus cremastogyne; (3) examining the potential of Alnus cremastogyne for remediating Pb-contaminated soils. The findings aim to establish theoretical foundations and provide data for advancing the application of phytoremediation in areas polluted with lead.

2. Materials and Methods

2.1. Experimental Site Conditions

The site designated for experimentation was situated at the Scientific Experimental Base of Sichuan Agricultural University (Chengdu Campus), with a geographic location of 103°51′36″ E and 30°42′9″ N. The average annual temperature is 16.8 °C, the average monthly maximum temperature is 25.9 °C (July), the average monthly minimum temperature is 6.9 °C (December), the extreme maximum temperature is 38 °C (July), and the extreme minimum temperature is −2 °C (January); the average cumulative yearly relative humidity reaches 84%, the average annual precipitation ranges from 759.1 to 1155.0 mm, and the average yearly total sunshine hour is 840.2 h.

2.2. Test Materials

2.2.1. Test Plants

On 25 March 2021, Alnus cremastogyne saplings of the same seed source (Alnus cremastogyne from Pingchang, Sichuan, China) were selected and transplanted into polyethylene pots, with one plant per pot. They were then watered with an appropriate amount of water. The seedlings were free of pests and diseases and robustly grown. Twenty-five plants were subsequently transplanted into polyethylene pots and subjected to conventional field management and acclimatization for six months. At this point, the average plant height was 145.6 ± 11.2 cm, with an average ground diameter of 12.3 ± 0.7 mm.

2.2.2. Planting Soil and Container

Polyethylene plastic pots were used as containers which have small holes in the bottom for drainage, a diameter of 35 cm at the mouth of the pot, a diameter of 25 cm at the bottom, and a height of 26 cm, with a dry soil mass of 8 kg per pot. The dry soil mass of each pot was 8 kg.

2.3. Experimental Programs

According to the actual situation of soil pollution in some areas of the Yangtze River Basin [20], the soil Pb stress treatments in this experiment were set at five levels of 0 (CK), 50 mg/kg (T1), 100 mg/kg (T2), 200 mg/kg (T3), and 400 mg/kg (T4), and Pb was applied as Pb(CH₃COO)² solution. To simulate the form of Pb emission in reality, the prepared Pb(CH₃COO)² solution was divided into 5 equal portions and applied to the soil 5 times, once every 15 d. A plastic disc was placed under the pot to collect the possible leakage of Pb solution and we poured it back into the basin, and CK was watered with an equal volume of deionized water. The experiment was conducted in a greenhouse to avoid the loss of Pb in the soil due to rainfall. To minimize the effect of the microenvironment in the greenhouse, the position of the planting pots was moved periodically. There were five replicates for each treatment, totaling 25 pots.

The plants were growing for 6 months. In September 2021, the height and diameter of the seedlings in each treatment were measured, and then, all of the plants were harvested, and the biomass, nutrient content, Pb content, and physiological indexes of resistance were measured according to the absorbing root, transporting root, main stem, branch, and leaf, respectively. The leaves used for these measurements were mature functional leaves. The root system of saplings was classified according to function: class 1–3 (non-woody) for absorbing roots and class 4–5 (woody) for transporting roots [21].

2.4. Measurement Indicators and Methods

2.4.1. Measurement of Growth Indexes

(1)

Determination of plant height/ground diameter: We measured the height of the plant with a tape measure (accuracy 0.1 cm); we measured the diameter of the soil with a caliper in two directions perpendicular to each other from the root neck and took the average (precision 0.1 mm).

Net growth of plant height/ground diameter = plant height/ground diameter at the end of Pb treatment-plant height/ground diameter of initial treatment.

(2)

Measurement of biomass: we harvested the whole plant of Alnus cremastogyne saplings in aboveground and belowground parts, washed them with deionized water, and dried them at a constant temperature of 65 °C until reaching a constant weight, and we measured the biomass.

