Phytotoxic Effects of Bisphenol A on Growth and Physiology of Capsicum annuum L.

Abstract

Bisphenol A (BPA) is a widely used chemical to produce raw materials in plastic production, which has led to its ubiquity in the natural environment and toxicity to both plants and humans. In this study, we evaluated the phytotoxic effects of BPA on the growth and physiology of pepper (Capsicum annuum L.), a globally cultivated horticultural plant. Our high-performance liquid chromatography (HPLC) result revealed that 0.5 mg/kg of BPA treatment did not lead to the accumulation of BPA in the leaves and fruits of pepper plants. The exogenous application of 5 mg/kg of BPA prominently inhibited pepper growth, while 0.5 mg/kg of BPA had no obvious effects on pepper growth. Additionally, our transcriptomic assay revealed that BPA-regulated gene expression is associated with photosynthesis and reactive oxygen species (ROS) signaling. Physiological and qRT-PCR assays further demonstrated that BPA reduced chlorophyll content and increased ROS levels by regulating the expression of genes related to chlorophyll synthesis and ROS production. Our transcriptomic data also elucidated the potential role of plant hormones, including brassinolides (BR), salicylic acid (SA), jasmonic acid (JA), and strigolactone (SL) in mediating BPA-induced phytotoxicity. Furthermore, BPA activated the N⁶-methyladenosine (m⁶A) modification to exert its toxicity. Collectively, our findings offer additional insights into the mechanisms through which BPA attenuates pepper plant growth, which might contribute new knowledge toward a better scientific assessment of BPA exposure risks in horticultural species.

1. Introduction

Bisphenol is known as a monomer in the synthesis of polycarbonate plastics and epoxy resin-based materials, with bisphenol A [2,2-Bis (4-hydroxyphenyl) propane, BPA] being the predominant bisphenol utilized in the industry [1]. BPA is subsequently used in the generation of various products, such as food storage containers, automotive components, electrical devices, safety equipment, and many more products [2]. It has been suggested that global demand for BPA will reach approximately 7.7 million metric tons, with expectations rising to 9.5 million metric tons by 2028 [3]. The heavy demand for BPA-based products in various industries has resulted in their infiltration into the natural ecosystem [4]. Research has shown that the concentration of BPA in dry soil varies from 2 to 140 g/kg across regions, including Europe, the United States, and South Korea [5], which could be due to the leaching of BPA from plastic- or resin-lined products [6]. Moreover, investigations have demonstrated the ubiquitous occurrence of BPA in various aquatic environmental samples, including surface water, seawater, groundwater, and sediments [7], and the highest BPA concentration detected in groundwater is 572.8 μg/L [8]. BPA is also detected in the atmosphere in many cities around the world [9]. Due to its pervasive presence and the potential for elevated concentrations in the ecosystem, BPA has been classified as an environmental contaminant of increasing concern, presenting risks to exposed living species, including humans, animals, and plants [4].

BPA has been reported to elicit numerous adverse health effects in humans [10]. Following ingestion, BPA undergoes rapid metabolism and elimination in humans, exhibiting a half-life of less than 6 h [11]. The majority of absorbed BPA is transformed into the gastrointestinal (GI) tract and liver into BPA-glucuronides (BPA-Gs) and, to a lesser extent, BPA-sulfate conjugates before entering systemic circulation and being excreted in conjugated forms via urine [12]. Consequently, BPA impacts various human organs such as the lungs, liver, and heart, leading to diseases including asthma [13], non-alcoholic fatty liver disease, cirrhosis [14], and diabetes [15]. Although BPA has not been classified as a human carcinogen by any regulatory or scientific organization, its adverse effects on human health have raised significant concerns regarding its status as a hazardous contaminant.

BPA also exhibits phytotoxicity in plants through various mechanisms, such as impairing photosynthesis, generating reactive oxygen species (ROS), and altering plant hormone levels [16,17]. In Glycine max, plant height and the fresh and dry biomass of stems and leaves show significant reductions after 7 days of BPA exposure [18]. Similar growth retardation is noted in Oryza sativa upon BPA exposure [19]. A plausible explanation for BPA’s impact on plant growth is its propensity to significantly suppress photosynthetic activity, as evidenced by its reduced chlorophyll content [20,21]. Exposure to exogenous environmental stressors can trigger ROS production, inflicting damage on essential biomolecules such as nucleic acids and proteins in plants. In Glycine max, BPA exposure activates antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), leading to elevated ROS levels and growth inhibition [22,23]. BPA exposure also alters the concentrations of phytohormones like indole-3-acetic acid (IAA), zeatin (cytokinin), and ethylene [24]. Pepper (Capsicum annuum L.) is an important vegetable crop grown globally. Notably, the mechanisms by which BPA affects pepper growth and development remain largely elusive.

