Abstract

   Recently, pollution caused by the heavy metal Cd has seriously affected
   the environment and agricultural crops. While Sinapis alba is known for
   its edible and medicinal value, its tolerance to Cd and molecular
   response mechanism remain unknown. This study aimed to analyze the
   tolerance of S. alba to Cd and investigate its molecular response
   mechanism through transcriptomic and physiological indicators. To
   achieve this, S. alba seedlings were treated with different
   concentrations of CdCl[2] (0.25 mmol/L, 0.5 mmol/L, and 1.0 mmol/L) for
   three days. Based on seedling performance, S. alba exhibited some
   tolerance to a low concentration of Cd stress (0.25 mmol/L CdCl[2]) and
   a strong Cd accumulation ability in its roots. The activities and
   contents of several antioxidant enzymes generally exhibited an increase
   under the treatment of 0.25 mmol/L CdCl[2] but decreased under the
   treatment of higher CdCl[2] concentrations. In particular, the proline
   (Pro) content was extremely elevated under the 0.25 and 0.5 mmol/L
   CdCl[2] treatments but sharply declined under the 1.0 mmol/L CdCl[2]
   treatment, suggesting that Pro is involved in the tolerance of S. alba
   to low concentration of Cd stress. In addition, RNA sequencing was
   utilized to analyze the gene expression profiles of S. alba exposed to
   Cd (under the treatment of 0.25 mmol/L CdCl[2]). The results indicate
   that roots were more susceptible to disturbance from Cd stress, as
   evidenced by the detection of 542 differentially expressed genes (DEGs)
   in roots compared to only 37 DEGs in leaves. GO and KEGG analyses found
   that the DEGs induced by Cd stress were primarily enriched in metabolic
   pathways, plant hormone signal transduction, and the biosynthesis of
   secondary metabolites. The key pathway hub genes were mainly associated
   with intracellular ion transport and cell wall synthesis. These
   findings suggest that S. alba is tolerant to a degree of Cd stress, but
   is also susceptible to the toxic effects of Cd. Furthermore, these
   results provide a theoretical basis for understanding Cd tolerance in
   S. alba.

   Keywords: Sinapis alba, transcriptome, Cd stress, physiological
   response

1. Introduction

   With developing industrialization and urbanization, heavy metal
   pollutants, including Cd, Cu, Pb, and Mn, have become a severe issue in
   soil [[42]1], because excess heavy metal levels in soil not only
   destroy the function of soil and inhibit the growth and development of
   plants and soil microorganisms that are intolerant to heavy metals, but
   also cause serious toxicity to crops and cash crops, resulting in a
   serious decline in crop yields [[43]2,[44]3]. Moreover, heavy metals
   can be transported along the food chain and negatively impact human
   health. Among these heavy metal pollutants, Cd pollution is the most
   globally distributed and causes the most serious threat to crops
   because Cd has very high mobility in soil-plant systems, leading to the
   accumulation of Cd in crops. Thus, determining the Cd tolerance of
   crops and revealing the tolerance mechanism to Cd in these crops have
   become a priority [[45]4,[46]5].

   Excessive Cd in plants has been found to have a negative impact on
   various aspects of plant physiology. It not only indirectly affects the
   synthesis and decomposition of sugar, protein, and fat, but also
   interferes with plant nutrition, hormone metabolism, and other
   essential activities. As a result, there is a shortage of substances
   and energy needed for plant growth, resulting in inhibited plant growth
   [[47]6,[48]7]. In addition, Cd has been found to induce the production
   of high levels of reactive oxygen species (ROS) in plants, which can
   impede growth, development, and reproduction. Moreover, excess ROS
   production can significantly increase lipid peroxidation, potentially
   leading to cell death [[49]8,[50]9]. The entry of heavy metals into
   plants also destroys the internal structure of chloroplasts, negatively
   impacting nutrient uptake, transfer, and chloroplast synthesis
   [[51]10].

   To reduce the levels of ROS caused by stresses, plants produce a
   variety of antioxidant enzymes, including superoxide dismutase (SOD),
   peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT). These
   enzymes are crucial for regulating osmotic pressure in the plant
   cytoplasm following abiotic stress. They also help stabilize
   biomolecular structures, decrease cellular acidity, and regulate
   cellular redox [[52]11,[53]12]. By converting excessive reactive oxygen
   species and free radicals into less toxic substances, antioxidant
   enzymes help maintain a balanced level of ROS in plant cells and
   provide protection against heavy metal stress [[54]13,[55]14]. There is
   a positive correlation between proline accumulation and plant tolerance
   to various abiotic stresses. Because of its metal-chelating properties,
   proline (Pro) acts as a molecular chaperone, an antioxidant defense
   molecule that clears ROS, and has signal transduction behavior that
   activates specific gene functions, which is critical for plant recovery
   from stress [[56]15]. In addition, recent studies have shown that
   certain transcription factors (TFs) play a role in plant responses to
   abiotic stress [[57]16]. For instance, some WRKY genes have been found
   to enhance plant tolerance to Cd or maintain metal ion homeostasis by
   regulating downstream functional genes. AtWRKY12 inhibits the
   expression of genes involved in plant chelator protein synthesis by
   directly binding to the W-box of the GSH1 promoter, indirectly
   repressing the expression of genes related to chelator protein
   synthesis in plants. However, it negatively regulates Cd tolerance in
   Arabidopsis thaliana [[58]17]. Another TF, MYB49, can directly bind to
   the promoters of bHLH38 and bHLH101, leading to the activation of IRT1,
   which is involved in the uptake of Cd. MYB49 also binds to the promoter
   regions of heavy-metal-associated HIPP22 and HIPP44, resulting in an
   up-regulation of their expression and subsequently increasing Cd
   accumulation [[59]18].

