Transcriptome Analysis Reveals Key Genes and Pathways Associated with Cadmium Stress Tolerance in Solanum aculeatissimum C. B. Clarke

Abstract

As a great economic Solanum with ornamental value and good adaptability, Solanum aculeatissimum is considered an excellent candidate for the phytoremediation of Cadmium-contaminated soils. However, there are no studies on the involvement of S. aculeatissimum in the response and tolerance mechanisms of cadmium (Cd) stress. In the present study, S. aculeatissimum was used for the first time for physiological and transcriptomic systematic analysis under different concentrations of Cd stress. The results showed that S. aculeatissimum was indeed well tolerant to Cd and showed Cd enrichment capabilities. Under the Cd stress treatment of 50 mg/kg (Cd6), S. aculeatissimum could still grow normally. At the 90th day of Cd stress, the amount of Cd content in different parts of the plant at the same concentration was in the order of root > stem > leaf. With the extension of the stress time up to 163 d, the trend of Cd content in each part was not consistent, and the results in the root (77.74 mg/kg), stem (30.01 mg/kg), leaf (29.44 mg/kg), immature fruit (18.36 mg/kg), and mature fruit (21.13 mg/kg) of Cd peaked at Cd4, Cd5, Cd1, Cd4, and Cd4, respectively. The enrichment and transport coefficients of all treatments were greater than 1. The treatment groups with the largest and smallest enrichment coefficients were Cd4 and CK, respectively. The treatment groups with the largest and smallest transport coefficients were CK and Cd4, respectively. Malondialdehyde (MDA), peroxidase (POD), and catalase (CAT) in the antioxidant system after Cd stress treatment were significantly increased compared to the untreated group. Under cadmium stress, by using real-time quantitative PCR, four genes (SaHMA20, SaL-AO, SaPrxs4, and SaPCs) were screened for possible correlations to Cd tolerance and absorption enrichment in S. aculeatissimum. The key DEGs are mainly responsible for the metabolic pathways of heavy metal ATPases, plastocyanin protein phytocyanins (PCs), peroxidases (Prxs), and ascorbate oxidase (AAO); these differential genes are believed to play an important role in Cd tolerance and absorption enrichment in S. aculeatissimum.

1. Introduction

Soil is an important environmental factor for human survival and the basis of agricultural production. Cadmium is a common pollutant element in heavy metal-contaminated soils and a non-essential element for the growth and development of plants and animals [1,2,3]. Under natural conditions, the content of cadmium in soil is very low, only 0.01–2 mg kg⁻¹, and the average background value of cadmium in soil in China is 0.097 mg kg⁻¹ [4]. However, cadmium is released directly or indirectly into the natural environment through industrial production, sewage irrigation, agricultural activities and atmospheric deposition, causing serious harm to human beings [5,6]. Humans are also inevitably exposed to Cd in the environment, and the effects of Cd on human health are chronic and potential Cd exposure can cause a variety of acute and chronic diseases. Overaccumulation of Cd in the human body can cause nephrotoxicity, bone damage, neurotoxicity, cardiovascular damage, diabetes, cancer, and other multi-organ system damage [7,8,9,10,11,12].

Phytoremediation is a new type of low-cost purification technology that purifies the soil through the absorption, filtration, volatilization, degradation, and stabilization of plants to render the environment harmless [13,14]. Many scholars at home and abroad have used phytoremediation technology to screen many tree species with strong resistance or enrichment ability against heavy metal pollution and use the characteristics of these plants to reduce heavy metal pollution in the environment to reduce the harm of heavy metals to the environment and human beings [15,16,17].

The heavy metal Cd also has effects on plants, causing morphological and physiological changes in plants. The toxicity of Cd reduces nutrient and water uptake and translocation, increases oxidative damage, and disrupts plant metabolism [18]. The first organ where heavy metal ions enter the plant is the root [19,20], and when Cd accumulates to a certain amount, it will cause the root to become shorter and thicker, leading to dysregulation of root metabolism, inhibition of the uptake of available substances, and nutrient deficiency in the plant [21]. When contaminated with high concentrations of Cd, plants grow slowly and short, with leaf chlorosis, curly leaves, and low germination rates [22,23]. Moreover, the root length, plant height, and stem diameter as well as the fruit yield and quality of the plants are reduced [24,25,26]. There was a significant concentration effect of Cd stress on C. annuum yield. Pepper yield decreases as the concentration of Cd contamination increases in the environment [27]. Under normal conditions, the production and scavenging of free radicals in plants is in a dynamic balance [28]. Heavy metal stress disrupts the dynamic balance and alters antioxidant enzyme activities. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activities in plants can further eliminate the production of oxidative stress induced by Cd stress [29,30].

Currently, related scholars have successfully revealed the tolerance mechanisms of heavy metal stress in Typha orientalis [31], Arabidopsis thaliana [32], Fagopyrum tataricum [33], Festuca elata [34], and Raphanus sativus [35] by RNA-Seq technology. Genes associated with Cd stress were analyzed and screened, including biosynthetic genes for chelating compounds, metal transport proteins, transcription factors, and antioxidant-related genes [36]. SaHMA3 and SaHsfA4ch in Sedum alfredii are associated with the Cd ion transport [37,38], and PeANN1 gene overexpression increased Cd enrichment in Populus euphratica [39].

Solanaceae are tolerant of and enriched for Cd-polluted environments. Some scholars have found that Solanaceae such as Solanum nigrum [40], Solanum tuberosum [41], and Lycopersicon esculentum [42] have certain tolerance and absorption enrichment effects in these environments. However, there is a lack of relevant studies applied to the remediation of environmental heavy metal pollution. There are also large differences in the ability to tolerate and enrich different heavy metals. As well as the adaptive changes in plant physiological metabolism under stress, Deng et al. [43] found that S. aculeatissimum can grow normally in lead–zinc mining areas. While domestic and foreign studies on S. aculeatissimum have focused on the chemical composition and pharmacological effects [44,45,46,47], cultivation techniques [48,49], water stress [50], high temperatures [51], and resistance to pest and disease stress [52,53], there has been less research on heavy metal stress. Therefore, it is of great theoretical and practical significance to carry out studies related to the tolerance, absorption, and enrichment capacity of heavy metals, such as Cd, in S. aculeatissimum.

In this study, the experiment was carried out on S. aculeatissimum seedlings under different concentrations of Cd mono-stress. The effects of Cd on the phenotypic characteristics, uptake and transport processes, and physiological adaptations of S. aculeatissimum were investigated. In this study, we combined the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways and gene ontology (GO) entries to investigate the molecular mechanisms of S. aculeatissimum tolerance to Cd stress. The study was conducted not only to provide a theoretical basis for the screening of woody plants with strong heavy metal enrichment capacity but also to provide a new approach for the phytoremediation of contaminated sites.

