Polyethylene terephthalate nanoplastics affect potassium accumulation in foxtail millet (Setaria italica) seedlings

Abstract

Background

As modern industrial activities have advanced, the prevalence of microplastics and nanoplastics in the environment has increased, thereby impacting plant growth. Potassium is one of the most crucial nutrient cations for plant biology. Understanding how polyethylene terephthalate (PET) treatment affects potassium uptake will deepen our understanding of plant response mechanisms to plastic pollution.

Results

In this study, we examined the impact of PET micro- and nanoplastics on foxtail millet seedling growth and potassium accumulation. Additionally, we measured reactive oxygen species (ROS) production, antioxidant enzyme activities, and the expression levels of the corresponding enzyme-encoding genes. Our findings indicated that the germination and seedling growth of foxtail millet were not significantly affected by exposure to PET plastics. However, the ROS levels in foxtail millet increased under these conditions. This increase in ROS led to the upregulation of several genes involved in K⁺ uptake and transport (SiHAK1, SiHAK2, SiAKT2/3, SiHKT2;2, SiHKT1;1, SiGORK, and SiSKOR), thereby increasing K⁺ accumulation in foxtail millet leaves. Further research revealed that higher K⁺ concentrations in plant leaves were correlated with increased expression of the antioxidant-related genes SiCAT1, SiPOD1, and SiSOD3, as well as increased activities of the corresponding antioxidant enzymes. This response helps mitigate the excessive accumulation and damage caused by ROS in plant cells after PET nanoplastic treatment, suggesting a potential stress response mechanism in foxtail millet against nanoplastic pollution.

Conclusions

Our research indicates that PET nanoplastic treatment induces the expression of genes related to K⁺ uptake in foxtail millet through ROS signaling, leading to increased K⁺ accumulation in the leaves. This process mitigates the ROS damage caused by PET nanoplastic treatment by increasing the expression and activity of genes encoding antioxidant enzymes. The present research has unveiled the K⁺ accumulation-related response mechanism of foxtail millet to PET nanoplastic treatment, contributing significantly to our understanding of both the potassium absorption regulation mechanism in plants and the broader impact of plastic pollution on agricultural crops. This discovery not only highlights the complexity of plant responses to environmental stressors but also underscores the importance of considering such responses when evaluating the ecological and agricultural implications of plastic pollution.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-024-06007-2.

Introduction

In recent years, plastic products have been extensively used in many fields, leading to a yearly increase in production. From 2000 to 2019, the amount of plastic waste worldwide doubled, reaching 353 million tons [1]. The degradation of plastic waste in natural environments is influenced by physical, chemical, and biological factors, which can deteriorate its structure and result in the formation of microplastics (smaller than 5 mm) and nanoplastics (smaller than 1 mm) [2–6]. Owing to their small size, large surface area, widespread presence, and resistance to biodegradation, these particles tend to accumulate in fungi and aquatic organisms. They can pass through the food chain, eventually endangering human health [4, 6].To date, numerous studies have focused on the impacts of micro/nanoplastics on marine and freshwater organisms, with their effects on terrestrial ecosystems garnering attention only in recent years. Microplastics and nanoplastics are also present in the soils of various terrestrial ecosystems [4, 7–9]. The annual input of microplastics and nanoplastics into farmland soils far exceeds that into the ocean [10]. In severely contaminated regions, the soil microplastic and nanoplastic contents may reach 60% [11, 12]. These tiny particles may directly affect plant health through toxicity, alter the soil structure, impact nutrient storage, and change the microbial community around the roots, thereby affecting plant growth and development [9]. Studies have also shown that these small plastics can facilitate the entry and uptake of heavy metal ions by plants, impacting seed germination, photosynthesis, and growth and causing stress in plant cells [13]. The extent of this effect is closely linked to the size and surface charge of the plastic particles [4, 9].

Polyethylene terephthalate (PET) is a thermoplastic polymer sourced from petroleum. Its durability, high transparency, and light weight have made it a prevalent choice for applications in agricultural films, packaging, and fibers worldwide [14]. The complex structure of PET makes it resistant to degradation. Recent studies have examined the effects of PET pollution on agricultural crops. The findings of these studies indicate that exposure to PET micro- or nanoplastics can instigate a sudden increase in reactive oxygen species (ROS), hinder seed germination, and decrease the biomass of plants such as Cicer arietinum L. (chickpea), Lepidium sativum L. (cress), and Allium cepa L. (onion) [15–17]. Like other plastic particulates, PET is also involved in the accumulation of heavy metals within plants. Abbasi et al. [18] reported that PET particles act as carriers for the absorption of heavy metals such as zinc (Zn), cadmium (Cd), and lead (Pb), transporting them into wheat roots.

Potassium (K⁺) is a vital nutrient ion for plants and is involved in numerous key biological processes [19]. The absorption and transport of plant K⁺ depend on two primary mechanisms: high-affinity K⁺ absorption, which is transporter dependent, and low-affinity K⁺ absorption, which relies on channels [20]. However, research exploring the impact of microplastics or nanoplastics on plant K⁺ uptake is scarce. Foxtail millet (Setaria italica), a crucial crop in arid and semiarid regions of Asia, has emerged as an ideal model plant for investigating stress resistance because of its remarkable tolerance to drought and barren environments, small genome size, self-pollination, and short life cycle [21, 22]. Potassium plays a critical role in plant cell turgor maintenance, stomatal movement, and osmotic balance [23], all of which are essential for stress tolerance in foxtail millet. Therefore, understanding how PET treatment affects potassium uptake can provide valuable insights into how millet responds to plastic pollution from a potassium nutrition perspective. This knowledge could pave the way for genetically improving crops to increase their resistance.

