The Alleviating Effect of Brassinosteroids on Cadmium Stress in Potato Plants: Insights from StDWF4 Gene Overexpression

Abstract

The potato is the fourth largest cultivated crop worldwide. Soil cadmium (Cd) pollution poses a significant threat to crop growth. Brassinosteroids (BRs) play a significant part in enhancing plant resistance against abiotic stresses. The DWF4 (dwarf4) gene is one of the rate-limiting enzyme genes involved in the synthesis of BRs. This study employed seedlings of transgenic potatoes overexpressing the StDWF4 gene (OE) and wild-type (WT) potatoes to clarify their alleviating effect on Cd stresses. The differences in phenotype, ultrastructure, physiological indicators, and plant hormone levels of Cd²⁺-treated potatoes were analyzed. The molecular mechanism of potatoes’ response to Cd²⁺ stress was revealed by transcriptomics. Results showed that the dry weight, fresh weight, plant height, root length, and stem diameter of OE potatoes under Cd stress were significantly higher than those of WT potatoes. Ultrastructural analysis revealed that the mitochondria, cell walls, and cell membranes of WT were more fragile than those of OE under Cd stress. The Cd²⁺ concentration in OE was always lower than that in WT, and both concentrations increased gradually as the duration of Cd²⁺ treatment was prolonged. The 24-epibrassionlide (EBL) content in OE was higher than that in WT. RNA-seq analysis manifested that the gene expression levels of OE and WT plants changed significantly under Cd²⁺ treatment. The differentially expressed genes (DEGs) were primarily connected to the moderation of the metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and plant hormone signal transduction. These findings indicated that overexpression of the StDWF4 gene in potatoes enhanced their alleviating effect on Cd stresses.

1. Introduction

When heavy metals, including Cd (cadmium), accumulate in the soil above a certain threshold, they can inhibit or abnormalize plant growth, resulting in symptoms such as fading green leaves, slowed growth, decreased biomass, and, in severe cases, even plant death [1,2]. Researchers have elaborated on the impact of heavy metals on plants at the phenotypic, metabolic, and molecular levels. With increased Cd stress duration, plant root growth is delayed, and lateral root growth is inhibited, leading to wilting and drying of the plant [3]. Leng et al. (2020) found that Cd treatment severely inhibited root development in mung bean seedlings. Chlorophyll content, which is essential for photosynthesis in plants, serves as an important indicator of heavy metal damage [4]. Studies have shown that chlorophyll content in maize declines significantly with increasing Cd concentration [5]. High Cd concentrations lead to the generation and excessive buildup of reactive oxygen species (ROS) in the cytoplasm, causing lipid peroxidation and the further production of malondialdehyde (MDA) [6,7]. BRs can bind to membrane proteins to scavenge a large amount of ROS produced by Cd stress, thereby alleviating the poisonous impacts of Cd on plants [8]. Additionally, Cd uptake through the plant’s root system alters endogenous hormone levels, enhancing the plant’s resistance to Cd stress by activating its resilient immune system [9,10]. Research has shown that the endogenous plant hormones BRs, ABA, GA₃, IAA, and cytokinin (CTK) change significantly under Cd stress [11].

BRs are indispensable in plant growth and development and adaptation to environmental adversity, such as drought, salt, and heavy metals [12,13,14]. BRs overcome heavy metal stress by increasing photosynthetic capacity, promoting the antioxidant system, promoting gene expression, and interacting with other phytohormones [15,16].

Plants under Cd stress suffer from growth inhibition and weakened metabolism [17]. EBL is an important class of biologically active BRs. It can activate nitric oxide (NO) production to alleviate cadmium stress in pepper plants [18]. Ahanger et al. reported that BRs and kinetin improved IAA, ABA levels, antioxidant enzyme activities, and enhanced Cd tolerance in tomatoes [19].

Furthermore, BRs contribute to the regulation of specific genes, thus boosting the resistance of plants exposed to heavy metal stress [20]. The DWF4 gene is responsible for producing C-22 hydroxylase, which serves as the initial rate-determining step in the BRs biosynthetic pathway of sterol synthesis in rapeseed and belongs to the CYP450 gene family [21]. Researchers found that overexpression of PscCYP716A1 improved the accumulation and transport capacity of Cd in poplar [22]. DELLA protein interacts with BRZ1 and promotes the accumulation of ROS to resist heavy metal stress [23]. Shi et al. (2016) discovered 509 differentially expressed genes in Salix integra after Cd stress, all of which were related to Cd detoxification, and many of them encoded heavy metal ATPases (HMAs) and ABC transporter proteins [24].

The potato (Solanum tuberosum L.) is the fourth largest cultivated crop globally. In our research, we obtained potato plants that overexpressed the StDWF4 gene, and their salt tolerance was preliminarily identified through morphology and physiological and biochemical indices [25]. However, the roles of the StDWF4 gene in alleviating the detrimental effects of heavy metal stress and its underlying mechanism have not yet been studied.

Therefore, in this study, we utilized wild-type (WT) and StDWF4-overexpressing (OE) potato plants under Cd treatment. We observed the phenotypic and ultrastructural changes of potatoes under Cd stress. The Cd²⁺ content, MDA content, antioxidant enzyme activities, chlorophyll, and hormone concentrations were measured to clarify the physiological response of potatoes to Cd stress. Transcriptome analysis of potato materials under different Cd treatments was conducted to elucidate the molecular mechanism by which StDWF4 gene overexpression responds to Cd stress and to emphasize the importance of BRs in potatoes alleviating detrimental effects on heavy metal stress.

