Uptake characteristics and plant hormone metabolic disruption of bisphenol A in pepper (Capsicum annuum L.) roots under soil exposure

Uptake characteristics and plant hormone metabolic disruption of bisphenol A in pepper (Capsicum annuum L.) roots under soil exposure | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 18 June 2025 V1 Latest version Share on Uptake characteristics and plant hormone metabolic disruption of bisphenol A in pepper (Capsicum annuum L.) roots under soil exposure Authors : Zhigang Huang , Qingmei Zhu , Jing Lu , Ze Liao , Qinyao Li , Qiuping Wang , Binglin Wu , Zhoubin Liu , Shaohua Zhu , and Zhoufei Luo [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175022846.60921505/v1 195 views 130 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Soil contamination by bisphenol A (BPA) has raised considerable ecological and environmental concerns, particularly due to its potential impact on plant growth. However, the interactive effects of BPA and different soil types on soil–plant systems remain poorly understood. Capsicum annuum L., a widely cultivated vegetable crop, was used as a model to systematically investigate the mechanisms of BPA uptake, translocation, and metabolic disruption in roots under varying soil types and BPA concentrations. Greenhouse experiments showed that BPA accumulation in pepper roots was highest in clay soil, significantly greater than in sandy or loamy soils. When BPA concentrations exceeded 10 mg/kg, root elongation and vitality were markedly suppressed, accompanied by enhanced antioxidant enzyme activity and elevated malondialdehyde content, indicating increased oxidative stress. Integrated transcriptomic and metabolomic analyses identified 995 differentially expressed genes and revealed significant disruptions in root metabolic processes. BPA stress altered the expression of genes related to the biosynthesis of hormone precursors and branched metabolites. Key pathways, including indole-3-acetic acid biosynthesis and hormone signal transduction, were significantly affected. These findings clarify the soil-dependent uptake and translocation patterns of BPA in pepper roots and provide important molecular insights into the plant’s adaptive and defense responses under BPA-induced stress. Uptake characteristics and plant hormone metabolic disruption of bisphenol A in pepper ( Capsicum annuum L.) roots under soil exposure Zhigang Huang a,1 , Qingmei Zhu a,1 , Jing Lu b , Ze Liao a , Qinyao Li a , Qiuping Wang a , Binglin Wu a , Zhoubin Liu c , Shaohua Zhu b , Zhoufei Luo a* a College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, PR China b Technology Center of Changsha Customs, Hunan Key Laboratory of Food Safety Science & Technology, Changsha 410004, PR China c Horticulture College, Hunan Agricultural University, Changsha 410128, PR China 1 Zhigang Huang and Qingmei Zhu contributed equally to this work. * Corresponding authors: Z.F. Luo: [email protected] Z.G. Huang: [email protected] Q.M.Zhu: [email protected] J. Lu: [email protected] Z. Liao: [email protected] Q.Y. Li: [email protected] Q.P. Wang: [email protected] B.L. Wu: [email protected] Z.B. Liu: [email protected] S.H. Zhu: [email protected] Z.G. Huang: [email protected] Highlights 1. BPA uptake by peppers is higher in viscosity soil than in sandy or loamy soil. 2. BPA concentration in soil porewater determines its plant bioavailability. 3. BPA alters expression of hormone-related genes via RNA-seq and qRT-PCR data. 4. BPA affects IAA, CK, and ABA precursor biosynthesis and metabolic pathways. 5. BPA disrupts IAA biosynthesis and plant hormone signal transduction pathways. Graphical abstract： not-yet-known not-yet-known not-yet-known unknown ABSTRACT Soil contamination by bisphenol A (BPA) has raised considerable ecological and environmental concerns, particularly due to its potential impact on plant growth. However, the interactive effects of BPA and different soil types on soil–plant systems remain poorly understood. Capsicum annuum L., a widely cultivated vegetable crop, was used as a model to systematically investigate the mechanisms of BPA uptake, translocation, and metabolic disruption in roots under varying soil types and BPA concentrations. Greenhouse experiments showed that BPA accumulation in pepper roots was highest in clay soil, significantly greater than in sandy or loamy soils. When BPA concentrations exceeded 10 mg/kg, root elongation and vitality were markedly suppressed, accompanied by enhanced antioxidant enzyme activity and elevated malondialdehyde content, indicating increased oxidative stress. Integrated transcriptomic and metabolomic analyses identified 995 differentially expressed genes and revealed significant disruptions in root metabolic processes. BPA stress altered the expression of genes related to the biosynthesis of hormone precursors and branched metabolites. Key pathways, including indole-3-acetic acid biosynthesis and hormone signal transduction, were significantly affected. These findings clarify the soil-dependent uptake and translocation patterns of BPA in pepper roots and provide important molecular insights into the plant’s adaptive and defense responses under BPA-induced stress. Keyword : Bisphenol A; pepper; root uptake; phytohormone metabolic; omics analyse Introduction Bisphenol A (BPA) is a common industrial chemical widely used in the production of polycarbonates, epoxy resins, flame retardants, unsaturated polyester resins, polysulfone resins, polyetherimide resins, and polyarylate resins (Trullemans et al., 2023). These materials serve as one of the primary raw materials for producing plastic products such as metal food cans, reusable plastic cups/bowls, medical devices, water supply pipelines, and construction materials. The extensive applications in manufacturing and food processing industries, combined with BPA’s chemical properties that enable it to migrate into surface water and soils, have led to environmental issues including soil contamination, water pollution, and bioaccumulation (Du et al., 2025; Chakraborty et al., 2021). Elevated BPA exposure has been documented to manifest neurotoxic and reproductive toxic effects across diverse animal models, resulting in developmental impairments in the nervous system and hormonal dysregulation (Zheng et al., 2024; Gokso̷yr et al., 2024). When BPA bioaccumulates in crops, it can enter the human body through the food chain, potentially disrupting endocrine and metabolic functions and