The aim of this work was to elucidate the toxicological impact of BPA and to delineate the molecular pathways implicated in the BPA-induced inhibition of pepper growth. Our high-performance liquid chromatography (HPLC) analyses indicate that the application of 0.5 mg/kg of BPA did not lead to its accumulation in pepper tissues, including leaves and fruits. Exposure to a concentration of 5 mg/kg BPA resulted in growth disturbances in pepper plants, evidenced by a reduction in both the fresh and dry biomass of leaves and roots. The transcriptomic assay further revealed that BPA regulated the transcriptional level of genes involved in photosynthesis, ROS signaling, and plant hormonal signaling. Our physiological assays also corroborated that BPA modulated photosynthesis and ROS levels by regulating the expression level of genes related to chlorophyll and ROS biosynthesis and metabolism. Beyond the established effects of BPA on phytohormones, our transcriptomic data elucidated the potential involvement of brassinolides (BR), salicylic acid (SA), jasmonic acid (JA), and strigolactone (SL) in mediating BPA’s toxic effects. Moreover, our findings demonstrated that BPA induces N⁶-methyladenosine (m⁶A) modifications, contributing to its toxicity. In summary, our study provides critical insights into the biotoxicity of BPA in pepper plants and establishes a theoretical framework for future evaluations of BPA toxicity in horticultural crops.

2. Materials and Methods

2.1. Plant Growth Conditions

Zunla-1 pepper seeds were surface-sterilized using 0.1% (w/v) potassium permanganate for 15 min and washed with deionized water four to five times. Subsequently, all seeds were germinated in the dark at 28 °C for five days. The germinated seeds were then transplanted into the soil (HAWITA, Shanghai, China, soil/vermiculite, 1:3), and each pot contained 120 g of soil. The pepper plants were cultivated in a growth chamber. To minimize the toxicity of BPA on Zunla-1 pepper plants, we first cultured pepper plants in the growth chamber for 30 days, then the 30-day-old pepper plants were used for BPA treatment and subsequent physiological assays.

2.2. BPA Treatment

Bisphenol A (BPA) (>99.8%, B802575, Macklin company, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) to create a stock solution with a concentration of 20 mg/L. The BPA stock solution was diluted in water to 50 mL, resulting in final concentrations of 0.5 mg/kg, 2 mg/kg, and 5 mg/kg of BPA (BPA/soil weight). Control samples were cultivated with BPA-free water. The Zunla-1 pepper plants were continuously exposed to BPA-contaminated soil. All photos were captured with a Nikon camera D300 (Nikon, Tokyo, Japan).

2.3. Determination of BPA Levels by High-Performance Liquid Chromatography (HPLC)

The 30-day-old Zunla-1 pepper plants were continuously exposed to control or 0.5 mg/kg BPA-contaminated soil for an additional 30 days under natural conditions. Then, the leaves and fruits of 60-day-old Zunla-1 pepper plants treated with control or 0.5 mg/kg BPA were collected for the HPLC assay. The leaf and fruit samples were ground into a fine powder, and 0.1 g power was collected and subsequently used for the HPLC assay. In total, 20 mL of methanol was added, and the samples underwent ultrasonication for 20 min. The resulting mixture was then centrifuged at 8000 rpm at 4 °C for 10 min, after which the supernatant was extracted for subsequent analysis. The concentration of BPA was quantified using an Agilent 1260 Infinity II HPLC system (Agilent Technologies, Santa Clara, CA, USA equipped with a UV–visible detector (VWD) and a ZORBAX SB-C18 column (4.6 × 250 mm). The standard concentration for BPA (B802576 for HPLC analysis, Macklin Company, Shanghai, China) was set at 0.1 mg/L. The mobile phase consisted of a methanol and water mixture in a ratio of 65:35 (v/v). The column was maintained at a temperature of 30 °C, with a flow rate of 0.3 mL/min. The injection volume was 10 μL, and detection was performed at a wavelength of 277 nm [25].

