Responses of Physiological Traits and Soil Properties in Pinus thunbergia and Euonymus japonicus Saplings under Drought and Cadmium (Cd) Stress

Abstract

Pinus thunbergii and Euonymus japonicus are two species commonly found in arid and semi-arid areas; however, their responses in terms of physiological traits and soil properties under drought and cadmium (Cd) stress are not clear. In this study, we carried out single and combined stress treatments consisting of drought and Cd on saplings of P. thunbergii and E. japonicus and investigated the responses in terms of the physiological traits and soil properties of both species. For both species, under single Cd stress, Cd was observed in both the xylem and phloem, while the root Cd²⁺ flow rate fluctuated at different levels of Cd stress. Under both single and combined stress, as the stress level increased, the abscisic acid (ABA) content of the leaves and roots increased significantly, while the indole-3-acetic acid (IAA) content of the leaves and roots decreased significantly. Moreover, the non-structural carbohydrate (NSC) content of the leaves, stems, and roots, as well as the leaf chlorophyll content, decreased significantly. Under drought stress, the xylem water potential and hydraulic conductivity significantly decreased, which was exacerbated by Cd stress; this led to a more significant decrease in water potential and hydraulic conductivity under the combined stresses. Meanwhile, no significant changes in the conduit lumen diameter and double-wall thickness were observed, except for the double cell wall thickness of the P. thunbergii tracheid, which increased. In addition, both the single stresses and the combined stress of drought and Cd induced significant changes in the soil properties of the two species, i.e., the ammonium nitrogen, nitrate nitrogen, and effective phosphorus of the soil increased significantly, and the increase in content was more significant under combined stress. The diversity of the soil microbial community of P. thunbergii saplings significantly increased, while no change was found in its microbial community abundance under the single stresses and combined stress; however, the diversity and abundance of the soil microbial community in E. japonicus saplings showed the opposite pattern, which indicates that the effect of Cd on soil microorganisms is more significant than the effect of drought. The activity of sucrase and catalase in P. thunbergii soil fluctuated under the single stress and combined stress when compared, and the activity of sucrase in the soil of the E. japonicus species decreased. However, its catalase activity increased significantly under the single drought and Cd stress and combined stress when compared. We found that the combined stresses exacerbated the effects of the single stress in both species. Our study provides more detailed information on the responses in terms of the physiological traits and soil properties of the two species under single and combined stress consisting of drought and Cd.

1. Introduction

Heavy metal contamination and drought stress are widespread environmental issues that represent a serious threat to plants [1,2]. Previous studies have shown that heavy metal ions aggravate drought stress and render plants more susceptible to death [3,4,5]. Cd is a toxic pollutant that can be absorbed by the roots and seriously affects plants’ survival and yields [6]. Moreover, plants grown in arid and semi-arid areas are always subject to heavy metal stress [7]. When plants are under the combined stress of Cd and drought, the negative effects on their growth and development could be more serious [8]. E. japonicus and P. thunbergii are commonly planted in arid and semi-arid areas and both have a high Cd adsorption capacity [9,10], with promising potential for heavy-metal-contaminated soil remediation. However, the effect on their physiological changes and soil properties under drought and Cd stress is not clear.

The Cd²⁺ flux rate in the plant roots reflects the rate of Cd uptake by plants [11,12]. Previous studies have focused on the differences in the Cd²⁺ flow rates of different species and ions, i.e., H⁺, Na⁺, and Ca²⁺ at one level of Cd stress, while investigations on the Cd²⁺ flow rate at different Cd concentrations are rare. Plant cell wall components are composed of pectin, hemicellulose, and cellulose and contain a large number of negatively charged functional groups (e.g., -COOH, -OH, -SH, etc.), which can effectively adsorb and immobilize heavy metal ions [13,14]. The leaf chlorophyll content reflects the photosynthetic ability of plants, and Cd affects the structure and function of chloroplasts through the inhibition of chlorophyll synthesis and photosynthetic electron transport [15], thereby influencing the photosynthetic efficiency. Furthermore, phytohormones like abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellic acid (GA), and jasmonic acid (JA) are useful in reducing the toxicity of Cd, and their concentration regulation serves as a helpful defense against abiotic stresses like heavy metals and drought [16].

Non-structural carbohydrates (NSC) play an important role in plant metabolism, osmoregulation, and water transport [17], such as the regulation of respiration, the maintenance of cellular elongation, xylem and phloem transport, signal passing, and defense [18]. They include starch and soluble sugars. NSC depletion is mainly due to an imbalance between carbon uptake and carbon demand via photosynthesis, also known as carbon starvation, which may also occur only in certain organs [19]. Decreased starch content usually occurs under short-term abiotic stress, while the soluble sugar content is usually maintained above a certain threshold [20]. Previous studies have shown that the changes in the NSC content during Cd and drought stress are different. Specifically, Cd stress reduces toxicity by affecting enzymes related to the synthesis of NSC, while drought results in redistributed NSC content in various parts of the plant. For example, under Cd stress, the NSC content of more than 70 species changed because Cd altered the sucrose phosphate synthase activity and starch phosphorylase activity, thereby influencing the content of soluble sugars and starch [21,22]. Under drought stress, the preferential allocation of carbon to glucose and fructose reflected an increased demand for soluble sugars for osmotic adjustment, thereby promoting the redistribution of the NSC in Hinoki cypress [23,24]. However, how the NSC content changes under the combined stress of Cd and drought has rarely been investigated.