posing long-term health risks such as reproductive disorders, developmental abnormalities, and hormone-related cancers (Qin et al., 2024). BPA has been frequently detected in sediments in Xiangjiang River Basin, southern China, with <LOD-53.90 ng/g (Liao et al., 2024). BPA contamination levels of 55.9 ng/g have been detected in agricultural soils in Spain (Perez, R.A., et al., 2017). Global soil monitoring results indicate that BPA-induced soil contamination has become widespread in agricultural ecosystems worldwide and has attracted significant attention. The pepper (Capsicum annuum L. ) is a widely distributed and highly popular Solanaceae crop, and is currently cultivated worldwide (Bulle, M., et al., 2024). According to data released by the Food and Agriculture Organization of the United Nations (FAO) (http://www.FAO.org), the global pepper production in 2023 reached 3.83×10⁷ tonnes. China ranked first in global pepper production, accounting for approximately 44.38% of the total output. As a high-value economic vegetable crop, pepper cultivation environments and yields have drawn significant attention. In conventional agricultural practices, plastic mulching is widely adopted to enhance soil temperature and suppress weed growth. Furthermore, as thermophilic crops, peppers demand significant irrigation water inputs. Notably, BPA is intentionally added to certain plastic mulch films during production processes to enhance material durability and flexibility (Lee et al., 2020). Consequently, these additives can leach into the soil during field application, resulting in BPA contamination of agricultural soils (Palsania et al., 2024). In summary, pepper cultivation systems demonstrate heightened vulnerability to BPA-contaminated irrigation water and soil exposure, with consequent bioaccumulation risks in plant tissues. Comprehensive elucidation of root uptake pathways, stress-response biomarkers, and phytohormone interference mechanisms under chronic BPA exposure during vegetative and reproductive stages is critical for establishing evidence-based thresholds in agricultural soil remediation protocols. This study focuses on Zhangshugang pepper, a traditional high-quality cultivar from Hunan, China, renowned for its superior flavor and consumer popularity. Under controlled greenhouse conditions, it systematically investigates BPA absorption and physiological toxicity responses in pepper roots throughout the entire growth cycle. Plant root systems serve as the primary organ for BPA uptake under soil exposure conditions, with absorption dynamics regulated by multiple interacting factors including plant species, growth stage, soil matrix composition, and BPA exposure concentration (Wang, et al., 2018). As a highly adaptable crop, pepper (Capsicum annuum L. ) thrives across diverse soil types, though its root absorption of BPA may be significantly influenced by soil particle composition and pH variations (Liu et al., 2024). In contaminated soils, BPA exists in two distinct fractions: dissolved in pore water and adsorbed onto soil particles. Current understanding remains limited regarding plant root preferential uptake between these two BPA phases. To systematically investigate BPA absorption patterns in pepper roots, the soil-root concentration factor (RCFsoil), which quantifies root absorption capacity from soil bound BPA, and the pore water-root concentration factor (RCFpw), which assesses uptake efficiency from dissolved BPA fractions, were employed (Liu et al., 2024). Additionally, the translocation stem concentration factor (TSCF) is uesd to evaluate BPA’s root-to-shoot translocation capacity (Bagheri et al., 2021). Elucidating these absorption-translocation mechanisms within the plant’s root-soil system provides critical insights for assessing exposure risks in BPA contaminated agricultural environments. Plant root systems physiological status plays a crucial role in the plant’s overall stress response (Zhang et al., 2018). Studies demonstrate that high BPA concentrations significantly inhibit soybean root growth. Under 6 mg/L BPA stress, the number of primary lateral roots, root surface area, and root length in soybeans decreased by 11.4%, 11.5%, and 22.7%, respectively (Li et al., 2018a). Additionally, BPA stress disrupts the antioxidant system in Arabidopsis roots, inducing abnormal accumulation of reactive oxygen species (ROS) and subsequently inhibiting root elongation (Bahmani et al., 2020). Research indicates that tetrabromobisphenol A, a BPA derivative, can elevate abscisic acid levels in plants and enhance stress resistance through activation of ABA signaling pathways (Yanhui et al., 2023). The metabolic disruptive effects of BPA on root growth are closely associated with phytohormone synthesis and signaling transduction pathways. BPA exposure interferes with the secretion of growth hormones in soybean roots and alters the ratios between growth hormones (auxins and cytokinins) and stress hormones (abscisic acid), thereby affecting root growth (Li et al., 2018b). Although existing studies have elucidated BPA’s interference mechanisms in root growth and physiological functions of some plants, systematic research remains lacking regarding the sensitivity of peppers, an economically significant crop, to BPA stress and their root physiological response mechanisms. It is important to systematically analyze phenotypic changes, antioxidant system parameters, and phytohormone metabolic responses in pepper roots under BPA stress. Integrated transcriptomic and metabolomic analyses will help clarify specific pathways through which BPA disrupts phytohormone signaling and reveal the potential disruptive effects of BPA exposure on pepper root development and physiological metabolism. The specific research objectives include: a) to analyze the effects of BPA application concentrations, soil types, and growth stages on BPA distribution and absorption at the soil-pore water-root interface; b) to decipher BPA translocation patterns in pepper plants and identify key factors influencing its translocation and accumulation; c) to assess BPA-induced interference effects on root growth parameters, antioxidant defense systems, and phytohormone; d) to integrate metabolomic and transcriptomic profiling to characterize root metabolite variations and gene expression changes under BPA stress, thereby identifying critical differential metabolites and associated regulatory pathways to elucidate molecular mechanisms of BPA