Polymer nanoparticles pass the plant interface.

Materials Dimethyl acrylamide (DMAm, 99 %; Sigma Aldrich 274135), Acrylic Acid (AA, 99 %; Sigma Aldrich 147230), (Methacryloyloxy)ethyl] trimethylammonium chloride (QDMAEMA, 75 % in H 2 O; Sigma Aldrich 408107), (Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS, 95 %; Sigma Aldrich 537284), 4,4′-azobis(4-cyanovaleric acid) (ACVA, 99%; Sigma Aldrich 11590), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 98%; Sigma Aldrich 341006), deuterated methanol (CD 3 OD, 99.8%; Sigma Aldrich 151947-10G-GL) and deuterium oxide (D 2 O, 99.9%; Sigma Aldrich 151882) were purchased from Sigma Aldrich (UK). DAAm (99%) was purchased from Alfa Aesar (UK; A15940.30). 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acyl hydrazide (BODIPY FL, 99 %) was purchased from ThermoFisher Scientific (UK; D2371). 4-((((2-Carboxyethyl)thio)carbonothioyl)thio)−4-cyanopentanoic acid (BM1433, 95%) was purchased from Boron Molecular (USA; BM1433). For protoplast preparation, cellulase “Onozuka RS” was purchased from Melford biolaboratories (Duchefa; C8003.0005) and pectolyase from Sigma Aldrich (P3026). 1 H NMR spectroscopy 1 H NMR spectra were recorded at 300 MHz on a Bruker DPX-400 spectrometer in either D 2 O for macro-CTA synthesis or CD 3 OD for all nanoparticle syntheses.

Size exclusion chromatography

SEC measurements were performed on a Varian 390-LC-Multi detector suite system fitted with Refractive Index (RI) and ultraviolet (UV) detectors ( λ = 309, 490 nm) equipped with a PLGel 3 μm (50 × 7.5 mm) guard column and two PLGel 5 μm (300 × 7.5 mm) mixed-D columns using DMF with 5 mM NH 4 BF 4 at 50 °C as the eluent at a flow rate of 1.0 mL min −1 . SEC data were calibrated against polystyrene standards and analysed using Cirrus v3.3 software. Transmission electron microscopy (TEM) TEM was performed using a JEOL 2000FX or JEOL 2100FX at 200 kV. TEM solution was typically made up at 0.1 mg mL −1 in water. Then, 10 μL of sample solution was dropped onto a carbon/formvar-coated copper grid placed on filter paper. After removing excess liquid, 10 μL of a 1% uranyl acetate solution was dropped onto the grid and left to dry. Synthesis of hydrophilic macro-chain transfer agents (macro-CTAs) A typical synthesis of a PDMAm 70 macro-CTA was as follows: dimethyl acrylamide (10 g, 100 mmol, 70 eq.), BM1433 (0.44 g, 1.4 mmol 1 eq.), ACVA (0.04 g, 140 μmol 0.1 eq.) were added to a round bottom flask and dissolved in water (24 mL) to give a 30% w/w reaction solution. A stirrer bar was added and then the flask was sealed and sparged with nitrogen for 20 min. The sealed flask was then immersed in an oil bath at 70 °C and left for 120 min after which it was removed from the oil bath and quenched by exposure to oxygen. Samples were then taken for 1 H NMR and SEC analysis followed by purification by dialysis and then lyophilisation to yield a yellow powder. The same procedure was followed for all other macro-CTAs.

Synthesis of diblock copolymer nanoparticles

A typical synthesis of a PDMAm 70 -PDAAm 50 was as follows: diacetone acrylamide (1 g, 6 mmol, 50 eq.), PDMAm 70 mCTA (0.85 g, 120 μmol 1 eq.), ACVA (3 mg, 12 μmol 0.1 eq.) were added to a round bottom flask and dissolved in water (7.4 mL) to give a 20% w/w reaction solution. A stirrer bar was added and then the flask was sealed and sparged with nitrogen for 20 min. The sealed flask was then immersed in an oil bath at 70 °C and left for 120 min after which it was removed from the oil bath and quenched by exposure to oxygen. Samples were then taken for 1 H NMR and SEC analysis. No further purification was performed for further experiments. The same procedure was followed for all other diblock copolymer nanoparticles (see Supplementary Table 1 ). BODIPY fluorophore attachment to polymer nanoparticles A typical attachment of BODIPY FL to PDMAm 70 -PDAAm 50 nanoparticles was as follows: EDC (240 μg, 0.6 μmol, 1 eqv) was added to PDMAm 70 -PDAAm 50 (100 mg, 0.6 μmol, 1 eq.) in water (10 mL) and stirred for 5 min. BODIPY FL (19 μg, 0.06 μmol, 0.1 eqv) dissolved in DMSO (15 μL) was then added and the solution was left to stir overnight. The solution was then purified by spin centrifugation against a 3k MWCO membrane. A sample was taken for SEC to confirm attachment of BODIPY to the polymer chains. The same procedure was followed for all other nanoparticles.

Arabidopsis thaliana root preparation

A. thaliana 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 h daylength.

Protoplast isolation

Protoplast solution (600 mM Mannitol, 2 mM MgCl 2 , 2 mM CaCl 2 , 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 Arabidopsis thaliana 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.

