PVC Inhibits Radish (Raphanus sativus L.) Seedling Growth by Interfering with Plant Hormone Signal Transduction and Phenylpropanoid Biosynthesis

Abstract

Polyvinyl chloride (PVC) is commonly employed as mulch in agriculture to boost crop yields. However, its toxicity is often overlooked. Due to its chemical stability, resistance to degradation, and the inadequacy of the recycling system, PVC tends to persist in farm environments, where it can decompose into microplastics (MPs) or nanoplastics (NPs). The radish (Raphanus sativus L.) was chosen as the model plant for this study to evaluate the underlying toxic mechanisms of PVC NPs on seedling growth through the integration of multi-omics approaches with oxidative stress evaluations. The results indicated that, compared with the control group, the shoot lengths in the 5 mg/L and 150 mg/L treatment groups decreased by 33.7% and 18.0%, respectively, and the root lengths decreased by 28.3% and 11.3%, respectively. However, there was no observable effect on seed germination rates. Except for the peroxidase (POD) activity in the 150 mg/L group, all antioxidant enzyme activities and malondialdehyde (MDA) levels were higher in the treated root tips than in the control group. Both transcriptome and metabolomic analysis profiles showed 2075 and 4635 differentially expressed genes (DEGs) in the high- and low-concentration groups, respectively, and 1961 metabolites under each treatment. PVC NPs predominantly influenced seedling growth by interfering with plant hormone signaling pathways and phenylpropanoid production. Notably, the reported toxicity was more evident at lower concentrations. This can be accounted for by the plant’s “growth-defense trade-off” strategy and the manner in which nanoparticles aggregate. By clarifying how PVC NPs coordinately regulate plant stress responses via hormone signaling and phenylpropanoid biosynthesis pathways, this research offers a scientific basis for assessing environmental concerns related to nanoplastics in agricultural systems.

1. Introduction

Over the past two decades, the escalating demand for plastics across various industries has led to a substantial increase in global plastic production [1]. Since plastics are a very versatile material with advantages such as cost-effectiveness, durability, and light weight, they have infiltrated every aspect of human life [2,3]. Because of their excellent processability, plastic products are extensively utilized in a wide range of applications, from consumer items and automotive components to construction materials and packaging [4]. However, the widespread use of plastics has made plastic waste management a more formidable challenge. Given that most plastics are not readily biodegradable in nature, plastic pollution has become an ongoing concern for the global ecological environment [5]. Existing waste management techniques, including mechanical recycling, landfilling, and incineration, all have significant limitations and are insufficient to address the growing issue of plastic pollution [6]. Over time, under the influence of environment factors, plastics degrade into smaller fragments. These particles are categorized as nanoplastics (less than 1000 nm) and microplastics (1 μm to 5000 μm) based on their size differences [7]. Research indicates that, when microplastics reach certain environmental quantities, they pose a threat to ecosystem stability and biodiversity. Moreover, they may also impact food security by contaminating the food chain [8]. What is even more worrying is that microplastics can absorb harmful substances from the environment. They act as carriers of pollutants and cause intricate toxicological impacts on living organisms [9].

Microplastics and nanoplastics exert a diverse array of complex effects on plants. For instance, in soils with a 0.5% ambient concentration of 100 μm polylactic acid–microplastics (PLA-MPs), the mineral and amino acid contents were significantly higher, and the growth indices and yields of rice were enhanced [10]. Conversely, in soils with 0.2% ambient concentration of 40–50 μm PVC, pumpkin plants exhibited a remarkable, dose-dependent decline in photosynthetic efficiency and chlorophyll content [11]. Due to their minute size, nanoplastics are more bio-permeable than microplastics and pose a greater potential risk [12]. A study revealed that, at concentrations ranging from 50 to 200 mg/L, 50 nm polystyrene (PS) nanoplastics significantly decreased biomass accumulation and induced oxidative stress in rice seedlings [13]. At doses of 0.1–1 g/L, PS-NPs of the same size inhibited the root development of onion seedlings [14] and had an adverse impact on the growth metabolism of cucumbers [15]. It should be emphasized that nanoplastics are small particles belonging to the category of nanoparticles, and there has been preliminary research on the application of nanoparticles in agriculture. They may serve as effective nanopesticides to boost food production and agricultural productivity [16], but they may also modify the genetic composition of plants to regulate, enhance, or suppress metabolic and regulatory processes [17].