phytotoxicity. Materials and methods not-yet-known not-yet-known not-yet-known unknown 2.1. Pot experiments of pepper The seeds of Zhangshugang pepper were provided by the National Research Laboratory for Specialty Vegetable Industry at Hunan Agricultural University. Three types of soil (viscosity soil, loamy soil, and sandy soil) were collected from pepper cultivation fields at Hunan Agricultural University in Changsha, Hunan Province, China (E113°08’, N28°17’). Detailed soil information can be found in Table S1. pepper seeds were germinated in a 30°C constant-temperature dark incubator, and uniformly grown seedlings were selected and transplanted into soil until the six-leaf stage. BPA particles were fully dissolved in acetone and mixed into the three soil types at concentrations of 1 mg/kg, 10 mg/kg, and 100 mg/kg, along with a blank control. Each treatment was replicated three times biologically. The treated soils were placed in a well-ventilated area for three days to allow complete evaporation of acetone before being used for pepper seedling cultivation. During the experiment, pepper seedlings were grown in a greenhouse and subjected to stress treatment for 15 days. The greenhouse maintained daytime temperatures at 30 ± 3°C and nighttime temperatures at 25 ± 3°C. The cultivation period spanned from May to August 2024. 2.2. BPA determination in pepper Pepper roots were sampled after 15 days of BPA stress treatment, thoroughly rinsed with deionized water to remove adhering soil particles, and blotted dry with filter paper. Plant samples were weighed, freeze-dried, and ground into fine powder. Additionally, soil and pore water samples were collected from pepper cultivation pots. All samples underwent BPA detection and quantitative analysis using previously reported analytical methods (Zhou et al., 2019). The details could be found in section 1.1 of the Supplementary Information (SI). 2.3. RCF and TSCF The RCF soil and RCF pw were employed as evaluation metrics. Root absorption capacity was assessed by calculating the ratio of BPA concentration in root tissues (on a fresh weight basis) to BPA concentration in the corresponding exposure medium (soil or pore water) (Liu et al., 2021). The TSCF is defined as the ratio of the contaminant concentration in the plant transpiration stream to its concentration in the soluble medium (pore water) (Bagheri et al., 2020). The RCF and TSCF values were calculated as follows: \(\text{RCF}_{\text{soil}}=\frac{C_{\text{root}}}{C_{\text{soil}}}\) (2-1) \(\text{RCF}_{\text{pore\ water}}=\frac{C_{\text{root}}}{C_{\text{pw}}}\)(2-2) \(\text{TSCF}=\frac{C_{\text{st}}}{C_{m}}\) (2-3) RCF soil represents the soil-to-root concentration factor, RCF pw represents the pore water-based concentration factor, C root denotes the BPA concentration in roots, Csoil denotes the BPA concentration in soil, TSCF represents the transpiration stream concentration factor, C st indicates the concentration of the compound in stem xylem solution, and C m indicates the concentration of the compound in the exposure media. 2.4. Antioxidant enzymes analysis Freshly pepper root samples were placed into 2 mL centrifuge tubes and immediately immersed in liquid nitrogen for rapid freezing. The frozen samples were then ground and pulverized using a high-throughput tissue grinder at 60 Hz.Then, according to the procedures of the enzyme activity detection kit for catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and malondialdehyde (MDA) content detection kit purchased from Beijing Hezi Shenggong Technology Co.The details could be found in section 1.2 of SI. 2.5. plant hormone analysis of pepper’ roots The levels of three plant hormone (indole-3-acetic acid (IAA), zeatin riboside (ZR), and abscisic acid (ABA)) in pepper roots between experimental and control groups (CK) were measured after 15 days of stress treatment. The cleaned roots were placed in 2 mL centrifuge tubes and ground into fine powder using a tissue homogenizer. Subsequently, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was employed to quantify these hormones in the samples. Detailed detection methods are described in section 1.3 of SI, and referenced from previous literature reports (Luo et al., 2021). 2.6. Transcriptome analysis Transcriptome profiling was conducted using the NovaSeq 6000 sequencing platform (Illumina) to investigate molecular response mechanisms of pepper roots under BPA stress. At 15 days post-stress treatment, 0.5 g of root tissues were collected from both the experimental group (100 mg/kg BPA) and the control group (three biological replicates per group), flash-frozen in liquid nitrogen, and homogenized. Total RNA was extracted using the TRizol Reagent Kit (Tiangen). RNA purity and concentration were assessed via Nanodrop 2000 spectrophotometer (Thermo Scientific), while RNA integrity was verified using the Agilent 2100 Bioanalyzer/LabChip GX system (PerkinElmer). Upon passing RNA quality control, library construction and sequencing were performed. Experimental details are described in Section 1.4 of SI. Post-sequencing data analysis was executed on the BMKCloud bioinformatics platform ( http://www.biocloud.net ). 2.7. Validation of RNA-Seq results using quantitative RT-PCR (qRT-PCR) Six candidate genes associated with IAA, ABA, and cytokinin pathways were selected for qRT-PCR validation. qRT–PCR was conducted in a 20-μL volume containing 1 μL of diluted cDNAs, 0.4 μL of the forward primer, 0.4 μL of the reverse primer, and SYBR qPCR Master Mix (Vazyme) under the following conditions: 95 ℃ for 180 s, followed by 40 cycles of 95 ℃ for 10 s, 60 ℃ for 30 s. The 2- △△Ct method was used to calculate relative expression levels. The expression trends of selected genes were analyzed to determine their consistency with transcriptomic profiling results. If the expression trends were consistent, the RNA-Seq results were considered reliable. 2.8. Metabolomic analysis To investigate differential metabolites in pepper roots under BPA stress, LC-MS-based metabolomic analysis was performed. After 15 days of BPA stress treatment, 0.5 g of root tissues were collected from both the experimental group (100 mg/kg BPA) and control group pepper seedlings (three biological replicates per group). 