Our research revealed that treating foxtail millet with 1 g/L PET nanoplastics did not suppress germination or seedling growth. However, it resulted in an increase in ROS and elevated leaf K⁺ levels. After exposure to PET nanoplastics, SiAKT2/3, SiSKOR, SiGORK, SiHAK1, SiHAK2, SiHKT1;1, and SiHKT2;2 were upregulated at 24–72 h post-treatment. The introduction of ROS scavengers reversed the upregulation of K⁺ uptake and transport-related genes and the increase in K⁺ accumulation in leaves induced by PET nanoplastic exposure. The higher concentration of K⁺ in plant leaves aids in inhibiting the excessive accumulation and damage of ROS in plant cells caused by PET nanoplastic treatment, which could be a potential response mechanism of foxtail millet to stress from nanoplastic pollution.

Materials and methods

Plant growth conditions and treatment

In the current study, the foxtail millet cultivar “Jingu21” was employed. Seeds were acquired from Professor Lizhen Zhang’s laboratory, located at the School of Life Sciences, Shanxi University, China. The PET microplastics and nanoplastics were purchased from Zhangmutou Suyuan Plastic Material Co., Ltd (Dongguan, China) and used after confirming their particle size.

Foxtail millet is typically cultivated under conditions with a photoperiod of 16/8 h light/dark, temperatures between 23 and 26 °C, light intensity of 50,000 lx, and relative humidity ranging from 30 to 50%. For the germination experiment, the seeds were sterilized as described before [24, 25]. We placed 25 seeds in each petri dish containing a seedbed of Whatman paper and were sterilely cultured in 10 mL of a 1 g/L PET micro or nano plastic solution. The concentration of plastic used in this study was similar to that used in previous reports [15–17, 26]. On the 8th day, the germination percentage was calculated as (the number of germinated seeds with roots longer than 2 mm / the total number of seeds used) * 100%. In this experiment, seeds were considered to have germinated if their roots exceeded 2 mm in length.

For soil culture, we evenly sowed the seeds in the seedling pot (vermiculite: nutrient soil = 1:2, with exchangeable potassium at 0.266 ± 0.013 mg/g soil). The addition of PET had no significant effect on the exchangeable potassium content in the soil. When the seedlings reached the two-leaf stage, the seedlings with consistent growth were selected and moved to a nursery pot containing the same amount of soil. After one week of continuous growth, the seedlings were irrigated with a quantitative 1 g/L PET micro or nano plastic solution (after 7 days treatment, finally equivalent to 0.2% w/w plastic/dry soil). Each pot was transplanted with 3 seedlings and each treatment was repeated with 3 pots. Leaf and root samples were collected after 7 days of treatment, frozen in liquid nitrogen and stored at -80 °C for subsequent experiments.

In the liquid culture experiment, we initially acclimated 10-day-old seedlings in a half-strength Murashige and Skoog (MS) liquid medium for 4 days. Subsequently, they were transferred to a half-strength MS solution supplemented with PET micro- or nanoplastics and varying concentrations of K⁺ for further treatment. To delve into the role of ROS within this context, the liquid medium was pre-conditioned with 50 mM tiron (O₂^•− scavenger) or 1 mM dimethylthiourea (DMTU; •OH scavenger) for 1 h before the introduction of PET nanoplastics. Leaves from the foxtail millet seedlings, after undergoing liquid culture for intervals of 24 h, 72 h, and 7 days, were harvested. These samples were then subjected to rapid freezing in liquid nitrogen before being stored at − 80 °C for subsequent biochemical analyses. Each experimental group included more than three biological replicates to ensure reliable and reproducible data.

Measurements of antioxidant enzyme activities and malondialdehyde, H2O2, and glutathione content

The leaf and root samples of “Jingu21” seedlings were harvested with or without PET microplastics or nanoplastics treatments as described above. The malondialdehyde (MDA) content (MDA-2-Y), the hydrogen peroxide (H₂O₂) content (H₂O₂-2-Y), the glutathione (GSH) content (GSH-2-W), the catalase (CAT) activity (CAT-2-Y), the superoxide dismutase (SOD) activity (SOD-2-Y), and the peroxidase (POD) activity (POD-2-Y) were measured using kits purchased from Suzhou Keming Biotechnology Co. ltd (China).

Measuring of the particle size and morphology observation by scanning electron microscope

After the sample solution was diluted with deionized water, the average diameter was determined by a laser particle size analyzer (Mastersizer 2000, Malvern, UK) for microplastics. For nanoplastics, the particle size and zeta potential were measured by a Malvern Zetasizer (Nano-ZS90, Malvern, UK). The surface morphology observation of particles was carried out using a Sigma 300 VP-Field Emission Scanning Electron Microscope (FE-SEM; Carl Zeiss, Germany).