In contrast to artificially modified agricultural nanomaterials, environmental nanoplastics are not intended for agricultural use. Their introduction into the soil may disrupt the normal physiological functions of plants and even pose a threat to human health through the food chain. These conflicting findings also suggest that, aside from their inherent characteristics, plant species influence the harmful effects of microplastics and nanoplastics on plants. Evidently, the impacts of microplastics and nanoplastics on plants are diverse and complex, and multiple factors contribute to these effects. There is still much to be learned about the potential effects of microplastics, especially nanoplastics, on human health and agroecosystems [18]. Currently, there is limited information on how nanoplastics affect humans [19]. Summarizing the trends in the effects of nanoplastics on plants is a crucial issue that demands immediate attention. Given the widespread presence of PVC as a major plastic type in the environment, it is essential to conduct systematic research on the principles governing the effects of PVC nanoplastics on crops and clarify their action mechanisms. This will enable scientists to accurately assess the ecological risks of nanoplastics and ensure the sustainable development of the agricultural sector. Therefore, a key problem to be addressed in the fields of environmental science and agroecology is a comprehensive examination of how PVC nanoplastics affect plant growth and development.

Radish is widely employed in environmental pollution evaluation due to its well-developed root system. In this study, we investigated the morphological, physiological, and biochemical alterations of radish under PVC concentrations of 5 mg/L and 150 mg/L. Additionally, transcriptomic and metabolomics techniques were utilized to uncover the molecular mechanism underlying the toxicity of PVC to radish. This study enriches the findings regarding the toxic impacts of nanoplastics on plants and paves the way for further generalization of the associated patterns. Simultaneously, it highlights the ecological and environmental risks of PVC and the imperative of preventing its contamination in farmland.

2. Materials and Methods

2.1. Experimental Design

Radish seeds (Raphanus sativus L.) were obtained from Runda Agricultural Science and Technology Development Center (Shenyang, China). The PVC (size 250 nm pellets) was supplied by HongCheng Plastic Material Co., Ltd. (Dongguan, China). Under the electron microscope, the size and particle diameter (Supplementary Figure S1) of PVC were basically consistent with the data provided by the company (SEM, ZEISS GeminiSEM 300, Oberkochen, Germany). Specificity test kits were sourced from Beijing Solarbio Technology Co., Ltd. (Beijing, China). This experiment used two treatments (5 mg/L of PVC, 150 mg/L of PVC) and one blank control group (CK).

2.2. Radish Cultivation and Growth Measurement

Floating radish seeds were discarded through flotation in sterile water. The remaining seeds were collected for further processing. PVC solutions were prepared at concentrations of 5 mg/L and 150 mg/L. Sterile water was used as the solvent. The seeds were surface-sterilized in 5% H₂O₂ for 3–5 min. Three sterile water rinses were performed. Excess moisture was removed using dry filter paper. Two layers of qualitative filter paper (12.5 cm; Hangzhou Special Paper Industry, Hangzhou, China) were placed in 90 mm Petri dishes. The papers were moistened with 5 mL test solution. Twenty-five seeds were distributed per dish. The dishes were sealed with parafilm. Incubation was conducted at 25 ± 1 °C for four days in dark. Three replicates were maintained per treatment. Germinated seeds were counted after incubation. Root lengths were measured to 1 mm accuracy. Stem lengths were recorded similarly.

