Unveiling the impact of polystyrene nanoplastics on tea seedlings: uptake, translocation, and oxidative stress‐mediated alterations

Abstract

Background

The accumulation of nano‐plastics in terrestrial environments raises urgent concerns for crop safety, but little is known about their bioavailability and impact in perennial plants like tea (Camellia sinensis L.). This study investigated the uptake and physiological effects of 80 nm polystyrene nano‐plastics (PS‐NPs) in hydroponically grown tea seedlings.

Result

Confocal laser scanning microscopy (CLSM) visualized PS‐NPs in root cortices and leaf tissues, confirming systemic translocation. Exposure to PS‐NPs (10–100 mg L⁻¹) for durations of 1, 3 and 7 days triggered concentration‐dependent oxidative stress and defense responses: root catalase (CAT) activity was initially suppressed by 69.7% (50 mg L⁻¹) but later induced 15‐fold, while leaf glutathione S‐transferase (GST) activity was significantly inhibited. Notably, theanine content in leaves increased by 77.6% at 10 mg L⁻¹ but decreased by 70.1% at 100 mg L⁻¹.

Conclusion

The findings demonstrate PS‐NPs uptake and their impact on tea seedling physiology and quality‐related metabolites, underscoring potential risks to perennial crops. © 2026 Society of Chemical Industry.

INTRODUCTION

Plastics, valued for their versatility, affordability, and high hardness under specific conditions, have become indispensable in numerous applications. ¹ Global plastic production has increased steadily over the years, reaching approximately 413.8 million tons by 2023.^{2, 3} Although plastics have revolutionized modern life, their widespread use has also contributed to serious environmental challenges.^{4, 5} Over time, the degradation of discarded plastics, especially nonrecyclable varieties, leads to smaller particles such as microplastics and nanoplastics (NPs).^{6, 7, 8} These contaminants infiltrate agricultural soils and aquatic environments, thereby entering the food supply chain. Their capacity to penetrate biological membranes and accumulate in tissues raises significant concerns regarding food safety and potential risks to human health.^{9, 10, 11}

NPs, defined as plastic particles smaller than 100 nm, ¹² exhibit higher bioavailability and potential toxicity than microplastics owning to their smaller size, thus raising serious ecological and health concerns. NPs retained in terrestrial ecosystems can be taken up by plants and translocated into their tissues. Such accumulation may impair plant growth and ultimately compromise crop quality and safety. For instance, polystyrene nanoplastics (PS‐NPs) have been shown to disrupt the growth and development of crops such as cucumber, rice, and lettuce by interfering with key physiological and biochemical processes.^{13, 14, 15} NPs can also alter physiological parameters and induce genotoxic effects, as demonstrated in onion under polyethylene and poly(vinyl alcohol) exposure. ¹⁶ These findings suggest that PS‐NPs accumulated in edible plant tissues may transfer along the food chain, posing potential risks to human health. While previous studies have confirmed NP uptake in various plants, including both dicotyledonous (e.g. lettuce) and monocotyledonous (e.g. rice) plants,^{15, 17} their effects on woody plant species remain scarcely studied.

Tea plants (Camellia sinensis L.), an important woody crop, warrant special attention due to their dual role as a key economic commodity and a globally consumed beverage source. ¹⁸ A recent study has reported micro‐/nanoplastic concentrations in tea plantation soils ranging from 254 to 1328 particles per kilogram, indicating a severe exposure risk for tea plants. ¹⁹ Despite this identified risk, current understanding remains incomplete regarding the uptake, translocation, and consequent physiological impacts of NPs in tea plants, particularly with respect to their effects on tea quality.^{20, 21} Addressing these knowledge gaps is essential for understanding the broader implications of NP pollution for human health, food safety, and global ecosystem integrity. ²²

The study reported here employed PS‐NPs, a colorless and transparent polymer derived from styrene monomers, as a model contaminant to investigate their uptake, accumulation, physiological effects, and impact on key quality‐related metabolites in tea seedlings. The distribution and accumulation patterns of PS‐NPs were visualized using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Concurrently, high‐performance liquid chromatography (HPLC) was used to analyze changes in major quality components, such as catechins and theanine, under PS‐NP stress. The findings provide mechanistic insights into how PS‐NP pollution affects crop quality formation in agricultural systems and offer a theoretical foundation for ensuring tea quality and safety.

