Effects of Microplastics on Growth and Physiological Characteristics of Tobacco (Nicotiana tabacum L.)

Abstract

The environmental effects caused by the proliferation of microplastics have received extensive attention. However, at this stage, there is a lack of research on the toxicology of microplastics and their impacts on plants. This study explored the effects of microplastics on the growth and physiological characteristics of tobacco. Using tobacco (Nicotiana tabacum L.) variety Yunyan87 and low-density polyethylene (LDPE, 13 μm particle size), different concentrations, which were 0 mg/L (CK), 10 mg/L (T1), 100 mg/L (T2), and 1000 mg/L (T3), were subjected to hydroponic experiments to study the effects of microplastics on the growth and physiological characteristics of tobacco. The results showed that microplastics had a certain inhibitory effect on the growth of tobacco. Among them, high concentrations of microplastics significantly inhibited the root architecture and growth characteristics. According to the performance observed with different treatments, the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) increased in the medium and high concentrations. In addition, the changes in catalase (CAT) and peroxidase (POD) activity were not obvious. Experiments showed that microplastics could significantly inhibit the growth and development of tobacco and reduce the accumulation of aerial parts and root biomass.

1. Introduction

Microplastics, as a new type of environmental pollutant, were first described in 2004 and usually comprise plastic particles with a particle size of less than 5 mm, present in the environment [1]. Plastic products are widely used because of their stable properties. According to statistics, from 1950 to 2015, plastic products amounted to approximately 6.3 billion tons globally, but only 6% to 26% of plastics were recycled [2]. Utilized plastic products are discarded in aquatic or terrestrial ecosystems. Plastic products can break down under the influence of light, heat, mechanical forces, and other factors, and often exist in the environment in the form of fibers, fragments, granules, films, etc. [3]. The growth of aquatic organisms and the survival of aquatic populations are seriously threatened by microplastics [4]. Terrestrial systems have received less attention than studies of microplastics in aquatic environments. Microplastics are more often in direct contact with organisms in aquatic environments. Due to the poor fluidity of the soil environment, the concentration of microplastics in the terrestrial environment will probably increase over time [2]. It was found that the abundance of microplastics in soil ecosystems is closely related to the long-term input of plastic products in agricultural production activities [5]. When microplastics enter the soil, they affect crop growth and pose a potential threat to crop safety, so there is an urgent need to understand the mechanisms of microplastics’ effects on plant growth [6].

Microplastics existing in the natural environment also have an impact on plants. Microplastics can enter plants through ecological cycles, and Li et al. [7] showed that lettuce can absorb microplastics and accumulate in roots and leaves. Bandmann et al. [8] found that tobacco BY-2 cells in cell culture could be associated with bound 20–40 nm polystyrene nanoparticles, assessing the potential bioaccumulation of microplastics in tobacco cells. Microplastics entering the body can lead to oxidative stress damage in plants, which can lead to biological cytotoxicity and genotoxicity [9,10]. Microplastics can directly affect plants by adsorbing to the seed epidermis or root cell wall pores, blocking the pores in the seed capsule, and retarding seed germination. In the late growth stage, microplastics accumulated on the root hairs disturb the normal absorption or transportation of water and nutrients by the seed or root system, thus leading to the suppression of plant growth and certain effects on plant traits [11,12]. Bosker et al. [13] found that microplastics blocked watercress seed coat stomata and inhibited seed germination and plant root growth. Studies found that both wheat [14] and onions [15] experienced stress due to microplastics, which changed their growth characteristics and reduced the accumulation of plant biomass.

In our present study, we hypothesized that low-density polyethylene microplastics would affect plant growth and physiological characteristics. To test our hypothesis, we conducted a hydroponics experiment by using tobacco (Nicotiana tabacum L.) variety Yunyan 87 as a model plant due to it often being cultivated with plastic mulching. Tobacco was exposed to low-density polyethylene microplastics at increasing doses (0 mg/L, 10 mg/L, 100 mg/L, and 1000 mg/L). Several commonly applied growth parameters (i.e., agronomic traits, root system conformation, chlorophyll, and resistance to stress, etc.) were used to assess the impacts of the microplastics on the growth of tobacco.

2. Materials and Methods: Photosynthetic Pigment

2.1. Experimental Design

To evaluate the effects of microplastics on plant growth and physiological properties, an experimental hydroponic culture of tobacco was designed, which allowed for more direct contact with microplastic particles. The experimental tobacco variety was tobacco (Nicotiana tabacum L.) variety Yunyan 87, and the seeds were provided by the Guizhou Academy of Tobacco Science. The microplastic was linear low-density polyethylene (LLDPE) powder with a particle size of 13 μm. With reference to the accumulation of microplastics in plants and the effect on plant growth [16], microplastics of this particle size can react microscopically on the plant surface, while the microplastic itself cannot be taken up by the plant. Gradients and high doses may amplify potential side effects that are overlooked and identify potential thresholds [17]. Therefore, we utilized an extreme concentration. Polyethylene powder was formulated with Hoagland nutrient solution (pH 5.5–6.5) at four concentrations of 0 mg/L, 10 mg/L, 100 mg/L, and 1000 mg/L, and polyethylene particles were uniformly dispersed in the liquid medium using an ultrasonic cleaner (KQ-500DE, Kunshan, China).