2.3. Enzyme Activities and Oxidative Stress Marker Analysis

Root tissues were aseptically collected from germinated radish seedlings using sterile forceps. Frozen root samples (100 mg) were homogenized in 1 mL ice-cold extraction buffer using a chilled mortar and pestle. Complete homogenization was achieved after 3 min of grinding. The resulting slurry was immediately transferred to pre-cooled 2 mL microcentrifuge tubes. Cellular debris was pelleted by centrifugation at 8000× g for 10 min at 4 °C. The supernatant was aliquoted into fresh pre-chilled tubes. All procedures were maintained at 0–4 °C to preserve enzyme activity. Peroxidase (POD) activity was determined using Doerge’s method [20]. Superoxide dismutase (SOD) activity was analyzed via Minami’s photochemical approach [21]. Catalase (CAT) activity was measured following Johansson’s UV absorption protocol [22]. Malondialdehyde (MDA) concentrations were quantified using the thiobarbituric acid (TBA) method [23] in plants exposed to 5 mg/L and 150 mg/L PVC treatments.

2.4. RNA Sequencing and Transcriptome Profiling

Total RNA was extracted from 0.1 g root samples using TRIzol reagent (ThermoFisher, 15596018, Waltham, MA, USA). RNA quantity and purity were assessed with a Qubit 3.0 fluorometer (ThermoFisher, Q33216) and Fragment Analyzer 5300 (Agilent, M5311AA, Santa Clara, CA, USA). High-quality RNA samples (RIN > 7.0) were selected for library preparation. mRNA was isolated from total RNA (2 μg) through two rounds of purification with mRNA capture Beads 2.0 (Yeasen, 12629ES, Shanghai, China). Sequencing libraries were constructed following standard protocols. Paired-end sequencing (PE150) was performed on an Illumina NovaSeq 6000 system (BIOTREE, Shanghai, China). Raw sequencing data were processed to remove low-quality reads, adapter sequences, and poly-N fragments. Clean reads were aligned to the reference genome using HISAT2 (V2.2.1). Gene expression levels were quantified as FPKM (fragments per kilobase of exon per million mapped reads).

2.5. Metabolite Extraction and LC-MS Analysis

Fresh radish root tips (25 mg) were flash-frozen and lyophilized, after which precisely weighed aliquots of dried tissue were transferred to EP tubes containing homogenization beads. Extraction solvent (methanol/acetonitrile/water = 2:2:1, v/v/v) containing isotope-labeled internal standards was added at specified ratios, followed by vortexing for 30 s. Homogenization was performed at 35 Hz for 4 min, with subsequent ice-water bath sonication for 5 min (repeat the experiment three times). Samples were incubated at −40 °C for 1 h, and then 400 μL aliquots were filtered through 0.22 μm plates using a positive pressure manifold (6 psi, 120 s) for protein precipitation. The resulting filtrates were collected for LC-MS analysis, while quality control (QC) samples were prepared by pooling equal volumes from all sample supernatants. Non-polar metabolites were separated using a Vanquish UHPLC system equipped with a Phenomenex Kinetex C18 column (2.1 × 100 mm, 1.7 μm; ThermoFisher, Waltham, MA, USA). Raw data were converted to mzXML format using ProteoWizard (V3.0) software. Metabolite identification was performed using BiotreeDB (V3.0) and BT-Plant (V1.1) databases through a custom R package (V3.3.5).

2.6. Statistical Analyses

To evaluate possible differences between treatments, SPSS 27.0 (IBM Corp., Armonk, NY, USA) was used to examine plant growth index experimental data using a one-way analysis of variance (ANOVA). Tukey’s test was used to examine differences between the control group and each PBV-OBS treatment group at the p < 0.05 level after the data had been evaluated for ANOVA. Prism 10.1.2 (GraphPad Software, San Diego, CA, USA) was used to visualize the data. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed on transcriptome metabolome data using R (ggplot2) and SIMCA. Fold change ≥ 2 (i.e., absolute value of log₂FC ≥ 1) and q value < 0.05 (q value is the corrected value of p value) (|log₂FC| ≥ 1 and q < 0.05) were the threshold criteria for screening differentially expressed genes and metabolites. Functional pathway enrichment analysis of differentially differentiated metabolite genes (DEGs) and differentially expressed metabolites (DEMs) was carried out using the R package. An analysis of functional pathway enrichment was conducted, and the enriched pathways were visualized.