   S. alba (2n = 24) is a versatile plant with various medicinal and food
   uses. It serves as an edible oil, edible vegetable, condiment, fodder
   and green manure source [[60]19]. In Europe, S. alba is well-known as
   the primary mustard variety. Recent research indicates that S. alba has
   potential applications as biodiesel. Additionally, S. alba rapeseed oil
   is utilized as a lubricant, while the seeds of S. alba are used in
   traditional medicine for their antitumor and antiviral effects
   [[61]20,[62]21]. These applications significantly enhance the medicinal
   and economic value of S. alba. Notably, S. alba exhibits remarkable
   tolerance to drought and high temperatures, making it particularly
   suitable for cultivation in arid regions. A high-quality genome
   sequence of S. alba has been released recently, affording a facility
   for its molecular analysis. However, its tolerance to Cd and the
   underlying molecular response mechanism remain elusive. In this study,
   we utilized phenotypic, physiological, and comparative transcriptomic
   analyses to investigate the Cd accumulation capability as well as the
   physiological and transcriptomic responses of S. alba under Cd stress.
   Our findings indicate that S. alba exhibits tolerance to
   low-concentration Cd stress. As a secondary amine, the content of
   proline was significantly increased under Cd stress, indicating that
   the degree of abiotic stress on plants was different. The
   differentially expressed genes (DEGs) induced by Cd stress were
   primarily enriched in metabolic pathways, plant hormone signal
   transduction (jasmonic acid and auxin), and the biosynthesis of
   secondary metabolites. Furthermore, our study revealed the presence of
   several transcription factors (TFs) associated with Cd tolerance, as
   well as the identification of two crucial DEGs involved in cell ion
   absorption and transporters: WRKY70 (Sal11g08240L) and MYB40
   (Sal12g05270L). These findings provide valuable insights into the
   molecular mechanism and physiological regulation of Cd tolerance in S.
   alba. This study also establishes a theoretical foundation for the
   utilization of S. alba in food processing and medicine.

2. Materials and Methods

2.1. Seedlings Treatment and Experimental Conditions

   A pure line of S. alba (accession number: BJ023) of a good seed yield
   was used in this study. The S. alba seeds were germinated in a sterile
   culture dish (9 cm in diameter). To enhance the germination rate of the
   seeds, two layers of qualitative filter paper were placed at the bottom
   of the petri dish to retain moisture. Additionally, a 1/4 Hoagland
   nutrient solution was replaced daily throughout this period. After one
   week, the seedlings were transferred to a black culture box filled with
   vermiculite (a non-nutritive soil) for duration of 14 days. During this
   time, irrigation was performed daily using a 1/2 Hoagland nutrient
   solution. After 14 days, the plants were divided into 4 groups, with 48
   plants in each group. Anhydrous CdCl[2] was dissolved in 1/2 Hoagland
   solution to prepare different concentrations of Cd solution (0.25
   mmol/L, 0.5 mmol/L, and 1.0 mmol/L). The amount of each solution added
   was 1 L. The CK group was irrigated with the same amount of 1/2
   Hoagland nutrient solution. The solution was changed once a day. After
   3 days of treatment, through the measurement of its physiological
   indexes in the early stage, it was observed that exposure to a 0.25
   mmol/L Cd solution for 3 days resulted in a significant increase in
   most antioxidant enzymes in the seedlings, with no obvious phenotypic
   changes. However, when treated with other concentrations of Cd solution
   for 3 days, most antioxidant enzymes in the body decreased
   significantly, and the phenotypic changes become more pronounced. The
   main objective of this study was to investigate the tolerance of S.
   alba to Cd. Therefore, in this study, the roots and leaves of S. alba
   treated with 0.25 mmol/L were selected for RNA-seq sequencing. The
   physiological indexes of the shoots of S. alba were measured after
   exposure to different concentrations of Cd (0 mmol/L, 0.25 mmol/L, 0.5
   mmol/L, and 1 mmol/L). The experiment was conducted in the tissue
   culture room of the School of Life Sciences at Guizhou Normal
   University. The temperature was maintained at a constant 19℃ throughout
   the day and night, with a 12 h light and dark cycle. The light
   intensity was set at 7000 Lux, and the relative humidity was maintained
   at 60%.

2.2. Determination of Cd Content in Roots and Shoots of S. alba

   After three days of treatment with different concentrations of Cd, the
   Cd content in roots and shoots of S. alba was determined. First, it was
   washed three times with tap water for ten minutes. Then, it was soaked
   in sterile deionized water for 20 min, and excess water was removed
   using absorbent paper. The tested S. alba plants were divided into
   shoots and roots. Both parts were dried in an oven at 37 °C. Then, 0.25
   g of the dried sample was weighed and placed into the digestion tank.
   To this, 8 mL of concentrated nitric acid and 2 mL of perchloric acid
   were added. The solution was digested at a variable temperature of 100
   °C in the digestion tank. After digestion, the solution was transferred
   to a 25 mL volumetric flask. The inner tank and cover were washed with
   a small amount of 1% nitric acid solution, ensuring at least 3 washing
   cycles. The solution was then pipetted into a volumetric flask using a
   glass rod and diluted to the mark with 1% nitric acid. Filtration was
   performed using a filter membrane and syringe, while a reagent blank
   experiment was conducted simultaneously. The standard curve was drawn
   using a mixed standard, and the content of Cd in S. alba was detected
   using AAS (atomic absorption spectrometry).