2.4.2. Determination of Nutrient Elements in each Organ of the Plant

The plant organs (absorbing roots, transporting roots, trunk, branches, leaves) were baked in an oven at 105 °C for 30 min, dried at 65 °C until reaching a constant weight, then crushed and ground according to the organs, and passed through a 1 mm sieve, and then, the elemental contents of N, P, and K were determined by distillation [22], molybdenum–antimony colorimetry [23], and atomic absorption spectrophotometry [24].

2.4.3. Determination of Pb Content in Plant Organs and Soil

Plant and soil samples (1.0 g) were powdered and sieved, then digested with nitric and perchloric acids. The lead content in individual plant organs was determined using a NexION 1000G inductively coupled plasma mass spectrometer (Shelton, CT, USA).

2.4.4. Determination of Physiological Indicators of Leaf Resistance

We chopped the clean leaves. The samples were then ground in a mortar while being cooled down with liquid nitrogen. A total of 10 mL phosphate buffer was used to homogenize the plant debris, which was then centrifuged at 10,000 r/min for 15 min. The supernatant was collected and assayed. The determination of SOD activity was carried out by using the nitrogen blue tetrazolium (NBT) method [25]. The activity of CAT was evaluated by quantifying the reduction in H₂O₂ [26]. POD activity was assayed by the oxidation of guaiacol under H₂O₂ [27]. The enzymatic activities of all antioxidants were expressed in units of fresh weight (FW) per gram of sample. The determination of H₂O₂ content was based on the formation of a [TiO(H₂O₂)]²⁺ complex between H₂O₂ and titanium ions [28]. Pro Content was determined by using sulphosalicylic acid and quantified by the acid ninhydrin colorimetric method [29]. The thiobarbituric acid heated colorimetric method was employed to ascertain the concentration of MDA and soluble sugars (SSs) [30]. All parameters were quantified according to the instructions provided in the kits (Jiancheng Bioengineering, Nanjing, China).

2.4.5. Data Statistics and Analysis

The data were collated using Excel 2016 software (Microsoft Office, Redmond, WA, USA). The data were subjected to one-way ANOVA (one-way ANOVA) using SPSS 22.0 statistical analysis software, with the significant level α set at 0.05. Comparisons were made by using the Duncan′s method. Finally, we used Origin 2021 to draw line graphs.

3. Results

3.1. The Growth and Pb Tolerance of Alnus cremastogyne Saplings

The growth of Alnus cremastogyne was significantly influenced by Pb. Specifically, the addition of Pb significantly inhibited (p < 0.05) the plant height, base diameter, and biomass of Alnus cremastogyne saplings, with a small effect when the Pb concentration was low (

Table 1. Growth of Alnus cremastogyne saplings at different soil Pb concentrations.

Pb Treatment mg/kg	Plant Height Increment cm	Base Diameter Increment cm	Absorbing Root Biomass g Plant−1	Transport Root BIOMASS g Plant−1	Stem Biomass g Plant−1	Branch Biomass g Plant−1	Leaf Biomass g Plant−1	Total Biomass g Plant−1
0	48.60 ± 1.40 a	2.40 ± 0.12 a	14.37 ± 0.79 a	10.41 ± 0.54 a	106.30 ± 4.07 a	25.06 ± 1.55 a	18.28 ± 2.52 a	174.41 ± 2.84 a
50	53.50 ± 2.50 a	2.50 ± 0.05 a	11.92 ± 1.85 ab	8.78 ± 1.37 ab	96.71 ± 5.05 ab	22.41 ± 3.99 ab	16.17 ± 1.85 ab	155.99 ± 9.64 ab
100	34.38 ± 2.79 b	2.19 ± 0.16 a	10.82 ± 0.95 abc	7.73 ± 0.62 ab	93.43 ± 11.71 ab	19.54 ± 1.99 ab	14.49 ± 1.05 ab	146.00 ± 14.59 ab
200	34.00 ± 4.62 b	1.73 ± 0.04 b	9.94± 0.77 bc	6.55 ± 1.24 b	81.92 ± 7.43 ab	18.98 ± 1.52 ab	13.94 ± 1.88 ab	131.32 ± 3.34 bc
400	26.50 ± 1.50 b	1.63 ± 0.13 b	7.80 ± 1.05 c	5.78 ± 0.63 b	73.37 ± 7.38 b	16.07 ± 0.89 b	10.90 ± 0.80 b	113.91 ± 8.24 c

Lowercase letters indicate significance, as detailed in the body of the article.