2. Materials and Methods

2.1. Plant Materials

In our prior research, we secured a transgenic OE line that overexpressed the StDWF4 gene and the wild-type ‘Zihuabai’ potato plants (WT) [25]. The stable expression level of the StDWF4 gene in OE potatoes was 3.18 times greater than that in WT, as detected by qRT-PCR. The seedlings were propagated in vitro in a liquid Murashige-Skoog (MS) medium and maintained in a light incubator with 16 h day (25 °C)/8 h night (18 °C) cycle, a light intensity of 6000 Lx, and environmental conditions at 80% relative humidity (RH). Seedlings that were 30 days old and of uniform growth were used for subsequent stress treatments. The original liquid medium was discarded and replaced with fresh MS liquid medium containing 100 μmol L⁻¹ CdCl₂·2.5H₂O for continued culture. Leaves and roots were separately collected at 0 h, 24 h, and 48 h treatments. All samples were collected at 9:00 p.m. Phenotypic analysis was performed on fresh material. Chlorophyll content was determined using leaves, while the remaining indicators were determined using roots. Roots of plants were dried at 80 °C for the determination of Cd²⁺ content, and the remaining samples were stored in the refrigerator at −80 °C for subsequent experiments. Ultrastructural observations were made of the roots of WT and OE lines that underwent treatment with 100 μmol L⁻¹ CdCl₂·2.5H₂O at 0 h, 48 h, and 96 h.

2.2. Observation of Roots Ultrastructure

Roots ultrastructure was analyzed following the protocols of [26].

2.3. MDA Content and Enzymatic Antioxidant Activities Determination

Determination of the MDA content and antioxidant enzyme activities was based on our previous techniques [27].

2.4. Ion Concentrations Detection

The Cd^{2 +} content in potato plants was determined according to Liu et al. [28]. The roots of both WT and OE potato plants were killed at 105 °C and then dried at 80 °C. A total of 0.1 g of each sample was digested with 10 mL of nitric acid at 120 °C until the tissues became transparent. Subsequently, the samples were fixed to 25 mL, and the Cd²⁺ content was detected using inductively coupled plasma emission spectroscopy (ICP-MS-7800, Agilent Technologies, Inc., Santa Clara, CA, USA).

2.5. Identification and Quantification of Plant Hormones

0.5 g of potato root tissue was rapidly ground into a powder with liquid nitrogen. The extraction and determination of plant hormones were carried out according to Liu et al. [29] and detected using liquid chromatography-tandem mass spectrometry (HPLC-MS, Agilent 1290, Santa Clara, CA, USA)/MS (QTRAP 6500, AB SCIEX, Marlborough, MA, USA).

2.6. Isolation and Analysis of Total RNA by Illumina Sequencing

Appropriate quantities of WT and OE potato roots were taken and ground to a powder, which was used to extract total RNA. The RNA quality was evaluated on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and verified using agarose gel electrophoresis. Then, it was employed for subsequent library construction. After isolating the total RNA, mRNA was enriched, subsequently cut into short fragments, and converted into cDNA using the reagent Kit (NEB #7530, New England Biolabs, Ipswich, MA, USA). Purified double-stranded cDNAs underwent end-repaired and were ligated to an adapter. The ligation reaction was purified and amplified by PCR. The whole sequencing process was conducted using an Illumina Hiseq 2500. The data have been uploaded to the SRA database of the National Center for Biotechnology Information (NCBI) (PRJNA1020451). The datasets generated during the current study are publicly available at NCBI, with BioSample accession: SAMN37523128 to SAMN37523145 and SRA accession: SRR26203916 to SRR26203933 (https://submit.ncbi.nlm.nih.gov/subs/bioproject/SUB13860213/overview, accessed on 5 March 2024).

2.7. Filtering of Sequences, Sequence Assembly, and Analysis of Unigene Expression

Raw data were filtered to remove sequences with >10% unknown nucleotides and >40% low-quality bases (Q-value ≤ 10) using Trimmomatic (v0.39) to ensure data integrity. High-quality reads were then mapped to the reference sequence (PGSC DM v4.03; Potato Genome Sequencing Consortium, 2021) using the HISAT2 aligner (v2.2.1), and transcript abundance was examined according to Bray et al. using StringTie (v2.1.7) [30]. Clean reads were assembled using Trinity (v2.13.2), according to Grabherr et al. [31]. Principal component analysis (PCA) was used to estimate the repeatability between samples. Gene expression levels were calculated using the fragments per kilobase of exon per million mapped reads (FPKM) [32]. In this study, a criterion of |FC| (|fold-change|) ≥ 2 and p ≤ 0.05 between samples were used to identify DEGs using the DESeq2 package (v1.36.0, https://bioconductor.org/packages/release/bioc/html/DESeq, accessed on 5 March 2024). Venn diagrams were plotted by Venny2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html, accessed on 5 March 2024). Heatmaps of the DEGs were plotted using Python Seaborn (http://seaborn.pydata.org/, accessed on 5 March 2024) and TBtools (v2.119), respectively [33].

2.8. Annotation and Clustering Analysis of DEGs

Annotation of the unigenes was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) with an e-value ≤ 10⁻⁵, respectively [34]. The gene cluster was assessed through Molecular Evolutionary Genetics Analysis (MEGA) 7.0.

2.9. Weighted Gene Co-Expression Network (WGCNA) Analysis

All differential genes were analyzed using the WGCNA algorithm in the R language package. The co-expression correlation matrices of all genes were calculated, and the neighbor-joining functions were constructed based on the gene network. Correlations between modules, sample expression patterns, individual module TFs, and module gene expression patterns were analyzed.

2.10. Assessment of Transcriptome Accuracy Through qRT-PCR

Fifteen genes were randomly selected for qRT-PCR analysis to authenticate the transcriptome results. The StEF-1α gene (GenBank accession No. AB061263) was used as an internal standard. RNA samples were purified via a reagent Kit (TIANGEN, Beijing, China, DP452). The specific primers were synthesized by Sangon Biotech (Shanghai, China) (Table S1). First-strand cDNA was synthesized using a reagent Kit (TIANGEN, KR118-03). The qRT-PCR gene expression was conducted using a reagent Kit (TIANGEN, FP205-03). The experiments were conducted on a LightCycler (LightCycler96 Real-Time PCR, Roche, BSL, Switzerland) according to the following protocol (2 min at 95 °C and 40 cycles of 5 s at 95 °C, 15 s at 60 °C, 30 s at 72 °C and 95 °C for 15 s, 60 °C for 1 min). The gene expression levels were then quantitated using the 2^−ΔΔCt quantitative method [35].