3. Results

3.1. Effects of PVC Stress on Radish Growth and Antioxidant System

Compared to the control group, the PVC treatments at concentrations of both 5 mg/L and 150 mg/L had no significant impact on radish germination (p > 0.05), with germination rates remaining at 99–100% (Figure 1a). Nevertheless, significant morphological alterations were noted in the development of shoots and roots. Under the 5 mg/L and 150 mg/L PVC treatments, shoot length was decreased by 33.7% (p < 0.05) and 18.0%, respectively (Figure 1b). Similarly, root length was reduced by 28.3% (p < 0.05) and 11.3% for the corresponding treatments (Figure 1c). The enzymatic responses demonstrated dose-dependent patterns. The POD activities increased by 60.8% (p < 0.01) at 5 mg/L but decreased by 23.1% at 150 mg/L (Figure 2a). The CAT activities increased by 737.5% and 264.1% at 5 mg/L and 150 mg/L, respectively (Figure 2b). The SOD activities were elevated by 51.8% (p < 0.05) and 20.6% at the respective concentrations (Figure 2c). MDA content, an indicator of oxidative stress, increased by 49.1% and 27.5% under the 5 mg/L and 150mg/L PVC treatments (Figure 2d).

3.2. Transcriptomic Analysis of PVC Stress Responses in Radish Roots

Transcriptomic analysis was conducted to investigate the toxicological mechanisms of PVC in radish root cells and elucidate the molecular basis for the observed morphological changes and alterations in antioxidant enzymes. Compared to control groups, treatment with 5 mg/L of PVC resulted in 4635 differentially expressed genes (DEGs), consisting of 2642 up-regulated and 1993 down-regulated genes. The higher concentration of 150 mg/L of PVC induced 2075 DEGs, with 1408 up-regulated and 667 down-regulated genes. KEGG pathway enrichment analysis revealed distinct response patterns between the two treatment groups. The 5 mg/L PVC treatment primarily affected metabolic pathways associated with stress responses, while the 150 mg/L treatment showed significant enrichment in specific pathways including the following: plant–pathogen interaction (93 up-regulated and 48 down-regulated DEGs), mitogen-activated protein kinase (MAPK) signaling (37 up-regulated and 10 down-regulated DEGs at 5 mg/L versus 50 up-regulated and 45 down-regulated DEGs at 150 mg/L), phenylpropanoid biosynthesis (33 up-regulated and 11 down-regulated DEGs), and plant hormone signal transduction (78 up-regulated and 56 down-regulated DEGs). These transcriptional changes demonstrate the dose-dependent molecular responses of radish roots to PVC stress. A closer look at the transcriptome results showed that the 5 mg/L treatment group had twice as many DEGs in the shared enriched pathways as the 150 mg/L treatment group, if not more. This suggests that the degree of pathway changes was much greater in the 5 mg/L treatment group than in the 150 mg/L treatment group (Figure 3). The expression of jasmonic acid-amino synthetase (JAR1, EC:6.3.2.52), phenylalanine ammonia-lyase (PAL, EC:4.3.1.24), and 4-coumarate--CoA ligase (4CL, EC:6.2.1.12) genes was down-regulated, whereas transcription factor MYC2 (MYC2), transcription factor TGA (TGA), regulatory protein NPR1 (NPR1), and shikimate O-hydroxycinnamoyltransferase (HCT, EC:2.3.1.133) genes were up-regulated (Figure 4).

3.3. Metabolomic Response After Treatment with PVC

A total of 1961 metabolites were consistently detected across all experimental groups (Supplementary Figure S2). Comparative analysis revealed 268 differentially accumulated metabolites (DAMs) in 5 mg/L PVC-treated radish roots versus controls, comprising 100 up-regulated and 168 down-regulated metabolites (Figure 5a). The 150 mg/L PVC treatment group exhibited 270 DAMs, with 82 up-regulated and 188 down-regulated metabolites (Figure 5b). Our findings showed a high enrichment of KEGG pathways associated with plant defense, including phenylpropanoid biosynthesis and alanine aspartate and glutamate metabolism (Figure 6).