). Pb concentration less than 50 mg kg⁻¹ promoted the plant height and basal diameter of Alnus cremastogyne saplings. The plant height, base diameter, and total biomass of Alnus cremastogyne saplings were significantly suppressed at Pb concentrations greater than 200 mg kg⁻¹ (Figure 1). In addition, the inhibition of root biomass was more significant. The total biomass of plants tended to decrease with increasing Pb concentration. When the lead concentration was below 100 mg kg⁻¹, the biomass of each organ of the plant was not significantly different from that of CK, which indicated that the effect of low Pb concentration on the growth of Alnus cremastogyne saplings was small.

3.2. Changes in Resistance Physiology in Alnus cremastogyne Saplings

In this study, we found that Pb significantly affected physiological resistance indicators in Alnus cremastogyne. Specifically, the activities of SOD (superoxide dismutase), POD (peroxidase), and CAT (catalase) tended to increase and then decrease with increasing Pb concentration (Figure 1). H₂O₂ (hydrogen peroxide) and MDA (malondialdehyde) levels increased significantly with increasing Pb levels in the soil. The activities of antioxidant enzymes (SOD, POD, and CAT) increased significantly when the Pb concentration was higher than 50 mg kg⁻¹. Pro (proline), SS (soluble sugar), and SP (soluble protein) contents increased and then decreased with the rise in Pb concentration. When the Pb concentration was lower than 50 mg kg⁻¹, there was no appreciable impact on the osmoregulation content (Pro, SS, and SP) (

Table 2. The peroxidation markers and antioxidant enzyme activities of Alnus cremastogyne saplings at different soil Pb concentrations.

Pb Treatment mg/kg	H2O2 mmol/g	MDA μmol/g	POD U/g	SOD U/g	CAT U/g	Pro μg/g	SS mmol/g	SP mg/g
0	4.64 ± 0.61 b	0.166 ± 0.014 c	94.40 ± 3.02 b	621.99 ± 12.54 b	7.13 ± 0.89 b	57.56 ± 4.05 c	0.429 ± 0.017 c	6.62 ± 0.63 b
50	6.89 ± 0.75 ab	0.189 ± 0.023 bc	157.92 ± 36.30 a	1137.44 ± 191.60 a	17.00 ± 2.97 a	75.52 ± 3.60 abc	0.588 ± 0.034 ab	10.70 ± 0.43 a
100	7.21 ± 1.06 ab	0.216 ± 0.014 ab	182.94 ± 32.65 ab	918.49 ± 128.49 ab	12.59 ± 1.97 ab	80.51 ± 10.05 ab	0.687 ± 0.046 a	11.98 ± 2.17 a
200	7.45 ± 0.74 a	0.223 ± 0.016 ab	174.21 ± 17.69 ab	794.29 ± 66.32 ab	11.27 ± 4.73 ab	63.15 ± 6.94 bc	0.652 ± 0.085 a	8.79 ± 0.50 ab
400	9.02 ± 0.85 a	0.240 ± 0.012 a	121.00 ± 15.24 ab	739.48 ± 154.19 ab	10.97 ± 1.84 ab	88.71 ± 7.01 a	0.484 ± 0.018 bc	8.69 ± 0.42 ab

Lowercase letters indicate significance, as detailed in the body of the article.

).

3.3. Pb Uptake and Accumulation Characteristics of Alnus cremastogyne Saplings

The Pb content of the roots, stems, branches, and leaves of Alnus cremastogyne saplings exhibited a notable increase in correlation with the soil Pb content, particularly when the soil Pb content exceeded 200 mg kg⁻¹ (

Table 3. The characteristics of lead accumulation in various organs of Alnus cremastogyne saplings at different lead levels in the soil.