2.4. Fresh and Dry Weight Measurements

The 30-day-old Zunla-1 pepper plants were treated with control or 5 mg/kg BPA for 4 days, then the leaves and roots were harvested. For fresh weight determination, the leaves and roots were washed with deionized water four times, followed by drying on a paper blot before weighing. For dry weight determination, the fresh samples were initially subjected to a drying oven at 105 °C for 15 min and subsequently dried at 75 °C until a constant mass was obtained.

2.5. Determination of Chlorophyll Content

The 30-day-old Zunla-1 pepper plants were exposed to either control or 5 mg/kg BPA for 4 days; chlorophyll content (SPAD units) in the leaves [26] was assessed utilizing a chlorophyll analyzer (TYS-B, Zhejiang TOP Cloud-agri Technology Co., Ltd., Zhejiang, China). For each treatment, two distinct leaves from eight individual plants were used for the measurements. All experimental procedures were conducted in triplicate to ensure biological reproducibility.

2.6. Determination of Hydrogen Peroxide (H₂O₂) and SOD, POD, and CAT Enzyme Contents

The assay kits for quantifying the levels of H₂O₂ (H₂O₂-2-Y), SOD (SOD-1-Y), POD (POD-2-Y), and Catalase (CAT) (CAT-2-W) enzymes were purchased from Suzhou Kemin Biotechnology Co., Ltd., Suzhou, China). Briefly, a 0.1 g leaf sample was collected for the assay. All procedures were performed following the manufacturer’s protocol. Absorbance was recorded at 415 nm for H₂O₂, 240 nm for CAT, 470 nm for POD, and 560 nm for SOD via the spectrophotometer (GENESYS, Thermo Fisher, Massachusetts, USA) to calculate the enzyme content.

2.7. Transcriptome Analysis

The 30-day-old Zunla-1 pepper plants were treated with control or 5 mg/kg BPA for 4 days; then, pepper leaves were collected for transcriptomic analysis by OE Biotech (Shanghai, China). Total RNA extraction was performed utilizing the TRIzol reagent (Invitrogen, California, USA), following the manufacturer’s guidelines. The concentration and purity of the RNA were assessed with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Massachusetts, USA). RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Subsequently, libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit. Transcriptome sequencing and analysis were executed on the Illumina Novaseq 6000 platform (Illumina, California, USA), generating 150 bp paired-end reads. Raw sequencing reads for each sample was aligned to the Zunla-1 genome [27] using the HISAT2 alignment tool [28]. Fragments Per Kilobase of the exon model per million mapped fragment (FPKM) values for each gene were calculated [29], and gene read counts were determined via HTSeq-count [30]. Differential expression analysis was carried out using the DESeq2 method [31], with a significance threshold set at q value < 0.05 and fold-change > 2 or fold-change < 0.5 to identify differentially expressed genes (DEGs). The DEGs were subsequently subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses. Heatmaps were generated via TBtools software (Version 2.3) [32].

2.8. Quantitative Real-Time PCR Analysis (qRT-PCR)

The 30-day-old Zunla-1 pepper plants were treated with control or 5 mg/kg BPA for 4 days; pepper leaves were harvested for qRT-PCR analysis. Total RNA was isolated utilizing the plant RNA extraction kit (DP432, TIANGEN Biotech, Beijing, China) following the manufacturer’s protocol. Subsequently, 1 μg of RNA was used for cDNA synthesis through the StartScript II First-strand cDNA Synthesis Kit (GenStar A224, China). The qRT-PCR assays were performed with the 2×RealStar Fast SYBR qPCR Mix (GenStar A304, Beijing, China) via the LightCycler^® 480 Instrument (Roche, Basel, Switzerland). The CaACTIN (Capana12g001934) gene was used as an internal control, and the relative gene expression level was determined using the 2^−ΔΔCT method. For each independent biological replicate, the relative expression level was averaged from three technical replicates. All qRT-PCR primers are listed in Table S1.

2.9. Statistic Analysis

One-way ANOVA analysis was conducted via GraphPad Prism (version 10.4.0), with statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001; ns indicates non-significant.