Under drought and Cd stress, a variety of physiological responses, such as changes in Cd²⁺ inflow and Cd content, changes in cell wall components, a reduction in NSC content, and changes in hormone content, have been observed [25,26,27]. In addition, Cd and drought stress can also cause a decrease in xylem hydraulic conductivity and water potential, leading to plant death [28,29]. Cd stress exacerbates the degree of drought stress in an additive way [4], making the plants more susceptible to morphological, physiological, and xylem anatomical changes.

Drought and Cd stress affect the physicochemical properties of soil, such as the nitrogen (N) and phosphorus (P) content, microbial composition, and soil enzyme activity [30,31]. On the one hand, drought directly affects plant physiological processes, such as photosynthesis and respiration, which in turn affect the diversity and abundance of the rhizosphere soil microorganisms [32]. On the other hand, the soil respiration rates and enzyme activity are significantly inhibited at different concentrations of Cd [33]. Therefore, the combined stress of the two may more significantly affect the soil microbial and enzyme activity [34]. Soil microorganisms are one of the most sensitive indicators of changes in the soil environment, and their community structure composition reflects the soil fertility, as well as the relationship between the soil and plants [35]. Soil sucrase enzyme and catalase enzyme activity can reflect the soil’s nutrient conversion capacity and the potential to supply nutrients to the plant roots [36]. However, heavy metal ions such as Cd²⁺ and Cu²⁺ can combine with enzyme molecules, leading to a decrease in enzyme activity and the formation of reactive oxygen, resulting in oxidative damage to proteins, lipids, and DNA and thus altering the microbial community structure [37]. The ectomycorrhiza (ECM) of the plant is crucial, forming a symbiotic structure with the plant root system, which plays an important role in promoting plant growth and nutrient uptake, the resistance of plants to abiotic stress such as heavy metals, and regulating the soil physicochemical properties, as well as influencing the microbial community of the root soil [38].In addition to altering the physicochemical properties of the soil, soil microorganisms can also regulate the ability of plants to cope with external stress through a variety of mechanisms [39]. Microbes can microbially regulate the plant’s morphological, physiological, and biochemical processes and molecular responses during drought stress as a means to enhance the plant’s resistance [40]. Additionally, microorganisms can promote plant growth and survival under heavy metal stress conditions because they reduce the toxicity of heavy metals to plants by consuming waste and converting complex waste into simple, non-toxic by-products/compounds [38,41]. At the same time, N, P, and other nutrients’ fate and form can vary greatly depending on the soil moisture and Cd stress; the N and P availability and mobility were found to be different under varying levels of drought and Cd stress [42,43].

In this study, we set up single and combined drought and Cd stress experiments and investigated the Cd uptake ability of the two species, by exploring the Cd content of the different organs, the flow rate of Cd²⁺ uptake in the roots, and the distribution of Cd in the phloem and xylem. Moreover, we investigated the physiological changes under drought and Cd stress, including the cell wall fractions, phytohormone content, leaf chlorophyll content, NSC content, xylem water potential, hydraulic conductivity, xylem conduit/tracheid diameter, and double-wall thickness. Furthermore, we investigated the patterns of nitrogen and phosphorus, the pH, the microbial community, and the sucrase and catalase activity in the soil around the roots of the two species. We hypothesized that more significant responses would be observed under combined stress than under single stress and that drought and Cd stress might have different effects on the different species’ traits.

2. Materials and Methods

2.1. Experimental Material

In June 2023, one- to two-year-old E. japonicus and P. thunbergii saplings that were ordered from nurseries were cultivated in a climate chamber. After washing the soil from the roots of the saplings with tap water and deionized water, the saplings were used for the hydroponic and pot experiments, respectively.

Pot experiment. Six groups were designed from a total of thirty healthy saplings of each species that were all of a similar size. The saplings were cultivated at a temperature of 21 °C to 26 °C in commercial nutrient soil, with 8 h of darkness and 16 h of artificial light per day; the light intensity was set to 4000 lx, and the humidity was kept at 60%. After micro-acclimation for one month, the saplings were treated with drought and/or Cd stress. Two watering patterns (well-watered and slight drought) and two Cd levels (50 mg/kg and 100 mg/kg) were set up; Cd stress was applied using CdCl₂ (analytically pure, Sinopharm Chemical Reagent Co, Tianjin, China). The soil water concentration of the well-watered group was 70%–80% of the maximum water-holding capacity of the soil, and the soil water concentration of the slight drought group was 20%–30% of the maximum water-holding capacity of the soil. The experiment consisted of six groups, each with 5 saplings as replications, namely a control (well-watered, no Cd stress, CK), well-watered + 50 mg/kg Cd²⁺ (50), well-watered + 100 mg/kg Cd²⁺ (100), drought (D), drought + 50 mg/kg Cd²⁺ (50D), and drought + 100 mg/kg Cd²⁺ (100D). After 28 consecutive days of Cd stress, the saplings were rinsed three times with tap water in the laboratory and samples were harvested.