Cation contents measurement

The measurement of cation content was performed by the Qidian Chemical Technology Service (Liaocheng, China) as previous described [27–29]. Plant samples were harvested and subjected to acid digestion. The contents of Na⁺, K⁺, Cd²⁺, Hg²⁺, and Pb²⁺ were measured using inductively coupled plasma optical emission spectrometry (ICP-OES 730, Agilent, USA). Soil exchangeable potassium was extracted by displacement with 1 M ammonium acetate (pH 7) and measured by ICP-OES [30]. The K⁺ absorption capacity of plastics was determined by adding 500 mg of PET nanoplastics to 5 mL of potassium solution, thoroughly mixing, allowing it to stand for 3 h, and then measure the K⁺ content of supernatant through ICP-OES.

qRT-PCR analysis

Quantitative real-time PCR (qRT-PCR) analysis was performed as described before [31–33]. The total RNA of foxtail millet leaves was extracted using TransZol™ UP Plus RNA Kit (TransGen Biotech, Beijing, China) and reversely transcribed using EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China). qRT-PCR was performed using the Perfect-Start Green qPCR SuperMix kit (TransGen Biotech, Beijing, China), and the results were quantitatively analyzed by the 2^−ΔΔCT method [34, 35]. The PCR cycling conditions were as follows: 94 °C for 30 s; 40 cycles of 94 °C for 15 s and 60 °C 30 s. SiAct2 (Seita.8G043100) and SiRNA POL II (Seita.2G142700) were used as the internal references [36]. All primers used in the experiment were synthesized by Sangon Biotech, Shanghai (Table S1). The specificity of these primers is checked by whether the melting curve has a single peak. Each experiment included three technical replicates and three biological replicates.

Statistical analysis

Statistical analysis of independent experiments was reported as mean ± standard error (SE). Student’s t-test analysis was performed on the data to determine the significant difference between the treatment group and the control. *P < 0.05 indicated significant difference, **P < 0.01 indicated extremely significant difference.

Results

Biological effects of PET microplastics or nanoplastics on foxtail millet

In this study, PET nanoplastics and microplastics were prepared by mechanically crushing white PET plastic sheets to the target size at Zhangmutou Suyuan Plastic Material Co., Ltd (Dongguan, China). The microplastics had a particle size of approximately 6.32 μm with a zeta potential of -28.8 ± 0.2 mV, and the nanoplastics were approximately 790 nm with a zeta potential of -18.5 ± 0.1 mV. We used a scanning electron microscope (SEM) to examine the PET microplastics and nanoplastics. Fig. S1 shows that these plastics had a spherical structure with a rough surface.

Healthy “Jingu21” foxtail millet seeds were subjected to germination analysis under PET plastic treatment. We opted for a PET microplastic and nanoplastic treatment at a concentration of 1 g/L (equivalent to 0.2% w/w plastic/dry soil) to streamline the experimental conditions. This specific concentration aligns with the upper limits of physiologically effective microplastic and nanoplastic treatment concentrations documented in the literature for plants such as Cicer arietinum L. (chickpea), Allium cepa L. (onion), Lepidium sativum L. (cress), and Cucurbita pepo L. (cucurbitaceae) [15–17, 26]. As shown in Fig. 1A and C, neither microplastic nor nanoplastic treatment affected the germination of foxtail millet. We then checked whether the PET plastic treatment affected seedling growth. As shown in Fig. 1B and C, all the plants presented similar height and leaf colors. However, treatment with nanoplastics led to increased MDA and H₂O₂ levels, whereas the GSH level, CAT activity, and POD activity decreased in the leaves (Fig. 1C). These findings suggest that oxidative damage occurs in plants under PET nanoplastic treatment. The addition of PET microplastics only increased the MDA content. We subsequently increased the treatment concentration (5 g/L and 10 g/L) and duration (14 days) of PET nanoplastic treatment, and the results revealed that they had no significant effect on the growth of foxtail millet seedlings (Fig. S2). On the basis of these results, we predict that short-term exposure to PET nanoplastics leads to an increase in ROS in plants but does not cause significant phenotypic changes.

Fig. 1.

Treatment with polyethylene terephthalate (PET) nanoplastics induced reactive oxygen species (ROS) germination and enhanced the activity of antioxidant enzymes, but it did not affect seed germination and growth of foxtail millet seedlings. (A) Treatment with 1 g/L PET micro- or nanoplastics did not impact the germination of “Jingu21” foxtail millet seeds. (B) Treatment with 1 g/L PET micro- or nanoplastics did not impact the morphology of two weeks old “Jingu21” foxtail millet seedlings as the growth parameters were not strictly measured. (C) The germination rate of seeds, height of seedlings, leaf hydrogen peroxide (H₂O₂) content, malondialdehyde (MDA) content, glutathione (GSH) content, catalase (CAT) activity, and peroxidase (POD) activity of two weeks old “Jingu21” foxtail millet under treatment with 1 g/L PET micro- or nanoplastics are shown. Values presented as mean ± SE (n = 3). * Significant differences compared to the control seedlings are indicated at P < 0.05

PET nanoplastic treatment enhances K+ accumulation in foxtail millet

Previous reports have shown that treatments with microplastics and nanoplastics increase the absorption of heavy metal cations by plants [13, 15]. To determine whether K⁺ cation absorption and transport are promoted by PET nanoplastics, we measured the K⁺ and Na⁺ contents in the leaves and roots of foxtail millet. As shown in Fig. 2A, B and Fig. S2, the nanoplastic treatment improved only the K⁺ content, not the Na⁺ content, in the leaves of the seedlings. There was no effect of microplastic treatment on the K⁺ and Na⁺ contents in either the leaves or the roots. Heavy metals such as Cd, Hg and Pb are the main heavy metal pollutants in foxtail millet-producing areas in central and western China [37–39]. We also assessed the changes in heavy metal ions such as Cd²⁺, Hg²⁺, and Pb²⁺ in foxtail millet leaves after 7 days of treatment with 1 g/L PET nanoplastics. The results indicated that the contents of these heavy metal ions remained unchanged (Fig. S2).