MATERIALS AND METHODS

Chemicals and reagents

PS‐NP beads (mean diameter ~ 80 nm) were purchased from Da'e Scientific Co. Ltd (Shanghai, China) and applied at a concentration of 10 mg mL⁻¹. Acetonitrile and methanol (for HPLC, ≥99.9%) were purchased from Tedia Co. (OH, USA). l‐Theanine (99.77%), gallic acid (GA, 99.96%), gallocatechin (GC, 98%), epigallocatechin (EGC, 99.84%), epigallocatechin gallate (EGCG, 99.49%), epicatechin (EC, 98.37%), and epicatechin gallate (ECG, 98.28%) were obtained from Shanghai Yuanye Bio‐Technology Co. Ltd. Caffeine (CAF, 98%) was purchased from Wuhan ChemFaces Biochemical Co. Ltd. The 0.22 μm filter membranes were purchased from ANPEL Laboratory Technologies (Shanghai) Inc.

Tea seedling cultivation and PS‐NP exposure

Fresh tea (Camellia sinensis cv. Shuchazao) seeds were obtained from the Hubei Raw Medicinal Materials Market, soaked in deionized water for 1 week, and cultivated in sterile pure vermiculite for 4 months under controlled conditions to produce uniform seedlings. Fifteen healthy 4‐month‐old tea seedlings (20 ± 2 cm in height) were acclimatized in a hydroponic system ²³ for 2 weeks before treatment with Rhodamine B‐labeled PS‐NPs (ca 80 nm in diameter, initial concentration: 10 mg mL⁻¹), which were diluted to three exposure concentrations (10, 50, and 100 mg L⁻¹). Seedlings were maintained at 25 ± 2°C with a 200 μmol m⁻² s⁻¹ photosynthetic photon flux density, 16 h/8 h light/dark cycle, and 70 ± 5% relative humidity throughout the experiment. Each treatment group (including controls without PS‐NPs) consisted of three independent replicates (one seedling per replicate vessel), with tea seedlings harvested at 1, 3, and 7 days post‐exposure (n = 3 per time point). The hydroponic cultivation system was adopted to standardize growth conditions and precisely determine the direct effects of PS‐NPs on tea seedlings, while subsequent studies will employ soil‐based exposure experiments to further evaluate their ecological impacts under realistic environmental conditions.

Plant sampling and localization of NPs in tea seedlings

CLSM analysis

Following 7‐day exposure to PS‐NP bead solutions, tea seedlings were thoroughly rinsed with deionized water. Fresh root, stem, and leaf tissues were collected and transversely sectioned into ca 100 μm thick slices using a razor blade. The sections were mounted on glass slides, hydrated with deionized water, and covered with coverslips. NP localization was visualized using a FluoView FV1000 confocal laser microscope (Olympus, Tokyo, Japan) with Rhodamine B fluorescence detection (excitation/emission: 570/590 nm). For each treatment concentration, we analyzed transverse sections from three independent biological replicates (different plants), with representative images selected for presentation. Image brightness and contrast were digitally adjusted using imaging software to optimize visualization.

SEM analysis

For SEM characterization, root, stem, and leaf stalk samples (particularly regions adjacent to primary veins) were excised, sectioned, and freeze‐dried for 12 h. Samples were mounted on copper sheets using conductive carbon tape and sputter‐coated with a 1 nm gold layer (Cressington 108 Auto, Ted Pella). High‐resolution imaging was performed using a Hitachi S‐4800 microscope operated at 2.5–30 kV in high‐vacuum mode with backscattered electron detection. Additional examinations of lateral root zones were conducted using environmental SEM (Hitachi S‐4800, Haisi Electronic Technology (Shanghai) Co. Ltd) under high‐vacuum conditions. Transverse sections from three biological replicates per treatment group were examined for SEM analysis. Digital images were captured at various magnifications to ensure comprehensive morphological characterization.

Detection of physiobiochemical indicators in tea seedlings

Biochemical assays

Tea seedlings were cultured for 1, 3, and 7 days in PS‐NP exposure solutions at concentrations of 0, 10, 50, and 100 mg L⁻¹ (80 nm). Leaves and root tissues were harvested and placed in a pre‐cooled porcelain mortar with phosphate buffer (9:1 mass ratio). The tissues were ground into homogenates in an ice–water bath, followed by centrifugation at 1697 × g for 10 min at 4 °C. The supernatants were analyzed for the enzymatic activities of catalase (CAT) and glutathione (GSH), as well as total protein (TP) and malondialdehyde (MDA) levels using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). Enzymatic activity was normalized to protein content, and all biochemical assays were performed in triplicate for each treatment group to ensure reproducibility.

Extraction and detection of theanine

Theanine extraction was performed using modified methods. Briefly, 100 mg of freeze‐dried sample powder was dissolved in 3 mL of deionized water and heated at 100 °C for 30 min. The mixture was then centrifuged at 12 840 × g for 20 min. The supernatant was filtered through a 0.22 μm membrane and subsequently analyzed using HPLC. ²⁴ Calibration curves were generated using theanine standards to ensure the accuracy of the measurements.