Hydroponic experiment. Twenty additional healthy and uniformly sized saplings were selected, transferred to plastic pots (12 cm in diameter and 15 cm in height) containing Hoagland’s solution, and cultured for 14 days. These saplings were divided into four groups: the Cd group with 50 μmol/L, 100 μmol/L, and 150 μmol/L CdCl₂ solution (abbreviated for 50, 100, 150) and the control group without CdCl₂ solution (abbreviated as CK). After 28 consecutive days of Cd stress, they were immersed in the test solution for 30 min, followed by measurement.

2.2. Measurement of the Root Cd²⁺ Flux

Root samples from every sapling were taken in hydroponics following a 14-day period of Cd stress. By using the noninvasive microtext technique (NMT), which can directly reflect the uptake of Cd²⁺, the Cd²⁺ flux was determined in the root meristem zone. The Cd²⁺ concentration (mmol/L level), the flow rate, and the direction of the flow into and out of the root measurement location were measured using a Cd²⁺-selective microsensor. Detailed information can be found in Li et al., 2024 [44]. The root Cd²⁺ flux of each seedling was measured for 200 s, and each repetition was performed two to three times.

2.3. Determination of Cd Content of Different Organs

For the determination of the Cd content of each sapling, five saplings from each group were selected, and samples were obtained from the middle parts of the main stem, the leaves, and almost the entirety of the roots. The roots were washed with EDTA solution, and about 0.1 g of the crushed root sample was transferred into a glass digestion tube, 10 mL of concentrated nitric acid was added, and the sample was soaked for 10 h and heated in a digestion furnace for 1.5 h at 80 °C and 3 h at 120 °C. Afterward, at 175 °C, the acid liquid was volatilized to about 1 mL. After cooling, the digested samples were diluted to 25 mL with a 1% solution of nitric acid. Inductively coupled plasma mass spectrometry (Agilent 7900, Santa Clara, CA, USA) was applied to measure the Cd content of the collected samples. The calculation of the Cd bioaccumulation factors (BCF) and transferring factors (TF) was performed using the following equations: BCF = Cd content in different organs (mg/kg)/Cd content in the nutrient solution (mg/kg), TF from roots to stems = Cd content in stems (mg/kg)/Cd content in roots (mg/kg), TF from stems to leaves = Cd content in leaves (mg/kg)/Cd content in stems (mg/kg).

2.4. In Situ Observation of Cd Distribution in Stem Xylem and Phloem

The middle part of the main stem of the sapling was selected and a 1-cm-long stem sample was cut into longitudinal sections with a thickness of 90 μm using a microtome (Leica SM2010R, Wetzlar, Germany) and freeze-dried using a freeze-dryer (LGJ-12A, Beijing, China). The Cd distribution in the stem xylem and phloem was determined by scanning electron microscopy (TESCAN, Prague, Czech Republic). Detailed information can be found in Li et al., 2024 [44].

2.5. Determination of Cell Wall Component Fraction

The samples were ground in liquid nitrogen, dissolved in 75% ethanol, and allowed to stand in an ice water bath for 20 min. The samples were then centrifuged at 8000× g for 20 min. The resulting precipitates were washed with ice-cold acetone, a methanol–chloroform mixture (1:1, v/v), and methanol, respectively. All steps were performed at 4 °C. After this, the extraction of pectin, hemicellulose, and cellulose from the cell wall was carried out. The absorbance of pectin, hemicellulose, and cellulose was determined by a microplate reader (Synergy H1,Sunnyvale, CA, USA). Specific steps can be found in Li et al., 2024 [44].

2.6. Determination of Leaf Chlorophyll Content

To determine the leaf chlorophyll content, 0.1 g of fresh leaves was collected from 3–5 saplings per group and extracted in 95% ethanol for 24 h. The extract was centrifuged at 8000 r/min for 10 min and the supernatant was taken. The absorbance of the extract was measured at 665 nm, 649 nm, and 470 nm, and the chlorophyll A, chlorophyll B, and carotenoid content was calculated according to the following formulas:

$${\mathrm{C}}_{\mathrm{a}}=13.95 \times { \mathrm{A}}_{665}- 6.88 \times {\mathrm{A}}_{649}$$

(1)

$${\mathrm{C}}_{\mathrm{b}}=24.96 \times { \mathrm{A}}_{649}- 7.32 \times {\mathrm{A}}_{665}$$

(2)

$${\mathrm{C}}_{\mathrm{c}}=\frac{1000{ \times \mathrm{A}}_{470 }- 2.05 \times { \mathrm{C}}_{\mathrm{a}}- 114.8{ \times \mathrm{C}}_{\mathrm{b}}}{245}$$

(3)

where C_a is the content of chlorophyll A, C_d is the content of chlorophyll, C_c is the content of carotenoids, A665 is the absorbance at the wavelength of 665 nm, A649 is the absorbance at the wavelength of 649 nm, and A470 is the absorbance at the wavelength of 470 nm.