Fig. 2.

Exposure to 1 g/L PET nanoplastics increased the potassium (K⁺) content in the leaves (A) of foxtail millet seedlings, but not in the roots (B), even when cultured in liquid media (C)

“Jingu21” foxtail millet seedlings were either soil-cultured or liquid-cultured for two weeks. Subsequently, they were treated with 1 g/L PET nanoplastics. The liquid culture media (with PET) was changed every two days. Leaves and roots were then harvested to measure Na⁺ and K⁺ contents using inductively coupled plasma optical emission spectrometry (ICP-OES). Values presented as mean ± SE (n = 3). * Significant differences compared to the control seedlings are indicated at P < 0.05.

Rillig et al. [9] suggested that plastic particles affect plant roots by altering the soil structure, changing the soil microbial community and root symbionts, and directly affecting nutrient transport and adsorption. Thus, to investigate whether PET treatment affects plant K⁺ accumulation by altering soil factors, we liquid-cultured foxtail millet and stressed it with 1 g/L PET plastics (Fig. 2C). Again, we found that PET nanoplastic treatment improved the K⁺ content, as was the case for the soil-cultured plants, but only when the K⁺ content in the culture solution was 10 mmol/L. Abbasi et al. [18] reported that PET plastics could directly adsorb Pb²⁺, Cd²⁺, and Zn²⁺ and help transport these three heavy metal cations to the wheat rhizosphere zone. However, exposure to PET nanoplastics did not result in the adsorption of K⁺ in different concentrations of K⁺ solution (Fig. S3). In conclusion, the accumulation of K⁺ in the leaves of foxtail millet under PET nanoplastic treatment was due to direct effects on the plant itself. This finding also indicates that plants have an intrinsic mechanism to increase K⁺ absorption in response to stressors such as nanoplastic exposure.

PET nanoplastic treatment induces the transcription of K+ channels and transporters

Plants assimilate and transport K⁺ through two primary mechanisms: high-affinity K⁺ transporters and low-affinity K⁺ channels [20]. Previous research has identified both types of proteins in foxtail millet [33, 40, 41]. In our study, we noted an increase in K⁺ in the leaves of foxtail millet when it was exposed to PET nanoplastics. To further investigate this phenomenon, we measured changes in the gene expression of K⁺ channels and transporters that are crucial for K⁺ absorption via qRT‒PCR [33, 40, 41]. As depicted in Fig. 3, exposure of foxtail millet to 1 g/L PET nanoplastics resulted in increased transcription of SiAKT2/3 (Seita.3G233000), SiGORK (Seita.7G111600), SiSKOR (Seita.4G110300), SiHAK1 (Seita.7G082300), SiHAK2 (Seita.2G427100), SiHKT1;1 (Seita.1G068700), and SiHKT2;2 (Seita.4G241700). However, the transcription levels of SiKAT3 (Seita.1G020100) and SiHKT1;2 (Seita.5G024400) decreased. In summary, our results showed that PET nanoplastic treatment induced the transcription of some K⁺ channels and transporters, increasing the K⁺ content in foxtail millet leaves. Many studies have shown that increased K⁺ content has positive effects on the ability of plants to resist some abiotic stresses [41, 42]. For example, under drought stress, a relatively high K⁺ content in plants helps maintain cell turgor, thus ensuring the normal physiological function of plants [43, 44]; under salt stress, K⁺ can regulate the ion balance and reduce the toxic effects of high salt concentrations on plants [41, 45]. On the basis of these findings, we propose the following hypothesis: an increase in K⁺ content may itself be a response mechanism of foxtail millet to nanoplastic stress.

Fig. 3.

The transcription level of K⁺ transport related genes in “Jingu21” foxtail millet seedlings under PET nanoplastics treatments. “Jingu21” foxtail millet seedlings were soil-cultured for two weeks. Subsequently, they were treated with 1 g/L PET nanoplastics. Leaves were harvested at 24 h, 72 h, and 7 days for further qRT-PCR analysis. The SiAct2 (Seita.8G043100) and SiRNA POL II (Seita.2G142700) were used as the internal references. In each gene, the transcription level of control at 24 h was assigned a value of 1 and the expression levels of other samples were transformed. Values presented as mean ± SE (n = 3). * Significant differences compared to the control seedlings are indicated at P < 0.05

PET nanoplastic treatment enhanced the leaf K⁺ content in foxtail millet seedlings in an ROS signaling-dependent manner