2.3. Determination of Physiological Indexes

   The physiological indicators followed the methodology described by Zuo
   et al. [[63]22]. To determine the chlorophyll content, approximately
   0.1 g of leaves was weighed and placed into a mortar. Then, 1 mL of
   extract (acetone:anhydrous ethanol = 2:1) and 10 mg of reagent one
   (chlorophyll content detection kit) were added, and the mixture was
   ground in dark conditions. The chlorophyll content in the leaves was
   subsequently determined according to the instructions provided by the
   chlorophyll content detection kit (Solarbio, Beijing, China). For the
   determination of proline content, approximately 0.1 g of leaves were
   weighed and placed into a mortar, and 1 mL of extract (proline content
   detection kit) was added for grinding. The proline content in the
   leaves was then determined following the instructions provided by the
   proline content detection kit (Solarbio, Beijing, China). The
   determination of MDA content involved weighing approximately 0.1 g of
   leaves and placing them into a mortar. Then, 1 mL of extract (MDA
   content detection kit) was added and fully ground. After centrifugation
   for 10 min, the supernatant was collected and placed on ice. The MDA
   content in the leaves was determined following the instructions
   provided in the MDA content detection kit (Solarbio, Beijing, China).
   Superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT),
   and peroxidase (POD) were also detected using the instructions provided
   in the kit (Solarbio, Beijing, China). Each group consisted of a
   control group and an experimental group, with three parallel control
   groups in each.

2.4. RNA Extraction, cDNA Library Preparation, and Transcriptome Sequencing

   The roots and shoots from seedlings of S. alba treated with 0.25 mmol/L
   CdCl[2] for 3 days were collected, and the total RNA was extracted
   using a commercial plant RNA extraction kit (Solarbio, Beijing, China).
   Three repeats per samples were prepared in this study. To confirm the
   quality of the extracted RNA, the concentration of RNA and RNA
   integrity were detected using a microplate reader and agarose gel
   electrophoresis (AGE). The qualified RNA was sent to Biomarker
   Technologies (Beijing, China) for RNA-seq sequencing. Then the cDNA
   libraries were obtained following the TruSeq RNA Sample Prep v2
   protocol (Illumina, San Diego, CA, USA). In total, 12 cDNA libraries
   were constructed and subjected to sequencing using the Illumina NovaSeq
   6000 platform.

   The raw data from RNA-seq were filtered using fastp (v0.20.1) with
   default parameters [[64]23]. Trimmomatic version 0.33 was utilized to
   obtain clean reads by removing reads containing adapters, poly-N, and
   low-quality bases. Subsequently, these clean reads were mapped to the
   recently released reference gene of S. alba [[65]24,[66]25] using
   Hisat2 (v2.2.1) with default settings. The transcript assembly was
   performed using StringTie (v2.2.1) software [[67]18]. The FPKM
   (fragments per kilobase per million bases) value of the gene was
   calculated using DESeq2 (v1.36.0) [[68]26]. DESeq2 (v1.36.0) was used
   to determine differentially expressed genes under the threshold of padj
   < 0.05 and |log2FoldChange| > 1. GO (Gene Ontology) enrichment and KEGG
   (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses of the
   DEGs were performed using OmicShare cloud platform tools
   ([69]https://www.omicshare.com/tools, accessed on 30 August 2023). The
   raw sequence data are available in NCBI-SRA
   ([70]https://www.ncbi.nlm.nih.gov/sra, accessed on 6 September 2021)
   under accession number PRJNA760315.

2.5. Verification of RNA-Seq Results

   The expression levels of DEGs in RNA-seq sequencing were validated
   using qRT-PCR analysis. For this verification, five up-regulated and
   five down-regulated genes were randomly selected. The actin gene
   (Sal05g04640L) of S. alba was used as the internal reference gene
   control in this study. A 20 μL qRT-PCR mixture was prepared using
   SYBR-GREEN fluorescent reagents (Solarbio, Beijing, China). The mixture
   consisted of 2 μL cDNA template, 10 μL 2xSYBR Green RT-PCR (RT-qPCR)
   Mix, 0.4 μL 10 μM Primer Forward, 0.4 μL 10 μM Primer Reverse, and 7.2
   μL ddH20. The primers of the selected genes were designed using NCBI
   primer-blast technology ([71]Table S2).

2.6. Data Analysis

   The significance of all data was determined via Excel and IBM SPSS
   Statistics 26 (SPSS Inc., Chicago, IL, USA) under the cutoff of p <
   0.05. The multiple comparisons were conducted using the LSD method (p <
   0.05). The error bar in chart indicates the SD, and the experiment was
   repeated three times (n = 3).

3. Results

3.1. Cd Tolerance and Accumulation in S. alba

   The S. alba seedlings were incubated for 2 weeks in 1/2 Hoagland
   solution and then treated with different concentrations (0.25 mmol/L,
   0.5 mmol/L, and 1 mmol/L) of CdCl[2] solution for 3 days. Compared with
   the CK, there was no significant change in shoots and roots under the
   0.25 mmol/L CdCl[2] treatment ([72]Figure 1), but seedling performance
   was seriously affected under 0.5 and 1.0 mmol/L CdCl[2] treatments. In
   addition, the root length and stem length of seedlings under 0.25
   mmol/L CdCl[2] were comparable to those of CK. Under the conditions of
   0.5 and 1.0 mmol/L CdCl[2], they all decreased significantly
   ([73]Figure 2), indicating that birch was tolerant to low
   concentrations of Cd stress.

Figure 1.