50 mg of powdered sample was weighed and mixed with 1200 μL of pre-chilled (-20°C) 70% methanol aqueous solution containing internal standards. The mixture was vortexed every 30 minutes (30 seconds each time, 6 times total), then centrifuged at 12,000 rpm for 3 minutes. The supernatant was discarded, and the solution was filtered through a 0.22 μm microporous membrane before being transferred to sample vials for ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry analysis. Detailed procedures could be found in Section 1.5 of SI. 2.9. Statistical analysis One-way analysis of variance (ANOVA) was utilized to evaluate the significant differences in the data of pepper. Statistical analysis was conducted using SPSS 22.0 software for Windows (IBM Corp., Armonk, NY). p < 0.05 was considered significantly different. Result and discussion 3.1 Distribution patterns of BPA in the “soil-pore water-root” ecosystem This study systematically investigated the accumulation characteristics of bisphenol A (BPA) in pepper seedlings over 15 days and its distribution patterns under three initial BPA concentration (1, 10, and 100 mg/kg) and three soil types (loamy soil, sandy soil, and viscosity soil). BPA concentrations in pepper roots and pore water showed highly significant variations (p < 0.01) across different soil types (Tables S2, Tabel S3). The cumulative concentration of BPA exhibited a positive correlation with its initial application rate, with a consistent three-phase distribution pattern across all treatments: soil > pore water > roots (FIGURE 1). As shown in FIGURE 1a, soil BPA concentrations increased significantly with higher initial application rates (1, 10, and 100 mg/kg), while differences among soil types under the same application rate were minimal. FIGURE 1b shows that the concentration of BPA in pore water also varies significantly between different soil types.FIGURE 1c shows that plant roots grown in Viscosity soil accumulated significantly higher BPA concentrations compared to those in Sandy and Loamy soils under identical BPA treatments.Existing studies suggest that soils with larger particle sizes exhibit stronger adsorption capacities for BPA (Xiao et al., 2020). In this study, despite the high clay content (46.6%) in Viscosity soil, its finer clay particles weakened BPA adsorption, may allowing more BPA to remain in pore water and subsequently be absorbed by pepper roots. In contrast, Sandy and Loamy soils, characterized by larger sand particles, showed stronger BPA adsorption, resulting in lower BPA concentrations in both pore water and roots. These findings indicate that pepper plants cultivated in viscous soil are more prone to BPA uptake from pore water. Different soil matrixes had a more significant effect on the bioavailability of BPA and its absorption by plant roots. Further studies will be conducted on the preference of pepper roots for BPA and uptake and transform tendency in different soils. FIGURE 1. The BPA concentrations in (a) pepper roots (ng/g dry weight, left scale), (b) soil (ng/g dry weight, left scale), and (c) pore water (ng/mL, right scale) after cultivating normally grown pepper seedlings in BPA-containing soil for 15 days(Error bars are standard deviations (n = 3, different letters indicate significant differences at p < 0.05 (a), p < 0.01 (b), and p < 0.001 (c) ). 3.2. Uptake and transform law of BPA in the ”soil-pore water-root” ecosystem To better elucidate the influence of soil sorption on BPA uptake and transform in root, two types of relative concentration factors (RCFs) were calculated: RCFsoil and RCFpore water. Both RCF soil and RCF pore water values differed significantly among the three soil types ( p < 0.01) (TablesS4, S5). As illustrated in FIGURE 2a and 2b, the RCF soil and RCF pore water values were highest in weakly alkaline Viscosity soil across all BPA concentrations. This disparity is likely attributed to the molecular structure of BPA.The two phenolic hydroxyl groups in BPA molecules tend to dissociate into phenolate anions under weakly alkaline conditions. These charged phenolate anions more readily form hydrogen bonds with water molecules, thus increasing its solubility (Monica et al., 2024). Consequently, BPA solubility increases markedly in weakly alkaline or alkaline environments. In contrast, under near-neutral pH conditions, BPA predominantly exists in its non-dissociated form, exhibiting lower solubility and stronger adsorption to organic matter or particulate surfaces. Collectively, these mechanisms suggest that alkaline soils facilitate BPA absorption by pepper roots. To investigate the transform patterns of BPA in pepper plants, the TSCF values under different soil cultivation conditions were illustrated. The TSCF under soil cultivation conditions prioritizes the concentration of pore water over the total soil concentration. All subsequent studies are based on TSCF pw . Significant differences (p < 0.05) in TSCF values were observed among the three soil types across varying BPA concentrations (Table S6). As shown in FIGURE 2c, TSCF values for pepper plants ranged approximately from 0.15 to 0.35. Previous studies have reported TSCF values for BPA in wheat, tomato, and maize stabilized at 0.1, 0.07, and 0.02, respectively (Bagheri et al., 2021). Compared to these plants, the comparatively higher TSCF values in pepper suggest that BPA is more readily transported via the transpiration stream through the xylem system to shoot tissues. At a BPA application concentration of 1 mg/kg, significant differences in TSCF values were observed among soil types ( p < 0.05), indicating that the soil matrix profoundly influenced BPA translocation to shoot tissues via the transpiration stream. However, as the BPA concentration increased to 10 mg/kg and above, differences in TSCF values across soil types gradually diminished and converged (FIGURE 2c). This phenomenon may be attributed to high-concentration BPA stress surpassing the detoxification capacity of pepper plants mediated by transpiration, resulting in the upward transport capacity of BPA tends to saturate and thereby slowing down the variation range of TSCF value.Sandy soil, characterized by the highest sand content, exhibited strong thermal insulation properties but poor water retention capacity. Elevated temperatures and high permeability in sandy soil may enhance plant transpiration rates, potentially increasing the efficiency of BPA translocation to shoots(Namiki et al., 2018). Consequently, differences in soil composition led to variations in BPA translocation efficiency, with pepper plants cultivated in Sandy soil were more easyly to transport BPA to the above-ground parts. FIGURE 2. Relationship between BPA concentration