2.11. Statistical Analysis

One-way ANOVA and Duncan’s multiple-range tests were used for statistical analysis. All experiments were analyzed using SPSS software (v22.0, SPSS, Inc., Chicago, IL, USA), at a statistically significant level (p ≤ 0.05). All measurements were replicated three times.

3. Results

3.1. Phenotypic Comparison of the Potatoes Under Cd Stresses

The phenotypes of WT and OE potatoes changed obviously as the duration of Cd²⁺ treatment increased (Figure 1). The dry and fresh weights of both WT and OE potatoes under Cd stress initially increased and then decreased. The OE potatoes exhibited a significant increase in both dry and fresh weights when compared to the WT potatoes, and the differences between them were statistically significant (

Table 1. Effects of Cd²⁺ stress on fresh and dry weight between WT and OE potatoes.

Treatment Time (h)	Leaf Fresh Weight (g/plant)		Leaf Dry Weight (g/plant)
	WT	OE	WT	OE
0	0.869 ± 0.054 c	1.309 ± 0.037 ab	0.086 ± 0.009 ab	0.096 ± 0.011 ab
24	1.036 ± 0.220 bc	1.592 ± 0.205 a	0.115 ± 0.027 ab	0.192 ± 0.023 a
48	0.709 ± 0.122 c	1.469 ± 0.183 a	0.072 ± 0.013 c	0.136 ± 0.020 b

Note: Significant differences among means for Cd²⁺ stress treatments were determined using Duncan’s test. The lowercases following the datum in each column represent statistically significant difference at p < 0.05. Bars = standard errors (n = 9, i.e., 3 biological repeats × 3 technical repeats), same as below.

). The results showed that Cd stress affected the plant height, root length, and stem thickness of both WT and OE potatoes to different degrees. With the prolongation of Cd stress, the plant height of both WT and OE potatoes first decreased and then increased, and the plant height, root length, and stem thickness of OE were significantly greater compared to those of the WT (

Table 2. Effects of Cd²⁺ stress on plant height, root length, and stem diameter of WT and OE potatoes.

Treatment Time (h)	Plant Height (cm)		Root Length (cm)		Stem Diameter (mm)
	WT	OE	WT	OE	WT	OE
0	11.54 ± 0.350 b	14.22 ± 0.561 a	9.90 ± 0.503 bc	11.68 ± 0.493 ab	1.36 ± 0.084 bc	1.56 ± 0.063 ab
24	9.42 ± 0.524 c	11.98 ± 0.831 b	9.06 ± 0.322 c	12.24 ± 0.684 a	1.16 ± 0.088 c	1.59 ± 0.078 ab
48	12.74 ± 0.567 ab	14.08 ± 0.546 a	9.42 ± 0.706 c	11.56 ± 0.555 ab	1.18 ± 0.030 c	1.71 ± 0.138 a

Note: Significant differences among means for Cd²⁺ stress treatments were determined using Duncan’s test. The lowercases following the datum in each column represent statistically significant difference at p < 0.05. Bars = standard errors (n = 9, i.e., 3 biological repeats × 3 technical repeats), same as below.

).

3.2. Cell Ultrastructure Changes of WT and OE Potato Roots Under Cd Stresses

Figure 2 presents the ultrastructure of potato roots without Cd stress treatment. WT potato roots (Figure 2a,b): Cellular architecture remains intact with uniformly thick cell walls and tightly appressed plasma membranes, showing no observable plasmolysis. Vacuoles maintain structural integrity. Even chromatin distribution with the intact nuclear envelope. There are a few short-fusiform chloroplasts containing intact starch grains. No swelling is observed, and the matrix remains homogeneous with a well-organized cristae. Ribosome attachment is sparse, and there is no significant dilation. Occasional lipid droplets are present in the cytoplasm. OE potato roots (Figure 2e,f): Similarly intact cellular structures with relatively uniform cell walls and tightly adhered plasma membranes are seen, with an absence of plasmolysis. Chromatin distribution is uniform, with an intact nuclear membrane. The morphology is well-preserved without swelling, and the matrix is homogeneous with a predominantly regular cristae arrangement. No apparent dilation and limited ribosome association are observed.

The cell ultrastructure of potato roots under Cd stress for 96 h has been shown in Figure 2. In Figure 2c,d, it is evident that the cells of the WT potato root system were heavily damaged. They exhibited heavily edematous and sparse cytoplasm, more intact cell walls, broken and lysed cell membranes, and moderate plasma-wall separation locally. The nucleus had sparse chromatin, an intact nuclear membrane, and a widened nuclear perinuclear space. The mitochondria in the cytoplasm were heavily swollen and enlarged, with uneven and localized lysis of the matrix and numerous broken and missing cristae. The rough endoplasmic reticulum (RER) was mildly expanded, with ribosomes shed from its surface. Observing Figure 2g,h, one can notice that the cells of OE potato roots were moderately damaged. They showed moderately edematous and sparse cytoplasm, a more uniform thickness of the cell wall, close apposition between the cell membrane and the cell wall, and no obvious plasma-wall separation. The structure of the vesicle membrane remained intact. The chromatin of the nucleus was uniformly distributed, and the nuclear membrane was intact. The mitochondria were not swollen, the matrix was uniform, and the cristae were slightly broken and shortened. The RER was not dilated, and a few ribosomes were attached to the surface.

3.3. Physiological Response of WT and OE Potatoes to Cd Stress Conditions

The leaf chlorophyll content in OE potatoes was considerably enhanced compared to WT under different degrees of Cd²⁺ treatments. At 48 h of Cd stress, the leaf chlorophyll content in WT and OE potatoes decreased significantly to 59.95% and 46.41% of the control, respectively, and these differences were significant (p < 0.05) (Figure 3A). The SOD, POD, and CAT activities of OE potato roots were higher than those of WT under different Cd stress treatment times (Figure 3D). Specifically, the SOD activities of WT and OE potato roots in the controls were 36.10% and 44.88% of those at 24 h of Cd stress and 46.66% and 54.08% of those at 48 h of Cd stress, respectively, showing significant differences (p < 0.05). The POD activities of WT potato roots displayed a rise and subsequent fall trend with the extension of Cd stress time, reaching a peak of 30.33% at 24 h. In contrast, the POD activities of OE potato roots gradually increased with the prolongation of Cd stress and reached their peak at 48 h, which was 45.39% of the control. The CAT activities of both WT and OE potato roots continued to increase as the degree of adverse stress deepened, with OE potatoes showing CAT activities 2.43 times higher than those of untreated WT potatoes. The MDA content in both WT and OE potato roots increased gradually, reaching its highest levels at 48 h of Cd stress. Specifically, the MDA content in WT and OE potato roots was 2.01 and 1.61 times higher than that of the control, respectively (Figure 3B).