2. Materials and Methods

2.1. S. aculeatissimum Seed Pretreatment

In this study, S. aculeatissimum was used. The S. aculeatissimum seeds were surface sterilized by soaking in 1% sodium hypochlorite for 5 min and then washed 3–5 times with distilled water. The seeds were surface sterilized by adding 400 mg L⁻¹ of gibberellin and then soaked in a water bath at 35 °C for 24 h. Seeds were sown in surface sterilized pots with a soil composition of humus/vermiculite/perlite = 3:1:1.

2.2. Experimental Design

The 0–20 cm topsoil in the arboretum of the Southwest Forestry University was used as the sample soil, and 3-month-old seedlings of S. aculeatissimum were used for pot experiments. The organic matter content was determined by the scorch loss method, total nitrogen and total phosphorus by the sulfuric acid–hydrogen peroxide decoction method, total potassium by flame atomic absorption spectrometry, nitrate nitrogen and ammoniacal nitrogen by leaching with potassium chloride, fast-acting potassium by leaching with ammonium acetate, and pH was determined by a pH meter, with reference to the method in Bao’s “Agrochemical Analysis of Soil” [54]. The basic physicochemical properties of the test soils are shown in

Table 1. Basic physical and chemical properties of the tested soils.

Soil Type	Organic Matter (g kg−1)	Total Nitrogen (g kg−1)	Total Phosphorus (g kg−1)	Total Potassium (g kg−1)	Nitrate Nitrogen (g kg−1)	Ammoniacal Nitrogen (g kg−1)	Fast-Acting Potassium (mg kg−1)	pH
Topsoil	58.19	6.14	6.03	2.29	0.14	0.009	12.24	6.99

. After determining the pH value in the soil, the screening value for Cd agricultural land soil contamination risk was based on the national standard GB15618-2018 [55]. Seven treatments with Cd content were set and each treatment was replicated 6 times. Among them, the no Cd concentration level was recorded as CK, and Cd content was added in sequence to concentrations of 3 mg kg⁻¹ (Cd1), 10 mg kg⁻¹ (Cd2), 20 mg kg⁻¹ (Cd3), 30 mg kg⁻¹ (Cd4), 40 mg kg⁻¹ (Cd5), and 50 mg kg⁻¹ (Cd6).

2.3. Soil Heavy Metal Treatment

Rectangular plastic pots of 50 cm long, 20 cm wide, and 15 cm high were used. The cultivation substrate was mixed soil [laterite/humus/vermiculite/perlite = 4:4:1:1 (all by volume)], and each pot contained 6 kg of substrate. No heavy metal was added to the control group, and Cd stress was applied to a total of 6 treatment groups: Cd 3 mg kg⁻¹, 10 mg kg⁻¹, 20 mg kg⁻¹, 30 mg kg⁻¹, 40 mg kg⁻¹, and 50 mg kg⁻¹. A total of 6 pots of each treatment were replicated, and 36 pots were treated with Cd heavy metal. The weighed CdCl₂-2.5H₂O solid powder of different concentrations corresponding to the 6 cadmium treatment groups were completely dissolved in water. The measured soil (6 × 6 kg) was poured into the pots at one time, and then water was poured into the pots. After mixing well, the soil was divided into 6 pots, and the soil without the CdCl₂-2.5H₂O solid powder was also divided into 6 pots and set as the control group. The soil was uniformly placed in a ventilated and cool place for 1 month for the experiment.

2.4. Plant Establishment and Management

The test plants were managed under conventional cultivation throughout the year, and the experiment was carried out in the arboretum of the Southwest Forestry University in Kunming, Yunnan Province. The climate type of the test site belonged to the subtropical plateau monsoon climate, with average annual maximum and minimum temperatures of 21.8 °C and 11.3 °C, respectively, and an average annual precipitation of 933 mm. Uniformly growing S. aculeatissimum seedlings were selected from plants that had been planted for three months (Figure 1). They were washed and planted in pots treated with Cd (performed in static equilibrium). There were 6 treatments under Cd stress, 6 pots each (2 plants per pot), and 42 pots in total (36 pots were in the treatment groups and 6 pots were in the control group). To prevent the loss of Cd ions by leaching, a potting mat is placed at the bottom of each cultivation pot. During watering, if there is any exudate, it is re-added to the pot.

2.5. Measurement of Seedling Growth Parameters

2.5.1. Determination of Morphological Indicators

Plant height and stem diameter were measured for each treatment group on the 15th, 30th, 45th, and 60th day after the experimental treatments. The plant height is the length from the soil scar to the base of the terminal bud of the main stem, measured with a tape measure. The stem diameter was measured with vernier calipers, at the soil scar of the plant. The diameter of the fruit was measured with vernier calipers. Biological replicates were counted in triplicate, and 4 seedlings were taken from each treatment for biological replicate measurements.

2.5.2. Observation of Morphological Indicators

The growth status of each treatment group was observed at the 30th, 50th, 90th, and 111th day after the experimental treatments, respectively. Six seedlings were taken from each treatment, and their leaf, flower, and fruit morphology and number were observed and recorded.

2.6. Content of Membrane Lipid Peroxidation and Antioxidant Enzyme Activity

After the 90th day of stress treatment, S. aculeatissimum was taken from the top of the 4th fully expanded leaf (counting down from the first fully expanded leaf). Samples were cut and mixed avoiding leaf veins, and three samples were randomly selected for the determination of malondialdehyde (MDA). After the 90th and the 163rd days of stress treatment, S. aculeatissimum was taken from the top of the 4th fully expanded leaf (counting down from the first fully expanded leaf). The samples were cut and mixed by avoiding leaf veins, and three samples were randomly selected for the determination of SOD, POD, and CAT.

MDA content was measured by taking 0.3 g of fresh leaves by grinding them in a 10% TCA within a frozen environment and then adding 0.5% TBA for determination, referring to the “Experimental guide to plant physiology” [56] by Li.

SOD was determined using the Suzhou Kemin kit method (Item No.: SOD-1-W); POD was determined using the Suzhou Kemin kit method (Item No.: POD-1-Y); and CAT was determined using the Suzhou Kemin kit method (Item No.: CAT-1-Y). A total of 1 mL of the kit extract was added to 0.1 g of the fresh sample, which was then homogenized in a frozen environment and centrifuged at 8000× g for 10 min at 4 °C. The corresponding reagents were then added into the supernatant resulting from the centrifugation for determination according to the kit instructions.

