Polymer nanoparticles pass the plant interface

Abstract

As agriculture strives to feed an ever-increasing number of people, it must also adapt to increasing exposure to minute plastic particles. To learn about the accumulation of nanoplastics by plants, we prepared well-defined block copolymer nanoparticles by aqueous dispersion polymerisation. A fluorophore was incorporated via hydrazone formation and uptake into roots and protoplasts of Arabidopsis thaliana was investigated using confocal microscopy. Here we show that uptake is inversely proportional to nanoparticle size. Positively charged particles accumulate around root surfaces and are not taken up by roots or protoplasts, whereas negatively charged nanoparticles accumulate slowly and become prominent over time in the xylem of intact roots. Neutral nanoparticles penetrate rapidly into intact cells at the surfaces of plant roots and into protoplasts, but xylem loading is lower than for negative nanoparticles. These behaviours differ from those of animal cells and our results show that despite the protection of rigid cell walls, plants are accessible to nanoplastics in soil and water.

Fig. 1. Synthesis of polymeric nanoparticles and subsequent uptake pathways explored.

The charges associated with each class of nanoparticle are colour coded throughout the manuscript: magenta = positively charged; gold = neutral; blue = negatively charged. This figure was created with Biorender.com.

Fig. 2. Certain nanoparticles penetrate intact roots.

a Confocal microscopy images for the penetration and distribution of polymeric nanoparticles in Arabidopsis root hair zones after one hour of treatment in nanoparticle solution (1 mg/ml) at room temperature. b Summary table of the different levels of root penetration observed. Good (✓✓), poor (✓), or none (✗). Penetration and accumulation were evaluated under a ZEISS 880 LSM. Maximum Z projections in 488 nm laser channel were analysed alongside the Z-slices and merged with brightfield images using ImageJ software. Scale bar = 20 μm. The images are representatives of experimental replicates (n = 3). Part of this figure was created with Biorender.com.

Fig. 3. Root cross-sections illustrate nanoparticle accumulations.

Cross-sections of root hair zones after uptake of small nanoparticles with (a) positive, (b) neutral, and (c) negative surface charges, respectively. Propidium iodide staining was used to outline the cellular structure (red) while the nanoparticles are in green, and areas of co-localisation appear yellow. ImageJ software was used to generate the orthogonal views from the Z-stack images. Scale bar = 20 μm. The images are representatives of experimental replicates (n = 3).

Fig. 4. Certain nanoparticles penetrate into root cell protoplasts.

a Confocal images for the penetration and distribution of polymeric nanoparticles with Arabidopsis protoplasts. b Summary table of the different levels of penetration observed. Good (✓✓), poor (✓), or none (✗). Penetration and accumulation were evaluated using a ZEISS 880 LSM. Maximum Z projections in the 488 nm laser channel were analysed alongside the Z-slices and merged with brightfield images using ImageJ software. Scale bar = 20 μm. The images are representatives of experimental replicates (n = 3). It was noted that the smallest positively charged nanoparticles appeared to have a cytotoxic effect with an increase in visible cell debris and rough, asymmetric cells compared to other nanoparticle systems. Part of this figure was created with Biorender.com.

Fig. 5. Accumulation of nanoparticles over time.

Confocal images for the penetration and accumulation of the smallest polymeric nanoparticles in Arabidopsis root hair zones over time. Penetration and accumulation were evaluated using a ZEISS 880 LSM. The images are representatives of experimental replicates (n = 3). Maximum Z projections in the 488 nm laser channel were analysed alongside the Z-slices and merged with brightfield images using ImageJ software. Scale bar = 20 μm.