   [74]Figure 1
   [75]Open in a new tab

   Seedlings performance of S. alba treated with different concentrations
   of CdCl[2] solutions. (A–D): the performance of shoots of S. alba
   plants treated with 0 mmol/L (CK), 0.25 mmol/L, 0.5 mmol/L, and 1
   mmol/L CdCl[2] solutions for 3 days. (E–H): the performance of young
   seedlings treated with 0 mmol/L (CK), 0.25 mmol/L, 0.5 mmol/L, and 1
   mmol/L CdCl[2] solutions for 3 days.

Figure 2.

   [76]Figure 2
   [77]Open in a new tab

   The changes in root and stem length of S. alba after treatment with
   different concentrations of CdCl[2]. (A): root length. (B): stem
   length. Different letters represent the significantly different groups,
   and significance was determined by multiple comparisons using Fisher’s
   least significant difference (LSD) method (p < 0.05). The error bar in
   chart indicates standard deviation (SD), and three replicates (n = 3)
   per sample are used to calculate the value of mean ± SD.

   To investigate the ability of Cd accumulation in S. alba, the atomic
   absorption spectroscopy method was used to determine the Cd content in
   S. alba after 3 days of treatment with CdCl[2] ([78]Figure 3A;
   [79]Table S1). The results show that the Cd content in dehydrated
   shoots and roots of S. alba was significantly elevated (LSD-test, p <
   0.01) with the increasing concentrations of CdCl[2]. In brief, the Cd
   contents in dehydrated shoots were 203.59 ± 5.20 mg/kg, 233.88 ± 3.68
   mg/kg, and 269.25 ± 2.88 mg/kg under the treatment with 0.25 mmol/L,
   0.5 mmol/L, and 1.0 mmol/L CdCl[2], respectively. Correspondingly, the
   Cd contents in dehydrated roots were 786.99 ± 21.45 mg/kg, 1065.29 ±
   31.30 mg/kg, and 1275.42 ± 39.75 mg/kg. However, the analysis of the
   transport coefficient (the ratio of the content of elements in the
   aboveground part to the content of the same element in the underground
   part) of Cd from S. alba showed that under the treatment of 0.25
   mmol/L, 0.5 mmol/L, and 1 mmol/L CdCl[2] ([80]Figure 3B), the transport
   coefficient of S. alba Cd was less than 0.5. This result shows that S.
   alba has a weak ability to transport Cd from the underground part to
   the aboveground part, which is not conducive to the recovery and
   utilization of heavy metals.

Figure 3.

   [81]Figure 3
   [82]Open in a new tab

   Cd accumulation and transport factors in S. alba under different Cd
   treatments. (A): Cd content. (B): Cd transport factor in different
   treatments. Different letters represent the significantly different
   groups, which were determined by multiple comparisons using Fisher’s
   least significant difference (LSD) method (p < 0.05). The error bar in
   the chart indicates the standard deviation (SD), and three replicates
   (n = 3) per sample were used to calculate the mean ± SD.

3.2. Physiological Responses to Cd in S. alba

   Some physiological indicators, including chlorophyll, SOD, APX, CAT,
   MDA, Pro, and POD, were employed to ascertain the extent of
   physiological responses to Cd stress in S. alba. S. alba exhibited a
   significant reduction in chlorophyll a, chlorophyll b, and total
   chlorophyll levels when subjected to different concentrations of Cd
   stress ([83]Figure 4). The decrease in chlorophyll content was
   particularly notable at Cd concentrations of 0.5 mmol/L and 1 mmol/L.
   Compared to the CK, the activities of SOD, POD, CAT, and APX were
   significantly induced (p < 0.05) under the treatment with 0.25 mmol/L
   CdCl[2], but obviously declined under the treatments with 0.5 mmol/L
   and 1.0 mmol/L CdCl[2] ([84]Figure 5A–D). We found that under 0.25
   mmol/L and 0.5 mmol/L CdCl[2] treatments, the content of Pro produced
   by plants under abiotic stress increased to threefold, while it
   decreased sharply under 1.0 mmol/L CdCl[2] treatment ([85]Figure 5E),
   indicating that when S. alba was subjected to Cd stress, Pro, as an
   important signal transduction factor, activated specific gene functions
   and is essential for plant recovery from stress. In addition, the MDA
   content in tested seedlings increased gradually with the Cd
   concentration ([86]Figure 5F), suggesting that Cd caused membrane lipid
   peroxidation in S. alba.

Figure 4.

   [87]Figure 4
   [88]Open in a new tab

   Chlorophyll content changes in S. alba seedlings under different
   concentrations of Cd treatment. (A): chlorophyll-a content. (B):
   chlorophyll-b content. (C): total chlorophyll content. Different
   letters represent significantly different groups determined by the LSD
   method (p < 0.05). The error bar in the chart indicates the standard
   deviation (SD), and three replicates (n = 3) per sample were used to
   calculate the mean ± SD.

Figure 5.

   [89]Figure 5
   [90]Open in a new tab

   Physiological indicators change in S. alba seedlings under different
   concentrations of Cd treatment. (A): SOD activity. (B): POD activity.
   (C): CAT activity. (D): APX activity. (E): Pro content. (F): MDA
   content. Different letters represent significantly different groups
   determined via the LSD method (p < 0.05). The error bar in the chart
   indicates the standard deviation (SD), and three replicates (n = 3) per
   sample were used to calculate the mean ± SD.