2.7. Cd Concentration Determination and Transfer Coefficient

The standard solution configuration and Cd concentration were determined according to the method of Bao, “Soil Agrochemical Analysis” [54]. After the 60th and 163rd days of cadmium stress treatments, fresh plant samples were dried in an oven at 105 °C for 30 min, and then dried at 60 °C until constant weight. Then, the dried plant samples were crushed and passed through a 100-mesh nylon sieve, 0.2 g of the dried plant samples were put into a microwave ablution tube, and then 1 drop of purified water, 3 mL of nitric acid, and 1 mL of hydrofluoric acid were added sequentially. After sample addition, the ablator tube was heated on a digester at 120 °C for 1 h. The tube was then transferred to a microwave ablator and heated for 75 min, and then placed again on a digester at 120 °C to reduce the liquid to 1 mL. After the acid depletion was completed, the volume was fixed to a 50 mL volumetric flask and fixed with 1% nitric acid to the scale line. A total of 3 replicates were taken for each sample, and 1 drop of pure water, 3 mL of nitric acid, and 1 mL of hydrofluoric acid were added. A blank control was made at the same time. The Cd content was determined by an inductively coupled plasma emission spectrometer (ICP-OES). Each sample was repeated three times and the average value was taken.

The enrichment factor [57,58] and the transport factor [59] were calculated as follows: enrichment factor = concentration of the element in the plant/concentration of the element in the soil; transport factor = heavy metal content in the plant above ground/heavy metal content in the plant below ground.

2.8. cDNA Library Construction, Deep Sequencing, and De Novo Assembly

After 60 days of Cd stress, the control and Cd6 seedlings were removed from the whole plant, rinsed twice with tap water and three times with ultrapure water, dried on filter paper, and separated into root, stem, and leaf parts, ensuring that each part was weighed with the same quality (0.2 g), and the weighted root, stem, and leaf of the same treatment group were mixed into the same 2 mL lyophilized tubes, quickly put into liquid nitrogen and stored in dry ice, and sent for sequencing (biozeron, Shanghai, China).

2.9. RNA Isolation

RNA was obtained from the roots, stems, and leaves of S aculeatissimum using the Total Plant RNA Extraction Kit (omega, Guangzhou, China) according to the instructions of the kit. RNA integrity was examined by 1% agarose gel electrophoresis. The concentration and purity of the extracted RNA were detected using Nanodrop2000.

2.10. Library Construction and Transcriptome Sequencing

Sequencing was performed using the Illumina Truseq^TM RNA sample prep kit method to construct libraries. The raw data will contain spurious sequences, which will affect the accuracy of the subsequent data. Therefore, the raw sequencing data were filtered to obtain high-quality sequencing data (clean data) to ensure the accuracy of the subsequent data. The Hisat2 software (v2.0.5) was used to compare the sequenced sequences after quality control with the specified reference genome. The software was run with default parameters to complete the comparison.

2.11. Gene Expression Level and Differential Analysis

After the comparison was completed, the Fragments Per Kilobase Million in each sample was used as the expression of that transcript, using the annotation file of the genome as a reference. For the screening of differential genes, the threshold value is generally set as |log2(FoldChange)| > 1 and qvalue < 0.005.

2.12. Functional Analysis of Differentially Expressed Genes GO and KEGG

The functional annotation of GO and KEGG terms was performed using the Cytoscape plugin BiNGO. GO and KEGG enrichment analyses were performed using the Goatools (v0.9.9) and KOBAS software (v3.0), respectively, to further understand the biological functions of DEGs.

2.13. Screening and qRT-PCR Validation of Cd Tolerance and Uptake Enrichment Genes in S. aculeatissimum

The genes related to Cd tolerance and absorption enrichment were screened by analyzing the differential gene KEGG enrichment and sequence comparisons with NCBI and combining with the existing literature reports. Based on the results of gene screening, sequences of genes related to Cd tolerance and translocation enrichment were identified from the transcriptome coding sequence files. Primers were designed using NCBI’s Primer-BLAST primer design online tool with the following design principle: the target fragment is 100~300 bp, primer length is 12~24 bp, and GC content is 40~60%.

The sequences of the corresponding genes were sent to the Synthesis Department of Beijing Prime Tech Biotechnology Co. Ltd., Kunming, China, for primer synthesis, and a total of 5 primers for SaHMA20, Sal-AO, and SaPrxs4 were synthesized. The Actin gene, which can be stably expressed in S. aculeatissimum, was selected as the internal reference gene [60].

Plant RNA extraction was performed using an RNA kit (Qiagen extraction of total plant RNA) for two parts, root and stem, in two treatment groups, CK and Cd6 of S. aculeatissimum, respectively. A total of 4 samples were taken. Each sample was replicated 3 times. The root and stem parts were selected for the follow-up test because the Cd was mostly concentrated in the root and stem parts and less in the leaves in combination with the same period.

The extracted RNA from the roots and stems of S. aculeatissimum was reverse-transcribed into cDNA using the Tiangen kit (Tiangen, Beijing, China). The expression of the candidate genes in the sequenced samples was verified by qRT-PCR using the LightCycler^® 480 system to be consistent with their expression in the sequencing results. Relative gene expression was analyzed using the method of 2^−ΔΔCT.

3. Results

3.1. Response of Plant Growth to Cd Stress

Changes in Phenotypes due to Cd Stress on S. aculeatissimum

In general, the growth and development of the different tissues of plants treated with Cd stress changed significantly in the control and treated groups. The changes in height, stem diameter, and number of leaves of S. aculeatissimum under seven different concentrations of Cd stress at four different times are shown in

Table 2. Changes in plant height at different times under different concentrations of Cd stress in S. aculeatissimum.

	Time (Day)	Plant Height (cm)
Treatment Group		15	30	45	60
CK		10.23 Cd	13.63 Cf	28.90 Bf	40.33 Ac
Cd1		17.30 Cab	17.97 Ce	43.17 Ba	52.37 Ab
Cd2		13.07 Dc	19.30 Cde	34.90 Bde	53.13 Ab
Cd3		13.93 Dc	23.83 Cc	36.43 Bcd	52.57 Ab
Cd4		14.93 Dc	26.07 Cb	37.77 Bc	52.67 Ab
Cd5		19.63 Da	32.07 Ca	40.50 Bb	57.07 Aa
Cd6		17.07 Cb	20.37 Cd	32.83 Be	51.20 Ac

Note: a one-way ANOVA was used, the results in the table are the mean values, capital letters mean that the difference reached a significant level (p < 0.05) at different times for the same concentration, and lowercase letters mean that the difference reached a significant level (p < 0.05) at different concentrations at the same time.

and

Table 3. Changes in stem diameter at different times under different concentrations of Cd stress in S. aculeatissimum.