According to previous studies, the dynamic regulation of K⁺ transport and channel genes in plants is often closely related to ROS signals. K⁺ and ROS jointly maintain cell homeostasis and enhance plant stress adaptability [46–48]. Our results revealed that exposure to PET nanoplastics led to elevated ROS levels in foxtail millet, as illustrated in Fig. 1. To test the hypothesis mentioned above, we sought to determine whether this accumulation of ROS contributed to the increased K⁺ content in foxtail millet. To investigate this, we applied the ROS scavengers Tiron and DMTU [49–51] prior to PET treatment and subsequently measured the K⁺ levels in liquid-cultured foxtail millet leaves. Figure 4 shows that Tiron and DMTU alone did not change the leaf K⁺ levels, but they counteracted the PET-induced K⁺ increase. In further experiments, we evaluated whether genes related to K⁺ uptake and transport, which were upregulated by PET exposure, were affected by ROS scavengers. In liquid-cultured foxtail millet, PET nanoplastic treatment alone upregulated the expression of SiHAK1, SiHAK2, SiAKT2/3, SiHKT1;1, SiHKT2;2, SiGORK, and SiSKOR, similar to that in soil-cultured plants (Fig. 5). The addition of ROS scavengers in the PET treatment matched the gene expression changes with the K⁺ content findings. The expression of genes was reduced when scavengers were present. Tiron alone or Tiron with PET treatments suppressed the expression of SiHKT2;2 and SiHKT2;1. The expression of SiAKT1 also decreased when the cells were cotreated with both ROS scavengers and PET (Fig. S4). SiKAT2 expression decreased after treatment with Tiron for 24 h or DMTU for 72 h alongside PET. These results suggested that PET nanoplastics altered the expression of genes involved in K⁺ uptake and transport under the control of ROS signaling, resulting in an increase in the K⁺ content in the leaves.

Fig. 4.

The PET nanoplastics treatment enhanced leave K⁺ content of “Jingu21” foxtail millet seedlings was depended on ROS signal. “Jingu21” foxtail millet seedlings were liquid-cultured with 10 mmol/L K⁺ for two weeks. The ROS scavenger Tiron (50 mmol/L) or DMTU (1 mmol/L) were added 1 h before 1 g/L PET nanoplastics treatment. The leaf K⁺ content was presented as mean ± SE (n = 3). * Significant differences compared to the control seedlings are indicated at P < 0.05

Fig. 5.

The PET nanoplastics treatment up-regulates K⁺ transport related genes in “Jingu21” foxtail millet seedlings were suppressed by ROS scavenger. “Jingu21” foxtail millet seedlings were liquid-cultured with 10 mmol/L K⁺ for two weeks. The ROS scavenger Tiron (50 mmol/L) or DMTU (1 mmol/L) were added 1 h before 1 g/L PET nanoplastics treatment. The SiAct2 (Seita.8G043100) and SiRNA POL II (Seita.2G142700) were used as the internal references. In each gene, the transcription level of control was assigned a value of 1 and the expression levels of other samples were transformed. Values presented as mean ± SE (n = 3). * Significant differences compared to the control seedlings are indicated at P < 0.05

K⁺ accumulation alleviates the ROS damage induced by PET nanoplastics

Tavakol et al. [47] reported that an optimized K⁺ supply is important for reducing ROS-related damage induced by osmotic stress, especially in drought-sensitive barley varieties. Given these findings, we investigated whether this phenomenon is also involved in the response mechanism of foxtail millet to PET nanoplastic stress. We conducted further experiments by culturing plants in solutions containing different concentrations of K⁺ (control: 10 mM K⁺, Low-K⁺: 0.1 mM, High-K⁺: 20 mM). We subsequently measured the K⁺ content, H₂O₂ production, and MDA content after the plants were exposed to 1 g/L PET nanoplastics. As depicted in Fig. 6A, the K⁺ content in the leaves of foxtail millet cultured in solutions with varying K⁺ concentrations was greater than that in the roots, and as the K⁺ concentration in the culture solution was increased, the K⁺ content in both the roots and the leaves also increased. PET nanoplastic treatment induced only K⁺ accumulation in the control and high-K⁺ groups, as mentioned previously. As shown in Fig. 6B and C, in the control group and the low-K⁺ culture group, PET treatment led to the accumulation of H₂O₂ and an increase in MDA levels. In the high-K⁺ group, the ROS damage in the foxtail millet exposed to PET nanoplastics was rescued by the high K⁺ supply (Fig. 6B and C).

Fig. 6.

High content of K⁺ in foxtail millet alleviates ROS damage induced by PET nanoplastics. “Jingu21” foxtail millet seedlings were liquid-cultured with 0.1 mmol/L K⁺ (low-K⁺), 10 mmol/L K⁺ (control), and 20 mmol/L (high-K⁺) for two weeks. Subsequently, they were treated with 1 g/L PET nanoplastics. The liquid culture media (with PET) was changed every two days. Leaves and roots were then harvested to measure K⁺ contents (A), leaf hydrogen peroxide (H₂O₂) content (B), and leaf malondialdehyde (MDA) content (C). Values presented as mean ± SE (n = 5). * Significant differences compared to the control seedlings are indicated at P < 0.05

Is this phenomenon related to the expression and activity of antioxidant enzymes? On the basis of previous reports, we selected catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) for measurement [52–55]. As shown in Fig. 7, PET treatment inhibited the activity of these enzymes, whereas high-K⁺ treatment alone did not affect their activities. Under combined treatment with high potassium and PET, the inhibitory effect of PET on enzyme activity was alleviated. This result explained the previously mentioned ROS-related findings (Fig. 6). Additionally, we investigated the expression of representative antioxidant enzyme-encoding genes, including SiCAT1 (Seita.4G286700), SiPOD1 (Seita.9G116200), and SiSOD3 (Seita.9G488200), as reported previously [25, 52]. At the gene expression level, the inhibitory effect of PET on enzyme activity is also related to the downregulation of antioxidant enzyme-encoding gene expression. High-K⁺ treatment relieved this inhibitory effect, providing support for the observed changes in activity (Fig. 7A). However, notably, under combined treatment with high K⁺ and PET, the gene expression levels of SiPOD1 and SiSOD3 were significantly increased, but their activities did not change.