4. Discussion

4.1. Germination and Growth Dynamics: PVC-Induced Stress Responses and Adaptive Strategies

Based on the findings of a study examining the effects of different plastics on plant germination, PVC exerted no distinguishable impact on seed germination. The results of the study on watercress exposed to PVC [24] indicated that the effect of MPs/NPs on seed germination disappeared after 24 h of exposure. Regardless of the size of the M/NPs or the exposure concentration, the germination rate was nearly 100% [25]. In this experiment, the germination rate of radish seeds under both the 5 mg/L and 150 mg/L PVC treatments was 99%, showing no significant difference compared to that of the control group. Similarly, small amounts of PS NPs (0.01–10 mg/L) did not significantly impede wheat germination [26], nor did biodegradable plastic leachate on watercress [27]. However, the effect of plastics on germination can vary depending on the size and type of plastic utilized, the plant species tested, and the time. For instance, some researchers have discovered that polystyrene (PS), polyethylene (PE), and polypropylene (PP) significantly inhibit the germination of Solanum lycopersicum L. seeds [28]. In this experiment, both PVC (although the 150 mg/L PVC treatment did not reach statistical significance (p > 0.05), a consistent trend was observed) dosages reduced the root and shoot length of radish, although the germination rate was not significantly affected. This finding aligns with the results of studies that have explored the impacts of PVC treatment on wheat [29], PS NP treatment on rice [13], and onion [14]. Research indicates that when concentrations of polyethylene, polypropylene, and polystyrene particles rise, so does their inhibitory effect on potatoes [30]. Simultaneously, various biological and physiological activities of plants have been reported to exhibit stimulatory responses. Hormesis occurs as a dose–response phenomenon, with high doses causing inhibition and low amounts causing stimulation [31]. In contrast to the previously stated results, it is important to note that, in this experiment, the inhibitory effect of 5 milligrams per liter of polyvinyl chloride treatment was stronger than that of 150 milligrams per liter. This non-monotonic dose–effect relationship may result from interactions between various factors, including the surface properties of the particles, the specificity of the plant species under test, and the various types of plastics [32]. From the perspective of physiological mechanisms, exposure to low concentrations may trigger the primary stress response in plants, leading to a reactive oxygen burst and disruption of hormone signaling, similar to the initial response to cadmium or salt stress [33,34]. At higher concentrations, plants may initiate secondary adaptive mechanisms to partially mitigate the toxic effects by activating antioxidant defenses or adjusting nutrient uptake strategies [35]. This dynamic equilibrium mechanism reflects the intricate regulatory network of plants in response to environmental stress [36], which also accounts for the variations in concentration effects observed in various studies. Therefore, to more accurately forecast their ecological risks, the assessment of the impact of plastic pollution on plants should take into account multi-dimensional aspects, such as exposure concentration, duration, plastic characteristics, and the plants’ own adaptation potential.

4.2. Antioxidant Defense Mechanisms: Enzyme System Activation Under PVC Stress

Peroxides and free radicals are typically generated in the root system when environmental conditions are adverse to root development. These substances pose a threat to the cell membrane structure and impede root activity, thereby reducing root biomass [37]. The coordinated action of SOD, CAT, and POD constitutes the primary mechanism by which plants have evolved efficient detoxification systems. This mechanism serves to reduce or eliminate reactive oxygen species (ROS) to acceptable levels and prevent membrane lipid peroxidation in response to oxidative stress [38]. SOD is the first enzyme to act on oxygen radicals. It mainly combines two O₂⁻ radicals to form O₂ and H₂O₂, thus regulating the amount of ROS in plants [39]. CAT, a key antioxidant enzyme in the plant defense system, decomposes H₂O₂ into H₂O and O₂, minimizing the damage caused by ROS [40]. POD is associated with the removal of H₂O₂ [41]. In this study, the activities of the three enzymes, POD, CAT, and SOD, showed a similar trend across all groups. The 5 mg/L PVC-treated group exhibited higher enzyme activity compared to the control group and the 150 mg/L PVC-treated group. This indicates that, similar to the investigation of the impact of PS-NPs on lettuce, oxidative stress is involved in the phytotoxicity mechanism of PVC NPs on radish [42]. In plant cells, MDA serves as an indicator of oxidation and lipid damage [43]. In this experiment, compared with the control group, the MDA content increased by 49.1% and 27.5% in the 5 mg/L PVC treatment group and the 150 mg/L PVC treatment group, respectively. It can be inferred that cell damage is mainly attributed to PVC.