Pb Treatment mg/kg	Pb Content in Absorbing Root mg/kg	Pb Content in Transport Root mg/kg	Pb Content in Stem mg/kg	Pb Content in Branch mg/kg	Pb Content in Leaf mg/kg	Remnant Pb Content in Soil mg/kg
0	22.83 ± 6.97 d	33.92 ± 5.93 d	2.37 ± 1.24 d	4.60 ± 2.12 ab	2.76 ± 0.43 b	45.40 ± 4.41 e
50	516.82 ± 55.46 d	211.95 ± 21.88 c	8.27 ± 2.61 cd	3.56 ± 0.85 b	5.61 ± 0.89 b	230.83 ± 7.21 d
100	1273.75 ± 275.94 c	503.29 ± 49.87 b	16.56 ± 3.73 bc	3.00 ± 0.55 b	8.52 ± 2.38 b	367.02 ± 16.68 c
200	1947.13 ± 199.10 b	841.19 ± 66.04 a	22.47 ± 5.23 b	4.75 ± 0.45 ab	12.22 ± 5.48 b	592.29 ± 10.55 b
400	2842.83 ± 157.26 a	911.27 ± 56.91 a	44.10 ± 1.62 a	7.78 ± 2.01 a	43.20 ± 24.17 a	924.26 ± 44.65 a

Lowercase letters indicate significance, as detailed in the body of the article.

). The Pb content of the belowground part was considerably higher than that of the aboveground part (Figure 2). The Pb content and the bioconcentration factor (BCF) of the roots were significantly higher compared to the other organs. Furthermore, this study revealed a significant decline in the transport factor (TF) of Alnus cremastogyne saplings in response to elevated soil Pb concentrations (

Table 4. Enrichment and translocation factors in various organs of Alnus cremastogyne seedlings with different soil lead levels.

Pb Treatment mg/kg	Absorbing Root BCF	Transport Root BCF	Stem BCF	Branch BCF	Plant BCF	Plant TF
0	0.49 ± 0.14 b	0.79 ± 0.21 b	0.049 ± 0.021 a	0.103 ± 0.049 a	0.063 ± 0.012 a	0.98 ± 0.12 a
50	2.26 ± 0.29 a	0.92 ± 0.10 ab	0.036 ± 0.011 a	0.015 ± 0.004 b	0.025 ± 0.005 ab	0.35 ± 0.04 b
100	3.52 ± 0.81 a	1.39 ± 0.18 a	0.045 ± 0.010 a	0.008 ± 0.002 b	0.024 ± 0.007 b	0.25 ± 0.05 b
200	3.29 ± 0.35 a	1.42 ± 0.11 a	0.038 ± 0.009 a	0.008 ± 0.001 b	0.021 ± 0.009 b	0.23 ± 0.02 b
400	3.09 ± 0.32 a	0.99 ± 0.11 ab	0.048 ± 0.001 a	0.009 ± 0.003 b	0.049 ± 0.029 ab	0.27 ± 0.02 b

Lowercase letters indicate significance, as detailed in the body of the article.

).

3.4. Characteristics of Nutrient Uptake by Alnus cremastogyne Saplings

Nitrogen content in the transport roots of Alnus cremastogyne saplings increased significantly with increasing soil Pb content; phosphorus and potassium content in the leaves and phosphorus content in the absorbing roots decreased significantly, and there were no significant changes in nutrient content in the branches and trunks (

Table 5. Changes in nutrient elements in Alnus cremastogyne saplings under different levels of lead treatments.