3.3. Gene Expression Patterns Responding to Cd Stress in S. alba

   To reveal the potential genes involved in the Cd tolerance in S. alba,
   we conducted a comparative transcriptome analysis to investigate the
   gene expression patterns of the control (CK) and CdCl[2] treated
   seedlings (Cd). Only when treated with 0.25 mmol/L Cd solution for 3
   days, most of the antioxidant enzymes in S. alba increased
   significantly, and the phenotypic changes were not obvious. Therefore,
   seedlings under the 0.25 mmol/L CdCl[2] treatment were used to check
   the gene expression change. In total, 12 cDNA libraries of S. alba (6
   libraries for roots and 6 libraries for shoots) were prepared. After
   trimming the adapters and removing the low-quality reads using FastQC
   with default parameters, 19,246,416–24,331,775 end-paired clean reads
   with a Q30 value > 94.34% (75.79 Gb clean data in total) were obtained
   from the 12 libraries. Then, the clean reads per sample were mapped to
   the recently released S. alba reference genome [[91]24]. The fragments
   per kilobase per million mapped fragments value (FPKM) of the genes was
   employed to assess the gene expression profiles. To verify the accuracy
   of the RNA-seq data, 10 genes (5 up-regulated and 5 down-regulated
   genes) were randomly selected to determine the relative expression
   levels using a quantitative real-time polymerase chain reaction
   (qRT-PCR) ([92]Table S2). The results of the qRT-PCR were consistent
   with those of RNA-seq analysis ([93]Figure S1), confirming the
   reliability of the RNA-seq results. Compared to the CK, a total of 37
   DEGs, comprising of 19 up-regulated and 18 down-regulated DEGs, were
   detected in shoots when exposed to Cd (CKs vs. Cds). In the comparison
   of roots of CK- and Cd-treated seedlings (CKr vs. Cdr), 542 DEGs (CKr
   vs. Cdr), including 137 up-regulated genes and 405 predominantly
   down-regulated genes (χ^2-test, p < 0.001), were identified ([94]Table
   S3). These results indicate that the genes in roots were likely prone
   to disturbance by Cd.

3.4. GO and KEGG Analyses of DEGs in Response to Cd Stress

   To investigate the role of DEGs after subjecting S. alba to Cd-induced
   stress, we conducted GO enrichment analysis on the identified DEGs. The
   analysis aimed to identify GO terms where the differential genes were
   significantly enriched compared to all annotated genes. The results of
   the GO enrichment analysis reveal significant enrichment of both
   up-regulated and down-regulated genes in biological process, cellular
   component, and molecular function categories in the comparisons of CKs
   vs. Cds and CKr vs. Cdr. The biological process section was mainly
   enriched in the categories of cellular process, single-organism
   process, and metabolic process. In the cellular component section, it
   was mainly enriched in the cell, cell part, membrane and membrane part
   categories. In the molecular function section, it was mainly enriched
   in the binding, catalytic activity, and nucleic acid binding
   transcription factor activity categories ([95]Figure 6). The results
   suggest that S. alba may up-regulate the production of antioxidant
   compounds in response to plant oxidative stress caused by Cd stress
   [[96]27].

Figure 6.

   [97]Figure 6
   [98]Open in a new tab

   Gene ontology analysis of S. alba under Cd conditions. (A,C)
   Up-regulated DEGs enrichment in shoots and roots. (B,D) Enrichment of
   down-regulated DEGs in shoots and roots.

   To investigate the roles and effects of DEGs on the biological pathways
   of S. alba under Cd stress, Kyoto Encyclopedia of Genes and Genomes
   (KEGG) enrichment analysis was performed. The analysis revealed that
   the top five KEGG terms of up-regulated DEGs in CKs vs. Cds were
   involved in the metabolic pathway, biosynthesis of secondary
   metabolites, plant hormone signal transduction, biosynthesis of amino
   acids, and MAPK signaling pathway–plant categories ([99]Figure 7A). The
   top five KEGG terms involved in down-regulated DEGs in CKs vs. Cds were
   found to be associated with metabolic pathways, biosynthesis of
   secondary metabolites, plant hormone signal transduction, oxidative
   phosphorylation, and pentose and glucuronate interconversions
   ([100]Figure 7B). Similarly, the top five up-regulated differential
   genes in CKr vs. Cdr were also related to metabolic pathways,
   biosynthesis of secondary metabolites, plant hormone signal
   transduction, ribosome, and carbon metabolism ([101]Figure 7C). The top
   five down-regulated DEGs enriched in KEGG pathways in CKr vs. Cdr were:
   “metabolic pathways”, biosynthesis of secondary metabolites,
   photosynthesis, carbon metabolism, and biosynthesis of amino acids
   ([102]Figure 7D). The differential genes of CKs vs. Cds and CKr vs. Cdr
   were significantly enriched in the hormone and metabolic pathways. This
   suggests that S. alba alleviates the toxic effect of Cd mainly through
   metabolic pathways and hormone synthesis after Cd stress [[103]28].

Figure 7.

   [104]Figure 7
   [105]Open in a new tab

   KEGG pathway enrichment analysis of significant DEGs. (A,C)
   Up-regulated DEG enrichment in shoots and roots. (B,D) Enrichment of
   down-regulated DEGs in shoots and roots.

3.5. Involvement of DEGs from Underground Parts in Phytohormone Signalings

   The functional analysis of the differentially expressed gene pathways
   revealed significant enrichment of DEGs in the plant hormone signal
   transduction pathway under Cd stress. Therefore, we conducted a
   detailed analysis of the DEGs related to this pathway, with special
   attention given to the 24 DEGs involved in the biosynthetic pathway of
   jasmonic acid and auxin primarily in the underground fraction
   ([106]Figure 8, [107]Table S4). In this study, we observed that of the
   total number of genes analyzed, 67% (16 genes) were down-regulated,
   while 33% (8 genes) were up-regulated. Interestingly, we identified
   eight DEGs that are directly associated with the biosynthesis of
   jasmonic acid. These DEGs included one gene encoding the JAR1 protein,
   one gene encoding the MYC2 protein, and six genes encoding the JAZ
   protein. A total of 16 DEGs were identified in the auxin biosynthesis
   pathway. Among these DEGs, five mainly encoded ARF proteins and seven
   mainly encoded GH3 proteins. The findings suggest that Cd stress may
   significantly impact the growth and development of S. alba.
   Furthermore, the DEGs involved in the jasmonic acid and auxin signaling
   pathways were mostly down-regulated in roots.

