DOI: 10.5061/dryad.cc2fqz69d
title: Polymer nanoparticles pass the plant interface

description: 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.
description: Image data: Methods
 <strong>Arabidopsis thaliana root
 preparation</strong> <em>A.
 thaliana</em> ecotype Columbia-0 was used for all nanoparticle
 uptake experiments. The seeds were surface-sterilised using successive
 washes in 10% Bleach or 70% ethanol prior to sowing onto sterile ½
 strength Murashige and Skoog (MS) medium (2.2 g/L MS supplemented with B5
 vitamins, 1% sucrose, 1% agar, pH 5.8). Seeds were stratified in the dark
 for two days at 4°C prior to germination for 5 days at 22°C with 12-hour
 day length. <strong>Protoplast
 isolation</strong> Protoplast solution (600 mM
 Mannitol, 2 mM MgCl2, 2 mM CaCl2, 10 mM KCl, 2 mM MES, 0.1% w/v bovine
 serum albumin) was prepared and adjusted to pH 5.5 with Tris-HCl, 0.2 µm
 filtered, and stored at -20°C until use. To isolate protoplasts, 5-day-old
 <em>Arabidopsis thaliana</em> roots were transferred into
 enzyme solution (1.5% w/v Cellulase RS, 0.1% w/v Pectolyase in protoplast
 solution) and chopped finely using a sterile blade. The solution with
 chopped roots was transferred to a 35 mm round Petri dish and incubated in
 the dark at 25°C with constant agitation for at least 2 hr. The resulting
 cell suspension was filtered sequentially through 70 μm and 40 μm meshes
 pre-soaked with enzyme solution. The protoplasts in solution were
 carefully transferred to polystyrene culture tubes and an equal volume of
 fresh protoplast solution added, before centrifugation at 300 g for 5 min
 at 4°C. The supernatant was discarded, the protoplasts resuspended in the
 same volume of protoplast solution and centrifugation repeated. The
 protoplasts were resuspended in 500 μL protoplast solution and used
 directly. <strong>Confocal
 Microscopy</strong> Intact
 <em>Arabidopsis</em> roots were incubated with nanoparticle
 samples for an hour before visualisation under fluorescence confocal
 microscopy. Seedling roots were dipped into 100 microlitres of the
 nanoparticle solution (all 1 mg/ml in water) or water in a microfuge tube.
 For confocal microscopy, root or protoplast samples were mounted on glass
 slides (1.0-1.2 mm thick) with a long cover glass (22x50 mm, 0.16-0.19 mm
 thick) before visualisation using a Zeiss LSM 880 instrument under 40x
 objectives lens with excitation laser at 488 nm and collecting emission at
 496 - 577 nm. The images were stacked and reconstructed by ZEN software
 and analysed using ImageJ software. After incubation with nanoparticles,
 roots were also stained with propidium iodide for 10 min and propidium
 fluorescence was visualised with excitation at 561 nm and emission
 collected from 580 – 718 nm.
description: The files are .jpg image files
 of excel data files for qPCR data. Files: Folder
 names begin with a symbol to indicate the charge type on the nanoparticle
 for that set of images. This is followed by the name of the polymer used
 to make that nanoparticle and then the tissue from which the image is
 taken (root tip, root hair zone or protoplast). The time course files are
 given in folders named according to the time of collection post-treatment.
 All images are saved as .jpg files.