3.4. Analysis of Cd²⁺ Concentrations

The Cd²⁺ content in WT and OE potato roots experienced a notable rise with the prolongation of Cd stress time (p < 0.05). However, under 24 h and 48 h of Cd²⁺ stress, the Cd²⁺ concentrations in the roots of OE decreased by 3.37% and 12.60%, respectively, compared with those of WT. The Cd²⁺ content in WT potato roots was consistently higher than that in OE potato roots under different degrees of Cd stress (Figure 3C).

3.5. Changes in Phytohormone Levels

Analysis of EBL content revealed distinct dynamic patterns in WT and StDWF4-OE potatoes under Cd stress. At 0 h Cd stress, EBL levels in OE potatoes were significantly higher than in WT (p < 0.05). Specifically, in both WT and OE potatoes, EBL content kept near-basal levels at 24 h (vs. 0 h control), and the former exhibited a sharp elevation by 48 h. However, at 48 h, OE potatoes exhibited a slight decline in EBL, falling below WT levels (Figure 3E). The EBL content in WT potato roots fell and then rose with the prolongation of Cd stress, reaching 4.40% and 12.81% of the control at 24 h and 48 h, respectively. In contrast, the EBL content in OE potato roots increased and then decreased, with values of 3.50% and 4.48% of the control at 24 h and 48 h, respectively. Therefore, it may be concluded that BRs synthesis and signal transduction-related genes upregulated and participated in the regulation of the Cd stress responses.

As can be seen in Figure 3F–I, the endogenous hormone content in both WT and OE potato roots changed under different Cd stress treatment times. Specifically, the IAA content in OE was 1.89, 4.12, and 2.48 times higher than in WT potato roots at 0 h, 24 h, and 48 h of Cd stress, respectively. With the prolongation of Cd stress, the 6-BA content in the root systems of both WT and OE gradually decreased. However, at 0 h and 48 h of Cd stress, the 6-BA content was higher in OE potatoes than in WT. The ABA content in the root system of OE potatoes showed a notable increase at 24 h of Cd stress, whereas it remained relatively unchanged in WT under different treatments. The GA₃ content in WT and OE potato roots showed insignificant changes.

3.6. Analysis of Transcriptome Sequencing and Comprehensive Gene Expression Profiling

To reveal the molecular mechanism of Cd alleviating effect in OE potatoes, transcriptome sequencing was performed in transgenic and WT potatoes after 0 h, 24 h, and 48 h of Cd stress. As shown in Table S2, the raw reads obtained from each treatment were 36.65–49.78 million, with Q20 ratios exceeding 97.38% and Q30 ratios exceeding 92.61% for all treatments. The GC content fell within the range of 42.32% to 42.81%. This suggests that the sequencing library possessed high quality and met the requirements for further analysis. Additionally, 87.77–89.22% of the clean reads were successfully mapped to the reference genome.

The correlation between biological replicates is an important indicator for assessing the reliability of the samples and the rationality of their selection. The correlation between the three biological replicates is shown in Figure 4A, where the biological replicates of all the samples are clustered together. The correlation coefficients of the samples within each group are shown in Figure 4B, indicating a high positive correlation among the samples. These results confirm the transcriptome data was of a high standard.

3.7. Differentially Expressed Gene Analysis

The DEGs of different comparison groups were analyzed by TBtools software and shown in Figure 5. The number of upregulated (UR) genes in the comparison groups of WT-0-vs-OE-0, WT-24-vs-OE-24, and WT-48-vs-OE-48 was 2366, 1414, and 1499, respectively. The number of downregulated (DR) genes was 2595, 3362, and 2949, respectively. A total of 793 UR and 1398 DR genes were found in the combined groups of WT-0-vs-OE-0, WT-24-vs-OE-24, and WT-48-vs-OE-48 (Figure 5E,F).

3.8. GO and KEGG Analysis of DEGs in OE and WT Potatoes

According to the GO annotation, the DEGs belonged to three major categories: biological processes, cellular components, and molecular functions (Figure S1). A total of 19,507 DEGs were enriched into the GO secondary classification in the comparisons of WT-0-vs-WT-24 and WT-0-vs-WT-48 (Figure S1A), and a total of 28,775 DEGs in the comparisons of OE-0-vs-OE-24 and OE-0-vs-OE-48 (Figure S1B). As shown in Figure S1C–E, the total number of DEGs in the comparisons of WT-0-vs-OE-0, WT-24-vs-OE-24, and WT-48-vs-OE-48 was 22,459 (9650 UR and 12,809 DR genes), 22,106 (5350 UR and 16,756 DR genes) and 20,196 (5707 UR and 14,489 DR genes), respectively.

Figure 6A shows significant variations in the expression levels of randomly selected plant hormone-related, Cd²⁺ transporter, and secondary metabolites biosynthesis genes between OE and WT plants, as quantified by FPKM values. The KEGG analysis manifested that these genes mainly participated in the regulation of metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and plant hormone signal transduction (Figures S2–S4).

3.9. TFs in DEGs of Potato Roots Under Cd Stress

Under Cd stress, 47 transcription factors (TFs) families comprising a total of 1588 individual TFs exhibited stress-specific expression patterns in the WT and OE potato. ARR-B, AP2-EREBP, and bHLH families had the greatest amount of DEGs, with 348, 189, and 158, respectively. The TUB, SAP, S1Fa-like, and CPP families had the least, with only one DEG each (Figure 6B).