2.2. Growing Conditions

Tobacco hydroponic experiments were conducted at the Guizhou Academy of Tobacco Science. The floating seedling method was used to cultivate tobacco. The seeds of tobacco were placed in the tobacco floating seedling matrix, treated with sowing fertilizer, and placed in an artificial climate box (photoperiod 12 h, light 270 μmol/(m²·s), temperature 25/20 °C (day/night), air humidity 70%). Plants were allowed to germinate and grow to the small cross stage, and we added a uniform seedling fertilizer. When the plants had grown 2 leaves and 1 bud, the most robust and consistent tobacco seedlings were selected. Then, different microplastic concentrations of 0 mg/L (CK), 10 mg/L (T1), 100 mg/L (T2), and 1000 mg/L (T3) were applied. Hydroponics was carried out in a 100 mL conical flask and 10 plants were cultured in each treatment. The tobacco culture is shown in Figure 1. Nutrient solution was supplemented every 2 days to confirm that the concentration of microplastics did not change during the experiment. The conical vials were randomly placed in the climatic chamber and moved in position every 3 days.

2.3. Measurement of Tobacco Growth Index and Biomass

Ten samples of tobacco plants were taken from each treatment after 27 d of the experiment, and the surface of tobacco was washed with ultrapure water. The number of leaves, plant height, stem diameter, maximum leaf length and width, aerial parts’ fresh weight biomass, and root fresh weight biomass were measured. The formula for calculating leaf area is

leaf area (cm²) = (leaf length × leaf width) × 0.6345

Seven samples of each treatment were selected using the root scanning analysis system (WinRHIZO, Regent, Canada) to measure the root length, root volume, root surface area, root projected area, root average diameter, number of root tips, number of root forks and number of root crosses, and other root system architecture traits.

2.4. Tobacco Physiological Indicators

Leaves with vigorous growth and metabolism were selected for the measurement of chlorophyll a content, chlorophyll b content, total chlorophyll content, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, and peroxidase (POD) activity. For each concentration, three samples were prepared. Among them, chlorophyll content was determined in a dark room and samples were kept on ice to prevent chlorophyll degradation. Chlorophyll was determined with a spectrophotometric method [18]. MDA content was measured using the thiobarbituric acid colorimetric method [19]. The SOD activity was measured with the nitrogen blue tetrazolium method [20]. The CAT activity was measured via the ultraviolet absorption method, and the POD activity was measured with the guaiacol method [21,22]. The content of antioxidant enzymes was also determined via the spectrophotometric method. These indicators were measured using a Multifunctional Enzyme Labeler (Synergy H4, Biotek, Winooski, VT, USA).

2.5. Data Processing

Data processing was performed using Microsoft Excel software version 2016 and SPSS software version 28.0. The experimental data were expressed as mean and standard deviation (SD). Statistical processing was performed using the Shapiro–Wilk test, followed by statistical processing using one-way analysis of variance (ANOVA) and a Bonferroni post hoc test at the p < 0.05 level. Graphs were produced with GraphPad Prism software version 9.0.0.

3. Results

3.1. The Effect of Microplastics on the Growth of Tobacco

The agronomic traits of tobacco under different treatments are shown in

Table 1. Agronomic characteristics of tobacco under different treatments.

Index	CK	T1	T2	T3
Number of Leaves	4.6 ± 0.5 a	4.7 ± 0.7 a	4.4 ± 0.5 a	4.4 ± 0.7 a
Plant Height (cm)	3.0 ± 0.3 a	3.1 ± 0.6 a	2.9 ± 0.8 a	2.2 ± 0.9 b
Stem Diameter (mm)	4.3 ± 0.3 a	4.2 ± 0.6 a	4.2 ± 0.4 a	3.7 ± 0.5 b
Maximum Leaf Area (cm2)	59.7 ± 13.2 a	59.1 ± 19.6 a	56.5 ± 22.0 a	25.2 ± 13.1 b

Note: Different letters after the data in the same row indicate that the difference between different treatments reaches a significant level (p < 0.05); the same applies below.