3. Results and Discussion

3.1. The 0.5 mg/kg BPA Treatment Does Not Trigger BPA Accmulation in Pepper Tissues

Under our growth conditions, we observed that 2 mg/kg of BPA treatment significantly inhibited the growth of pepper plants, and 5 mg/kg of BPA exhibited a more pronounced effect, while 0.5 mg/kg of BPA did not induce obvious growth defects under our growth conditions (Figure S1). Consequently, we treated 30-day-old pepper plants with a substantially lower concentration of BPA (0.5 mg/kg) to enable the harvesting of pepper fruit and evaluated the accumulation of BPA in the leaves and fruits of pepper plants. Following an additional 30 days of continuous exposure to control or 0.5 mg/kg BPA-contaminated soil under natural conditions, we then collected the leaves and fruits of 60-day-old pepper plants treated with control or 0.5 mg/kg BPA to perform the HPLC assay. The detection of the standard BPA occurred at a 12 min retention time (Figure 1A). Nonetheless, there was no significant accumulation of BPA in the leaf (Figure 1B) and fruit tissues (Figure 1C) of BPA-exposed pepper plants at the same retention time. This may suggest that BPA penetration into pepper plants is limited, unlike in soybeans [33], or that BPA cannot be transported to shoots or fruits [16]. Alternatively, the BPA hydroxyl group can interact with binary ions or compounds (such as sulfates and glucuronic acid) in the soil and form conjugated estrogens, which would lead to the degradation of BPA during the long-term growth of pepper plants [34]. It was also possible that BPA displayed a short residence time of less than 5 days in the pepper tissues [11,16]. Notably, we detected some unknown metabolites; this may indicate that BPA triggers an accumulation of unknown metabolites to relieve or enhance their toxicity [16]. Taken together, our HPLC data indicate that 0.5 mg/kg of BPA treatment might not lead to the accumulation of BPA in pepper tissues. However, it is necessary to explore whether a higher concentration of BPA treatment triggers BPA accumulation.

3.2. BPA Inhibits Pepper Growth

Subsequently, we examined the impact of BPA (5 mg/kg) on the physiological development of Zunla-1 pepper plants. The 30-day-old pepper plants were treated with 5 mg/kg BPA; then, the fresh and dry weights of both roots and leaves were quantified. Our findings indicate that BPA exposure results in lodging and leaf damage after 4 days of treatment (Figure 2A). A prominent decrease in both the fresh and dry weight of leaves and roots was observed in 5mg/kg BPA-treated pepper plants (Figure 2B,C). While previous studies suggest that low doses of BPA could enhance plant growth [16], our findings demonstrate that even low concentrations of BPA can detrimentally inhibit the biological growth of pepper plants (Figure 2), likely due to their varying sensitivity to environmental contaminants.

3.3. The Transcriptome Assay Reveals the BPA-Mediated Signaling Pathways

To elucidate the potential regulatory mechanisms underlying the toxicological effects of BPA exposure on pepper plants, a comprehensive transcriptomic analysis was performed in Zunla-1 pepper leaves that were treated with control or 5 mg/kg BPA for 4 days. Our transcriptome analysis identified 14691 significantly differentially expressed genes (DEGs) in BPA-treated pepper leaves (Table S2). Among these DEGs, 6361 genes were upregulated, and 8330 genes were downregulated (Figure 3A). Gene Ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway assay revealed the involvement of these DEGs in diverse developmental and adaptive processes, including environmental adaptation, photosynthesis, plant hormone signal transduction, ROS signaling, and epigenetic regulation (Figure 3B,C). Additionally, a couple of DEGs associated with environmental adaptation (Capana02g002739, Capana01g000185, Capana00g003942, Capana09g000934, Capana11g000743, Capana06g001506) and cell death (Capana02g001430, Capana12g000060, Capana00g002858, Capana00g004413, Capana02g000475) were differentially regulated upon BPA treatment (Figure 3D), corroborating with our observations (Figure 2). KEGG analysis also highlighted metabolic pathways related to starch and sucrose, glutathione, porphyrin, flavonoid, phenylalanine, fatty acid, propanoate, glycosphingolipid, and amino acid metabolism, implicating the potential role of secondary metabolites and amino acid in BPA-induced toxicity (Figure 3C). Notably, the upregulated and downregulated genes were associated with specific GO and KEEG pathways, indicating the complexity of BPA-induced toxicity in pepper plants (Figure S2). Given the scarcity of studies on BPA’s effects in plants [17], particularly regarding transcriptomic alterations in BPA-induced growth inhibition in peppers, our GO and KEGG analyses identified biological processes such as photosynthesis, hormonal response, and ROS signaling, which are consistent with observations in other species [16,17]. Additionally, our data unveiled further regulatory mechanisms and epigenetic modifications (Figure 3C). In summary, our transcriptomic data provide novel and critical insights into the gene expression alterations induced by BPA treatment in pepper plants.