The top ten families with the highest number of TFs were analyzed. The results showed that, at 24 h exposure to Cd stress in WT potatoes, the transcription factor ARR-B had the highest percentage of 22.03%, followed by the AP2-EREBP and bHLH families, with percentages of 12.99% and 10.73%, respectively. The percentages of the ARR-B, AP2-EREBP, and bHLH transcription factor genes gradually decreased, accounting for 19.74%, 14.47%, and 9.21%, respectively. The transcription factors with the highest proportions at 24 h and 48 h of Cd stress in OE potatoes were the same as those in WT potatoes, but the number of genes in the transcription factor families was higher than in WT potatoes. Without stress, the ARR-B transcription factor family had the highest percentage of 19.60%, followed by the AP2-EREBP and bHLH families in WT and OE potatoes. The variations in TF percentages under 24 h of stress were insignificant compared to those at 0 h. At 48 h of Cd stress, the ARR-B transcription factor family had the highest percentage of 19.65%, followed by the bHLH and WRKY families, with percentages of 12.66% and 10.48% (Table S3).

3.10. DEGs Related to Phytohormone Signaling of Potato Plants Responded to Cd Stresses

The WT_0_vs_OE_0 group exhibited a total of 4961 DEGs, of which 47.7% were UR genes and 52.3% were DR genes. With the aggravation of Cd²⁺ stress, the number of DEGs decreased in both OE and WT potatoes. Under 100 μmol/L CdCl₂·2.5H₂O, the number of DEGs in OE and WT potatoes were as high as 4322 and 2731 at 24 h and decreased to 3953 and 2472 at 48 h Cd²⁺ treatment, respectively (Figure 5G).

We conducted a KEGG analysis on the DEGs of WT and OE potatoes, focusing on the top 20 pathways. Notably, the highest enrichment was observed in the biosynthesis of secondary metabolites and metabolic pathways in WT and OE plants at 24 h and 48 h of Cd²⁺ stress (Figures S3 and S4).

Analysis of DEGs associated with phytohormone signaling after Cd stress in WT and OE potatoes revealed that 57 DEGs were screened in WT potatoes, with 38 upregulated and 19 downregulated expressions (Table S4). In OE potatoes, 72 signaling-related DEGs were screened, with 31 UR and 41 DR DEGs (Table S5). Among these, 28 genes functioned together in WT and OE potatoes (Figure 6C).

3.11. WGCNA Analysis of OE and WT Potatoes Under Cd Stresses

WMCNA was used to identify key genes. By grouping genes with similar expression patterns across samples, 19,857 genes were classified into 19 modules (Figure 7A). The outcomes of the correlation analysis among the modules are shown in Figure 7B. The p-value for assessing the correlation between any two modules was determined using the student’s t-test, with a lower p-value signifying a greater degree of similarity between them. Additionally, to identify modules that were significantly correlated with a particular sample for subsequent studies, a sample expression pattern analysis was performed (Figure 7C). The TFs in each module were statistically analyzed to reveal the regulatory mechanisms of TFs on Cd stress in OE and WT potatoes (Figure 7D). Among them, the light-green module contained the highest number of TFs (216). We further analyzed the top 20 KEGG-enriched pathways. Based on the KEGG annotation analysis, metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction exhibited enrichment in the dark orange 2 module. On the other hand, metabolic pathways, biosynthesis of secondary metabolites, and phenylpropanoid biosynthesis were enriched in the light green module (Figure 7E,F).

3.12. qRT-PCR Validation

Fifteen hormone-related DEGs were confirmed through qRT-PCR. Among these, four were important in the biosynthesis and signaling pathway of BRs (CPD, CYP85A1, DWF4 and BZR1), two were ETH-related expressed genes (ERF4 and EIN3), two were CTK-related expressed genes (CKX6 and LOG8), one was GA₃-related expressed gene (GIDIB), one was IAA-related expressed gene (SAUR36), two were ABA-related expressed genes (PYL4 and PYL8) and three were Cd²⁺ stress resistance-related genes (ABCB1, PCR2 and HMA2) from both the OE and WT plants. The expression profiles of the majority of the selected genes aligned with the outcomes of RNA-sequencing (RNA-seq), thereby affirming the credibility of the transcriptome data (Figure 8).

4. Discussion

It is well-known that BRs can alleviate environmental stresses on plants [36,37]. In our prior research, we discovered that overexpressing the StDWF4 gene can markedly improve the salt tolerance of potatoes [25]. However, the regulation of adversity by BRs is a highly complex process [38]. The molecular mechanisms by which BRs regulate Cd stress need to be thoroughly investigated.

Plants under heavy metal stress suffer from growth inhibition and weakened metabolism [39]. BRs are principal in alleviating heavy metal stress. It has been found that BRs can bind to membrane proteins to scavenge the large amounts of ROS produced by Cd stress, thereby alleviating the side effects of Cd on plants [8]. EBL is an important class of biologically active BRs [40]. Many studies have shown that exogenous BRs, including EBL, have significant effects on phenotypes, physiological and biochemical indices, and molecular levels in plants under adversity stress, including heavy metal stress [41,42]. EBL has also demonstrated its ability to enhance the Cd stress tolerance of Triticum aestivum [3], pepper [19], Lycopersicon esculentum [43], Raphanus sativus [44], etc.

Researchers also found that heterogeneous overexpression of PeDWF4 in tobaccos can increase the endogenous brassinolide content, thereby enhancing heavy metal stress resistance by increasing the content of proline and soluble protein, as well as SOD and POD activities and chlorophyll content [45]. Also, the PscCYP716A1 gene, when overexpressed in poplar, significantly improved endogenous BRs content and enhanced cadmium accumulation and transport capacity [22]. The BRs receptor gene BRI1 upregulates the expression of transcription factors, leading to increased levels of endogenous BRs, which in turn alleviates heavy metal stress [46,47]. The interplay between the DELLA protein and BRZ1 improves the activities of ROS-scavenging enzymes, leading to improved heavy metal stress resistance [23]. The interaction of BRs-mediated transcription factors with other phytohormones under heavy metal stress has also been studied. Nevertheless, the relationship between the content of endogenous BRs and the ability of plants to tolerate cadmium, as well as the underlying mechanism, needs further investigation.