	Time (Day)	Stem Diameter (mm)
Treatment Group		15	30	45	60
CK		2.86 Bb	3.72 Bab	5.27 Ac	5.40 Ab
Cd1		3.35 Cb	4.11 Bab	5.92 Aab	6.36 Aa
Cd2		3.23 Db	4.09 Cab	5.14 Bc	5.53 Ab
Cd3		3.09 Cb	3.61 Bb	5.17 Ac	5.25 Ab
Cd4		3.33 Cb	4.21 Ba	5.47 Abc	5.47 Ab
Cd5		4.91 Ca	4.15 Dab	5.92 Bab	6.69 Aa
Cd6		4.31 Ba	4.09 Bab	6.47 Aa	6.57 Aa

Note: A one-way ANOVA was used. The results in the table are the mean values, capital letters mean that the difference reached a significant level (p < 0.05) at different times for the same concentration, and lowercase letters mean that the difference reached a significant level (p < 0.05) at different concentrations at the same time.

, and Figure 2. There was no inhibitory effect on the growth of S. aculeatissimum in the CK group. It is worth noting that all treatment groups showed the same trend of change in height of S. aculeatissimum at different periods and were higher than the control group. A short period of Cd treatment can promote an increase in the stem diameter of S. aculeatissimum. On the 15th day of Cd treatment, the stem diameter of S. aculeatissimum was greater in all treatment groups than in the control group. However, with the prolongation of the stress time, the stem diameter of S. aculeatissimum was inhibited. On the 30th day of Cd stress, the stem diameter of Cd3 was smaller than that of the control, at the 45th day, the Cd2 and Cd3 stem diameters were smaller than that of the control, and on the 60th day, the Cd3 stem diameter was also smaller than that of the control. With increasing stress time under Cd treatment, the number of leaves increased to different degrees in all treatment groups. The highest number of leaves was found on the 60th day, and the number of leaves ranked in order of size was Cd1 > Cd2 > Cd5 > CK > Cd4 = Cd6 > Cd3. The flowering and fruiting of S. aculeatissimum at six different concentrations of Cd stress at four different times are shown in

Table 4. Morphological changes of S. aculeatissimum after Cd stress.

	Time (Day)	Morphological Changes
Treatment Group		30	50	90	111
CK		Normal leaves	Normal leaves, 2 flowers, 1 open, with immature green fruit	6 flowers, 3 open, 2 fruit, 1.88 cm in diameter	16 flowers, 10 open, 9 fruit, 1.5 cm in diameter; 6 yellow fruit
Cd1		Normal leaves	Normal leaves, 5 flowers, 1 open	14 flowers, 4 open, 2 fruit, 1.72 cm in diameter	20 flowers, 5 open, 6 fruit, 1.6 cm in diameter; 2 yellow fruit
Cd2		Normal leaves	Normal leaves, 1 flower, 0~1 open, 1 fruit	15 flowers, 5~6 open, 6 fruit, 1.1 cm in diameter	19 flowers, 6~7 open, 9 fruit, 1.25 cm in diameter; 1 yellow fruit
Cd3		Normal leaves	Normal leaves, 1 flower, 0~1 open	9 flowers, 1~2 open, 2 fruit, 0.43 cm in diameter	14 flowers, 2~3 open, 4 fruit, 0.3 cm in diameter; no yellow fruit
Cd4		Normal leaves	Normal leaves, 7 flowers, 0~2 open	14 flowers, 3~5 open, 3 fruit, fruit diameter 1.16 cm	17 flowers, 3~5 open, 6 fruit, 1.90 cm in diameter; 1 yellow fruit
Cd5		Normal leaves	Normal leaves, 2 flowers, 0~2 open, 1 fruit	9 flowers, 3~5 open, 4 fruit, fruit diameter 1.28 cm	13 flowers, 3~5 open, 7 fruit, 1.85 cm in diameter; 1 yellow fruit
Cd6		Normal leaves	Normal leaves, 2 flowers, 1~2 open	14 flowers, 9~11 open, 4 fruit, fruit diameter 0.87 cm	19 flowers, 9~11 open, 9 fruit, 0.86 cm in diameter; 1 yellow fruit

Note: The total number of flowers, blooms, and fruit are based on repeated counts from the previous time period.

. All treatment groups exhibited no significant toxicity symptoms at the different times. After the 30th day of Cd treatment, the leaf morphology was normal and not significantly different than that of the control group. After the 50th day of Cd treatment, S. aculeatissimum had entered the flowering stage. A large number of flower buds appeared and some plants had flowered. Immature fruit appeared for the first time in the CK group and the Cd2 group was the earliest to fruit. After 90 days of Cd treatment, S. aculeatissimum fruiting was observed at all concentrations of treatment. With the prolongation of time, after 111 days of Cd treatment, the control group had the highest number of mature fruit with six ripe fruit and the average fruit diameter reaching 1.5 cm, the Cd4 group had the largest average fruit stem reaching 1.90 cm, and the Cd3 group had no mature fruit.

3.2. Cd Absorption and Transport by S. aculeatissimum

3.2.1. Cd Content of Plant Organs

Plants can transport heavy metals from the environment into the plant through absorption enrichment, which is influenced by the nature of the soil, type of pollution, and environmental factors. Due to the different plant species and heavy metal species, the absorption and accumulation capacities may vary widely [61,62]. The variations of Cd content in each part of S. aculeatissimum at six different concentrations of Cd treatment at 90 days and 163 days are shown in Figure 3A,C. The trend of Cd content in different parts of S. aculeatissimum in all treatment groups was consistent and higher than that in the control group. On the 90th day of Cd stress, the magnitude of Cd content in different parts of the same concentration was in the order of root > stem > leaf. At the 163rd d of Cd stress, the trend of Cd content in each part was not consistent. The Cd content of roots peaked at 77.74 mg·kg⁻¹ in the Cd4 group, the Cd content of stems peaked at 30.01 mg kg⁻¹ in the Cd5 group, the Cd content of leaves peaked at 29.44 mg·kg⁻¹ in the Cd1 group, and the Cd content of immature fruit and mature fruit peaked at 18.36 mg·kg⁻¹ and 21.13 mg·kg⁻¹, respectively, in the Cd4 group.