, and the difference in the number of leaves of tobacco plants was small, among which the T2 and T3 treatments yielded lower results than the CK and T1 treatments. The tobacco plant height and stem diameter reached a maximum with the CK treatment of 3.0 cm and 4.3 mm, respectively. Plant height and stem diameter were significantly different between the CK, T1, and T2 treatments and T3 treatment. Compared with the CK treatment, the maximum leaf area of the tobacco in the T1, T2, and T3 treatments was 1%, 5%, and 58% lower, respectively. The difference between the T3 treatment and the CK, T1, and T2 treatments was significant.

In Figure 2, it can be seen that different concentrations of microplastics also had some effect on the aerial parts’ biomass and root biomass accumulation of the tobacco plant. Medium and low concentrations of microplastics had a lower effect on the accumulation of aerial parts’ fresh weight biomass. The high concentration of microplastics inhibited the accumulation of aerial parts’ fresh weight biomass, and the difference between the T3 treatment and CK, T1, and T2 treatments was significant. As the concentration of microplastics increased, the plants showed a trend in which microplastics first promoted and then inhibited the overall root growth. Compared with the CK treatment, the T1 treatment showed the promotional effect of microplastics on the root fresh weight biomass, which reached the maximum value under the T1 treatment. The T2 and T3 treatments were inhibited by microplastics to varying degrees, among which the T2 and T3 treatments were significantly different from the CK and T1 treatments.

3.2. The Effect of Microplastics on the Root Architecture of Tobacco

In the hydroponic environment, the mean values and differences in the root architecture indexes of different treatments were as shown in Figure 3 and Figure 4. The root average length for the T1, T2, and T3 treatments was 13%, 34%, and 68% lower than that for the CK treatment, which was 254.05 cm, respectively. The difference between the T3 treatment and CK and T1 treatments was significant. The root average volume under the T1 and T2 treatments was 10% and 19% lower than that under the CK treatment, which was 0.31, and that under the T3 treatment was 68% lower than that under the CK treatment. With the increase in the microplastics concentration, the root surface area and projected area of tobacco also decreased, and the CK treatment had the highest value, with mean values of 31.21 cm² and 9.93 cm², respectively. The differences between the T3 and CK, T1, and T2 treatments were significant. There was a minor significant difference in the root average diameter among different treatments. The root average diameter reached the maximum value of 0.45 in the T2 treatment, followed by the T1 treatment. Microplastics had a certain degree of influence on the root average diameter. The overall trend of the number of root tips, number of root forks, and number of root crosses was similar, wherein the T3 treatment was significantly different from the CK and T1 treatments and the maximum values were all found in the CK treatment (842.29, 995.43, and 71.14).

3.3. Effects of Microplastics on Chlorophyll Content and Stress Resistance of Tobacco Leaves

As can be seen from Figure 5, the chlorophyll content of tobacco leaves increased first and then decreased with the increase in the microplastics concentration, and the variability of each treatment was small. The total chlorophyll content of tobacco in the T2 treatment was the highest, and the increase in the T1, T2, and T3 treatments compared with the CK treatment was 29%, 36%, and 7%, respectively. Chlorophyll a content was also the highest in the T2 treatment, with increases of 35%, 40%, and 10% in the T1, T2, and T3 treatments, respectively, compared to the CK treatment. The chlorophyll b content increased by 25% in both the T1 and T2 treatments compared to the CK treatment, while the content did not change in the T3 treatment compared to the CK treatment.

There were different fluctuation trends in MDA content and the activity of three antioxidant enzymes (SOD, CAT, and POD), in the different treatments, but their differences were small. The MDA content for the CK and T1 treatments was similar, with mean values of 10.55 nmol/g and 10.12 nmol/g, respectively. Meanwhile, the MDA content under the T2 and T3 treatments slightly increased to 10.87 nmol/g and 11.63 nmol/g (Figure 6). The SOD activity under the CK and T1 treatments was similar, with mean values of 209.61 U/g and 209.12 U/g, respectively. Moreover, the SOD activity for the T2 and T3 treatments was also similar, with increases of 29% and 37% compared to the CK treatment. With the increase in the microplastics concentration, the POD activity showed a trend of first decreasing and then increasing. T1, T2, and T3 showed increases of 24%, 46%, and 6% compared to the CK treatment, and the lowest value of 129 ΔOD470/min/g was reached in the T2 treatment. CAT activity increased by 10% and 21% in the T1 and T2 treatments compared to the CK treatment, while it decreased by 43% in T3 and reached the lowest value of 57.37 μmol/min/g. An overall trend of increasing followed by decreasing was observed.