2.7. Determination of Phytohormone Content (ABA, GA3, IAA, JA)

The roots and leaves of 3–5 saplings per group were collected for the determination of the phytohormone concentrations (ABA, GA3, JA-Me, IAA) using an enzyme-linked immunosorbent assay (ELISA), as described by Yang et al. [45].

2.8. Determination of NSC Content

The NSC concentration was determined as described by Liu et al., [46]. For every group, 3–5 saplings were assessed. The leaves, stems, and roots of the saplings were collected and dried in an oven at 105 °C for 30 min, followed by drying at 65 °C for 48 h. Soluble sugars and starch were then extracted and determined. Specific steps can be found in Li et al., 2024 [44].

2.9. Determination of Xylem Hydraulic Traits

The xylem hydraulic traits include the xylem potential and xylem hydraulic conductivity. Every three days, the midday leaf water potential of each seedling was monitored using a pressure chamber (PMS Instruments, Albany, OR, USA). For the xylem hydraulic conductivity measurement, a 2-cm-long stem was collected underwater from the middle parts of 3–5 saplings per group, and the xylem-specific hydraulic conductivity (K_s) was measured using a Sperry apparatus, where the pressure drop of a degassed KCl solution (0.01 mol/L) generates a certain water flow in the stem.

$${\mathrm{K}}_s=\frac{\mathrm{F} \times \mathrm{L}}{\Delta \mathrm{P} \times \mathrm{A}}$$

(4)

where F is the velocity of the water flow through the stem, L (m) is the length of the stem, ΔP (MPa) is the pressure difference generating the water flow in the stem segment, and A is the xylem area of the cross-section of the stem segment.

2.10. Determination of Xylem Anatomical Traits

A 2-cm-long sample was selected from the middle of the main stem and the roots of 3–5 saplings per group, and then stored in 70% alcohol and later rehydrated in pure water for 1.0 h. After the rehydration process, the samples were slightly dried and fixed on a microtome (Leica RM2126RT, Leica Camera AG, Wetzlar, Germany). Sections with a thickness of 20 μm were sliced and stained with 1% safranin for 2 min and subsequently dehydrated in 50%, 75%, 95%, and 100% ethanol for several seconds. For the observation and collection of microscopic images, a light microscope (Olympus CX43, Olympus Corporation, Tokyo, Japan) was utilized. A pie-shaped xylem area containing at least 200 consecutive conduits was selected and the conduits’ double-wall thickness and diameter were measured using the ImageJ 1.53 software (National Institutes of Health, Bethesda, MD, USA).

2.11. Determination of Soil Nutrient (N, P) Content and Enzyme Activity

The soil samples used for the soil nutrient content evaluation were collected near the roots of 3–5 saplings and assessed regarding the NY/T 1121-2006 soil testing standards (MoA, 2006). Briefly, the ammonium nitrogen (NH₄⁺-N) content was determined by the potassium chloride extraction–indigo phenol blue colorimetric method; the nitrate nitrogen (NO₃⁻-N) content was determined by the phenol sulfonic acid colorimetric method. The available phosphorus (AP) content was determined by sodium bicarbonate extraction–molybdenum antimony colorimetry. The soil catalase activity was determined by potassium permanganate titration, and soil sucrase was determined by 3,5-dinitrosalicylic acid colorimetry.

2.12. Structure of Rhizobacterial Community

Soil DNA was extracted and determined from 0.5 g low-temperature lyophilized rhizosphere soil of 3–5 saplings, according to the user manual of the FastDNA™ SPIN Kit for Soil (MP Biomedicals LLC, Santa Ana, CA, USA).

2.13. Statistical Analysis

Data were statistically analyzed using IBM SPSS Statistics 26 (IBM Corp., Almonk, NY, USA). Two-way analyses of variance were performed to analyze the effects of drought, Cd, and their combined stress on the Cd content, phytohormone content, leaf chlorophyll content, NSC content, and xylem hydraulic and anatomical traits, as well as the soil nutrient (N, P, K) content, rhizobacterial community and enzyme activity. The differences between the mean values were compared by Duncan’s tests at a significance level of p < 0.05. The software OriginPro 2023b (OriginLab Corp., Hampton, MA, USA) was used to generate graphs.