Under harsh environmental conditions, an excessive amount of ROS is generated, and the antioxidant system struggles to completely eliminate them. This leads to a strong oxidative burst in the plant, which enhances the synthesis of malondialdehyde (MDA) in the plant, causing DNA damage, as well as protein and lipid peroxidation [44]. In this experiment, although the antioxidant enzyme activities of the treated groups were generally higher than those of the control group, the MDA content remained elevated. This may suggest that the stress induced by PVC NPs excessively increased ROS levels. Specifically, the scavenging capacity of the antioxidant system was lower than the rate of ROS production. As a result, although some ROS were scavenged, they still accumulated, damaging the cell membrane and leading to an increase in the MDA content.

4.3. Phytohormone Signaling Networks: Modulation and Crosstalk Under PVC Stress

Nanoplastics may reduce crop disease resistance as they might cause defense-related genes to be expressed less often [45]. Transcriptome analysis indicated that the 5 mg/L treatment group had at least twice as many differentially expressed genes (DEGs) in these shared pathways compared with the 150 mg/L group. These results suggest that pathway alterations were more prominent in the 5 mg/L treatment (Figure 3). Significant enrichment of KEGG pathways associated with plant defense, including phenylpropanoid biosynthesis and phytohormone signaling, was detected.

Phytohormones are crucial in regulating plant development and stress responses. Under unfavorable conditions, plants activate specific signal transduction pathways mediated by these hormones to effectively respond to stress [46,47]. Hormone interactions frequently determine the growth, development, and stress responses of plant roots [48]. As signaling molecules, phytohormones regulate root morphology and growth through processes such as synthesis, metabolism, transport, and signaling in roots [49,50]. Jasmonates (JAs), key plant defense hormones, are synthesized from α-linolenic acid via oxidation, cyclization, and acyl chain shortening. They are involved in various plant responses, ranging from defense against biotic and abiotic stresses to growth, flowering, and senescence [51]. JAs enhance the activity of antioxidant enzymes [52]. Additionally, as a defense hormone, JAs can participate in several plant growth processes, including the inhibition of root growth [53]. Based on the metabolome data of the experiment, the JA content in the 5 mg/L treatment group was significantly higher than that in the control group. Meanwhile, the root length was most strongly inhibited, and the antioxidant enzyme activity was at its highest, which was in line with the findings of previous research.

Salicylic acid (SA) serves as a plant’s defense mechanism against biotic stressors and is also crucial for physiological functions related to disease resistance and plant immunity [54]. Plants attempt to avoid growth inhibition by restricting SA synthesis because excessive SA levels may disrupt physiological processes. This occurs when resources are redirected towards defense responses, leading to the accumulation of reactive oxygen species and the disruption of other phytohormone signals [55]. In the present investigation, following PVC treatment, the SA concentration was down-regulated. The DNA-binding bHLH transcription factor MYC2, as an activator of JA-induced root growth inhibition and an inhibitor of root development, plays a vital role in JA signaling [56]. The transcriptome gene expression analysis of this experiment revealed that MYC2-related genes were significantly up-regulated (Figure 4), and root growth was inhibited, which was consistent with the findings of other studies. These findings indicate that PVC stress influences the development of radish roots by interfering with the signal transduction pathways of JA and SA.