Extraction and detection of catechin/caffeine

Catechin and CAF were extracted from 100 mg of tea root samples using 1 mL of 80% methanol solution, followed by ultrasonic extraction for 30 min at room temperature. The mixture was then centrifuged at 12 840 × g for 20 min, and the supernatant was collected and filtered through a 0.22 μm membrane. The extraction process was repeated, and the combined supernatants were analyzed using HPLC.^{25, 26} Calibration curves and internal standards were used for quantitative accuracy.

Statistical analysis

All experiments were conducted with three biological replicates and repeated three times. Statistical analyses were performed using SPSS 26.0 (IBM, Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). Duncan's test was applied, followed by Tukey's HSD post hoc test, to identify significant differences between treatment groups. Data are presented as the mean ± standard deviation (SD), with statistical significance set at P < 0.05. Pearson's correlation analysis was used to evaluate relationships between variables.

RESULTS AND DISCUSSION

Fluorescence signal of NP beads in tea roots

To ensure batch‐to‐batch consistency, we verified the particle size distribution of the beads using dynamic light scattering and SEM. Fluorescence intensity was measured with a spectrofluorometer calibrated against standard fluorescent beads. All solutions were prepared with ultrapure water and stored according to the manufacturer's guidelines to prevent degradation and maintain bead integrity. The beads, labeled with Rhodamine B, exhibited excitation/emission peaks at 540/580 nm, emitting orange fluorescence. SEM characterization (Fig. 1) confirmed their spherical morphology and uniform size distribution, consistent with the specified diameter of 80 nm. This fluorescent labeling enabled the visualization and tracking of PS‐NP accumulation in tea seedling roots, stems, and leaves.

Figure 1.

SEM images of 80 nm NP.

No fluorescence signals were detected in the roots of control seedlings at the specified excitation wavelengths (Fig. 2(a1)–(b3)). In contrast, distinct fluorescence signals from PS beads were observed in the cortical tissue of treated seedling roots (Fig. 2(c1)–(h3)). The fluorescence intensity exhibited a concentration‐dependent pattern (Fig. 2). Quantitative analysis revealed significantly higher intensities at 10 and 100 mg L⁻¹ compared to 50 mg L⁻¹ (mean intensity ± SD: 10 mg L⁻¹, 52.4 ± 4.3 AU; 50 mg L⁻¹, 34.8 ± 5.1 AU; 100 mg L⁻¹, 72.7 ± 3.9 AU; P < 0.05). The strongest signals were detected in cortical cells of roots exposed to 100 mg L⁻¹, suggesting transport of PS beads through these cells (Fig. 2(h3)).

Figure 2.

Accumulation of 80 nm fluorescently labeled NP beads by tea seedling roots. Five‐month‐old tea seedlings were exposed to 0, 10, 50, and 100 mg L⁻¹ suspensions of NP beads labeled with Rhodamine B for 10 days. Global transverse section images (A) and partial transverse section images (B) of tea root uptake of NP beads were analyzed under bright‐field conditions and in orange channels using CLSM. (a1–a3, b1–b3) Transverse sections of roots untreated with NP beads. (c1, e1, g1, d1, f1, h1) Bright‐field images showing transverse sections of roots treated with NP beads labeled with Rhodamine B. (c2, e2, g2, d2, f2, h2) Orange channel images of transverse sections used to detect Rhodamine B fluorescence at 570 nm excitation and 590 nm emission. (d2, f2, h2) Aggregates of NP beads at varying concentrations observed in the intercellular spaces of the epidermis moving towards the cortex. (a3, c3, e3, g3, b3, d3, f3, h3) Merged images, where ‘a3’ corresponds to ‘a1 + a2’, ‘c3’ to ‘c1 + c2’, and so forth.

Figure S1(a)–(f) shows the adsorption and accumulation of 80 nm PS beads on lateral roots. This observation aligns with the findings of Gao et al., ²⁷ who reported that plant roots secrete viscous substances to facilitate nutrient absorption. It is hypothesized that tea roots secrete mucus ²⁸ which could encapsulate NP beads, aiding their infiltration into plant cells. NPs appear to penetrate the plant through gaps in the cell wall, where they are compressed into irregular shapes due to the rigidity of the plant cell walls. This deformation may lead to the physical or chemical binding of the beads to tissue components, as observed in the tissue gaps (Fig. S2). A strong fluorescence signal was observed at the junction of lateral and secondary roots, indicating the multiple potential pathways for NP absorption. ²⁹ Microscopic images showed that PS beads accumulated around root cracks and penetrated the primary roots (Fig. S1). However, no PS beads were detected in the vascular bundles, likely due to the barrier function of the Casparian strip in root tissues. ³⁰ SEM analysis confirmed the presence of NP beads in the pores of root epidermal cells.