3. Results

3.1. Cd Uptake and Accumulation in P. thunbergii and E. japonicus Saplings

The roots of the P. thunbergii and E. japonicus saplings showed a steady instream flow of Cd²⁺ between 0 and 200 s (Figure 1), and the mean rate of Cd²⁺ influx fluctuated significantly (p < 0.05). At 100 mmol/L Cd, the fastest average rate of Cd²⁺ influx of both woody species’ roots, the roots’ Cd²⁺ flux rate in E. japonicus was 199.57 pmol/cm²·s, which was twice as high as for P. thunbergii. At a 150 mmol/L concentration, the rate of Cd²⁺ influx was significantly inhibited (p < 0.05) in the two species. Moreover, the Cd accumulation in the roots, stems, and leaves of P. thunbergii and E. japonicus in the different groups was in the order of roots > stems > leaves (

Table 1. Cd content of leaves, stems, and roots from different groups of P. thunbergii and E. japonicus saplings, different lowercase letters (a, b, and c) indicate significant differences (p < 0.05).

		P. thunbergii			E. japonicus
Groups	Roots	Stems	Leaves	Roots	Stems	Leaves
CK	1.23 ± 1.02 c	0.27 ± 0.12 b	0.29 ± 0.02 b	1.76 ± 1.15 c	0.21 ± 0.14 b	1.22 ± 0.82 a
50	18.16 ± 4.08 b	1.61 ± 0.36 b	0.80 ± 0.58 b	6.55 ± 3.68 ab	0.66 ± 0.42 b	1.73 ± 1.65 a
100	42.75 ± 8.42 a	12.93 ± 7.94 a	9.65 ± 3.42 a	8.96 ± 4.00 a	3.03 ± 1.81 ab	2.83 ± 1.92 a
D	1.91 ± 1.24 c	0.40 ± 0.19 b	0.93 ± 0.86 b	0.91 ± 0.79 c	0.18 ± 0.02 b	0.96 ± 0.38 a
50D	22.14 ± 5.00 b	2.46 ± 1.64 b	1.25 ± 0.11 b	3.05 ± 1.73 bc	4.20 ± 3.41 a	3.07 ± 1.69 a
100D	10.75 ± 9.80 bc	8.82 ± 2.13 a	1.42 ± 0.75 b	4.96 ± 2.68 abc	3.44 ± 1.20 ab	2.43 ± 1.55 a

). For both the single Cd stress and combined stress, the Cd content of all organs increased significantly with increased Cd levels in both species (p < 0.05). The TF from the roots to the stems and from the stems to the leaves was higher in E. japonicus than P. thunbergii, and the TF from the roots to the stems was increased significantly in P. thunbergii with increased Cd levels. The BCF of the roots, stems, and leaves decreased in P. thunbergii, but the BCF of the roots, stems, and leaves had no significant change in E. japonicus (

Table 2. TF and BCF of roots, stems, and leaves in P. thunbergii and E. japonicus saplings from different groups, different lowercase letters (a, b) indicate significant differences (p < 0.05).

Species	Parameter	50	100	50D	100D
P. thunbergii	TF from roots to stems	0.08 ± 0.02 a	0.40 ± 0.17 a	0.16 ± 0.14 a	1.26 ± 0.72 b
	TF from stems to leaves	0.57 ± 0.42 a	0.62 ± 0.08 a	0.74 ± 0.04 a	0.15 ± 0.04 a
	BCF for roots	0.27 ± 0.19 ab	0.43 ± 0.08 a	0.39 ± 0.16 a	0.11 ± 0.09 b
	BCF for stems	0.03 ± 0.01 b	0.15 ± 0.05 a	0.05 ± 0.03 b	0.09 ± 0.02 b
	BCF for leaves	0.02 ± 0.01 ab	0.09 ± 0.03 a	0.03 ± 0.02 ab	0.01 ± 0.007 b
E. japonicus	TF from roots to stems	0.14 ± 0.11 a	0.56 ± 0.39 a	1.37 ± 1.16 a	0.97 ± 0.69 a
	TF from stems to leaves	1.63 ± 0.01 a	1.42 ± 1.10 a	1.16 ± 0.76 a	0.73 ± 0.24 a
	BCF for roots	0.13 ± 0.07 a	0.09 ± 0.04 a	0.06 ± 0.03 a	0.05 ± 0.03 a
	BCF for stems	0.01 ± 0.002 a	0.03 ± 0.01 a	0.08 ± 0.06 a	0.03 ± 0.02 a
	BCF for leaves	0.04 ± 0.01 a	0.03 ± 0.01 a	0.06 ± 0.03 a	0.02 ± 0.01 a

). In addition, we observed that under different concentrations of Cd stress, the phloem and xylem of both the P. thunbergii and E. japonicus saplings were distributed with Cd (Figure S1).

3.2. Physiological Responses of P. thunbergii and E. japonicus Saplings under Cd and Drought Stress

Under drought conditions, Cd affected the cell wall fraction proportion. Under the compound stress, the pectin proportion decreased significantly in the roots and leaves of P. thunbergii, while the E. japonicus saplings showed the opposite. The pectin proportion had no significant change in the stems of P. thunbergii, while that in the E. japonicus saplings increased significantly (p < 0.05). The cellulose and hemicellulose proportions of the roots, stems, and leaves of the two species had significantly different trends (Figure 2, p < 0.05). Meanwhile, the chlorophyll a and carotenoid content decreased with the increasing Cd concentration under Cd and drought stress in the two species (Figure S2). In addition, the ABA content was significantly higher and the IAA content was significantly lower in both the roots and leaves of both species (Figures S3 and S4, p < 0.05).