4.4. Phenylpropane Biosynthesis: Metabolic Reprogramming in Response to PVC Stress

Many plants and certain microbes possess the phenylpropanoid biosynthesis pathway, a crucial secondary metabolic pathway that generates a vast number of secondary metabolites [57]. Using phenylpropanes as precursors, numerous flavonoids, isoflavonoids, lignans, and other compounds are synthesized [58]. Thus, the phenylpropanoid biosynthesis pathway is one of the primary sources of flavonoid synthesis. The enzyme that regulates the rate of phenylpropanoid biosynthesis, phenylalanine ammonia-lyase (PAL), affects the subsequent biosynthesis of lignin, anthocyanins, and flavonoids [59]. PAL can convert L-phenylalanine into ammonia and trans-cinnamic acid [60]. Subsequently, 4-coumarate: CoA ligase (4CL) further catalyzes the synthesis of flavonoids, lignin precursors, and other biomolecules [61]. This enzyme controls the quantity of lignin and flavonoids and further impacts plant growth [62,63]. Lignin, a phenolic polymer derived from phenylalanine, is an essential component of plant secondary cell walls [64]. As a vital physical barrier, lignin mainly protects cellular protoplasts from various abiotic stresses and prevents external impacts by reducing the permeability of the cell wall. Proanthocyanidins, anthocyanidins, flavonols, and flavonoids are instances of flavonoids that possess antioxidant properties and can scavenge reactive oxygen species [65].

In this study, the majority of the genes encoding PAL and 4CL were down-regulated due to the decrease in SA content. Resources were utilized for lignin production, and the majority of the genes encoding CCR and HCT were up-regulated. Nine flavonoid contents were found to be up-regulated in both the low and high PVC treatment groups. The up-regulation of these flavonoids and the alterations in genes regulating the enzymes involved in phenylpropane biosynthesis (Figure 4) suggest that radish may disrupt secondary metabolism to enhance its defense against oxidative damage induced by PVC stress. Due to resource limitations, plants must make a trade-off between growth and defense, depending on both internal and external factors [66]. Consequently, growth indicators such as radish root and shoot length decreased as resources were redirected towards defense. Meanwhile, in seawater, Wegner et al. found that 30 nm nano-polystyrene can rapidly form millimeter-scale aggregates. This indicates that NPs can form millimeter-scale (mm-scale) aggregates in ecosystems [67], and smaller NPs are more likely to penetrate plant structures [68]. The formation of aggregates may explain why the inhibitory effect of 150 mg/L PVC in the treatment group is less pronounced than that of 5 mg/L PVC in the treatment group. This might lead to a reduction in plant uptake and a weaker inhibitory effect on radish compared to that of the 5 mg/L treatment group. This can be an area for in-depth research in the future.

5. Conclusions

In this study, the toxicity mechanism of PVC NPs on radish development and physiological metabolism was systematically revealed. During short-term (4-day) exposure, treatments at 50 mg/L and 150 mg/L significantly decreased the root and shoot lengths. Meanwhile, higher antioxidant enzyme activity and an increased MDA concentration were observed, indicating continuous oxidative damage. A combined multi-omics analysis showed that PVC NPs interfered with the phenylpropane biosynthesis pathway by disrupting the JA and SA signaling, leading the plant to reallocate resources from growth to defensive metabolism (such as lignin accumulation). However, this defensive remodeling did not completely alleviate oxidative stress, and plant development was still inhibited by the high MDA concentration. The dispersion and aggregation of nanoparticles at different concentrations might account for the fact that low concentrations of PVC NPs exhibited more pronounced toxic effects than high-concentration treatments. The results of this study indicated that even low concentrations of PVC NPs could pose a threat to crops, although laboratory conditions could not precisely replicate the complex farmland environment. This research provides a theoretical basis for assessing the long-term impacts of nanoplastics on terrestrial ecosystems, which is essential for the development of sustainable agriculture. Future research on the uptake and transport of PVC NPs by radishes is necessary to gain a better understanding of how nanoplastics affect crops.