The changes in Cd content in the soil and plants of S. aculeatissimum at the 90th and 163rd day under different concentrations of Cd treatment are shown in Figure 3B,D. With the increase in Cd treatment concentration, the trend of Cd content in the soil and plants was the same in all treatment groups of S. aculeatissimum, which was higher than in the control group. With the increase in treatment concentration, the Cd content in the soil gradually increased, and the highest content was 142.80 mg kg⁻¹ in the Cd5 group under Cd stress on the 90th day. The Cd4 group had the highest content at 165.86 mg kg⁻¹ under Cd stress on the 163rd day, followed by the Cd5 group, which was 152.08 mg kg⁻¹ on the 163rd day.

3.2.2. Enrichment Transport Characteristics

Table 5. Bioconcentration and translocation factor of S. aculeatissimum on day 163.

Treatment Group	Enrichment Factor	Translocation Factor
CK	2.33 d	10.77 a
Cd1	4.12 ab	7.42 b
Cd2	4.27 ab	3.05 c
Cd3	3.80 bc	1.51 c
Cd4	4.58 a	1.13 c
Cd5	3.26 c	1.14 c
Cd6	2.49 d	1.53 c

Since the Cd content of the flowers was not measured, the enrichment coefficient and transport coefficient did not take into account the Cd content of the flowers. Different letters indicate that the difference reached a significant level (p < 0.05).

shows the changes in enrichment and transport coefficients at 163 days for different concentrations of Cd treatments. The trend in enrichment coefficients was the same for all treatment groups and was higher than in the control group. The trends in the transport coefficients were the same in all treatment groups and were lower than in the control group. The enrichment and transport coefficients were above 1 for all treatments. The enrichment coefficients were in the order of Cd4 > Cd2 > Cd1 > Cd3 > Cd5 > Cd6 > CK and the transport coefficients were CK > Cd1 > Cd2 > Cd6 > Cd3 > Cd5 > Cd4, in order.

3.3. Effect of Cd Treatment on Membrane Lipid Peroxidation and Antioxidant Enzymes

To assess the physiological response under Cd stress, MDA at 90 d was further measured and was higher in all treated groups than in the control group (Figure 4). This result indicated that the membrane lipid peroxidation level was maintained at a high level under Cd stress. In addition, SOD, POD, and CAT were significantly increased in the treated samples compared to the control under Cd stress at 90 d. With the extension of time, POD and CAT were still significantly increased at the 163rd d compared to the control. It indicates that antioxidant enzymes are involved in the elimination of free radicals and play a key role in the protection of the membrane system.

3.4. Transcriptome Sequencing, De Novo Assembly, and Functional Annotation

Table 6. Statistical table of sample sequencing data evaluation.

Sample	Total Reads	Clean Reads	Total Bases	Error Rate (%)	Q20 (%)	Q30 (%)	GC Content
CK	34,513,646	22,329,418	3,305,425,613	0.04	97.46	91.58	47.87
Cd6	31,622,016	19,480,982	2,895,836,234	0.04	97.95	93.09	45.92
Average	33,067,831	20,905,200	3,100,630,924	0.04	97.71	92.34	46.90

Q20, Q30: the percentage of bases with Phred values greater than 20 and 30, respectively, to the overall bases were calculated.

shows the statistical results of transcriptome sequencing data and sequencing quality information for unstressed and Cd-stressed S. aculeatissimum, respectively. The average GC content was 46.90%, and the percentage of Q30 bases in all two samples was not less than 92.34%. The sequencing quality could be considered very reliable if the Q30 is above 80%.

After removing the low-quality sequences, the two samples were compared with the genomic data of S. aculeatissimum and the results are shown in

Table 7. Mapping ratio statistics.

Sample	Clean Reads	Mapped Reads	Mapped Rate (%)
ck	22,329,418	11,870,318	53.16
Cd6	19,480,982	13,675,649	70.20

. The samples had the reads of the genome of S. aculeatissimum matching between 53.16 and 70.20%.

3.5. Analysis of Differentially Expressed Genes between Different Treatment Groups

The volcano scatter plot of differential genes of the S. aculeatissimum transcriptome is shown in Figure 5, where A and B are the scatter plot and volcano plot, respectively, of Cd6 vs. CK. The volcano plot can be used to infer the overall distribution of differential genes and to screen the differential genes, and the threshold value of this experiment was set to |log2(FC)| ≥ 1 and qvalue < 0.05. A total of 4597 genes were up-regulated and 2271 genes were down-regulated in Cd6 compared with CK.

3.6. GO Function Classification of DEGs

To explore the potential functions of these genes, the functions of the S. aculeatissimum DEGs were classified using GO belongs. Thus, these DEGs were classified into 84 functional groups, including :”biochemical processes” (BP, 59 subclasses), “cellular components” (CC, 10 subclasses), and “molecular functions” (MF, 15 subclasses) (Figure 6, Table S1). In the group of biological processes, most GO terms clustered into the regulation of biological quality, nucleic acid phosphodiester bond hydrolysis, RNA modification, regulation of hormone levels, hormone metabolic process, and hormone metabolism. For the cellular component group, the top five subcategories were plastid, chloroplast, plastid envelope, thylakoid, and chloroplast thylakoid, and among the molecular functional groups, hydrolase activity, acting on ester bonds, nuclease activity, endonuclease activity, UDP-glycosyltransferase activity, and glucosyltransferase activity were the dominant subcategories.

3.7. Functional Analysis of the KEGG Pathway of DEGs

To characterize the biological pathway response to Cd stress, all DEGs were assigned to the KEGG database for functional annotation. Overall, all DEGs with significant matches were mapped to 223 KEGG pathways. These pathways belong to five categories, namely Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, and Organismal Systems, which are further divided into 28 subcategories (Figure 7, Table S2). It can be seen that Cd stress caused the highest number of DEGs in these five categories: global and overview maps, translation, signal transduction, transport and catabolism, and immune system of metabolic pathways.