The absorption of NPs by plants necessitates overcoming physiological and chemical barriers, such as size exclusion limits, which can vary depending on the plant species, growth stage, and environmental conditions. ³¹ The concentration‐dependent effects observed in this study underscore the varying impacts of NPs at different exposure levels, with significant physiological and biochemical changes occurring at higher concentrations. It should be noted, however, that treatments such as 100 mg L⁻¹ may exceed environmentally relevant levels typically found in agricultural systems. In practical scenarios, NP exposure in crops is influenced by factors such as environmental accumulation, agricultural practices, and local contamination sources. Previous research has shown that tobacco cells can absorb 20–40 nm plastic microspheres via endocytosis, but not 100 nm beads. ³² In contrast, root cracks and transpiration pull play key roles in facilitating the absorption and transport of NPs in tea seedlings. ⁴ Another study reported that 100 nm PS microplastics are taken up by strawberry seedlings, altering their physiological and biochemical parameters. ³³

The accumulation of NPs in cortical cells and intercellular spaces suggests potential interactions with components of the root extracellular matrix, likely facilitated by root exudates such as mucilage. Their penetration through cell wall gaps indicates a size‐dependent mechanism, where NPs smaller than the species‐specific size exclusion limits can bypass physical barriers. The observed deformation of these particles further implies that mechanical compression by rigid cell walls contributes to their entry into plant tissues. The absence of fluorescence signals in the vascular bundles underscores the role of the Casparian strip as a selective barrier, limiting the systemic transport of NPs beyond the cortex. Although the hydroponic system used in this study provides a controlled environment for assessing NP uptake, natural soil conditions introduce additional variables, including soil composition, microbial communities, and environmental fluctuations. Future research should explore NP absorption under soil‐based conditions to better understand their behavior and transport in realistic agricultural settings.

Fluorescence signal of NP beads in aerial parts of tea seedlings

Tea stems and leaves from the control group exhibited no detectable autofluorescence at the specified excitation wavelengths (Figs 3(a1)–(b3) and 4(a1)–(b3)). In contrast, the treated groups showed fluorescence signals in the intercellular spaces of stems and leaves, with the most intense signals observed in stems exposed to 100 mg L⁻¹ suspensions (Fig. 3(g1)–(h3)). In leaves, the highest fluorescence intensity was recorded in the 50 mg L⁻¹ treatment group (Fig. 4(e1)–(e3),(f1)–(f3)), while the 100 mg L⁻¹ group showed weaker signals than the 50 mg L⁻¹ group but stronger signals than the 10 mg L⁻¹ group. Environmental NP levels are influenced by factors such as macroplastic degradation rates, the use of treated wastewater, and industrial runoff. Although the 10 mg L⁻¹ concentration may better reflect environmentally relevant exposure levels, these findings highlight the importance of assessing whether chronic exposure to low doses could lead to cumulative effects comparable to those caused by short‐term exposure to higher concentrations.

Figure 3.

Accumulation of 80 nm fluorescently labeled NP beads by tea seedling stems. Five‐month‐old tea seedlings were exposed to 0, 10, 50, and 100 mg L⁻¹ suspensions of NP beads labeled with Rhodamine B for 10 days. Global transverse section images (A) and partial transverse section images (B) of tea stem uptake of NP beads were analyzed under bright‐field conditions and in orange channels using CLSM. (a1–a3, b1–b3) Transverse sections of stems untreated with NP beads. (c1, e1, g1, d1, f1, h1) Bright‐field images showing transverse sections of stems treated with NP beads labeled with Rhodamine B. (c2, e2, g2, d2, f2, h2) Orange channel images of transverse sections used to detect Rhodamine B fluorescence at 570 nm excitation and 590 nm emission. (d2, f2, h2) Aggregates of NP beads at varying concentrations observed in the intercellular spaces of the epidermis and cortex. (a3, c3, e3, g3, b3, d3, f3, h3) Merged images, where ‘a3’ corresponds to ‘a1 + a2’, ‘c3’ to ‘c1 + c2’, and so forth.

Figure 4.

Accumulation of 80 nm fluorescently labeled NP beads by tea seedling leaves. Five‐month‐old tea seedlings were exposed to 0, 10, 50, and 100 mg L⁻¹ suspensions of NP beads labeled with Rhodamine B for 10 days. Global transverse section images (A) and partial transverse section images (B) of tea leaf uptake of NP beads were analyzed under bright‐field conditions and in orange channels using CLSM. (a1–a3, b1–b3) Transverse sections of leaves untreated with NP beads. (c1, e1, g1, d1, f1, h1) Bright‐field images showing transverse sections of leaves treated with NP beads labeled with Rhodamine B. (c2, e2, g2, d2, f2, h2) Orange channel images of transverse sections used to detect Rhodamine B fluorescence at 570 nm excitation and 590 nm emission. (d2, f2, h2) Aggregates of NP beads at varying concentrations observed in the intercellular spaces of the epidermis. (a3, c3, e3, g3, b3, d3, f3, h3) Merged images, where ‘a3’ corresponds to ‘a1 + a2’, ‘c3’ to ‘c1 + c2’, and so forth.