in pepper roots (C root ) and BPA concentration in soil (C soil ) and pore water (C pw ) (a), changes of RCF values in different soils(b), changes of TSCF values of peppers in different soil (c). To investigate BPA uptake in the root-plant ecosystem, the relationships among BPA concentrations in roots, soil, and pore water were analyzed using a linear regression model. In . As shown in FIGURE 3, sandy soil and viscosity soil, the correlation between BPA concentrations in roots and pore water was the highest (R 2 = 0.9379 in sandy soil, 0.9561 in loamy soil, and 0.9809 in clay soil), whereas weaker correlations were observed between root and soil BPA levels (R 2 = 0.8968, 0.9208, and 0.8567, respectively). Combined with the results in FIGURE 1, the BPA concentration in pepper roots was comparable to that in soil pore water. This indicates that the accumulation trend of BPA in pepper root system shows a more similar change pattern with the concentration of BPA in soil pore water. Collectively, within the ”soil-pore water-root” system, the BPA concentration in soil pore water serves as a critical determinant of its bioavailability for plant uptake. FIGURE 3. Relationship between BPA concentration in pepper roots (C root ),soil (C soil )and pore water (C pw ). 3.3. Response of plant root physiochemical parameters to BPA exposure Physiochemical parameters of pepper when exposed to BPA were investigated. As shown in FIGURE 4a and 4b, under the treatment of 1 mg/kg BPA concentration, the length of the main root of the treated pepper was slightly larger than that of the CK group. When the BPA concentration was more than 10 mg/kg, BPA had a significant inhibitory effect on the length of the main root of the pepper. The phenotypes of the roots in different soils showed similar patterns. The paraffin sections of pepper roots after BPA treatment are shown in FIGURE S1. The results demonstrated intensified staining coloration in root tissues of BPA-treated groups, suggesting potential cell wall lysis or structural disruption induced by BPA stress. As shown in FIGURE 4c, BPA treatment had a certain effect on the root vitality of pepper, which was measured by the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) (Aljayan et al., 2025). Pearson correlation analysis of TTC reduction across soil types revealed no statistically significant differences between Sandy and Loamy soils ( p > 0.05). However, Viscosity soil exhibited significant correlations with sandy soil ( p < 0.05) and highly significant correlations with loamy soil ( p < 0.01) (Table S7). Low-concentration BPA treatment (1 mg/kg) induced a marginal increase in TTC reduction compared to CK, suggesting transient stimulation of root metabolic activity. Conversely, BPA concentrations exceeding 10 mg/kg resulted in progressive decline of TTC reduction levels, indicating impaired root vitality. At 100 mg/kg BPA treatment, TTC reduction in Sandy, Loamy, and Viscosity soils decreased by 68.68%, 76.18%, and 65.75% respectively relative to CK, demonstrating severe root vitality impairment. These findings establish a critical threshold concentration (10 mg/kg BPA) for significant phytotoxic effects on pepper root systems across varied soil matrices. FIGURE 4. Phenotype of pepper roots after 15 days of BPA stress (a), statistical graph of pepper root length after BPA stress in different soils (b), and statistical graph of pepper root vitality after 15 days of BPA stress (c). 3.4. BPA responses of antioxidant systems The most pronounced uptake and accumulation of BPA in roots occurred in viscosity soil. Thus, subsequent investigations focused on BPA stress in viscosity soil. The antioxidant system serves as a critical biomarker for evaluating plant stress responses, primarily characterized by key enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and MDA content (Zhou et al., 2021). In FIGURE 5a, BPA exposure induced membrane lipid peroxidation in pepper seedlings, triggering MDA production. While 1 mg/kg BPA caused minor alterations in MDA levels, 100 mg/kg treatment provoked a marked increase. CAT activity demonstrated significant upregulation in medium-to-high BPA concentration groups (FIGURE 5b). Both POD (FIGURE 5c) and SOD (FIGURE 5d) activities exhibited concentration-dependent enhancement under BPA stress, though their activities at 72 h showed progressive attenuation compared to 48 h measurements. Under the three different soil conditions, the trends of MDA and enzyme activities levels were generally consistent. In addition, there were differences in the time when SOD and POD activity reached peak under different concentrations of BPA. Among them, the 10 mg/kg treatment group reached peak at 72 hours, while the 100 mg/kg treatment group reached peak at 48 hours.This temporal shift suggests accelerated activation of defense mechanisms under high-concentration BPA stress. Collectively, these findings demonstrate that BPA significantly induces membrane lipid peroxidation, prompting plants to modulate antioxidant enzyme activities as a countermeasure against oxidative damage. The observed enzymatic kinetic patterns further indicate concentration-dependent temporal optimization of stress response pathways. FIGURE 5. The root MDA contents (a), CAT value (b), POD value (c) and SOD activity (d) in three types of soil. not-yet-known not-yet-known not-yet-known unknown 3.5. plant hormone responses in pepper roots under BPA stress plant hormone play a central regulatory role in plant responses to environmental stress. This study investigated the effects of BPA exposure on three root-related plant hormone (IAA, ABA, and ZR). The results are presented in FIGURE 6. FIGURE 6a demonstrates a biphasic response of IAA content in pepper roots following 15-day BPA exposure: under low-concentration BPA treatment, IAA levels increased, suggesting potential promotion of root growth, whereas high-concentration treatment significantly reduced IAA content, indicating probable inhibition of auxin-mediated root development regulation. This trend aligns with previously reported literature (Li et al., 2018). FIGURE 6b reveals that ZR content exhibited a concentration-dependent trend similar to IAA: low-dose BPA exposure enhanced ZR accumulation, whereas high concentrations suppressed it. As a cytokinin-class phytohormone, ZR regulates cell division/elongation and modulates photoassimilate translocation. These results suggest coordinated regulation by IAA and ZR in pepper root