3.8. DEGs Related to Heavy Metal Transport and Detoxification under Cd Stress

A total of 69 heavy metal-related transporter protein genes (Table S3, 46 in up-regulation and 23 in down-regulation) were screened in S. aculeatissimum transcripts and were significantly expressed under Cd stress (Table S3). Among these identified transporter proteins, the largest transporter protein family was the ATP-binding cassette (ABC) transporter protein family with 22 DEGs (16 in up-regulation and six in down-regulation), followed by the glutathione S-transferase (GST) family with 16 DEGs (12 in up-regulation and four in down-regulation). The isoprenylated plant protein (HIPP) family had six DEGs (three up-regulated and three down-regulated) and the cyclic nucleotide-gated channel (CNGC) family had five DEGs (three up-regulated and two down-regulated). The plant tolerance protein (MTP) family had four DEGs (one up-regulated and three down-regulated), the ZIP family had four DEGs (three up-regulated and one down-regulated), the natural resistance-associated macrophage protein NRAMP family had three DEGs (one up-regulated and two down-regulated), and the iron chelating reductase (FRO) family had two DEGs (two up-regulated). Only one up-regulated DEG was found in the metallothionein (MT) family. Two DEGs (one up-regulated and one down-regulated) were found in the heat shock transcription factor (HSF) family, the vesicular iron transport protein 1 (VIT1) family, and the cation/proton exchange (CAX) family.

3.9. Cd-Tolerant, Uptake Enrichment-Related Gene Screening in S. aculeatissimum

In this study, genes related to Cd tolerance and uptake enrichment that have been reported in NCBI were collected and compared with the genes that were significantly different from those screened in this experiment. The genes that may be associated with Cd tolerance and uptake enrichment were further screened in combination with the reported literature. The top 20 significantly different genes screened for Cd treatment are shown in Figure 8 and the top 5 genes were further screened for subsequent experiments. The genes evm. TU.LG02.1499, evm.TU.LG01.3435, evm.TU.LG09.1763, evm.TU.LG08.586, and evm.TU.LG10.886 were tentatively hypothesized to be possibly associated with Cd stress in S. aculeatissimum and they were listed as candidate genes for Cd stress response. NCBI was used for CD sequence alignment and the differential genes evm.TU.LG02.1499, evm.TU.LG01.3435, evm.TU.LG09.1763, evm.TU.LG08.586, and evm.TU.LG10.886 associated with Cd stress were screened and named SaHMA20, SaL-AO, SaPrxs4, SaPCs, and SaPrxs2, respectively.

3.10. Validation of DEG Results by qRT-PCR Analysis

The qRT-PCR technique was used to further analyze the expression trends of the above candidate genes in Cd-stressed S. aculeatissimum and to compare the trends with the expressions of sequencing results for mutual validation.

Based on the results of qRT-PCR (Figure 9), the expression patterns of four genes were consistent with RNA-seq. The qRT-PCR validation analysis proved that the RNA-seq data were reliable. Except for SaPrxs2, the expression trends of the remaining four genes in the transcriptome data of S. aculeatissimum showed consistency. In the roots and stems of S. aculeatissimum at 60 days of Cd stress, SaL-AO and SaPrxs4 were up-regulated in the roots and stems of Cd6, respectively, while SaHMA20 and SaPCs were down-regulated in the roots and stems of Cd6, respectively. SaPrxs2 was up-regulated in stems and down-regulated in roots.

4. Discussion

Plant growth is a comprehensive expression of the morphology of the plant metabolic process, and the amount of growth can visually reflect the degree of plant exposure to adversity [63]. When a certain amount of heavy metals enters the plant, it will affect the plant height, stem diameter, leaf number, flowering, and fruiting. In this experiment, the growth of S. aculeatissimum was normal and there was no significant Cd damage. With the increase in Cd concentration and the prolongation of stress time, the trend of change in height and stem diameter of S. aculeatissimum was not the same. Under Cd stress, the plant height of S. aculeatissimum peaked at different times in the high concentration treatment group (Cd5) and decreased in the higher concentration (Cd6) group, which is in accordance with the phenomenon of “low promotion and high inhibition” of plant response to heavy metal injury [64]. And this phenomenon is that the effect of Cd on plant growth shows a significant quantitative effect, also known as the excitation effect [65]. With the prolongation of Cd stress treatment, stem diameter peaked in the Cd5 group, consistent with plant height. Moreover, plant height is one of the most important indicators to evaluate plant tolerance to Cd toxicity [66,67,68,69]. Thus, it is shown that S. aculeatissimum can be grown in Cd-contaminated areas and has the potential to be used for phytoremediation. Leaf number is one of the important indicators of the effects of heavy metal stress on plants to reflect the stress [70]. In this study, it was found that the increase in concentration promoted the leaf number of S. aculeatissimum and did not show an inhibitory effect as the duration of stress increased. Different concentrations of Cd stress have different toxic effects on the plants. Wang et al. [71] found that gymnosperm spruce pollen is highly sensitive to Cd stress. Cd strongly disrupts endosomal organelles and induces cytoplasmic vacuolization in pollen tubes. The results of this study showed that the leaves of S. aculeatissimum were normal in all treatment groups under Cd stress, no significant toxic effects were observed, and the leaves moved smoothly from nutritional to reproductive growth. All treatment groups were able to flower normally at the 50th day of Cd stress, with the highest number of flowers in Cd1 and the first appearance of immature fruit in the CK, Cd2, and Cd5 groups, indicating that moderate Cd stress could promote the entry of S. aculeatissimum seedlings into reproductive growth.

When a plant is stressed by adversity or undergoes senescence, membrane lipid peroxidation occurs. The final breakdown product of membrane lipid peroxidation is MDA, which reflects the degree of stress injury to the plant [72,73] and is one of the important markers of damage to the membrane system [74]. Higher MDA levels indicate stronger stress injury to plants [75]. In this study, we found that the MDA content of S. aculeatissimum decreased with increasing Cd stress concentration after reaching a peak in the Cd5 group. In studies on potatoes [76] and tomatoes [77] of the Solanaceae family, it was found that MDA content increased under Cd stress, and the higher the Cd concentration, the higher the MDA content. Peng et al. [78] found that the MDA content of S. nigrum roots gradually increased with Cd concentration. It reached a peak at 600 μmol L⁻¹ and the content decreased at 1200 μmol L⁻¹. The MDA content of leaves gradually increased with Cd concentration and peaked at 1200 μmol L⁻¹. The MDA content of the roots was consistent with the results of the leaves of S. aculeatissimum in this study. No significant decrease in MDA content in potato leaves was found in studies on S. tuberosum [79] and S. nigrum [80] in the Solanaceae family at low concentrations of Cd stress. However, a significant increase in MDA content in the leaves was found when Cd concentrations reached 25 mg kg⁻¹ in the treated soil and 30 μMol of the nutrient solution. This could be the result of differences between plant species and concentration settings.