4. Discussion

Root architecture indicators include root length, root volume, root projected area, root surface area, number of root tips, number of root crosses, and number of root forks, which can reflect the spatial shape and distribution of the root system in the growing medium. In this study, with the increase in the microplastics concentration, the total root length, root volume, root projected area, root surface area, number of root tips, number of root crosses, and number of root forks all showed a decreasing trend. Microplastics had a highly significant effect on the root architecture, and the lateral roots of the stressed tobacco were obviously stunted; the number of root forks and crosses in the roots of the plants treated with high concentrations of microplastics were significantly reduced, and the development of root hairs was limited; microplastics significantly affected the number of root tips, the root vitality decreased, and we observed decreased proliferation ability of the meristem. This indicates a significant inhibitory effect of microplastics on the root growth of tobacco. This is because, during hydroponics, microplastics accumulated on the root surface and formed white feathery complexes with root secretions [23]. These substances were attached to the surface of the root system and could not be absorbed into the body due to the limitation of the pores of the cell wall. These substances adhere to the root surface and cannot be absorbed into the body due to the restriction of cell wall pores (5–50 nm), and they accumulate in layers around the cells, causing a physical blockage and limiting root water uptake, which in turn interferes with the normal development of root hairs, reduces root permeability, and limits the overall root growth [24,25,26]. Previous studies have shown some stressing effects of microplastics on the root growth of watercress [12] and wheat [13], which is consistent with the results of the present study.

In addition, the average root diameter of tobacco in this study reached a maximum at medium concentrations (T2), but the difference was not significant. This phenomenon had certain differences from existing studies. van Weert et al. [17] and Meng et al. [27] showed that the increased competition between developed roots and microorganisms at the beginning of hydroponics or stimulated by the decrease in nutrient status caused an increase in plant root diameter. The later stages of culture were more affected by microplastics and the effect of persistent stress on nutrient uptake, which, in turn, affected the continued root development [28].

In this study, the stem diameter, maximum leaf area, and aerial parts’ biomass showed a decreasing trend with the increasing microplastics concentration. Meanwhile, the plant height and number of leaves decreased significantly at medium and high concentrations. This is mainly due to the significant negative effect of the microplastics concentration on plant N uptake. The lack of N in the plant leads to a decrease in stomatal conductance, transpiration, and assimilation rate, which results in a slower rate of biomass accumulation [23]. Previous studies found that the microplastics concentration was negatively correlated with plant N uptake and physiological performance, and biomass accumulation was inhibited [14], which is consistent with the results of the present study.

In this study, the MDA content increased in the medium- and high-concentration treatments, and the SOD content increased in the medium- and high-concentration treatments. Meanwhile, the POD content decreased in the medium- and high-concentration treatments, and the CAT content decreased in high-concentration treatments. Whereas the medium- and low-concentration treatments caused plants to consume a large amount of POD to ensure intracellular homeostasis, the high-concentration treatment caused them to consume a large amount of CAT to scavenge ROS. This suggests that plants are subjected to environmental stress and have elevated levels of superoxide radicals and reactive oxygen species (ROS) in the body, causing oxidative damage to cells and tissues [29,30] and increased levels of MDA substances that reflect the extent of damage to cell membranes [31]. The plant body produces a series of antioxidant enzymes (SOD, POD, CAT) that synergistically scavenge ROS [32]. The variability of the chlorophyll content of tobacco was low among the treatments in this study, and the study of Boots et al. on ryegrass showed that chlorophyll was also not significantly affected [33]. In this study, the incubation time was short, and the plants were stressed mainly in the roots, and the resulting oxidative damage had not yet accumulated in the leaves.

5. Conclusions

The stress effect of microplastics imposed a significant limitation on the accumulation of biomass in tobacco plants. There was some significant variability in root fresh weight and aerial part fresh weight biomass between treatments with different microplastics concentrations. The present study on tobacco exposed to microplastics showed that the root length, root volume, root projected area, and number of root tips tended to decrease with increasing microplastics concentration. Polyethylene microplastics (particle size of 13 μm) were able to affect the root architecture of tobacco and significantly inhibit root growth and development. In addition, the stem diameter and maximum leaf area also tended to decrease with increasing microplastics concentration, indicating that microplastics had a significant negative effect on the growth characteristics of tobacco. In particular, both the aerial parts’ and root fresh weight biomass were highly significantly different from those in the CK treatment at a 1000 mg/L concentration.

Polyethylene microplastics had an effect on the growth and physiological characteristics of tobacco. Despite the lower variability of chlorophyll in tobacco leaves, it would cause certain oxidative damage, increased MDA content, and fluctuated antioxidant enzymes to synergistically scavenge ROS. Among them, the content of SOD increased in the treatment involving microplastics stress. The 10 mg/L and 100 mg/L concentration treatments consumed POD, and the 1000 mg/L concentration treatment consumed CAT to scavenge ROS. In the future, we will continue to carry out comparative studies on different plants and microplastics with different particle sizes to further explore the effects of microplastics on the physiological characteristics of plants.