Fig. 7.

High content of K⁺ alleviates the down-regulation of ROS scavenging enzyme genes caused by PET treatment and promotes the ROS scavenging in Foxtail millet. “Jingu21” foxtail millet seedlings were liquid-cultured with 10 mmol/L K⁺ for two weeks. Then they were treated with 10 mmol/L K⁺ (control), 20 mmol/L K⁺ (high-K⁺), 10 mmol/L K⁺ with 1 g/L PET nanoplastics (PET (nm)), and 20 mmol/L K⁺ with 1 g/L PET nanoplastics (high K⁺+PET (nm)). The liquid culture media was changed every two days. After 7 days, leaves were then harvested for the measurement of ROS scavenging enzyme activity and qRT-PCR analysis. (A) The catalase (CAT) activity, peroxidase (POD) activity, and Superoxide dismutase (SOD) are shown. The SiAct2 (Seita.8G043100) and SiRNA POL II (Seita.2G142700) were used as the internal references. In each gene, the transcription level of control was assigned a value of 1 and the expression levels of other samples were transformed. Values presented as mean ± SE (n = 3). * Significant differences compared to the control seedlings are indicated at P < 0.05

On the basis of the above results, it could be concluded that greater leaf K⁺ accumulation helps plants alleviate the damage caused by ROS, which provides a preliminary explanation for the physiological significance of K⁺ accumulation in plants subjected to PET nanoplastic treatment.

Discussion

In this study, we explored the impact of PET plastics, pollutants generated by modern industrial development, on foxtail millet. As shown in Fig. 8, our observations indicated the following: (1) Exposure to PET nanoplastics did not hinder germination or seedling growth in foxtail millet but led to the accumulation of ROS. (2) qRT‒PCR experiments revealed alterations in the transcription levels of several genes associated with K⁺ uptake and transport following exposure to PET nanoplastics, resulting in increased K⁺ content in the leaves. (3) The accumulation of leaf K⁺ and the upregulation of specific genes were attributed to ROS signaling, which was triggered by PET nanoplastics. This leaf K⁺ accumulation assisted in mitigating the ROS damage induced by PET nanoplastic treatment in foxtail millet.

Fig. 8.

PET nanoplastics treatment induces K⁺ transport related genes expression and K⁺ accumulation in leave of “Jingu21” foxtail millet seedlings depend on ROS signaling. K⁺ accumulation, as one of the mechanisms by which plants respond to PET treatment, helps alleviate oxidative damage caused by PET treatment by enhancing the expression level and activity of antioxidant enzyme genes

PET nanoplastic treatment does not affect foxtail millet germination or seedling growth but leads to the accumulation of ROS

In our experiments, we chose 1 g/L as the PET nanoplastic treatment concentration, which is commonly used in laboratory studies. This concentration helps researchers observe and simulate environmental effects under extreme conditions or evaluate the removal efficiency of nanoplastics and the effectiveness of ecological purification technologies [15–17, 26, 56–58]. We conducted a preliminary investigation into the impact of 1 g/L PET nanoplastics on the germination and seedling growth of foxtail millet. Our findings revealed no discernible influence (Fig. 1). We also tested higher PET concentrations (5 g/L and 10 g/L) and longer durations (14 days) of PET nanoplastic treatment but still observed no significant effect on the growth of foxtail millet (Fig. S2).

The accumulation of microplastics and nanoplastics in plants affects their growth characteristics, morphological changes, photosynthesis, oxidative stress and antioxidant defense systems [59]. Germination is the initial phase of plant development and is characterized by heightened sensitivity to environmental stress. While numerous studies have demonstrated that microplastics and nanoplastics can affect germination [15, 60, 61], others have reported that they have no effect on certain plant species [62, 63]. Similarly, the literature presents contradictory perspectives on the impact of plastics on seedling growth [26, 64–66]. This might be because the effects of microplastics and nanoplastics are contingent upon the type of plastic, the plant species, and various environmental factors [67]. Sun et al. [68] noted that positively charged nanoplastics caused more ROS accumulation and inhibited growth more than negatively charged nanoplastics did. In our study, the PET nanoplastics (surface charge of -18.5 ± 0.1 mV) and microplastics (surface charge of -28.8 ± 0.2 mV) employed possessed a negative charge. According to previous reports, negatively charged nanoparticles exhibit electrostatic repulsion with their own negatively charged cell membranes [68, 69]. Compared with those of positively charged nanoparticles, the interactions between negatively charged particles and cell membranes are relatively weak, reducing the likelihood of cell damage or stress responses. This may lead to less disruption of normal physiological processes, which aligns with our findings.

Although no significant growth phenotype was observed in foxtail millet seedlings treated with PET nanoplastics, we found that 1 g/L PET nanoplastics increased H₂O₂ and MDA levels in the seedlings. This was coupled with lower GSH levels and reduced activity of antioxidant enzymes CAT and POD, suggesting stress in the plants.