Nutrient Elements	Plant Organs	Pb Treatment mg/kg
		0	50	100	200	400
N (g/kg)	Absorbing root	7.07 ± 0.15 a	6.32 ± 0.60 a	6.08 ± 0.23 a	6.90 ± 0.79 a	7.81 ± 0.72 a
	Transport root	3.53 ± 0.87 b	2.79 ± 0.29 b	3.46 ± 0.42 b	4.17 ± 0.18 b	7.08 ± 0.21 a
	Trunk	3.09 ± 0.71 a	1.58 ± 0.37 ab	1.59 ± 0.16 ab	1.14 ± 0.40 b	2.52 ± 0.76 ab
	Branch	3.78 ± 0.85 a	3.67 ± 0.96 a	2.55 ± 0.72 a	3.26 ± 0.71 a	4.45 ± 0.05 a
	Leaf	11.91 ± 2.96 a	7.24 ± 1.23 a	12.34 ± 1.84 a	10.11 ± 0.95 a	9.95 ± 0.22 a
P (g/kg)	Absorbing root	1.02 ± 0.16 b	1.15 ± 0.10 ab	1.16 ± 0.08 ab	1.03 ± 0.13 ab	1.44 ± 0.05 a
	Transport root	0.77 ± 0.03 a	0.88 ± 0.13 a	0.83 ± 0.05 a	0.75 ± 0.02 a	0.75 ± 0.04 a
	Trunk	0.70 ± 0.04 a	0.67 ± 0.04 a	0.62 ± 0.05 a	0.64 ± 0.05 a	0.56 ± 0.05 a
	Branch	0.81 ± 0.05 a	0.97 ± 0.10 a	0.89 ± 0.05 a	0.94 ± 0.10 a	0.78 ± 0.01 a
	Leaf	1.27 ± 0.19 a	0.76 ± 0.09 b	0.78 ± 0.04 b	0.67 ± 0.06 b	0.82 ± 0.18 b
K (g/kg)	Absorbing root	1.27 ± 0.19 a	0.76 ± 0.09 b	0.78 ± 0.04 b	0.67 ± 0.06 b	0.82 ± 0.18 b
	Transport root	41.96 ± 5.15 a	55.91 ± 6.84 a	49.50 ± 7.04 a	51.69 ± 4.01 a	43.58 ± 1.51 a
	Trunk	33.08 ± 3.22 b	45.37 ± 3.21 a	43.97 ± 2.03 a	43.42 ± 1.79 a	39.33 ± 4.40 ab
	Branch	45.85 ± 4.93 a	49.18 ± 1.56 a	54.45 ± 2.03 a	57.11 ± 4.44 a	51.49 ± 1.32 a
	Leaf	108.49 ± 23.63 a	66.30 ± 4.81 b	66.28 ± 5.76 b	69.03 ± 5.17 b	73.10 ± 18.84 ab

Lowercase letters indicate significance, as detailed in the body of the article.

).

4. Discussion

In this study, the growth of plant height and diameter of Alnus cremastogyne saplings tended to increase and then decrease in response to Pb stress. The results demonstrate the positive effects of low concentrations of lead on the growth of Alnus cremastogyne saplings. Nevertheless, the growth of Alnus cremastogyne saplings was inhibited at Pb concentrations higher than 100 mg kg⁻¹. This may be because the low concentration of Pb will induce the nutrient elements in the stressed plants to participate in their physiological processes more effectively [31]. The roots of Alnus cremastogyne saplings play a role in nutrient uptake and fixation of the plant body [32]. In this study, with the aggravation of Pb stress, the biomass of the underground part of Alnus cremastogyne saplings decreased significantly and more than that of the aboveground part, which may be because Pb entered the root system and inhibited the cell division of the root system, and at the same time, disturbed the nutrient uptake of the root system, resulting in the inhibition of root system growth [33]. In the soil, the root system is the first to be exposed to Pb, and a large amount of Pb accumulates in the root system, causing it to suffer more damage from Pb stress than the aboveground part [34]. In this study, with the aggravation of Pb stress, the biomass of the aboveground parts of Alnus cremastogyne saplings first increased and then decreased, which may be because the low concentration of Pb triggered the protective effect of the plant, which accelerated plant growth to reduce the relative concentration of Pb in the plant body [35]. It is also possible that low concentrations of Pb induce hormesis, which promotes plant growth under low concentrations of Pb stress and inhibits plant growth under high concentrations of Pb stress [36].