The presence of NPs in the intercellular spaces of stems and leaves indicates their potential transport via apoplastic pathways, likely driven by capillary action and transpiration pull associated with water movement. Their accumulation in stomatal pores and intercellular gaps may interfere with gas exchange and reduce photosynthetic efficiency, as suggested by observed cellular alterations. This physical obstruction could also trigger localized stress responses, including changes in antioxidant enzyme activity, reflecting the plant's physiological response to NP exposure.

SEM analysis showed the presence of agglomerated NP beads within stem cross‐sections (Fig. 5(C),(D)), confirming their uptake by roots and subsequent translocation to leaves (Fig. 5(A)–(D)). These beads appear to persist in leaf tissues without significant degradation, indicating the potential for long‐term accumulation and highlighting the need for further investigation. The presence of NPs in tissue gaps may reduce cellular porosity and hinder nutrient transport. Moreover, the embedding of dispersed NPs on stomatal surfaces could compromise tissue integrity, though this requires further verification. These findings suggest that NPs can accumulate in the aerial parts of tea seedlings. However, in natural conditions, translocation dynamics may vary depending on factors such as soil‐bound NPs, plant transpiration rates, and environmental stressors. Field‐based studies are essential to better understand these interactions under realistic agricultural scenarios.

Figure 5.

SEM images of 80 nm NP beads localized in the roots, stems, and leaves of tea seedlings. Five‐month‐old tea seedlings were exposed to 100 mg L⁻¹ suspensions of NP beads labeled with Rhodamine B for 10 days. (A) NP beads observed in the intercellular spaces of root epidermal cells. (C) Aggregates of NP beads on the outer cortex of the stem. (E) NP beads embedded in the vein spaces of leaves. (B, D, F) Enlarged views of regions indicated by red squares in (A, C, E), respectively.

Physiological and biochemical assessment of tea seedlings

Quantitative analysis of theanine content

Theanine, a key compound in tea quality formation and nitrogen metabolism, is primarily synthesized in tea plant roots and subsequently transported to the aboveground young tissues. Thus, its concentration remains higher in root tissues (Fig. 6(A1)). Compared to the control group, theanine levels significantly increased in both roots and leaves under 10 and 50 mg L⁻¹ NP treatments (roots: +26.9% and +20.6%; leaves: +77.6% and +60.1%; P < 0.05) (Fig. 6(A1)). However, a marked reduction was observed in the leaves of the 100 mg L⁻¹ group (−70.1%; P < 0.05) (Fig. 6(A1)). The elevated theanine content at lower NP concentrations suggests a stress‐induced metabolic adjustment rather than a beneficial effect, potentially driven by the activation of amino acid biosynthesis in response to mild oxidative stress. The decline at higher concentrations (100 mg L⁻¹) indicates metabolic disruption, likely due to oxidative stress and impaired nutrient transport.

Figure 6.

Physiological indexes of tea roots and leaves exposed to PS‐NPs. (A1) Theanine content in tea roots and leaves cultured in different exposure solutions. (A2) CAF and catechin contents under different exposure conditions. (B1, B2) CAT activity in tea seedling roots and leaves. (C1, C2) GST activity in tea seedling roots and leaves. (D1, D2) MDA levels in tea seedling roots and leaves.

Quantitative analysis of polyphenols and caffeine content

Given the higher abundance of catechins and CAF in tender tea shoots, this study focused on analyzing their content changes in fresh leaves under NP stress. Under 50 and 100 mg L⁻¹ NP exposure, GC content significantly increased by 98.65% and 140.17%, respectively (P < 0.05), while the 10 mg L⁻¹ group showed a marked decrease of 44.24% (P < 0.05) (Fig. 6(A2)). EGC levels decreased in the 10 and 100 mg L⁻¹ groups (−33.61% and −12.09%, P < 0.05), but increased by 16.56% in the 50 mg L⁻¹ group (P < 0.05) (Fig. 6(A2)). Importantly, GA, EGC, and CAF contents were significantly elevated in the 50 mg L⁻¹ treatment (+30.34%, +16.56%, and +24.15%, respectively; P < 0.05) (Fig. 6(A2)). Overall, the changes in catechin and CAF contents were limited to specific compounds and concentrations, with no clear concentration‐dependent trend. For instance, GC content increased significantly at 50 and 100 mg L⁻¹, while EGC showed a slight increase only at 50 mg L⁻¹ and decreases at 10 and 100 mg L⁻¹. GA and CAF were elevated only at 50 mg L⁻¹. Notably, at the highest concentration (100 mg L⁻¹), most tested compounds either showed minimal changes or returned toward control levels. These sporadic fluctuations suggest that PS‐NPs, at the tested concentrations and exposure duration, do not exert a strong or consistent impact on the catechin and CAF profiles of tea leaves. This finding is consistent with previous reports, ³⁴ which indicated that NP beads exhibit low short‐term toxicity and have limited effects on these tea constituents.