3.4. BPA Suppresses Chlorophyll Biosynthesis and Increases ROS Levels

Our transcriptomic analysis revealed that photosynthesis was implicated in BPA-induced toxicity (Figure 3B). Specifically, the expression levels of several genes associated with chlorophyll biosynthesis (Capana00g000503, Capana00g004560, Capana10g000065), photosystem complexes (Capana00g001154, Capana00g001213, Capana00g001869, Capana00g004571), and chlorophyll-binding proteins (Capana00g000031, Capana00g002793, Capana00g002794, Capana01g000647, Capana01g002398) were downregulated by BPA (Figure 4A). Conversely, the transcriptional level of the chlorophyll degradation gene (Capana00g003817) was upregulated (Figure 4A). To verify our transcriptomic results, we quantified the chlorophyll concentration in both control and BPA-exposed pepper leaves. The control leaves displayed no notable variation in chlorophyll content, whereas the BPA-treated leaves demonstrated a significant decrease in chlorophyll levels (Figure 4B). Moreover, our qRT-PCR analysis revealed a downregulation of genes involved in chlorophyll biosynthesis and photosystem complexes in BPA-exposed pepper leaves, along with an upregulation of genes associated with chlorophyll degradation, corroborating our transcriptomic results (Figure 4C).

ROS, especially hydrogen peroxide (H₂O₂), are known to cause chloroplast damage and impair photosynthetic efficiency [35]. Thus, we assessed whether H₂O₂ levels were affected by BPA treatment. Our results revealed that BPA exposure led to an increase in H₂O₂ levels in pepper leaves (Figure 4D). Corresponding to the elevated H₂O₂ levels induced by BPA, we observed increased levels of H₂O₂ biosynthetic enzymes, including POD and SOD, while the level of the scavenger enzyme CAT was decreased (Figure 4E–G). Consistent with these findings, our transcriptomic results showed that the expression of H₂O₂ biosynthetic genes, such as RESPIRATORY BURST OXIDASE HOMOLOG (RBOH, Capana03g000595, Capana08g001513) and APOPLASTIC PEROXIDASE (PRX, Capana01g002939), was upregulated upon BPA treatment (Figure S3). In contrast, the expression of ASCORBATE PEROXIDASE (APX, Capana08g000304, Capana02g002557, Capana04g002111), referring to key enzymes involved in H₂O₂ scavenging, was suppressed upon BPA treatment (Figure S3). Indeed, our qRT-PCR result further indicated that BPA regulated the expression of these genes to trigger ROS production in pepper leaves (Figure 4H). Our qRT-PCR findings also revealed an upregulation of the SOD enzyme gene (Capana05g002104) and a downregulation of the CAT enzyme gene (Capana02g002452) (Figure 4H). Collectively, these results indicate that BPA impairs photosynthesis by modulating genes associated with photosynthetic processes and ROS production.