SOD, POD, and CAT are important antioxidant enzymes for plant adaptation to heavy metal stress [81]. They may protect the membrane system by synergistically scavenging free radicals in the plant when it is stressed [81,82]. When plants are contaminated with Cd, corresponding changes in SOD, POD, and CAT activities occur, but the effect varies in different plant species. In this study, we found that SOD and CAT both showed a trend of increasing and then decreasing at the 90th day of Cd stress, but POD changed less. This indicates that excessive ROSs may have been produced in S. aculeatissimum during this phase. These ROSs were cleared by increasing SOD activity, while H₂O₂ produced by this or other pathways was cleared by CAT. The CAT content gradually increased at high concentrations of Cd stress, indicating that CAT in S. aculeatissimum leaves had strong resistance to high concentrations of Cd stress. POD showed a gradual increase in Cd stress, probably because S. aculeatissimum alleviated the toxic effects of Cd on the organism itself by increasing POD content. As the treatment time increased, POD activity increased and SOD activity decreased, and in the leveling-off phase, among treatment groups, CAT activity decreased and peaked in the Cd4 group. This indicates that CAT in S. aculeatissimum leaves has a weaker resistance to high Cd stress with prolonged stress time. This may be due to some limitations in the Cd enrichment capacity of S. aculeatissimum itself. It showed a decrease in CAT activity under high concentrations of Cd stress.

As a more vigorous plant, S. aculeatissimum is highly enriched for Cd. In the present study, the root Cd content peaked in the Cd5 group on the 60th day of S. aculeatissimum growth with increasing Cd concentration, and the overall plant absorbed the largest amount of Cd, which is consistent with the findings of Zhou et al. [83]. The Cd content of the Cd1, Cd2, Cd3, and Cd4 groups of S. aculeatissimum roots gradually increased on the 163rd day of Cd treatment, while it decreased significantly in the Cd5 group, and the Cd content of stems, leaves, and fruit increased accordingly. This indicates that the Cd content in the lower part of the ground was transported to the above-ground part. At the seedling stage Cd5 group treatment, the Cd content in S. aculeatissimum plants was >100 mg/kg. During the fruiting period in the Cd2 group, Cd3 group, Cd4 group, Cd5 group, and Cd6 group treatments, the Cd content in S. aculeatissimum was over 100 mg kg⁻¹ and the enrichment and transport coefficients were >1. Cd hyper-enriched plants are defined as plants with a threshold value of 100 mg kg⁻¹ of Cd in the body and both bioconcentration factor and translocation factor should be greater than 1 [84,85,86], thus indicating that S. aculeatissimum is a Cd hyper-enriched plant.

The enrichment factor is used to evaluate the magnitude of the plant’s ability to take up heavy metals into its body and the translocation factor is used to evaluate the plant’s ability to transport heavy metals from the roots to the above-ground parts [87]. In this study, the transport coefficients of Cd treatment in S. aculeatissimum were all greater than 1, indicating that it was mainly enriched in the above-ground part for Cd. The enrichment coefficient was greater than 1 under Cd treatment, indicating that the plant had good enrichment of Cd and was able to absorb Cd from the environment into the plant. Ashrafzadeh et al. [88] found that the enrichment factor for Cd was greater than 1 for all the nine varieties of potatoes and only Summer Delight showed an enrichment factor less than 1 (0.95) and potatoes showed a strong enrichment capacity. Lima et al. [89] found a Cd enrichment factor of 14.02 for tomatoes in cultivated soils in southeastern Brazil. This indicates that the enrichment capacities of the different Solanaceae materials were all significantly different from each other. This study obtained a total of 25,225 high-quality genes by transcriptome sequencing and comparison with the reference genome, and 6868 differential genes were identified by Cd6 vs. CK. Annotation of these differential genes revealed a large number of gene concentrations in the GO database on functions such as regulation of biological quality and plastid and hydrolase activity. This indicates that S. aculeatissimum-related life activities are more active. However, this may also be caused by heavy metal stress on S. aculeatissimum. The six pathways of nucleic acid phosphodiester bond hydrolysis, plastid, chloroplast, thylakoid, chloroplast thylakoi, and hydrolase activity in GO enrichment were similarly annotated in studies by Li [90] and Deng [91]. These studies indicate that photosynthesis and enzyme activity play a greater role in Cd stress conditions in S. aculeatissimum. The KEGG database annotation contains more genes for global and overview maps, translation, signal transduction, transport and catabolism, immune system, and other functions. Among them, He et al. [92] also found a higher number of differential genes enriched in the translation metabolic pathway in a study of Luan seedlings remediating Cd-contaminated soil.

In the present study, we found the most differential genes enriched in the translation pathway of the Genetic Information Processing metabolic pathway in the Cd6 vs. CK group, which is consistent with the results of previous studies.

The main antioxidant systems involved include antioxidant enzymes, glutathione transferase, oleuropein lactone biosynthesis, and hydrolase activity. The nucleic acid phosphodiester bond hydrolysis was related to osmoregulatory substances and light- and respiration-related plant organ plastid, chloroplast, thylakoid, and chloroplast thylakoid pathways. Thus, it is hypothesized that S. aculeatissimum, in response to heavy metal stress, maintains intracellular redox homeostasis, controls material transport channels, and repairs damaged cell walls by regulating the expression of genes related to the antioxidant system. Thus, the expression of genes related to the antioxidant system can maintain the balance of intracellular redox status, control the material transport channels, and repair the damaged cell wall, thus resisting the stress and improving the resistance.