Additionally, we acknowledge that this study focused only on theanine, catechins, and CAF as representative quality‐related metabolites, based on their well‐established roles as core indicators of tea quality. Other free amino acids (e.g. glutamic acid, arginine, aspartic acid) and additional quality components (e.g. soluble sugars, organic acids, aroma precursors) were not analyzed. This constitutes a limitation of the present study. Future research employing comprehensive metabolomic approaches (e.g. liquid or gas chromatography–mass spectrometry‐based untargeted metabolomics) is warranted to fully elucidate the impact of PS‐NPs on the overall quality profile of tea leaves.

Dynamic responses of CAT activity in tea seedlings under PS‐NPs stress

The exposure of hydroponic tea seedlings to PS‐NPs induced complex, concentration‐ and time‐dependent alterations in CAT activity, with distinct patterns observed between roots and leaves (Fig. 2(b1),(b2)). These responses indicate a spatially and temporally coordinated, yet nonlinear, antioxidant defense mechanism against NP‐induced stress.

In roots, the most pronounced adaptive response was triggered by the medium‐concentration treatment (50 mg L⁻¹). CAT activity was initially significantly suppressed by 69.7% (P < 0.05) at day 1, suggesting an acute physical or chemical shock that temporarily impaired enzyme function or synthesis. However, a robust recovery and defense induction followed, with activity increasing significantly by 1.6‐ and 15‐fold at days 3 and 7, respectively (Fig. 2(b1)). This shift from initial inhibition to subsequent upregulation reflects the activation of a transcriptional or post‐translational antioxidant program in response to sustained oxidative pressure. ³⁵ In contrast, the low‐concentration treatment (10 mg L⁻¹) elicited a distinctly different pattern from the medium‐concentration treatment. No statistically significant changes were observed during the first 3 days. By day 7, although the mean CAT activity showed a threefold increase, this change remained nonsignificant relative to the control (P > 0.05, two‐way ANOVA). The high‐concentration treatment (100 mg L⁻¹) induced a distinctly different pattern: no significant change at day 1 was followed by a significant decrease of 85.3% (P < 0.05) at day 3, with a return to baseline by day 7 (Fig. 2(b1)). This pattern may indicate severe metabolic disruption or a strategic shift to non‐CAT antioxidant pathways under extreme stress.

In leaves, the CAT activity dynamics were more attenuated and asynchronous, suggesting a possible indirect, systemically acquired response that may be mediated by root‐to‐shoot signaling (Fig. 2(b2)). Under 10 mg L⁻¹ NP stress, leaf CAT activity was significantly inhibited at days 1 and 7 (by 66.9% and 56.7%, P < 0.05), but showed a transient increase of fivefold (P < 0.05) at day 3. This ‘suppress–activate–suppress’ pattern suggests an early warning signal, a mid‐term defense mobilization, and a possible long‐term resource reallocation under mild chronic stress. The 50 mg L⁻¹ treatment induced a classic triphasic response: no initial change at day 1; no significant change in CAT activity relative to the control (P > 0.05, two‐way ANOVA), despite a numerical decrease of 2.7‐fold at day 3; and a significant decline of 61.4% (P < 0.05) at day 7, potentially marking the transition from active defense to metabolic exhaustion or systemic damage. For the 100 mg L⁻¹ group, a slight but nonsignificant increase at days 1–3 was followed by a drastic decrease of 62.0% (P < 0.01) at day 7. This likely reflects an initial systemic alarm triggered by severe root impairment, culminating in a collapse of leaf metabolic and antioxidant capacity due to catastrophic disruption of water and nutrient supply.

The differential spatiotemporal patterns of CAT activity underscore a ‘stress perception–signal transduction–dynamic defense’ model. Roots, as the primary contact site, exhibited direct and rapid responses, ranging from acute shock (50 mg L⁻¹, day 1) to adaptive upregulation or severe inhibition depending on concentration and duration. Leaves, conversely, displayed delayed and often biphasic or triphasic responses, which could be interpreted as secondary oxidative stress potentially propagated via systemic signals (e.g. reactive oxygen species (ROS), abscisic acid) from damaged roots, although direct evidence for such signaling remains to be established.

These results collectively demonstrate that NP toxicity is not a simple function of concentration, but a dynamic process determined by the interplay of exposure dose, duration, and the plant's capacity for spatial redistribution of defensive resources. ³⁶ The early root response is predictive, while the later leaf response integrates the systemic physiological cost of NP exposure.