The NSC content of the roots, stems, and leaves in the two species decreased gradually with the increase in the Cd levels. Under drought stress, the NSC content decreased significantly by 42.04% compared with the CK in the roots (Figure 3, p < 0.05). Under the combined stress, the NSC content decreased significantly in the stems and roots of E. japonicus and in the roots of P. thunbergii, while the NSC content was not significantly changed in the stems and leaves of P. thunbergii. In the leaves of P. thunbergii and in the roots, stems, and leaves of E. japonicus, the starch content was greater than the soluble sugar content, while, in the stems and roots of P. thunbergii, the soluble sugar content was greater than the starch content.

Under combined stress, the xylem water potential and hydraulic conductivity decreased significantly with the increasing Cd concentration in both species (Figure 4a,b and Figure S5, p < 0.05), but there were no significant changes in the xylem conduit lumen diameter (Figure 4e,f, p > 0.05). Moreover, the double-wall thickness of the P. thunbergii tracheid increased significantly (p < 0.05); however, there were no significant changes in the double-wall thickness of E. japonicus conduits (Figure 4c,d, p > 0.05).

3.3. Response of P. thunbergii and E. japonicus Soil Properties to Drought and Cd Stress

The soil physicochemical properties of the two species saplings changed significantly under different Cd concentrations, drought, and combined stress. The soil NH₄⁺-N and NO₃⁻-N content and pH increased significantly with increased Cd levels in the two species (Figure 5a,b,d,f,h, p < 0.05); these changes under drought stress were similar for P. thunbergii, while different decreases were found for E. japonicus. Regarding the AP content, there was no change with increased Cd levels in E. japonicus and P. thunbergii, but, under drought stress, the AP content increased significantly for P. thunbergii, while the E. japonicus saplings showed the opposite. It decreased significantly in P. thunbergii, while it increased significantly in E. japonicus under combined stress (Figure 5d,h, p < 0.05).

The rhizosphere soil samples from each group of P. thunbergii had 4648 OTUs (Figure 6a), and the rhizosphere soil of E. japonicus had 2610 OTUs under the different groups (Figure 6b). The soil microbial communities of P. thunbergii and E. japonicus had different dominant phyla (Figure 6c,d). The dominant phyla of the soil microorganisms at the phylum level in P. thunbergii were Proteobacteria, Bacteroidetes, Acidobacteria, and Chloroflexi, while the dominant phyla of the soil microorganisms at the phylum level in E. japonicus were Proteobacteria, Proteobacteria, Acidobacteria, and Chloroflexi, respectively. The dominant phyla of the soil microorganisms at the genus level in P. thunbergii were Devosia, Sphingomonas, Pseudomonas, and Nitrospira, while the dominant phyla of the soil microorganisms at the genus level in E. japonicus were Rhodanobacter, Rhizomicrobium, Burkholderia–Caballeronia–Paraburkholderia, and Devosia, respectively. In addition, significant changes in the soil microbial community diversity (p < 0.05), but not the community abundance (p > 0.05), were observed in P. thunbergii under compound stress, while the opposite was found in the soil microbial community diversity and abundance of E. japonicus (Figure 6e–l). Furthermore, there was greater similarity in the microbial community structure between the CK and D groups, and the 50 and 50D groups showed a similar microbial community, as did the 100 and 100D groups, demonstrating that Cd stress has a greater effect on the soil microbial community structure compared with drought (Figure S6).

The sucrase activity in the soil around the roots of P. thunbergii saplings increased, both under single stress and combined stress, with the sucrase activity of the 100, D, and 100D groups significantly increased (p < 0.05) compared to the CK group (Figure 7a). For E. japonicus, the soil sucrase activity of the 100, 50D, and 100D groups decreased significantly (p < 0.05) (Figure 7b). The soil catalase activity of P. thunbergii roots alone did not change significantly (p > 0.05), neither in the single Cd stress nor the drought group, while, in the 50D group, the catalase activity increased significantly (Figure 7c, p < 0.05). However, for E. japonicus, the catalase activity of the soil around the roots significantly increased (p < 0.05) under single stress and combined stress compared with the CK group (Figure 7d).

4. Discussion

4.1. Cd Uptake and Accumulation in P. thunbergii and E. japonicus Saplings

In this study, the Cd content of both woody species increased with the increasing Cd concentration, and the responses under the combined stress were similar to those under single Cd stress, which is in agreement with a previous study and indicates exacerbated effects [47]. In addition, the highest Cd accumulation was found in the roots of both species, whereas the Cd in the leaves was the lowest, which is in agreement with the findings for other woody species [48]. This is probably because the Casparian band impedes Cd’s translocation from the roots [49]. Moreover, we found that P. thunbergii had a higher BCF in the roots than E. japonicus, which was possibly due to its xylem resin canals, which could be advantageous for Cd adsorption [8] and also the adsorption of its ECM.