The difference in oxidative damage between nanoplastics and microplastics may be because nanoplastics have a larger surface area and stronger adsorption capacity than microplastics do. Owing to their smaller particle size, nanoplastics more easily adsorb onto plant roots and directly damage plant roots or spread within plants, leading to physical damage and ROS accumulation [68, 70–72]. In summary, our study did not reveal a significant impact of PET microplastics or nanoplastics on seed germination or plant growth. However, we observed an increase in plant ROS levels under PET nanoplastic treatment, indicating that the plants were subjected to a certain level of stress.

PET nanoplastic treatment induced the expression of genes related to K⁺ uptake and transport and increased the leaf K⁺ content

K⁺ plays an important role in plant growth, development, defense, immunity, signal transduction, and substance transport [73]. Recent studies have revealed a close relationship between K⁺ and ROS during the plant stress response [47, 74]. In our study, we examined the effects of different concentrations (1 g/L, 5 g/L, and 10 g/L) of PET nanoplastics on the K⁺ content in foxtail millet seedling leaves. The results revealed that all the treatments led to K⁺ accumulation in the leaves of the foxtail millet seedlings (Fig. S2). On the basis of these results, combined with those of previous studies, we chose 1 g/L PET nanoplastics as the treatment concentration for subsequent experiments. After seven days of exposure to 1 g/L PET plastics, we measured the Na⁺ and K⁺ levels in the leaves and roots of the seedlings. We found that only treatment with PET nanoplastics increased the K⁺ levels in the leaves, whereas the Na⁺ levels in the leaves and the Na⁺ and K⁺ levels in the roots remained unchanged (Fig. 2A and B). Micro- or nanoplastics and heavy metals exist widely in the soil environment. Micro- or nanoplastics entering the soil may change the pore structure, water retention and aeration of the soil and affect the migration and availability of metal ions [75]. Many studies have shown that micro- or nanoplastics can promote the uptake of cations, including heavy metal ions, in plants [13, 76, 77]. Therefore, the finding that PET nanoplastics also increase K⁺ in foxtail millet is not unexpected. We further determined changes in the Cd²⁺, Hg²⁺, and Pb²⁺ heavy metal ion contents in foxtail millet leaves under the treatment conditions of this study. The results revealed that the contents of these heavy metal ions in foxtail millet leaves did not change after 7 days of treatment with 1 g/L PET nanoplastics (Fig. S2). This finding is inconsistent with the results reported in some studies [13, 76, 77]. However, Wang et al. [78] noted that the addition of microplastics reduced the accumulation of Cd in plants. Nikoumaram and Sepehri [79] also reported that microplastics reduced and balanced cadmium toxicity in millet seedlings. In these studies, the duration and concentration of microplastic treatment varied. In further studies, extending the PET treatment duration and increasing the concentration of PET treatment while exploring whether PET treatment affects the gene expression related to heavy metal ion absorption will help explain the phenomena we observed.

Reports suggest that micro- or nanoplastics can affect how plants absorb ions, either directly by changing gene expression and altering physiological processes or indirectly by changing soil properties [9, 77]. We found that PET nanoplastic treatment can also increase the K⁺ content in millet leaves under liquid culture conditions but only when the solution contains 10 mmol/L K⁺ (Fig. 2C), which confirmed that nanoplastics do not increase leaf K⁺ when the soil conditions are changed. K⁺ accumulation only occurred in the 10 mmol/L K⁺ mixture-cultured plants, suggesting that both high-affinity and low-affinity types of K⁺ absorption mechanisms are involved in this response. Additionally, research has indicated that PET microplastics and nanoplastics can directly attract cations in liquid, which helps plants absorb them [18]. We examined this possibility and found no significant K⁺ adsorption from solutions with different concentrations of nanoplastics (Fig. S3). In conclusion, PET nanoplastics cause K⁺ to accumulate in leaves by affecting mainly the plant itself.

Numerous studies have reported that an increase in potassium content is important for plants to resist abiotic stresses [41, 42]. We propose that the change in K⁺ accumulation might constitute a strategy for foxtail millet to respond to PET nanoplastic treatment.

We first explored whether PET treatment affects the expression levels of genes related to K⁺ absorption. Previous reports indicate that numerous K⁺ channels and transporters are involved in the uptake and transport of K⁺ in foxtail millet [33, 40, 41]. We found that genes such as SiHAK2, SiAKT2/3, SiHAK1, SiHKT1;1, SiHKT2;2, SiGORK, and SiSKOR presented elevated expression, whereas SiKAT3 and SiHKT2;1 presented reduced expression after 24 and 72 h. After 7 days, their expression returned to levels similar to those of the control group, suggesting that the effect of PET nanoplastics on gene expression is transient. Among the genes analyzed, SiHAK1, SiHAK2, SiHKT1;1, and SiHKT2;2 belong to the high-affinity K⁺ uptake system, whereas SiAKT2/3 is a K⁺ channel associated with the low-affinity uptake system. The expression of K⁺ transport and channel genes is closely related to ROS signaling. When the external K⁺ concentration is low, the level of ROS in plants increases, promoting root hair elongation and K⁺ absorption [80, 81]. Exogenous application of H₂O₂ can increase the K⁺ content in Arabidopsis by regulating the expression of the K⁺ transporter HAK5 [48, 82]. Our study results support this conclusion; even without low-K⁺ stress, the accumulation of ROS caused by PET nanoplastic treatment can regulate K⁺ absorption-related protein expression and promote K⁺ accumulation in plants. SiGORK and SiSKOR are outward-rectifying K⁺ channels. In Arabidopsis, ROS O₂.⁻ induced K⁺ outward-rectifying current, which was related to GORK gene splicing [83]. Our findings reveal increased expression of SiGORK, necessitating further investigation into potential posttranscriptional regulation. As an outward-rectifying channel, SKOR participates in facilitating K⁺ secretion from the xylem parenchyma cells of roots toward the xylem vessels [84], whereas the inward-rectifying channel AKT2 also mediates phloem K⁺ loading and unloading [85, 86]. These two channels are instrumental in the long-distance transport of K⁺, particularly in the translocation of K⁺ from roots to shoots. The upregulation of these two genes helps explain why only the leaf K⁺ content increased. In summary, PET nanoplastic treatment affects the expression of genes related to plant K⁺ uptake, leading to K⁺ accumulation in the leaves. Given the role of K⁺ in plant stress responses, this could represent a strategy employed by foxtail millet to respond to nanoplastic stress.