Among the five differentially expressed genes screened in this study, the relative expression in the stems was consistent with the trend of transcriptome changes. The trend of SaPrxs2 in the roots was not consistent with that in the stems. In a study of Cd stress in willow, Wang [93] found that the pattern of expression changes in the roots, stems, and leaves in the same gene were partially consistent, and it is speculated that it may be that the expression of these genes is not obvious in the roots. It is also possible that the above genes are not the key genes regulating heavy metal Cd stress, and the specific gene functions need to be further verified. Due to the lack of conclusive evidence of heavy metal tolerance and detoxification-related genes in S. aculeatissimum, we referred to the genes related to Cd response, transport, and detoxification in the annotated information of the S. aculeatissimum transcriptome sequence on the one hand when selecting the target genes. On the other hand, we referred to the existing literature for studies on heavy metal tolerance and detoxification-related genes in hyperaccumulator and model plants. The screened genes corresponded to heavy metal ATPase (HMA), plastocyanin proteins (PCs), peroxidases (Prxs), and ascorbate oxidase (AAO). HMA is a family of transmembrane metal transport proteins. It can pump a variety of cations across the membrane against its electrochemical effects. The metal-binding structural domain in the transmembrane helix allows binding and interaction with specific metal ions (Cd²⁺ and Pb²⁺) [94]. HMA plays a huge role in the transport and detoxification of Cd in C. annuum [95], Sedum aizoon [96], Populus trichocarpa [97], wheat [98], and rice [99]. Wu [100] found that the expression of HMA3 was higher in the lower part of sorghum treated with Cd stress than in the above-ground parts and that HMA3 was involved in the detoxification process of Cd in sorghum. In this study, SaHMA20 was found to be the down-regulated gene. Expression was suppressed in both the roots and stems of S. aculeatissimum treated in the Cd6 group. The expression in the stem was higher than that in the root. Plastocyanin (PC) is a copper-containing electron transfer protein located on the inner surface of the vesicle-like membrane [101,102]. It is involved in a variety of biological processes, including plant subdivision and recombination, photosynthesis, reproductive pollination, and stress response to the environment [103,104,105,106,107]. Phytocyanins (PCs) are plant-specific type I blue copper proteins. It is a member of the copper oxygen-reducing protein (Cupredoxins) superfamily. The PC gene in plants is involved in the process of response to other abiotic stresses, such as drought, salt damage, and metal ions, in addition to the composition of the electron transport chain [108]. Ezaki et al. [109] showed that AtBCB, a gene associated with aluminum stress in southern mustard, inhibits aluminum uptake and prevents aluminum toxicity. Wu et al. [110] found that the ENODL gene BcBCP of thick-leaved spinach chicory (Boea crassifolia) is a drought tolerance-related gene. Overexpression of this gene in tobacco improved photosynthetic efficiency and antioxidant enzyme activity in transgenic plants. In this study, SaPCs were found to be lower in Cd6 with S. aculeatissimum roots and stems than in the control group. The relative expression of this gene was consistent with the changing pattern of down-regulation information shown by SaPCs in the transcriptome. The involvement of this gene in the regulation of Cd stress has not been reported. Therefore, further validation of the function of this gene is needed.

Prxs activity is altered in plants after exposure to heavy metal stress. Wang et al. [111] found that differentially expressed genes and differentially expressed metabolites of Prxs in S. nigrum were involved in cell wall biosynthesis upon Cd stress, and the cell wall biosynthesis pathway plays a key role in the detoxification of Cd. In the present study, two Prxs-related genes, SaPrxs4 and SaPrxs2, were involved. SaPrxs4 was overexpressed in Cd6 S. aculeatissimum roots and stems. By the pattern of transcriptome changes, SaPrxs2 was overexpressed only in the S. aculeatissimum stems and less than the control in the roots. This may be related to the differences in different sites.

Ascorbic acid is an organic small molecule that resists oxidative stress and maintains the dynamic balance of redox outside the plant protoplasm. Ascorbate oxidase (AO) catalyzes the oxidation of ascorbic acid to produce dehydroascorbic acid and regulates the redox state outside the plastid [112]. Several authors have discovered that AAO responds to abiotic stresses.

Garchery et al. [113] et al. found that the inhibition of AO expression under water deficit conditions affected plant carbon transport and increased tomato yield. Batth et al. [114] found that OsAAO2 was one of the most responsive genes to stress in rice branching tissues, while OsAAO3 and OsAAO4 could show high expression in root tissues in response to salt and drought stress. ATAAO1 was highly up-regulated in aboveground and roots, while AtAAO3 showed high expression at all developmental stages. ZmAAO3 was the most stress-responsive gene observed in maize in cold and drought stresses. OsAAO2, OsAAO3, and OsAAO4 from rice, AtAAO1 and AtAAO3 from Arabidopsis, and ZmAAO3 from maize could be good candidates for breeding for resistance transgenes. In the present study, we found that the gene expression of ascorbate oxidase SaL-AO in Cd stress in S. aculeatissimum was consistent with the transcriptome results. This gene may be a heavy metal tolerance and detoxification-related gene. The involvement of this gene in the regulation of Cd stress has not been reported, and further verification of the function of this gene is needed. Based on the results of the variation of Cd content in the roots and stems of S. aculeatissimum, SaHMA20, SaL-AO, SaPrxs4, and SaPCs showed consistent trends in the expression of the S. aculeatissimum transcriptome data.

We analyzed the expression of the above four genes related to Cd transport and detoxification in different tissues of S. aculeatissimum. Although the functions of these genes in Cd tolerance and detoxification in S. aculeatissimum have not been confirmed, a large number of candidate genes were obtained through this study. It can provide a reference for further study of the functions of these genes.

5. Conclusions

Phenotypic analysis showed that cadmium tolerance of S. aculeatissimum was strong and the cadmium stress growth of S. aculeatissimum seedlings was not inhibited. At 50 mg kg⁻¹ Cd stress, plant growth can be normal; the same concentration in many plants could result in growth inhibition or death. S. aculeatissimum can be grown in Cd-contaminated soil and participate in soil remediation. It was clarified that different organs of S. aculeatissimum enriched and transported cadmium content differently. In the early stage of the experiment, the order of organs absorbing cadmium content was root > stem > leaf. At the later stage of the experiment, the highest Cd uptake was found in roots and the lowest in immature fruit under high Cd treatment. The analysis of physiological indices revealed that S. aculeatissimum maintained high MDA content and SOD, POD, and CAT activities in the early stage of Cd stress. The toxicity of Cd to S. aculeatissimum was alleviated by increasing SOD, POD, and CAT activities. Transcriptome analysis identified 6868 DEGs under Cd stress, of which 4597 were up-regulated and 2271 were down-regulated. Combining GO functional annotation and KEGG functional annotation, we conclude that the metabolic pathways of heavy metal ATPase (HMA), plastocyanin protein PCs, Prxs, and AAO may play a role in the tolerance and uptake of Cd ion enrichment in S. aculeatissimum. The ATP-binding cassette (ABC) family of transporter proteins, the glutathione S-transferase (GST) family, and the isoprenylated phycobiliprotein (HIPP) family of transporter protein genes slowed down the toxic effects of Cd on S. aculeatissimum and were involved in the transport of Cd ions. In summary, S. aculeatissimum is not only highly tolerant to Cd²⁺ but also has a complex set of molecular response mechanisms. Although the test subjects in this study are only individuals of this species, it is enough to prove that S. aculeatissimum is a candidate species for heavy metal remediation in the Solanaceae family with great potential.

DEGs that may be involved in regulatory pathways were also identified in this study. This is the first systematic study of cadmium tolerance and enrichment of cadmium in Solanaceae. it may provide important insights into the complex mechanisms of tolerance and detoxification in plants. It provides theoretical guidance for the resistance to heavy metal stress in S. aculeatissimum.