The dynamic pattern of Cd²⁺ uptake by the P. thunbergii roots was similar to that of E. japonicus. It fluctuated with the increase in the Cd concentration; specifically, the uptake of Cd²⁺ increased with the increase in the Cd concentration in the low Cd concentration range, while, when the concentration exceeded a certain range, the inhibition of the Cd²⁺ flux occurred due to the protein damage associated with Cd absorption [50]. At higher concentrations, both species were able to take in a large amount of Cd²⁺, even though the Cd²⁺ flow rate was decreased, which proves that both species have a high Cd²⁺ uptake capacity. The maximum Cd²⁺ flux rate of the E. japonicus roots was two times faster than that of P. thunbergii. Interestingly, the Ks of the former was about twice that of the latter, demonstrating that the Ks may be correlated with the root Cd²⁺ flux, while the exact mechanism behind the higher Cd²⁺ flux of E. japonicus needs further investigation.

We found Cd in both the xylem and the phloem of the two species, which was in agreement with previous findings [44]. However, it was impossible to quantify the Cd concentrations of the xylem and phloem in this study. Therefore, future studies could be conducted to quantify the Cd content in different tissue types of plants under different Cd stress levels.

4.2. Physiological Responses of P. thunbergii and E. japonicus under Cd and Drought Stress

During the accumulation of Cd in a species, the cell wall acts as the primary barrier, and the pectin in the cell wall plays an important role in Cd fixation [51]. Previous studies have shown that the pectin content rises under heavy metal stress, which is in agreement with the findings of our study, which showed a significant rise in pectin content under Cd and combined stress [52]. Therefore, the increase in the pectin content of the cell wall would prevent more Cd from entering the cells, thus reducing the toxicity of Cd. The content of cellulose and hemicellulose in the cell wall showed diverse changes, which may have been the result of the adaptation of the plant cell wall components to drought and Cd stress, while the specific mechanism needs to be further investigated.

The significant decrease in the chlorophyll content is attributed to the fact that Cd prohibits the synthesis of δ-aminol evulinic acid or inhibits the activity of prochlorophyllate reductase [53,54]. The decline in the chlorophyll content under combined stress in our study was more significant than that in the case of the single Cd stress, which is consistent with others’ results [55]. The reason may be that the compound stress intensifies the destruction of the chloroplast ultrastructure of the plant leaves [56].

In our study, the IAA content decreased significantly and the ABA content increased significantly under the single Cd and drought stress, as well as the combined stress. Under Cd stress, membrane lipid peroxidation is enhanced, which accelerates the decomposition of IAA, leading to a decrease in IAA content [57], which is consistent with the results of this study. Meanwhile, the GA3 content of both species was reduced under drought and Cd stress, probably due to its depletion, reducing the content of endogenous NO, preventing the synthesis of Cd transporter proteins, reducing the Cd uptake, and decreasing the toxicity of Cd [58]. Similar results were also observed by Kaminek et al. [59]. Under drought and Cd stress, the ABA content increased with the increase in the Cd content, and ABA could mitigate the toxic effects of Cd by regulating the synthesis of chelating peptides and alleviating the toxicity of Cd by modulating the antioxidant system [60]. Moreover, the leaf stomata close during drought in isohydric species [61], leading to an increase in ABA content [62]. The changed JA-ME content in both species lead to a decrease in glutathione (GSH) content, which reduces Cd stress [63]. In addition, JA-ME improves drought tolerance by regulating stomatal closure and increasing the organic osmoprotectants and antioxidative enzyme activity, which also effectively alleviates heavy metal damage by increasing the levels of antioxidative enzyme activity and secondary metabolites [64]. Our results regarding the phytohormone content confirm their regulatory role as protective substances against Cd stress.

Under single Cd stress, only the P. thunbergii roots showed a significant decrease in NSC content, probably due to the decreased activity of related enzymes, which is consistent with a previous study [65]. Moreover, under single drought stress, only the P. thunbergii roots’ NSC content significantly decreased, which was probably due to the redistribution of the NSC content during drought [66]. In our results, the NSC content significantly decreased under combined stress, which may have been due to the drought and Cd stress affecting the osmotic pressure and osmoregulation of P. thunbergii and E. japonicus and inducing a stress response, whereas the sustained increase in the soluble sugar content in the leaves, as well as the high content in the roots and stems, indicated that the two species had strong self-regulation and defense abilities against Cd [67].

As Cd enters the cell, it can bind and interact with proteins and phospholipids on the cell membrane, altering the permeability of the cell membrane, leading to a decrease in the permeability of the cell membrane to water molecules and thus reducing the osmotic pressure [29], which leads to a decrease in the water potential of the xylem. Drought stress also significantly reduces the xylem water potential, which is in agreement with Gauthey et al. [68]. In addition, the decrease in water potential under drought and Cd stress may be due to the stimulation of the overall antioxidant defense system by phytohormones such as salicylic acid at enzymatic and non-enzymatic levels, as well as the regulation of the osmotic pressure by proline, soluble sugars, and organic acids, thus increasing the plant’s resistance to combined Cd and drought stress [69]. Cd disrupts the cell membrane system and reduces the activity of aquaporins, which reduces the hydraulic transport function of the xylem [70], and drought exacerbates this change by hindering hydraulic transport, thus reducing the hydraulic conductivity of the xylem of the species.