Figure 8.

   [108]Figure 8
   [109]Open in a new tab

   Transcriptional changes in DEGs involved in plant hormone biosynthesis
   and plant hormone signal transduction in shoots and roots. The heatmaps
   from left to right are CK, Cds, and Cdr in shoots and roots.

3.6. Effect of Cd on Antioxidant Enzyme Activities of and Pro S. alba Cell
Wall Synthesis, and Transcription Factor

   Previous studies have demonstrated that SOD, CAT and Pro antioxidant
   enzymes have the ability to counteract the reactive oxygen species
   (ROS) produced during heavy metal stress by cleaning plant cells,
   thereby aiding in detoxification at the physiological level
   [[110]29,[111]30]. In our current investigation, we observed that S.
   alba exposed to Cd stress exhibited eight DEGs associated with the
   biosynthesis of SOD, CAT, and Pro antioxidant enzymes in the roots.
   Specifically, we identified one down-regulated DEG involved in SOD
   biosynthesis, two DEGs (one up-regulated and one down-regulated)
   involved in Pro biosynthesis, and five DEGs (three up-regulated and two
   down-regulated) involved in CAT biosynthesis ([112]Figure 9, [113]Table
   S5).

Figure 9.

   [114]Figure 9
   [115]Open in a new tab

   Transcriptome network in shoots and roots. The heatmaps from left to
   right are CK, Cds, and Cdr in shoots and roots.

3.7. Analysis of TFs of S. alba during Cd Stress

   Li et al. [[116]31] demonstrated that transcription factors contain
   numerous phosphorylation sites, which play a crucial role in regulating
   heavy metal stress by controlling the expression of downstream genes.
   In this study, the response of S. alba to Cd stress resulted in the
   identification of 12 down-regulated DEGs from the WRKY transcription
   factor family, 16 DEGs from the MYB transcription factor family (9
   up-regulated and 7 down-regulated), and 6 DEGs from the bHLH
   transcription factor family (2 up-regulated and 4 down-regulated) in
   the roots ([117]Figure 9, [118]Table S5).

3.8. Screening of DEGs Related to Transport and Absorption of Cd in S. alba

   ABC transporter proteins are transmembrane transporter proteins that
   have important functions in phytohormone transport, lipid metabolism,
   detoxification of exogenous toxins, and plant stress resistance
   [[119]32]. In this study, we identified a total of 10 DEGs in the
   roots, with 3 DEGs belonging to the ABCA transporter protein family, 1
   DEG belonging to the ABCB transporter protein family, and 5 DEGs
   belonging to the ABCC transporter protein family ([120]Figure 9,
   [121]Table S5). In relation to the cell wall, the phenylpropanoid
   biosynthesis pathway is associated. In the roots, two DEGs (one
   up-regulated DEG and one down-regulated DEG) were found to be involved
   in the biosynthesis of Cinnamoyl-CoA in the phenylpropanoid
   biosynthesis pathway. Additionally, five DEGs (one up-regulated DEG and
   four down-regulated DEGs) were found to be associated with the
   biosynthesis of coniferyl alcohol ([122]Figure 9, [123]Table S6). Upon
   exposure to Cd stress, S. alba exhibited a significant down-regulated
   of DEGs involved in cell wall and antioxidant enzyme biosynthesis. This
   suggests that the plant has a low tolerance to Cd.

4. Discussion

   When plants are exposed to Cd stress, an excessive amount of Cd can
   trigger the generation of excess ROS. These excess ROS can lead to
   lipid peroxidation, disruption of the biofilm system, and damage to
   proteins, carbohydrates, and other biological macromolecules [[124]33].
   Research has shown that plants possess an antioxidant enzyme system
   consisting of enzymes such as Pro, SOD, CAT, APX and POD. This system
   works synergistically to scavenge accumulated active oxygen free
   radicals under stress conditions, thereby reducing membrane lipid
   peroxidation and preventing heavy metal poisoning in plants
   [[125]34,[126]35,[127]36,[128]37].