responses to BPA stress, with both hormones showing significant concentration-dependent variations. In contrast, ABA content progressively increased with BPA concentration (FIGURE 6c), indicating activation of enhanced stress signaling pathways under high BPA exposure. While ABA (a pivotal stress hormone), has been documented to improve stress tolerance in pepper plants, its accumulation may simultaneously inhibit root growth through trade-off effects between defense and development (Dong et al., 2021). The precise regulatory mechanisms through which BPA stress inhibits root growth remain to be fully elucidated. Although the current study observed significant alterations in phytohormone levels in pepper roots under BPA stress, the underlying regulatory pathways require further mechanistic investigation. Therefore, integrated multi-omics analyses combining metabolomic and transcriptomic approaches are necessary to systematically investigate the response mechanisms of pepper roots at both metabolic and gene expression levels. FIGURE 6. Concentrations of IAA (a), ABA (b), and ZR (c)(different letters indicate significant differences at p < 0.05 (a), p < 0.01 (b), and p < 0.001 (c)). not-yet-known not-yet-known not-yet-known unknown 3.6. Transcriptome analysis not-yet-known not-yet-known not-yet-known unknown 3.6.1. RNA-seq and data quality control To characterize the gene expression profiles in pepper roots under BPA stress, this study performed transcriptome sequencing of root tissues from both BPA-treated (R-BPA) and control (R-CK) groups after 15 days of BPA exposure. Sequencing data quality was evaluated based on the percentage of bases with Q-score ≥30, with all samples exhibiting 93% of sequenced data), demonstrating high data quality and integrity. Clean reads were aligned to the reference genome (Capsicum annuum.Zunla-1 v2.0.genome.fa) using HISAT2 software. All samples showed insert sizes ranging 300-500 bp (reflecting good library uniformity) and mapping rates of 89.95%-93.31%. Detailed alignment statistics are provided in Table S8. 3.6.2 Transcriptome annotation and DEG recognition Gene expression levels in both treated and control groups were quantified using FPKM values. PCA analysis (FIGURE 7a) and OPLS-DA (FIGURE 7b) revealed significant transcriptomic differences in pepper root tissues under 100 mg/kg BPA soil stress. The three replicate samples from both CK and treated groups clustered together, indicating good intra-group reproducibility. Differential expression analysis was performed by calculating Fold Change (FC) and False Discovery Rate (FDR) for all expressed genes. Using thresholds of FC≥2 and FDR<0.01, we identified 5,202 differentially expressed genes (DEGs). Compared with the CK group, the treated group showed 2,370 significantly upregulated genes and 2,832 significantly downregulated genes (FIGURE 7c). Cluster analysis results of all DEGs are presented in FIGURE 7d. not-yet-known not-yet-known not-yet-known unknown FIGURE 7. PCA of differentially expressed genes (DEGs) in pepper roots (a); orthogonal partial least squares discriminant analysis (OPLS-DA) of DEGs (b); volcano plot showing significance and fold change of DEGs (c); hierarchical clustering heatmap of DEGs (d). 3.6.3. Gene function annotation and analysis DEGs were functionally classified based on the Clusters of Orthologous Groups (COG) database, with the classification results and their proportions shown in FIGURE 8a. There were 12 functional categories containing over 50 enriched genes. Among these, genes associated with “secondary metabolites biosynthesis, transport and catabolism” were the most abundant, accounting for 13.38% of the total, suggesting this process may play a significant role in the response to BPA stress. Detailed annotation information is provided in Table S9 and Table S10. Gene ontology (GO) enrichment analysis was performed for all DEGs (FIGURE 8b). In the Biological Process category, DEGs were primarily enriched in three core functional modules: metabolic processes, cellular processes, and biological regulation. Hierarchical clustering analysis revealed that specific terms such as the auxin-activated signaling pathway (GO:0009733) showed highly significant enrichment ( p < 0.001). Within the Cellular Component classification, enriched terms demonstrated strong association between DEGs and integral components of membrane (GO:0016021), with this GO term annotating 978 genes, suggesting BPA treatment primarily induces alterations in membrane composition and structure in pepper roots. Such changes may affect intercellular hormone communication and disrupt root growth and development. At the Molecular Function level, DEGs showed significant enrichment in three protein functional characteristics: catalytic activity, binding activity, and transporter activity, indicating BPA stress likely mediates physiological changes by regulating the expression of diverse functional proteins, thereby influencing overall physiological functions and stress response capabilities in pepper plants. KEGG pathway enrichment analysis (FIGURE 8c) systematically elucidated the metabolic and signal transduction networks involved in BPA stress-induced DEGs. Among the top 18 most significant pathways (q < 0.20), three key metabolic regulatory modules were identified: the plant-pathogen interaction pathway (ko04626) was significantly enriched with 250 DEGs ( p <0.05); the plant hormone signal transduction pathway contained 150 enriched DEGs ( p <0.05); and the phenylpropanoid biosynthesis pathway also showed significant enrichment. These results suggest that BPA stress may coordinately disrupt three core regulatory modules, plant hormone signaling, pathogen interaction pathways, and secondary metabolism, thereby compromising the plant’s overall defense response capacity. FIGURE 8. COG analyse and annotation (a), GO analyse and annotation (b), KEGG analyse and annotation (c). 3.6.4. qRT-PCR verification To validate the reliability of the RNA-seq data in this transcriptomic study, six DEGs associated with IAA, CK, and ABA were selected for qRT-PCR analysis (FIGURE 7). The candidate genes included: YUC protein genes (Capana08g002820, Capana08g002823), TDC protein gene (Capana07g001621), CKX protein genes (Capana00g000659, Capana04g000295), and NCED protein gene (Capana00g000349). Results demonstrated that under 100 mg/kg BPA soil exposure, YUC protein genes were upregulated while TDC, CKX, and NCED protein genes were downregulated. This expression pattern was consistent with the transcriptional changes observed in RNA-seq, confirming the high reliability of the transcriptomic sequencing results. FIGURE 9. BPA affects the expression of plant-related genes. 