Therefore, we concentrated on the content of endogenous BRs, Cd²⁺ concentration, and transcription levels to elucidate the regulating mechanism of BRs in Cd alleviating effects in transgenic and WT potatoes.

4.1. Physiological Levels Response to Cd Stress of OE and WT Plants

Chlorophyll is an essential pigment for the absorption and conversion of light energy during photosynthesis and serves as an indicator of photosynthesis strength. The photosynthesis activity was enhanced by the application of BRs in rice to resist the adverse effects of cadmium [48]. Heavy metal Cd stress affects enzymes involved in photosynthetic pigment synthesis, induces stomatal closure, disrupts chlorophyll synthesis, and ultimately leads to the weakening of the plant’s photosynthetic capacity [49]. Therefore, to a certain degree, plant chlorophyll content can reflect the degree of damage plants suffer under Cd stress. Studies have found that Cd stress inhibits photosynthetic activity and chlorophyll synthesis in spinach [50]. In this study, we observed that the chlorophyll content in both WT and OE potato leaves gradually decreased as Cd stress levels increased. However, at different treatment times, the chlorophyll content in OE potato leaves was consistently elevated compared to that in WT leaves, indicating that while Cd stress-induced damage to the chloroplast structure of both WT and OE potatoes, the overexpression of StDWF4 in potatoes was able to overcome the negative influence of Cd stress on photosynthesis in potato leaves to a certain extent.

When plants are under stress, excessive ROS are generated in their cells. When the ROS content exceeds a certain threshold, the antioxidant system in plants is activated to resist the effects of stress [51]. SOD, POD, and CAT work together to construct a full antioxidant network [52]. Dai et al. found that the SOD and POD activities of mulberry leaves under Cd stress increased and then decreased with the increase in Cd concentration [53]. Faizan et al. observed that SOD, POD, and CAT activities rose significantly in rice under Cd stress [54]. In this study, the SOD and CAT activities of WT potatoes showed a gradual increase with the extension of stress, while the POD activity showed increasing and then decreasing with the extension of Cd stress. The SOD, POD, and CAT activities of the root system of the OE potato all escalated gradually with the extension of Cd stress. Since the root system is the first to encounter Cd stress, which subsequently generates a large amount of ROS, it activates the activities of antioxidant enzymes as a way to remove ROS and reduce the damage caused by the damage sustained from Cd stress on potatoes. Furthermore, the SOD, POD, and CAT activities of OE potato roots were higher than those of WT under different Cd stress treatment times. This is consistent with the results reported by Li et al. [55], which indicate that the antioxidant enzyme activities of Hibiscus syriacus significantly increased under Cd stress.

The MDA content in plants under adversity stress can reflect the electrolyte leakage due to altered membrane permeability and the peroxidative damage suffered by the plant [56]. Cd stress leads to peroxidation of the plasma membrane, and therefore, high concentrations of Cd induce the production of large amounts of MDA [57]. It has been found that with increasing concentration of the heavy metal Cd, the MDA content increases, and the degree of membrane permeability damage intensifies. In the present study, when compared with the control, the MDA content in both WT and OE potato root systems was elevated with increasing Cd stress levels. However, the MDA content in WT roots was higher than that in OE potatoes, which may be due to the fact that OE potatoes are more Cd-tolerant than WT under Cd stress. The OE potatoes mitigated injuries caused by Cd stress to the membrane lipid system by boosting the function of antioxidant enzymes, resulting in a decreased MDA content. Han et al. also found that MDA content in oilseed rape was significantly elevated, and the degree of membrane lipid peroxidation increased at Cd stress concentrations of 0.5 and 6 mg/kg [51].

The enrichment of DEGs in the ‘biosynthesis of secondary metabolites’ (e.g., phenylpropanoids, flavonoids) directly correlates with enhanced antioxidant capacity in OE plants. For instance, CHS, ANS, and DFR upregulated in OE roots (Figure 6A), driving flavonoid synthesis. These metabolites scavenge ROS [58], reducing oxidative damage and MDA accumulation (Figure 3B). Concurrently, DEGs in ‘phenylpropanoids metabolism’ (e.g., PAL5) were induced in OE (Figure 6A), boosting enzymatic ROS detoxification and aligning with elevated SOD/POD/CAT activities (Figure 3D).

Plant roots absorb Cd from the soil and accumulate it, and different plants respond differently to Cd uptake and accumulation [59]. It was found that the Cd²⁺ content in plants increased with the Cd concentration in the environment [60]. In this study, the Cd²⁺ content in WT and OE potato roots showed a significant increase with the extension of Cd stress time. At different Cd stress treatment times, the Cd²⁺ content in OE was always higher than in WT, indicating that OE potatoes have a stronger Cd alleviating effect than WT. Liu et al. treated two types of tobacco at a Cd stress concentration of 50 μmol L⁻¹. The Cd²⁺ content in the root system of the Cd-sensitive tobacco was higher than that in the Cd-tolerant type. As the Cd-tolerant tobacco accumulated less Cd, the ultrastructural damage to its root system was also lower than that of Cd-sensitive tobacco [61]. This aligns with the observations made by Wang et al. in Kentucky bluegrass as well [62]. Charfeddin et al. found that, with the prolongation of the time of Cd stress, the Cd²⁺ content in wild-type potatoes was higher than that in transgenic potatoes [63]. This was attributed to the overexpression of StDREB1 and StDREB2 in transgenic potatoes, which increased their tolerance to Cd in the culture medium.

Therefore, we speculated that the overexpression of StDWF4 in potatoes enhanced the chlorophyll content and the enzymatic activities of antioxidants while decreasing the content of MDA and Cd²⁺, thus triggering the Cd alleviating effect in OE potatoes.

4.2. Phytohormone Levels Regulation of Cd Stresses in OE and WT Plants

EBL, a highly active synthetic analog of BRs, plays a crucial role in a range of developmental processes, encompassing the promotion of cell elongation and division, as well as root development. It is intricately involved in the modulation of numerous stress responses [64]. BRs can regulate the intracellular absorption of ions in plant cells and significantly decrease the accumulation of heavy metals. They can also bind to membrane proteins to scavenge excess ROS produced, thus mitigating the side effects of heavy metals on plants [36,37]. Exogenous application of EBL led to changes in the levels of protein, proline, and MDA, the activities of antioxidant enzymes, as well as the expression patterns of genes in BRs synthesis and those responsive to heavy metals, enabling rice to resist heavy metals [65].