Previous studies have shown that under drought stress, the xylem conduit diameter decreases significantly [71]. This change is even more significant when coupled with Cd stress [72], and the decrease in the conduit diameter helps to increase the plant’s hydraulic transport capacity [73]. However, in our study, for both species, there was no significant change in the diameter of the conduit; at the same time, the conduit double-wall thickness of E. japonicus did not change significantly, which may have been due to the relatively shorter period of exposure to Cd or the relatively low Cd content. The double-wall thickness of the xylem tracheids of P. thunbergii significantly increased, which may have been due to the toxicity of Cd, which increases the lignin content and accelerates the lignification of the xylem, leading to an increase in the tracheid thickness [74].

4.3. Response of Soil Properties around Roots of P. thunbergii and E. japonicus to Drought and Cd Stress

Ammonium N and nitrate N significantly increased in the soil around the roots of both P. thunbergii and E. japonicus, which may have been because drought leads to the slowing down of the nitrogen transformation processes in the soil and hence the accumulation of ammonium N and nitrate N. In addition, nitrogen’s transformation by microbes may be limited by drought; on the other hand, Cd also inhibits the microbial transformation of nitrogen, thereby causing the accumulation of ammonium N or nitrate N [75]. In addition, some studies have shown that Cd can promote ammonia oxidation and nitrification reactions in the soil, leading to an increase in ammonium nitrogen and nitrate nitrogen in the soil [76], which is consistent with the results of this study. Under combined stress, the soil AP content decreased in the P. thunbergii saplings, while the soil AP content increased in the E. japonicus saplings; this effect may be species-specific and also depend on the ECM. As conifer species are typical ectomycorrhizal fungal species with a large number of microbial communities in their root systems, their ECMs not only develop a mutually beneficial symbiotic relationship with the host plant, but also change the soil pH and N and P content and increase the soil enzyme activity [77], which improves the stability and resistance of the soil environment and reduces stress damage.

Root secretions and soil physicochemical properties can shape the structures of specific bacterial communities [78], which could be one of the reasons for the different microbial structures in the rhizosphere soil of P. thunbergii and E. japonicus in our study. Cd induced and promoted the growth of soil microorganisms from Proteobacteria, Acidobacteria, and Actinobacteria at the phylum level, which are considered to be tolerant to heavy metals [79,80], and it can maintain root nutrient uptake and the rhizosphere soil microbial balance, improve the soil environment, and increase plant resistance. Moreover, the similarity in the microbial community structure between the CK and D groups, between the 50 and 50D groups, and between the 100 and 100D groups indicates that the effect of heavy metals on soil microorganisms is more significant than the effect of drought, which is consistent with previous findings [81,82]. Moreover, changes in the microbial communities in plant rhizosphere soils are also closely related to the inter-root secretions, which was not investigated, representing a limitation of our study.

In our study, with the increased Cd stress levels, the soil sucrase and catalase activity of both species increased to varying degrees, except for the sucrase activity of E. japonicus. Previous studies have shown that under both single stress and combined stress, the content of ammonium nitrogen, nitrate nitrogen, and AP increases in the soil, and the increase in the microbial diversity also increases the enzyme activity [33,83,84], which is consistent with our results.

5. Conclusions

Our study showed that for both species, under single Cd stress, Cd was observed in both the xylem and phloem, while the root Cd²⁺ flow rate fluctuated at different levels of Cd stress and the Cd content increased significantly with the increasing Cd level. Under both single and combined stress, the ABA content of the leaves and roots increased significantly, while the IAA content of the leaves and roots decreased significantly with the increase in the stress level. Moreover, the NSC content of the leaves, stems, and roots, as well as the leaf chlorophyll content, decreased significantly. In addition, the xylem water potential and xylem hydraulic conductivity significantly decreased under drought stress and combined stress in both species, while the xylem conduit diameter and double-wall thickness showed no change, and the double-wall thickness significantly increased only in P. thunbergii. Furthermore, drought, Cd, and combined stress changed the soil properties of the two species. The soil ammonium nitrogen, nitrate nitrogen, and effective phosphorus increased significantly in both species, and the increase was more significant under combined stress. The diversity of the soil microbial communities in the roots of P. thunbergii increased significantly but the abundance did not change, and the activity of sucrase and catalase was increased to different degrees, whereas the abundance of the soil microbial communities in E. japonicus increased significantly but the diversity had no change, and the activity of soil sucrase decreased while that of catalase increased significantly. We found that the responses of the P. thunbergii and E. japonicus saplings under the combined stress of Cd and drought were more significant than those under single stress, drought stress exacerbated Cd’s toxicity in these species, and Cd stress exacerbated the degree of drought in these two species.