Confocal microscopy Intact

Arabidopsis 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 (22 × 50 mm, 0.16–0.19 mm thick) before visualisation using a Zeiss LSM 880 instrument under ×40 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 PI for 10 min and propidium fluorescence was visualised with excitation at 561 nm and emission collected from 580–718 nm. Quantitative PCR A set of genes known to respond to abiotic and disease stresses 32 (Supplementary Fig. 10 ) were selected for reverse transcription quantitative PCR (RT-qPCR). Primers were designed (Supplementary Table 2 ) and reaction conditions optimised for all steps in the protocol. Arabidopsis thaliana Col-1 seeds were grown on agar plates (half-strength MS salts plus 0.5% sucrose). At seven days old, seedlings were treated with nanoparticles for 90 min, then flash-frozen in liquid nitrogen and stored at −80 °C. There were three experimental treatments—positively-, negatively- and neutrally charged nanoparticles—and a control group (water only). Three biological replicates were performed. 20 mg of tissue per sample was homogenised using the TissueLyser II Sample Disruptor (Qiagen). Three sterile metal beads were added to each sample and disrupted for 2 min at 25 Hz frequency, followed by incubation on dry ice for 5 mins, then another disruption at the same settings. Total RNA was isolated using the Monarch Total RNA Miniprep Kit (New England BioLabs; #T2010) with the manufacturer-recommended on-column DNase treatment. Elution was done in 100 µl nuclease-free water. Nucleic acid concentrations were quantified, and contamination assessed using the NanoDrop ND-1000 Uv-Vis Spectrophotometer (ThermoFisher Scientific). All samples had a 260/230 ratio ≥2 and a 260/280 ratio ≥2, and were stored at −80 °C. RNA integrity was assessed using the 2100 Bioanalyzer Instrument (Agilent); all samples had a RIN number >5. RNA was stored at −80 °C. 1 μg total RNA per sample was reverse transcribed using the ProtoScript II First Strand cDNA Synthesis Kit (New England BioLabs; E6560L) following manufacturer’s instructions in a 20 μL reaction volume with the included Random Primer Mix (60 μM). The resultant cDNA was diluted 10-fold with nuclease-free water and stored at −20 °C. qPCR reactions were performed in 96-well plates (ThermoFisher Scientific) with a Stratagene Mx3005P system (Agilent Technologies) using SYBR Green JumpStart Taq ReadyMix (Sigma Aldrich; S4438). Reactions were in 10 μl volumes containing 500 nM of each primer, 0.5× SYBR Green mix, and 1 μl of cDNA; each qPCR reaction was run in triplicate. Reactions were performed as follows: 94 °C initial denaturation for 2 min, preceded by 40 cycles of 94 °C for 15 s and 51 °C or 53 °C for 1 min, denaturation at 95 °C for 1 min, 55 °C for 30 s and a final denaturation step at 95 °C for 30 s. The constitutively expressed genes UBIQUITIN10 (UBQ10 , AT4G05320) or TAP42 INTERACTING PROTEIN OF 41 KDA (TIP41 , AT4G34270) were used as endogenous controls for normalisation. Experimental genes tested assessed were: FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1 , AT2G19190), PHOSPHITE-INSENSITIVE 1 (PHI1 , AT2G21870), NDR1/HIN1-LIKE (NHL10 , AT2G35980) and WRKY1 (AT2G04880). All primer specificities were assessed with an in silico specificity screen using Primer-Blast (NCBI). Amplicon sizes are between ~70 to 280 bp. Primers are in gene exons, except the UBQ10 primers which fall in the 3’UTR. All primer pairs had PCR efficiencies of between 90 and 110%. Data was outputted into MxPro qPCR Software (Agilent). The Cq threshold was set to 1000, and outliers omitted from analysis if deviation from mean Cq was >0.5. The Pfaffl Method of normalisation was used and statistical analysis performed in R Studio.

Materials Dimethyl acrylamide (DMAm, 99 %; Sigma Aldrich 274135), Acrylic Acid (AA, 99 %; Sigma Aldrich 147230), (Methacryloyloxy)ethyl] trimethylammonium chloride (QDMAEMA, 75 % in H 2 O; Sigma Aldrich 408107), (Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS, 95 %; Sigma Aldrich 537284), 4,4′-azobis(4-cyanovaleric acid) (ACVA, 99%; Sigma Aldrich 11590), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 98%; Sigma Aldrich 341006), deuterated methanol (CD 3 OD, 99.8%; Sigma Aldrich 151947-10G-GL) and deuterium oxide (D 2 O, 99.9%; Sigma Aldrich 151882) were purchased from Sigma Aldrich (UK). DAAm (99%) was purchased from Alfa Aesar (UK; A15940.30). 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acyl hydrazide (BODIPY FL, 99 %) was purchased from ThermoFisher Scientific (UK; D2371). 4-((((2-Carboxyethyl)thio)carbonothioyl)thio)−4-cyanopentanoic acid (BM1433, 95%) was purchased from Boron Molecular (USA; BM1433). For protoplast preparation, cellulase “Onozuka RS” was purchased from Melford biolaboratories (Duchefa; C8003.0005) and pectolyase from Sigma Aldrich (P3026).

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