In this study, compared to the WT, the transgenic plants exhibited an elevated EBL content, as shown in Figure 3E. Consequently, the biosynthesis and metabolism of BRs were modulated in the OE potatoes. The overexpression of the PtoDWF4 gene led to insignificant variations in BR content in approximately half of the transgenic lines, as reported by Shen et al. [66]. Furthermore, the ectopic expression of PeCPD in wild-type Arabidopsis did not increase the concentrations of BRs [67]. However, under Cd stress, the concentration of EBL in OE potatoes was markedly elevated compared to that in the WT at both 0 h and 24 h. In the WT roots, the EBL content decreased and then increased with the prolongation of Cd stress, while in the OE potatoes, an opposite trend was observed. This may be due to the fact that WT potatoes were more fragile than OE potatoes under Cd stress, and the former activated conserved transcription factors (e.g., bHLH, ERF), which bind promoters of BRs-related genes (e.g., DWF4, BZR1), triggering their transcriptional upregulation within 48 h. This is part of the primary defense signaling cascade, where gene expression serves as an immediate ‘alert’ response [68]. Feedback regulation in OE potatoes may also be another reason for these results. BZR1 directly binds to the promoter regions of BRs biosynthesis genes (e.g., CPD, DWF4), suppressing their expression and forming a feedback inhibition [69].

The observed delay between rapid BRs biosynthesis/signaling genes upregulation at 24 h and peak BRs accumulation at 48 h under Cd stress stems from multi-layered regulation: (1) Transcriptional activation precedes metabolism: Signal transduction and TF activation (e.g., OsMYC2 in rice, BrMYB116 in Brassica rapa) occur rapidly, inducing downstream genes (e.g., JA biosynthesis peaking at 24 h) which then stabilize BRs enzymes for later metabolite production [70,71]. (2) Protein maturation requires time and face inhibition: Newly transcribed mRNAs must be translated, and proteins (e.g., DWF4) undergo post-translational modifications, folding, localization, and assembly. Cd-induced ROS oxidatively inactivates nascent enzymes, delaying functional complex formation until antioxidant systems (e.g., GST/GSH) restore redox homeostasis (24–36 h) [72]. (3) Resource allocation prioritizes immediate defense: BRs biosynthesis involves multi-compartment steps with rate-limiting enzymes (e.g., DWF4) [69]. Under Cd stress, resources shift to primary metabolism and detoxification (e.g., antioxidants, Cd sequestration), diverting precursors away from BRs. Proteomics shows specialized metabolism enzymes like BRs are upregulated later than primary metabolism enzymes [73]. Therefore, rapid gene induction (24 h) prepares the enzymatic machinery, while metabolic delays ensure resources first support immediate survival (e.g., antioxidant bursts, Cd sequestration); subsequent BR accumulation (48 h) then drives growth recovery.

Other phytohormones are also crucial for plant growth and development despite their low content in the plant. These hormones can interact with BRs and thus regulate the physiological and biochemical mechanisms in plants [74]. It has been found that Cd stress changes the content of endogenous hormones as well as the balance between them, ultimately affecting the plant’s life cycle [75].

IAA is involved in plant cell division, morphogenesis, and environmental responses [76]. Studies have shown that Cd treatment results in a notable reduction in IAA level, gene expression downregulation in the synthesis pathway, and enhancement in IAA oxidase activity [77]. In this study, the IAA level in WT under 24 h of Cd stress was lower than that in the control group. This was presumed to be due to the WT’s weak Cd alleviating effect, which inhibited its growth and blocked the synthesis pathway under Cd stress. In contrast, the IAA content of Cd-tolerant OE potato material remained constant with the prolongation of Cd stress, consistent with previous studies in wheat [9].

Many studies have shown that ABA acts as a stress hormone. Plants promote stomatal closure and cause an increase in ABA content to prevent excessive water loss, thus enhancing the detrimental impacts of Cd stress on plants [78]. In this research, the level of ABA in OE potatoes rose considerably as the duration of Cd²⁺ treatment was prolonged. This increase helped maintain stomatal closure and water balance, reduced Cd content in tissues, and thus mitigated the damaging effects of Cd stress. In contrast, the ABA content in WT potatoes showed non-significant changes compared to the control. These findings further confirmed that OE potato is more Cd-tolerant than WT.

6-BA is the first synthetic CTK and is vital in improving plant stress tolerance [79]. In this research, the 6-BA level in WT roots was slightly reduced as the duration of Cd²⁺ treatment was prolonged. Similarly, the 6-BA content in OE potato roots showed a decreasing trend, which was hypothesized to be caused by the significant increase in ABA content after 48 h of treatment.

In summary, heavy metal-tolerant materials maintain a balance in phytohormone levels through interactions, thereby alleviating Cd stress.

4.3. Transcriptional Levels Regulation of Potato Response to Cd Stresses

The concentration of EBL was elevated, and alterations were observed in the BRs signaling pathway in OE potatoes. A majority of the DEGs were linked to metabolic and cellular activities within biological processes. The groups of WT-0-vs-OE-0, WT-24-vs-OE-24, and WT-48-vs-OE-48 collectively highlighted enrichment in metabolic processes, cellular processes, single-organism processes, stimulus regulation, and general biological processes (Figure S1). The DEGs between OE and WT were primarily enriched in the biosynthesis pathway of secondary metabolites. Significantly, the BRs biosynthesis genes (DWF4, CPD, and CYP85A1) and the signal transduction gene BZR1 were upregulated in the OE. Upregulated BZR1 in OE potentiates BRs signaling (Figure 8A), elevating EBL (Figure 3E) and corroborating higher enzyme activities (Figure 3D). Simultaneously, PYL4/PYL8 and SAUR36 DEGs (Figure 6A and Figure 8F) modulate stomatal closure and root architecture, reducing Cd uptake and sustaining growth (Table 2). It has also been reported that the transcript levels of the BRs signaling pathway are increased in transgenic poplar and Arabidopsis lines [66,67].