In summary, the CAT activity responses across different concentrations and time points reveal several key patterns: (i) low concentration (10 mg L⁻¹) induced minimal early changes but a late, nonsignificant increase in roots, while leaves exhibited a ‘suppress–activate–suppress’ triphasic pattern; (ii) medium concentration (50 mg L⁻¹) triggered the most pronounced adaptive response in roots, with an initial 69.7% suppression (day 1) followed by a 15‐fold induction (day 7), whereas leaves showed progressive suppression over time; and (iii) high concentration (100 mg L⁻¹) caused delayed suppression in roots (85.3% at day 3) followed by recovery, while leaves displayed a drastic 62.0% decrease at day 7. These patterns indicate that roots respond directly and rapidly to PS‐NPs stress, while leaves exhibit systemic, delayed responses that reflect the integrated physiological cost of exposure.

GST activity in response to NP exposure

Glutathione S‐transferase (GST), a crucial detoxification enzyme, catalyzes the conjugation of GSH with endogenous or xenobiotic electrophiles. This conjugation enhances the solubility and subsequent transport of toxic compounds across cellular membranes, promoting their compartmentalization or excretion from plant tissues. ³⁷ In roots, the response of GST activity to PS‐NP stress was time‐ and concentration‐dependent. On day 1, while the low concentration (10 mg L⁻¹) elicited no significant change, both medium and high concentrations induced a significant suppression, with reductions of 77.0% and 52.4%, respectively, compared to the control (P < 0.05). However, this initial inhibition was transient. Two‐way ANOVA revealed no significant main effect of concentration at days 3 and 7 (P > 0.05). Post hoc comparisons confirmed that GST activity in all treatment groups returned to levels statistically indistinguishable from the control at these time points, despite notable numerical decreases (e.g. a 44.0% mean reduction under high concentration at day 7). In leaves, a contrasting spatial response was observed. By day 3, GST activity was significantly inhibited under all concentrations (P < 0.05), with reductions of approximately 12.6%, 19.0%, and 35.0% for low, medium, and high concentrations, respectively. By day 7, the pattern diverged: the inhibitory effect of the high concentration was no longer statistically significant, whereas significant suppression persisted in the low‐ and medium‐concentration groups, with decreases of about 37.1% and 47.8%, respectively (P < 0.05).

These results demonstrate a complex spatiotemporal regulation of GST activity under PS‐NP stress. The transient inhibition in roots suggests a rapid acclimation or compensatory response in the detoxification system. In leaves, the sustained inhibition at low‐to‐medium concentrations, coupled with the recovery under high concentration by day 7 may indicate a concentration‐dependent shift in detoxification strategy or a differential sensitivity of leaf GST isoforms. The concurrent modulation of CAT (as previously reported) and GST activities highlights the integrated nature of the plant's antioxidant and detoxification defense systems against microplastic‐induced stress. The underlying mechanisms, potentially involving ROS signaling or direct interference with enzyme function, warrant further investigation.

In summary, the GST activity responses across different concentrations and time points reveal distinct spatial and temporal patterns: (i) in roots, GST activity was transiently suppressed at day 1 under medium (77.0% reduction) and high (52.4% reduction) concentrations, but returned to control levels by days 3 and 7, indicating a rapid acclimation or compensatory response; (ii) in leaves, GST activity was significantly inhibited at day 3 across all concentrations (12.6–35.0% reductions), suggesting a systemic detoxification response; and (iii) by day 7, leaf GST inhibition persisted under low and medium concentrations (37.1% and 47.8% reductions, respectively), but recovered under high concentration, indicating a concentration‐dependent shift in detoxification strategy. These findings highlight that roots can rapidly acclimate to PS‐NP stress, whereas leaves experience more sustained detoxification system modulation, particularly under low‐to‐medium concentration exposure.

MDA levels in response to NP exposure

MDA is employed as a key physiological indicator to quantitatively assess plant stress resistance and cellular aging, with its concentration correlating positively with the intensity of oxidative stress. ³⁸ In roots, MDA levels exhibited a biphasic response to PS‐NP exposure. A significant accumulation was observed at days 1 and 3 across all concentrations tested, with increases of approximately 21.5%, 30.5%, and 13.3% at day 1, and 43.8%, 43.9%, and 65.4% at day 3 for the low, medium, and high concentrations, respectively. This initial rise was followed by a pronounced decrease at day 7, with levels significantly reduced by about 45.5%, 31.9%, and 17.2% compared to the control. MDA levels in tea leaves significantly decreased after 1 day of exposure at all concentrations (−41.65%, −33.79%, −29.71%, P < 0.05), suggesting a suppression of lipid peroxidation. However, after 7 days of exposure, MDA levels significantly increased in the 10 and 100 mg L⁻¹ groups (+13.62%, +16.89%, P < 0.05), indicating the onset of oxidative stress. The initial reduction in MDA levels suggests a temporary suppression of lipid peroxidation, while the subsequent increase reflects the development of oxidative damage due to prolonged exposure. This dynamic response highlights the complex interplay between antioxidant defenses and the persistent generation of ROS triggered by NP stress. ³⁹