   Pro can form a complex with Cd^2+ called Cd-Pro, which helps transport
   Cd^2+ out of plant cells in vitro [[129]38]. This reduces the toxic
   effect of Cd on plant cell bodies. Additionally, Pro also plays a
   crucial role in scavenging ROS induced by heavy metals in plants. It
   helps regulate cell osmotic pressure and redox [[130]39]. SOD, the
   first antioxidant enzyme, plays a vital role in scavenging reactive
   oxygen species. It can rapidly convert superoxide anion radical to
   H[2]O[2] and O^2. Then, H[2]O[2] is further converted to water and
   oxygen with the help of catalase (CAT), peroxidase, and the
   ascorbate-glutathione cycle system. Both SOD and APX are important in
   scavenging oxygen free radicals, preventing them from damaging the
   composition, structure, and function of cells. They play a crucial role
   in protecting cells from oxidative damage [[131]40,[132]41]. In this
   study, the effect of treating S. alba with different concentrations of
   CdCl[2] on the activities of the three antioxidant enzymes (SOD, POD,
   and CAT) in the leaves was investigated. The results show that when the
   plants were treated with 0.25 mmol/L CdCl[2] for three days, the
   activities of SOD, POD, and CAT increased significantly. However, as
   the concentration of CdCl[2] increased to 0.5 mmol/L and 1 mmol/L, the
   activities of these enzymes decreased significantly, even lower than
   those in the control group. Additionally, compared to other antioxidant
   enzymes, APX activity and Pro content also decreased significantly at
   0.5 mmol/L and 1 mmol/L. However, at 0.5 mmol/L, the APX activity and
   Pro content were still higher than those in the CK group ([133]Figure
   5). As one of the important protective enzymes in organisms, SOD can
   effectively scavenge oxygen free radicals. POD is a common
   oxidoreductase in plants that can prevent the accumulation of hydroxyl
   groups in plants. CAT is an enzyme scavenger and one of the key enzymes
   in the biological defense system. Under appropriate stress conditions,
   the synthesis of antioxidant enzymes in plants plays a role in
   promotion, but when it exceeds a certain limit, it is not conducive to
   the growth of plants, causing biological disorders. Exceeding the
   maximum tolerance range of the enzyme, the activity of the enzyme is
   greatly reduced, and the accumulation of reactive oxygen species
   endangers the plant [[134]42]. In addition, a total of eight DEGs were
   identified to be involved in the biosynthesis of SOD, CAT and Pro
   antioxidant enzymes. Among these DEGs, one was down-regulated and
   associated with SOD biosynthesis, while two DEGs (one up-regulated and
   one down-regulated) were involved in Pro biosynthesis. Furthermore,
   five DEGs (three up-regulated and two down-regulated) were found to be
   associated with CAT biosynthesis ([135]Figure 9, [136]Table S5).

   Excess Cd can lead to abnormal cell division and chromosome variation,
   resulting in a decrease in the activity of protochlorophyll reductase,
   which directly affects the biosynthesis of plant chlorophyll
   [[137]43,[138]44]. Previous studies have shown that when Vigna radiata
   L., Brassica juncea, and Ficus parvifolia were exposed to Cd stress,
   the unsaturated fatty acids on the cell membrane underwent a series of
   oxidation reactions, ultimately producing malondialdehyde (MDA). MDA is
   one of the end products of membrane lipid peroxidation, and its content
   can be used as one of the indicators to investigate the severity of
   cell stress. Its main damage leads to membrane lipid peroxidation and
   damages the structure of biofilm, mainly the cytoplasmic membrane, so
   that the structure and function of the cell membrane are damaged and
   the permeability of the membrane is changed, thus affecting the normal
   progress of a series of physiological and biochemical reactions. MDA
   has a negative effect on mitochondrial respiratory chain complexes and
   key enzyme activities in mitochondria. High levels of MDA can
   exacerbate cell membrane damage [[139]45,[140]46,[141]47]. In our
   study, when S. alba was subjected to Cd stress, there was a significant
   decrease in chlorophyll a, chlorophyll b, and total chlorophyll with
   increasing Cd concentration, while the MDA content significantly
   increased ([142]Figure 4 and [143]Figure 5). These findings suggest
   that S. alba is sensitive to Cd, as evidenced by changes in antioxidant
   enzyme activity, malondialdehyde content and chlorophyll content.

   Previous studies have reported that applying methyl jasmonate (MeJA)
   from an external source has shown a significant reduction in Cd
   concentration in both root and shoot tissues. Additionally, it has been
   observed that MeJA inhibited the expression of AtIRT1, AtHMA2, and
   AtHMA4 genes in the underground part after exposure to Cd stress
   [[144]48]. Jasmonic acid has the ability to decrease the expression of
   genes that facilitate the uptake and translocation of Cd, resulting in
   a decrease in Cd concentration in root cell sap and alleviating Cd
   stress. This ultimately enhances plant tolerance to Cd [[145]49]. When
   S. alba was subjected to Cd stress, only eight DEGs related to the
   biosynthesis of jasmonic acid were identified in the roots. These DEGs
   mainly encoded proteins such as JAR1, MYC2, and JAZ, with most of them
   being down-regulated genes. These down-regulated DEGs could be
   attributed to the higher sensitivity of S. alba to Cd ([146]Figure 8,
   [147]Table S4).

   Auxin plays a crucial role in facilitating the transport of heavy
   metals to the cytoplasm through the apoplast. It has the ability to
   initiate H^+—ATPase, which generates an electrochemical gradient in the
   plasma membrane. This gradient either opens the cation channel or
   activates the anion transporter, resulting in the influx of metal
   cations [[148]50]. Additionally, auxin influences the DELLA protein,
   indirectly regulating the balance of ROS or eliminating ROS, thereby
   enhancing the plant’s uptake of heavy metal elements. Our study
   identified 16 DEGs in the roots that are involved in the biosynthesis
   of auxin, primarily encoding ARF and GH3 proteins ([149]Figure 8,
   [150]Table S4).