Flavonoids are known to eliminate ROS levels [36]. Our transcriptome analysis revealed lower expressions of flavonoid biosynthetic-related genes, including Capana01g000365, Capana01g000487, Capana01g000494, Capana01g002745, Capana01g002912, Capana02g002763, Capana03g000892, Capana05g000114, Capana05g002107, Capana05g002274, Capana09g002173, Capana09g002174, Capana10g000647, Capana10g001337, and Capana12g000350 (Figure S4), potentially leading to higher ROS levels (Figure 4D). Enhanced ROS levels can also induce the lipid peroxidation of polyunsaturated fatty acids (PUFAs), generating reactive carbonyl species (RCS) [37]. RCS can interact with DNA and proteins, forming advanced lipoxidation end products (ALEs), thereby impairing the molecular functionality of various proteins and disrupting plant developmental and adaptive processes [37]. Genes encoding ALDO/KETO REDUCTASE (AKR) and ALDEHYDE DEHYDROGENASE (ALDH), key enzymes involved in RCS detoxification, including Capana03g003802, Capana03g004289, Capana04g001277, Capana09g001701, Capana02g000850, Capana06g000973, Capana09g000320, Capana09g000321, Capana00g004964, Capana02g002642, Capana06g000973, Capana09g000320, and Capana09g000321 were downregulated in response to BPA (Figure S5), ultimately promoting RCS accumulation and increasing BPA sensitivity.

Consistent with the previously documented effects of BPA on photosynthesis through a reduction in chlorophyll content and photosynthetic systems (PS I and PS II) [16,17], our study also demonstrated that BPA impedes photosynthesis by downregulating genes related to chlorophyll synthesis and photosynthetic systems, while simultaneously upregulating genes involved in chlorophyll degradation (Figure 4A–C). It has been reported that BPA exposure reduces stomatal size, thereby reducing nutrient uptake and leading to an inadequate supply of nutrients critical for chlorophyll synthesis [38]. Additionally, BPA can inhibit carbon assimilation, resulting in an accumulation of surplus electrons in electron transport chains and the excessive excitation of PS II reaction centers [24]. However, the precise mechanisms by which BPA inhibits photosynthesis in peppers require further investigation.

Our data indicate that BPA induces the production of ROS (Figure 4D) by increasing the content of ROS-generating enzymes such as POD and SOD while suppressing the scavenging enzyme CAT level (Figure 4E–G). Transcriptomic analyses further reveal the involvement of ROS metabolic genes, including RBOH, APX, and PRX, in BPA-induced ROS generation (Figure 4H and Figure S3). The phosphorylation of RBOHs is crucial for ROS production [39], though it remains unclear whether BPA affects the phosphorylation status of RBOH to modulate ROS production in peppers. Additionally, we delineated the role of flavonoids in the BPA-stimulated production of ROS (Figure S4). Nonetheless, elucidating how BPA influences ROS levels via flavonoids necessitates further study. Furthermore, it is interesting to explore the role of RCS in modulating the molecular functions of proteins that may be implicated in BPA-induced toxicity. Investigating the impact of BPA on genetic mutants related to ROS production, metabolism, and signaling will also significantly enhance our understanding of the underlying mechanisms of ROS signaling-mediated BPA toxicity in peppers. In addition to the role of ROS in inhibiting photosynthetic processes [35], ROS also facilitates the degradation of BPA [23], thereby reducing BPA concentration, which may explain our HPLC results (Figure 1). Nonetheless, the specific mechanisms by which ROS mediates the breakdown of BPA in pepper plants necessitate further elucidation.

3.5. Plant Hormone Pathways Are Involved in BPA-Induced Toxicity

Phytohormones play pivotal roles in the regulation of plant development and stress responses. Previous studies have implicated several phytohormones, including auxin, gibberellin (GA), cytokinin, abscisic acid (ABA), and ethylene, in BPA-induced growth suppression [16,17,40]. Consistently, our transcriptomic analysis also indicated the involvement of phytohormones in mediating BPA toxicity (Figure 5 and Figure S6). The homeostasis of phytohormones is tightly controlled through their biosynthesis, transport, and signaling transduction. Our transcriptomic findings revealed that BPA modulated a couple of genes associated with the biosynthesis and signal transduction of brassinolides (BR), salicylic acid (SA), jasmonic acid (JA), and strigolactone (SL) (Figure 5). Furthermore, a similar regulatory mechanism for auxin, cytokinin, GA, ABA, and ethylene was also observed (Figure S6), consistent with observations in other plant species [16,17,40].