3.7. Metabolome analysis 3.7.1 Metabolomics data quality control Metabolomic analysis was conducted using the blank control group (R-CK, 0 mg/kg) and high-concentration BPA treatment group (R-BPA, 100 mg/kg). Overlay analysis of total ion chromatograms (FIGURE S2) and QC sample correlation analysis (FIGURE S3) demonstrated high reproducibility and reliability of the detection data. Principal component analysis (PCA) results (FIGURE 10a) revealed clear separation between the treatment and CK groups, indicating significant metabolic differences before and after BPA stress. Clustering of samples from the same treatment confirmed good experimental reproducibility. The variance explanation rates were 48.35% for the first principal component (PC1) and 14.26% for the second principal component (PC2). Orthogonal partial least squares-discriminant analysis (OPLS-DA) of both groups yielded a model (FIGURE 10b) with Q2=0.94. The clear separation between treatment groups in OPLS-DA results was consistent with PCA findings, confirming significant metabolic differences between BPA-treated and control groups. 3.7.2. Differential metabolites analysis Comparative analysis between treated and control groups identified 1,076 differential metabolites (DMs), including 567 significantly upregulated (FC≥2, VIP>1) and 509 significantly downregulated (FC≤0.5, VIP>1) metabolites. Cluster analysis of all DMs is presented in the heatmap (FIGURE S4). Primary classification (FIGURE 10c) revealed that amino acids and their derivatives constituted the highest proportion (35.78%) of detected metabolites, highlighting their crucial role in both basal metabolism and stress responses in plant roots. Additionally, benzene and its substituted compounds (9.94%) and organic acids (9.76%) represented substantial fractions, suggesting potential involvement of aromatic compounds and carbon metabolism-related substances in complex physiological regulation under stress conditions. Other components including alkaloids (5.20%) and alkanolamines (3.53%) also showed notable abundance, further demonstrating the complexity and diversity of metabolic responses in pepper roots under BPA stress. Mong these differential metabolites (DMs), 385 (35.8%) were classified as amino acids and derivatives, while 107 (9.9%) belonged to benzene and substituted derivatives. FIGURE 10d displays the top 10 upregulated and top 10 downregulated DMs, including 16 secondary metabolites (8 upregulated and 8 downregulated). These secondary metabolites primarily consisted of amino acids and their derivatives, alkaloids, and benzene derivatives. The two substances showing the most significant fold-changes were short peptides: Tryptophylglycine (Log 2 FC = 11.13) and Met-Abu-OH (Log 2 FC = -8.50). These findings demonstrate that BPA stress primarily affects pepper root cell growth by disrupting the expression levels of secondary metabolites. 3.7.3 KEGG pathway enrichment analysis KEGG pathway annotation and classification of DMs identified 213 metabolites mapped to 65 distinct signaling pathways across ** categories, with the six pathways containing the highest number of annotated DMs shown in FIGURE 10e. The two pathways with the most differential metabolite annotations were metabolic pathways (ko01100) and biosynthesis of secondary metabolites (ko01110). In plants, IAA, CK, and ABA are functionally linked to tryptophan metabolism, nucleotide metabolism, and carotenoid metabolism respectively (Wu et al., 2023; Parry et al., 1991; Woodward et al., 2005). Further analysis revealed 18 DMs associated with these three metabolic pathways and involved in the biosynthesis or activity regulation of these three plant hormone, with detailed information provided in Table S11. not-yet-known not-yet-known not-yet-known unknown FIGURE 10. PCA analysis of the metabolome in pepper roots (a); OPLS-DA analysis of the metabolome data from pepper roots (b); classification and relative abundance of metabolites detected in pepper roots (c); top 10 upregulated and downregulated differential metabolites(d); the top 6 KEGG annotation pathways in pepper (e). FIGURE 11. presents cluster analysis results of the 18 DMs. Both L-tryptophan and indoleacetaldhde showed significant downregulation (FIGURE 11a). As direct precursors for IAA biosynthesis, their decreased levels would suppress IAA production. Notably, both coumarin and IAA originate from the shikimate pathway, while gramine shares the tryptophan metabolic pathway with IAA (Harbome et al., 1982; O’Donovan et al., 1963). The upregulation of coumarin and gramine may thus indirectly alter IAA biosynthesis through metabolic flux redistribution. These findings demonstrate BPA inhibits root IAA levels via dual mechanisms: suppressing precursor availability and promoting competitive pathway metabolites. Regarding cytokinin biosynthesis, isopentenyladenine serves as the pivotal intermediate derived from dephosphorylation of isopentenyladenosine monophosphate. Downregulation of 2’-deoxyadenosine and ribose-1-phosphate (FIGURE 11b) would impair nucleotide synthesis, consequently reducing IPA accumulation and ultimately inhibiting zeatin riboside production (Åstot et al., 2000). In ABA metabolism, geranyl diphosphate serves as a key intermediate in carotenoid-derived ABA biosynthesis, while mevalonate, a product of the mevalonate pathway, contributes indirectly to ABA production (Milborrow et al., 2000; Cutler et al., 1999). The stress-signaling molecule L-proline accumulated under elevated ABA levels (FIGURE 11c), activating plant defense responses (Tseng et al., 2013). These findings demonstrate that BPA stress alters the expression levels of precursors and shunt metabolites involved in the biosynthesis of IAA, CK, and ABA, thereby modulating the production levels of these three plant hormone. not-yet-known not-yet-known not-yet-known unknown FIGURE 11. Analysis of differentially expressed metabolites (DEMs) relative to IAA (a), CK (b) and ABA relative (c). 