PET nanoplastic treatment-induced increases in the leaf K+ content depend on the ROS signal

ROS are important signaling molecules in plants that enable them to respond to various types of stress. Alterations in cellular ROS levels can modify the structure and function of numerous proteins, subsequently affecting multiple signal transduction pathways [87–89]. Previous studies have shown that the expression of K⁺ transporter and channel genes is closely related to ROS signaling [46–48]. On the basis of these findings, we speculate that the observed increase in K⁺ channel and transporter genes could be attributed to ROS signaling. To investigate this possibility, our findings were validated through the external addition of ROS scavengers. Two ROS scavengers, Tiron and DMTU, were used in this study according to previous reports [49–51]. The results of the present study revealed that the addition of ROS scavengers led to the disappearance of PET-induced regulation of K⁺ channel and transporter protein genes, which inhibited the increase in leaf K⁺ content (Figs. 4 and 5), confirming our hypothesis. Moreover, the addition of only the ROS scavengers changed the expression of some genes. For example, Tiron decreased the level of SiHKT1;2 (Fig. S4). These results also suggest that the activity of K⁺ channels and transporters is controlled by ROS signaling (Fig. 6). The ROS molecule H₂O₂ enhanced the current amplitude and activation kinetics of SKOR [49]. The increase in K⁺ content caused by PET nanoplastic treatment, in addition to being due to the upregulation of K⁺ channels and transport genes, may also involve direct regulation of proteins by ROS, necessitating further research.

In this study, we also investigated the physiological significance of K⁺ accumulation in foxtail millet leaves in mitigating the effects of PET nanoplastic treatment by focusing on the relationship between K⁺ and ROS. As shown in Figs. 6 and 7, the oxidative damage caused by PET treatment was relieved by increasing the activity of antioxidant enzymes through high-K⁺ treatment. A high K⁺ supply also alleviated the inhibitory effect of PET nanoplastic treatment on the expression of these genes (Fig. 7). Notably, the expression levels of the SiPOD1 and SiSOD3 genes, which we chose to measure, significantly increased under combined treatment with high K⁺ and PET, which was inconsistent with the changes in enzyme activity. This inconsistency may be due to post-transcriptional regulation of antioxidant enzyme-encoding genes. Omics analysis under nanoplastic treatment will help address this question.

In summary, our results validate the previously proposed hypothesis that a change in K⁺ content is indeed an important strategy for foxtail millet to cope with nanoplastic stress.

Conclusion

In conclusion, our study revealed that 1 g/L PET nanoplastic did not affect the germination or growth of foxtail millet. However, this treatment increased the production of ROS, and this change temporarily increased the transcription levels of some genes related to K⁺ uptake and transport, such as SiHAK1, SiHAK2, SiAKT2/3, SiHKT1;1, SiHKT2;2, SiGORK, and SiSKOR. This led to more K⁺ accumulation in foxtail millet leaves. Further studies revealed that higher K⁺ concentrations could help foxtail millet alleviate the damage caused by PET nanoplastic treatment by increasing the expression level of several genes encoding antioxidant enzymes and their enzyme activity. This process may represent one of the mechanisms by which foxtail millet responds to nanoplastic stress. Our research helps us understand how foxtail millet reacts to PET nanoplastics and how this treatment affects K⁺ absorption, which will help deepen our understanding of the impact of plastic pollution on crops.

Electronic supplementary material

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Author contributions

BZ, YW, ZL, and LZ designed the experiments. YG, LL, SD, YF, YC, YD, GW, SX, and HZ performed the experiments. YG, LL, and SD prepared Figs. 1, 2 and 4, and 5. LL, YF, YC, and YD prepared the Fig. 3. GW, SX, and HZ prepared the Fig. 6. BZ, YW, YG, PY, WZ, and LZ analyzed the data and wrote the manuscript. All authors read and reviewed the manuscript.

Funding

This work was supported by Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province (No.20230003), National Natural Science Foundation of China (No.32272012, 32000210), Research Project Supported by Shanxi Scholarship Council of China (No.2021-015), the open funding of State Key Laboratory of Sustainable Dryland Agriculture, Shanxi Agricultural University (No.YJHZKF2108) to BZ. The Science and Technology Innovation Young Talent Team of Shanxi Province (No.202204051001019) to ZL and BZ. The Doctoral Research project of Shanxi Agricultural University (No. 2021BQ40) to HZ. The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

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Competing interests

The authors declare no competing interests.