Transcription factors have important roles in plant responses to adversity. It was found that heavy metal Cd stress affects the expression of transcription factors in plants [80]. Wu et al. found that transcription factors in tobacco seedlings, including WRKY, MYB, bHLH, and NAC families, were differentially expressed under the stress of Cd [81]. It was also noted that bHLH transcription factors played crucial roles in the tolerance of maize and Arabidopsis to Cd stress [82,83]. In this study, ARR-B, AP2-EREBP, bHLH, and WRKY transcription factors in OE potatoes were significantly expressed, and expression levels were higher than in the WT. This suggests that these transcription factor families may serve vital functions in potato alleviating effects on Cd stress and that different transcription factor families respond differently to Cd stress.

Photosynthesis is the main way of energy production in plants, and it has been shown that the process of photosynthetic bio-carbon fixation plays a key part in Cd stress [84]. In total, 21 DEGs related to photosynthetic biochar fixation were screened in WT potatoes, and 37 DEGs related to this process were screened in OE potatoes. This suggests that Cd stress can induce genes involved in photosynthetic biochar fixation to regulate photosynthesis in potatoes and enhance their alleviating effect on Cd. Exogenous application of EBL can also boost photosynthesis by enhancing the chlorophyll content, thereby enhancing resistance to Cd stress [85].

Phytohormone signaling was crucial in regulating the molecular mechanisms underlying Cd tolerance [80]. In this study, many DEGs related to BRs, IAA, 6-BA, ABA, and GA₃ biosynthesis and signaling pathways were significantly upregulated or downregulated in WT and OE potatoes. This indicates that overexpression of StDWF4 in potatoes responds to Cd stress by regulating multiple hormone pathways.

It has been demonstrated that overexpression of CYP85A1 in tobacco promotes root development because of an increase in endogenous BRs, which further enhances drought tolerance in tobacco [86]. Additionally, overexpression of BZR1 in tomatoes was found to positively regulate the expression of the ABA biosynthesis gene NCED1, resulting in an increase in ABA content and improved resistance to cold stress in the overexpressing tomatoes [45]. In this research, overexpression of the StDWF4 gene increased the content of BRs by heightening the expression levels of genes involved in BRs synthesis, namely DWF4, CPD, and CYP85A1, as well as the BRs signal transduction-related gene BZR1. This increase in BR content subsequently improved the potato’s resistance to Cd stress. Xian et al. discovered that under Cd stress, BZR1 was upregulated in Cd-tolerant Kentucky bluegrass [80].

The auxin response factor (ARF) transcription factors play a pivotal role in regulating the response to Cd stress [58]. In a study by Cui et al., it was observed that the external application of IAA decreased the Cd concentration in both the leaves and roots of Poa pratensis [87]. It was reported that overexpression of MdIAA24 increased auxin concentration and boosted tolerance to Cd stress in apples [88]. SAUR (Small auxin-up RNA) is a key component of auxin response genes [89]. The present analysis indicated that the SAUR36 gene was upregulated in OE potatoes, but its expression levels declined with prolonged Cd treatment (Figure 6A). This suggests that auxin regulation is also associated with the degree of stress in Cd-tolerant plants.

Cd transporters in plants are also important for tolerance to Cd stress. Exogenous Mel and BRs promoted the expression of SlPCR2 (PLANTCADMIUM RESISTANT 2) and SlHMA3 (HEAVY-METAL-ASSOCIATED 3) genes and protected tomatoes from Cd stress by facilitating Cd transport and detoxification [90]. Wang et al. found that most members of the ABCB family genes were upregulated in the Cd-tolerant variety of Kentucky bluegrass under Cd stress [73]. In this study, the lower Cd²⁺ content in OE roots (Figure 3C) links to DEGs in ‘ABC transporters’ and ‘metal ion binding’. ABCB1, HMA2, and PCR2, upregulated in OE (Figure 8G), mediate vacuolar Cd sequestration [58], limiting cytosolic toxicity. WGCNA further tied the ‘dark orange 2’ module (enriched for ABCB1/MDR-type transporters) to Cd²⁺ efflux (Figure 7E), explaining reduced root Cd accumulation.

4.4. A Plausible Network Responding to Cd Stress in Potatoes Transgenic for the StDWF4 Gene

In this study, a molecular network responsive to Cd stress was constructed in OE potatoes based on a comprehensive analysis encompassing phenotypic, physiological, and biochemical traits, hormone levels, and transcriptome profiling (Figure 9). This network comprises three primary components: Firstly, transgenic plants elevated BRs content by upregulating the expression of DWF4, CPD, CYP85A1, and BZR1 genes under Cd stress. The increased endogenous BRs content promoted potato growth and development as well as mitigate the damage to root ultrastructure caused by Cd stress. In addition, the transgenic potatoes responded to Cd stress by increasing antioxidant enzyme activities, decreasing MDA content, and improving their antioxidant capacity, which in turn affected the root Cd²⁺ content. Finally, the promotion or inhibition of the relevant genes led to changes in the content of endogenous plant hormones such as IAA, 6-BA, ABA, and GA₃, which responded to Cd stress and alleviated stress injury to a certain extent. Therefore, a series of changes in phenotypes, physiology, and biochemistry, as well as transcriptomic levels in the StDWF4 gene-overexpressing potatoes, may help alleviate the damaging effects of Cd stress.

5. Conclusions

This study manifested that enhancing the expression of StDWF4 in potatoes resulted in an elevated level of EBL, which subsequently induced phenotypic and molecular variations. These changes included increases in plant stature, root elongation, stem girth, fresh mass, and dry mass, as well as alterations in ultrastructure, Cd²⁺ concentration, MDA content, chlorophyll content, and antioxidant enzyme activities. The expression levels of genes participated in the production, breakdown, and signaling pathways of IAA, 6-BA, ABA, GA_3, and BRs regulated phytohormone levels in reaction to Cd stress in transgenic plants. These results suggested that endogenous BRs could alleviate Cd stress by regulating BR synthesis and signal transduction in potatoes.