Incorporating field trials that replicate realistic NP distribution and soil–plant interactions would provide more accurate assessments of their impacts in agricultural settings. The observed physiological and biochemical changes align with the effects of short‐term exposure to NPs. However, this study has certain limitations, including its exclusive use of a hydroponic system and focus on early‐stage seedling responses. Regarding the exposure concentrations used in this study (10–100 mg L⁻¹), we acknowledge that they are higher than typical environmentally relevant levels. Nevertheless, they fall within the range commonly employed in NP exposure studies to ensure detectable uptake and effects over a short experimental period. The 10 mg L⁻¹ treatment may better reflect extreme contamination scenarios, while chronic low‐dose exposure in natural settings could lead to cumulative effects that are not captured in this short‐term study. Therefore, future research should prioritize long‐term, low‐concentration exposure experiments under more realistic field‐ or soil‐based conditions to better assess the ecological risks of PS‐NPs in agricultural systems. Such studies should also examine the interactions between PS‐NPs and tea seedlings in complex soil matrices, evaluate the potential trophic transfer of NPs in agricultural ecosystems, and explore their combined effects with co‐occurring pollutants (e.g. pesticides or heavy metals) to better understand multi‐stressor interactions in realistic scenarios.

CONCLUSIONS

This study demonstrated the uptake, transfer, and effects of PS‐NPs in tea seedlings, highlighting their entry through roots, transport to shoots, and the resulting impact on physiological and biochemical processes. Exposure to PS‐NPs resulted in alterations to theanine, CAF, and catechin levels, as well as changes in antioxidant enzyme activities, MDA, and TP levels. Although PS‐NPs induced oxidative stress, as indicated by elevated MDA levels at specific time points, the overall antioxidant defense system appeared to prevent severe or persistent oxidative damage under the tested conditions. It is important to note that the observed changes in quality‐related metabolites (e.g. increased theanine and catechin contents at certain concentrations) should be interpreted as stress‐induced metabolic adjustments or early adaptive responses rather than beneficial or promotive effects. These findings raise important ecological and human health concerns regarding NP contamination in agriculture. The study highlights the need for further research on interactions of NPs with plants in soil systems, their long‐term effects, and potential mitigation strategies. Nevertheless, this study has certain limitations, including the exclusive use of a hydroponic system, the lack of soil microbial interactions, and the short‐term exposure duration. Future field‐based studies are needed to validate these findings under more realistic environmental conditions.

AUTHOR CONTRIBUTIONS

Tingting Shen: Conceptualization, formal analysis, investigation, data curation, writing – original draft. Fei Liu: Methodology, validation, data curation, writing – review and editing. Rui Zhang: Methodology, validation, formal analysis. Tianyuan Yang: Provided raw material tea seed seedlings. Ping Zhang: Formal analysis, visualization. Xiuhong Zhou: Conceptualization, formal analysis. Rimao Hua: Methodology. Jianfa Zong: Data curation, writing – review and editing, visualization. Weiting Jiao: Resources, conceptualization, formal analysis, writing – review and editing.

CONFLICT OF INTEREST

The authors declare that they have no conflicting interests.

Supporting information

Figure S1. Localization of 80 nm fluorescent polystyrene (PS) beads in transverse section of tea main and lateral roots after exposure to a 10 mg L⁻¹ solution of Rhodamine B labeled PS beads; Images of tea root transverse sections were acquired using the orange channel to detect Rhodamine B fluorescence (excitation: 570 nm, emission: 590 nm). Panels (a–f) show enlarged views of the main root and associated root hairs, revealing strong fluorescent signals on the root hairs surrounding the main root; Panels (g–i) present cross‐sectional views at sites where root hairs were observed, demonstrating strong fluorescence in cells of the root epidermal layer. (Scale bar: a, b, c, 100 μm; d, e, f, 20 μm; g, h, i, 100 μm).

Figure S2. Scanning electron microscopy (SEM) images of 80 nm nano‐plastic beads in the leaves of tea plants. Tea seedlings were exposed to 100 mg L⁻¹ 80 nm PS‐NPs for 7 days. The images show that irregular PS‐NPs were adsorbed onto the surface of cell wall pores and intercellular spaces. Most of the PS‐NPs appeared to be physically entrapped within extracellular structures (e.g., cell wall fibrils or surface depressions), with no clear evidence of intracellular internalization.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.