Transcription factors (TFs) are a class of proteins that bind to a specific DNA sequence of downstream targets, thus regulating gene transcriptional levels. Many TF families, such as MYB, WRKY, and NAC, have been identified in various plant species, and they play indispensable roles in response to environmental stress and plant hormones [41,42,43]. Plant hormones display very complicated interactions under stress conditions [44]. Our transcriptomic data revealed the involvement of various plant hormones in BPA-indued growth inhibition (Figure 5 and Figure S6). However, how pepper plants integrate these hormonal signals to finely adjust the growth inhibition triggered by BPA is largely unclear. One possibility is that TFs serve as the integrators to coordinate complicated hormonal pathways, as we identified that a couple of genes belonging to the MYB family (Capana01g002912, Capana01g004167, Capana02g003351, Capana03g000696, Capana03g002269, Capana03g002680), WRKY family (Capana03g002635, Capana04g001820), and NAC family (Capana05g000569, Capana06g001387) responded to multiple hormones, and were also regulated by BPA (Figure S7). However, the precise mechanism of these TF-mediated hormone responses to BPA needs to be clarified in further studies. Taken together, our findings suggest that BPA can modulate the biosynthesis and signaling transduction of various plant hormones, thus adjusting their cellular and physiological responses to BPA.

3.6. BPA Affects N⁶-Methyladenosine (m⁶A) Gene Expression

Plants adapt to extreme environmental stresses by modulating their gene expression at multiple layers, including transcription, post-transcriptional RNA processing, translation, post-translational regulation, and epigenetic modifications. N⁶-methyladenosine (m⁶A) is the most prevalent chemical modification identified in eukaryotic mRNAs, playing a crucial role in plant stress responses [45]. The addition and removal of m⁶A modifications are mediated by methyltransferases (termed “writers”) and demethylases (termed “erasers”), respectively. Moreover, m⁶A modifications on mRNAs are recognized and interpreted by YT521-B HOMOLOGY (YTH) domain m⁶A-binding proteins (termed “readers”), which govern the fate of mRNAs, including their stability, splicing, transport, and translation [46]. Our transcriptomic data revealed that the VIRILIZER (VIR) writer gene (Capana03g001422), along with EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ETC) reader genes (Capana01g002051, Capana12g000186) and ALKBH10B eraser genes (Capana03g003454, Capana05g000874, Capana08g001916), were regulated by BPA (Figure 6A), suggesting that BPA activates the m⁶A regulatory mechanism in pepper plants. To corroborate our transcriptomic findings, we conducted a qRT-PCR to verify the expressions of these genes; we observed a consistent expression pattern for BPA-regulated m⁶A genes including three additional writer genes mRNA ADENOSIN METHYLASE (MTA) (Capana09g001711), METHYLTRANSFERASE B (MTB) (Capana05g002563) and CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30 (CPSF30) (Capana02g001428), and one ALKAH9A eraser gene (Capana02g002595) (Figure 6B). Transcriptome-wide m⁶A-seq and RNA-seq experiments demonstrate that m⁶A modifications influence mRNA stability [45,46]. Our data indicate that BPA regulates the TFs involved in the plant hormone pathway (Figure S7), increasing the possibility that BPA activates m⁶A modifications to stabilize these TFs, thereby modulating plant hormone signaling pathways and BPA toxicity. However, this requires further investigation. Additionally, m⁶A modification regulates mRNA translation efficiency under stress conditions [46], suggesting that the translation efficiency of hormone-responsive TFs may also be affected by BPA treatment due to their altered m⁶A status. It is also possible that BPA directly regulates plant hormone biosynthesis and signaling via m⁶A modifications [47], thus (de)activating plant hormonal response to BPA (Figure 5 and Figure S6). The exact pathways through which BPA modulates the dynamics of m⁶A via writers, readers, and erasers to modulate pepper growth require further exploration.

4. Conclusions

In this study, we examined the cytotoxic effects of BPA and elucidated the molecular mechanisms underlying the BPA-induced inhibition of pepper growth. Our results revealed that 0.5 mg/kg of BPA treatment did not trigger BPA accumulation in the leaves or fruits of pepper plants. Nevertheless, exposure to 5 mg/kg BPA significantly suppressed pepper growth and led to a notable reduction in biomass. Transcriptomic analysis and qRT-PCR assays further demonstrated that BPA modulated the transcriptional level of genes associated with photosynthesis, ROS homeostasis, phytohormonal signaling, flavonoid biosynthesis, RCS metabolism, and m⁶A modifications, thereby exerting its phytotoxic effects. In summary, our study offers novel and significant insights into the biotoxicity of BPA in horticultural plants.