Pb stress causes the dysregulation of oxidative metabolism in plants, and the dynamic balance of intracellular reactive oxygen species (ROS) generation and scavenging is disrupted, while excessive accumulation of ROS leads to oxidative stress [37]. H₂O₂ in ROS is a pleiotropic molecule which can be used as a signaling molecule to participate in the expression of a variety of adversity-related genes in order to improve plant resistance [35]. In this study, the H₂O₂ content in the leaves increased significantly with increasing Pb stress, indicating that the antioxidant enzyme system in the plant cells was no longer able to maintain the dynamic balance of H₂O₂ production and scavenging under high Pb stress, resulting in accelerated ROS accumulation [38]. MDA is the final product of lipid peroxidation and is a significant indicator of the degree of plant adversity stress [39]. In this study, the MDA content continued to increase with the increasing degree of Pb stress, which could be attributed to the Pb stress-induced free radical reactions and membrane lipid peroxidation in the plant, leading to severe damage to the cell membrane structure, thus accumulating a large amount of MDA [40].

The characteristics of Pb enrichment and translocation vary among plants [41]. In this study, under the same level of Pb stress treatment, the Pb content of the absorbing and transporting roots was much higher than that of the aboveground parts, which indicates that plants enriched most of the Pb in the roots. This may be because plants rely on both active and passive transport for Pb uptake, and after Pb enters the root system, it is immobilized in the cell wall and cannot be transported to the aboveground part [42]; it has been shown that most of the Pb is distributed in the plant root system in the acetic acid-extractable and hydrochloric acid-extractable states [43,44], which is one of the reasons why the Pb content of the roots of the plant is much higher than that of the aboveground part. When Pb is transported from the underground part to the aboveground part, the greater the enrichment capacity of the former organ for Pb, the less Pb is present in the subsequent organ [45]. In this study, Alnus cremastogyne saplings had a low transport coefficient for Pb, which may be because most of the Pb was enriched by the root system, resulting in less Pb content in the aboveground part.

In addition to tolerance, metal elements’ uptake and translocation capacity is another criterion for determining whether a tree species has a potential for remediation. The ability of trees to accumulate heavy elements can be expressed in terms of BCF, which is also an important parameter in determining phytoremediation potential [46,47]. Experiments have shown that the BCF of Alnus cremastogyne saplings was around 0.036, indicating that Alnus cremastogyne saplings have a weak capacity for Pb uptake and accumulation. Another important indicator for assessing phytoremediation potential is TF, which is used to assess the ability of plants to transfer heavy metals from roots to stems [48,49]. The TF of Alnus cremastogyne saplings under Pb stress had a maximum of 0.980, which did not meet the requirement of a Pb hyper-enriched plant transfer coefficient of greater than 1, indicating that Alnus cremastogyne saplings have a low translocation capacity for Pb.

N is an important component of chlorophyll, and N deficiency in plants causes symptoms such as smaller and yellowing leaves [50]. Pb deficiency in plants will inhibit root growth and cause symptoms such as necrosis of leaf margins and weak branches [51]. K deficiency in plants causes symptoms such as lightening of leaf color and leaf abscission [52]. With the aggravation of Pb stress, the content of nutrients in each organ of Alnus cremastogyne saplings did not change much, indicating that Pb stress had little effect on the process of nutrient absorption in Alnus cremastogyne saplings. There was no significant change in the P content in organs except leaves, which may be because elemental P improves the buffering of the cytosol, keeps the protoplasm stable, and improves the plant’s adaptability to Pb stress [53].

In summary, Alnus cremastogyne is not a Pb hyper-enriched plant and does not have an outstanding translocation capacity for Pb, and its biomass will be suppressed under high Pb stress. There are some shortcomings in our experiment. Our research utilized a pot experiment, which reduces susceptibility to environmental variables and experimental inaccuracies, ensuring that the data acquired are unbiased, dependable, reproducible, and comparable. However, pot experiments cannot fully simulate the complex interactions between different factors under natural conditions. Therefore, future research efforts should focus on experiments with natural environments.

5. Conclusions

In general, Alnus cremastogyne is not a Pb hyper-enriched plant and its ability to transport Pb is not outstanding. The plant height, base diameter, and total biomass of Alnus cremastogyne were significantly suppressed under high Pb stress. However, under low Pb stress, Alnus cremastogyne grew well and had a high wood production capacity. Since Alnus cremastogyne is a widely distributed and fast-growing tree species, it can be used as a silvicultural species in environments where the soil contains less than 50 mg kg⁻¹ of Pb.