3.8. Integrated transcriptomic and metabolomic analysis plant hormone play crucial regulatory roles in root cell growth and development during pepper’s response to BPA stress. Therefore, integrated transcriptomic and metabolomic analyses of these three plant hormone were conducted based on the KEGG database. Detailed data for all key metabolites and genes are provided in Table S12 and Table S13. As shown in FIGURE 12a., the integrated analysis of IAA synthesis pathways (ko00380) revealed significantly differential responses of two major IAA biosynthetic routes under BPA stress. The indolepyruvate-dependent pathway was markedly activated, evidenced by significant upregulation of key enzymes L-tryptophan-pyruvate aminotransferase and indole-3-pyruvate monooxygenase genes, despite minimal changes in the intermediate metabolite indole-3-pyruvate (FC=1.69, VIP=1.25), suggesting this pathway may maintain a dynamic equilibrium between synthesis and metabolism. Conversely, the indole-3-acetaldehyde-dependent pathway was substantially suppressed, showing downregulated expression of related enzyme genes (aromatic-L-amino-acid decarboxylase, aromatic-L-tryptophan decarboxylase, aldehyde dehydrogenase) accompanied by significant reduction of the intermediate metabolite indole-3-acetaldehyde (FC1). Furthermore, decreased aromatic-L-amino-acid decarboxylase activity may also impair serotonin synthesis, thereby further diminishing this branch’s contribution to IAA production. Collectively, BPA treatment significantly inhibited the tryptamine-dependent IAA synthesis pathway while enhancing the indolepyruvate-dependent route. FIGURE 12b. presents an integrated analysis of plant hormone signal transduction pathways (ko04075). In the IAA signaling pathway, genes encoding AUX/IAA proteins were significantly downregulated. As AUX/IAA proteins form heterodimers with auxin response factors (ARFs) to repress auxin-responsive gene expression (Cho et al., 2024), their downregulation may attenuate ARF inhibition, thereby enhancing IAA signal responsiveness as a compensatory mechanism for reduced IAA biosynthesis. For CK signaling, downregulation of both AHP proteins (which phosphorylate B-ARRs to activate response genes) and B-ARR proteins collectively suppressed cytokinin signal transduction, consequently restricting cell division, differentiation and growth (Xie et al., 2018). Concurrent downregulation of A-ARR proteins (negative regulators of B-ARRs) further weakened this feedback inhibition. Regarding ABA signaling, upregulated SnRK2 and ABF genes activated ABA-responsive elements to initiate stress responses, while downregulated PYR/PYL receptors released inhibition on PP2Cs (Fujita et al., 2012). The resulting PP2C overexpression suppressed SnRK2 activity, preventing excessive ABA-responsive gene activation (Wang et al., 2024). PA stress triggered a coordinated hormonal response characterized by preferential activation of the indolepyruvate-dependent IAA biosynthetic pathway alongside suppression of the tryptamine-dependent route. Concurrently, both IAA and cytokinin signaling pathways were inhibited, while ABA signaling was activated yet tightly regulated through feedback mechanisms. This integrated response highlights the plant’s ability to reprogram hormonal pathways, balancing growth restriction with stress adaptation. FIGURE 12. IAA synthesis pathway (a) and plant hormone signal transduction pathway (b) for IAA, Cytokinin and ABA; +p represent phosphorylation; -p represent dephosphorylation; +u represent ubiquitination. Conclusion This study systematically elucidated the distribution characteristics of BPA within the “soil-pore water-root” ecosystem and revealed its disruptive effects on root metabolic processes. The results indicated that peppers grown in silt loam were more sensitive to BPA exposure, and BPA concentration in soil pore water was the key determinant of its bioavailability and phytotoxicity. BPA exhibited a typical non-linear dose response relationship in pepper roots, characterized by low-dose stimulation and high-dose inhibition. When BPA concentrations exceeded 10 mg/kg, root development was significantly suppressed, accompanied by elevated levels of ROS and MDA, indicating enhanced oxidative stress and membrane system damage. In terms of hormone response, pepper plants downregulated growth-promoting hormones (IAA and ZR) and upregulated stress-related hormones (ABA) to enhance their stress adaptation. Transcriptomic analysis revealed that BPA interfered with key regulatory pathways such as hormone signal transduction, plant-pathogen interaction, and secondary metabolism, thereby reshaping the root’s defense response network. Metabolomic analysis further showed that BPA stress altered the expression levels of hormone precursors and branch metabolic products, which in turn regulated the synthesis and signaling of IAA, CK, and ABA. Integrated analysis demonstrated that the IPA-dependent IAA biosynthesis pathway was activated, while the IAAld-dependent pathway was suppressed. Meanwhile, AUX/IAA protein expression was significantly downregulated, which may serve as a compensatory mechanism for reduced IAA biosynthesis. CK signaling was inhibited, whereas ABA signaling was activated but subject to negative feedback regulation. 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Keywords bisphenol a development hormones pepper phytohormone metabolic root uptake Authors Affiliations Zhigang Huang Hunan Agricultural University View all articles by this author Qingmei Zhu Hunan Agricultural University View all articles by this author Jing Lu Hunan Key Laboratory of Land and Resources Evaluation and Utilization View all articles by this author Ze Liao Hunan Agricultural University View all articles by this author Qinyao Li Hunan Agricultural University View all articles by this author Qiuping Wang Hunan Agricultural University View all articles by this author Binglin Wu Hunan Agricultural University View all articles by this author Zhoubin Liu Hunan Agricultural University View all articles by this author Shaohua Zhu Hunan Key Laboratory of Land and Resources Evaluation and Utilization View all articles by this author Zhoufei Luo [email protected] Hunan Agricultural University View all articles by this author Metrics & Citations Metrics Article Usage 195 views 130 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Zhigang Huang, Qingmei Zhu, Jing Lu, et al. Uptake characteristics and plant hormone metabolic disruption of bisphenol A in pepper (Capsicum annuum L.) roots under soil exposure. Authorea . 18 June 2025. DOI: https://doi.org/10.22541/au.175022846.60921505/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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