   Cd primarily exists as Cd^2+ ions in the soil, which can be easily
   absorbed by plant roots, especially in regions with root hairs. These
   ions accumulate in various organs of the plant due to their biological
   mobility [[151]51]. Cd^2+ is transported to the xylem through both
   apoplastic and symplastic pathways in the root epidermis and root
   cortex. Once Cd^2+ enters the xylem, it is efficiently transported to
   the stems and leaves [[152]52]. Many transport factors or genes
   involved in synthesis of the cell wall and lignin play an important
   role in the process of Cd^2+ transport, absorption, and accumulation.
   ABC transporters are a class of transmembrane transporters that play a
   crucial role in various plant processes, including hormone transport,
   lipid metabolism, detoxification of toxins, and stress resistance. In
   A. thaliana, AtABCG36 expels intracellular Cd to the outside of the
   cell, reducing the harm caused by Cd toxicity [[153]53]. AtABCB25,
   located on the mitochondrial membrane, enhances the tolerance of
   Arabidopsis plants to Cd when overexpressed [[154]54]. AtABCC1,
   AtABCC2, and AtABCC3 transport Cd from the cytoplasm into vacuoles,
   thus minimizing the damage caused by Cd toxicity to plants [[155]55].
   In this study, we identified a total of 10 DEGs belonging to the ABC
   transporter family in the roots. Among them, three DEGs belonged to the
   ABCA transporter family, one DEG belonged to the ABCB transporter
   family, and five DEGs belonged to the ABCC transporter family
   ([156]Figure 9, [157]Table S5). Most of these genes were
   down-regulated, with only two up-regulated DEGs. This down-regulation
   may be attributed to the evident toxic effects of Cd on S. alba. In the
   phenylpropanoid biosynthesis pathway, two DEGs, Sal02g36790L and
   Sal06g23960L, were identified to be involved in the biosynthesis of
   cinnamoyl-CoA, which is responsible for secondary wall formation in
   plants [[158]56]. Additionally, five DEGs (one up-regulated and four
   down-regulated) were found to be associated with the biosynthesis of
   coniferyl alcohol, which is an important component of lignin that
   participates in the formation of the cell wall [[159]57].

   TFs serve an important function in the regulation of gene expression in
   plants, particularly during plant stress responses [[160]58]. Among
   these TFs, WRKY transcription factors have been found to have a high
   affinity for a specific DNA cis-acting element called the W-box, which
   is located in the promoter region. By binding to this element, WRKY TFs
   regulate the expression of downstream genes and actively participate in
   the response to Cd stress [[161]59]. The overexpression of TaWRKY70 in
   transgenic Arabidopsis was found to enhance Cd tolerance [[162]60]. In
   this study, one WRKY70 gene (Sal11g08240L) was predicted be
   down-regulated, and all WRKY differential genes also showed a
   down-regulated pattern. Additionally, studies have demonstrated that
   overexpression of MYB40 leads to the expression of plant chelating
   enzymes and ABCC1 and ABCC2, which encode the main vacuolar plant
   chelating protein transporters. This, in turn, increases the tolerance
   of plants to heavy metals [[163]61]. In our study, one MYB40 gene
   (Sal12g05270L) was predicted to be up-regulated ([164]Figure 9,
   [165]Table S5). These findings suggest that the identified regulatory
   elements may contribute to the response of S. alba to Cd stress.

5. Conclusions

   In this study, S. alba was exposed to different concentrations of
   CdCl[2] (0.25 mmol/L, 0.5 mmol/L, and 1 mmol/L) for three days. The
   activity and content of antioxidant enzymes and chlorophyll exhibited a
   decreasing trend as the concentration of heavy metals increased. RNA
   sequencing (RNA-seq) was employed to analyze the gene expression
   profiles of S. alba treated with CdCl[2] (0.25 mmol/L) for 3 days. The
   results reveal that 37 and 542 differentially expressed genes (DEGs)
   were identified in leaves and roots, respectively. These DEGs were
   predominantly expressed in roots. The DEGs induced by Cd stress were
   mainly enriched in metabolic pathways, plant hormone signal
   transduction, and biosynthesis of secondary metabolites. In addition,
   we identified several transcription factors (TFs) associated with Cd
   tolerance, and highlighted two key DEGs involved in cell ion absorption
   and transporters, specially WRKY70 (Sal11g08240L) and MYB40
   (Sal12g05270L). These findings offer valuable insights into the
   molecular mechanisms and physiological regulation processes of S. alba
   in response to Cd. Furthermore, they provide useful knowledge for
   enhancing the yield and quality of S. alba under Cd conditions while
   also serving as a theoretical foundation for food processing and
   medical treatment.

Supplementary Materials

   The following supporting information can be downloaded at:
   [166]https://www.mdpi.com/article/10.3390/genes14122224/s1, Figure S1.
   RT-PCR verification of the expression levels of 10 DEGs (five
   up-regulated and five down-regulated) determined by RNA-seq. Table S1.
   Cd content of heavy metal after three days of treatment. Table S2.
   Primers used in experiment of gene expression by RT-PCR. Table S3.
   Statistics on the number of up-regulated genes down-regulated genes in
   CKs vs. Cds and CKr vs. Cdr. Table S4. DEGs involved in plant hormone
   biosynthesis and plant hormone signal transduction in plants. Table S5.
   Antioxidant enzyme, ABC transporter protein and transcription factor in
   root and shoot. Table S6. phenylpropanoid biosynthesis.
   [167]Click here for additional data file.^ (734.7KB, zip)

Author Contributions

   Conceptualization, T.P. and T.Z.; methodology, T.Y. and S.F.; software,
   L.Y.; validation, M.C. and X.D.; investigation, T.Y.; writing—original
   draft preparation, M.C. and T.Y.; writing—review and editing, L.G. and
   B.Z.; supervision, T.P.; project administration, T.Z.; funding
   acquisition, H.W. and B.Z. All authors have read and agreed to the
   published version of the manuscript.

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The sequence data in raw format can be accessed at NCBI-SRA
   ([168]https://www.ncbi.nlm.nih.gov/sra, accessed on 6 September 2021)
   using the accession number PRJNA760315.

Conflicts of Interest

   The authors declare no conflict of interest. This study did not involve
   any human or animal research participant data. The plant sample tested
   in this study was not an endangered species, and the collection of
   samples did not cause any environmental problems.

Funding Statement

   This work was supported by the Precursor Projects of Guizhou Province
   for Biological. Breeding Supporting by Science and Technology in 2022
   (QKHZC2022ZD031), National Natural Science Foundation of China (grant
   no. 32260460).

Footnotes

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References