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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Abstract

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Introduction

Cerium oxide nanoparticles (nanoceria) are widely used in living organisms, from basic research to bioengineering, due to their distinct catalytic reactive oxygen species (ROS) scavenging ability1,2,3. Nanoceria have ROS scavenging abilities due to a large number of surface oxygen vacancies that alternate between two oxidation states (Ce3+ and Ce4+) 4,5,6. The Ce3+ dangling bonds effectively scavenge ROS while the lattice strains at the nanoscale promote the regeneration of these defect sites via redox cycling reactions7. Nanoceria have also been recently used for studying and engineering plant function8,9. Plants under abiotic stress experience accumulation of ROS, causing oxidative damage to lipids, proteins, and DNA10. In A. thaliana plants, nanoceria catalytic scavenging of ROS in vivo leads to improved plant photosynthesis under high light, heat, and chilling stresses8. Applying nanoceria to soil also increases shoot biomass and grain yield of wheat (Triticum aestivum)11; canola (Brassica napus) plants treated with nanoceria have higher plant biomass under salt stress12.

Nanoceria offer bioengineers and plant biologists a nanotechnology-based tool to understand abiotic stress responses and enhance plant abiotic stress tolerance. Nanoceria's in vivo ROS scavenging capabilities are independent of plant species, and the facile delivery into plant tissues has the potential to enable broad application outside of model organisms. Unlike other genetically-based methods, nanoceria do not require generating plant lines with the overexpression of antioxidant enzymes for higher ROS scavenging ability13. Leaf lamina infiltration of nanoceria to plants is a practical approach for lab-based research.

The overall goal of this protocol is to describe 1) the synthesis and characterization of negatively charged poly (acrylic) acid nanoceria (PNC), 2) the delivery and tracking of PNC throughout leaf cells, and 3) the monitoring of PNC-enabled ROS scavenging in vivo. In this protocol, negatively charged poly (acrylic) acid nanoceria (PNC) are synthesized and characterized by their absorption spectrum, hydrodynamic diameter, and zeta potential. We describe a simple leaf lamina infiltration method to deliver PNC into plant leaf tissues. For in vivo imaging of nanoparticle distribution within mesophyll cells, a fluorescent dye (DiI) was used to label PNC (DiI-PNC) and observe the nanoparticles via confocal fluorescence microscopy. Finally, we explain how to monitor in vivo PNC ROS scavenging through confocal microscopy.

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Protocol

1. Growing A. thaliana Plants

  1. Sow A. thaliana seeds in 5 cm x 5 cm disposable pots filled with standard soil mix. Put 32 of these pots into a plastic tray filled with water (~ 0.5 cm depth) and transfer the plastic tray with the plants into a plant growth chamber.
    1. Set the growth chamber settings as follows: 200 µmol/ms photosynthetic active radiation (PAR), 24 ± 1 °C day and 21 ± 1 °C night, 60% humidity, and 14/10 h day/night light regime, respectively.
  2. Thin each pot to leave only one individual plant after one week of germination. Take note to keep the seedlings with similar size in each pot.
  3. Water the pots by pouring tap water directly on the plastic tray once every two days. Grow the plants for four weeks. A. thaliana plants are ready for further use.

2. Synthesis and Characterization of PNC

  1. Weigh 1.08 g of cerium (III) nitrate and dissolve it in 2.5 mL of molecular biology grade water in a 50 mL conical tube.
  2. Weigh 4.5 g of poly (acrylic) acid and dissolve it in 5 mL of molecular biology grade water in a 50 mL conical tube.
  3. Mix these two solutions thoroughly at 2,000 rpm for 15 min using a digital vortex mixer.
  4. Transfer 15 mL of ammonium hydroxide solution (7.2 M) to a 50 mL glass beaker.
  5. While stirring at 500 rpm, add the mixture from Step 2.3 dropwise to the ammonium hydroxide solution and stir at 500 rpm at room temperature for 24 hr in a fume hood.
  6. Cover the beaker with a piece of paper to avoid the substantial loss of solution during the overnight reaction.
  7. After 24 h, transfer the resulting solution to a 50 mL conical tube and centrifuge it at 3,900 x g for 1 h to remove any possible debris and large agglomerates.
  8. Transfer this 22.5 mL of supernatant solution into three 15 mL 10 kDa filters and fill the remainder of the filter with molecular grade water to make a total dilution of 45 mL.
  9. Purify the supernatant solution from free polymers and other reagents with a benchtop centrifuge by adding the supernatant to a 15 mL 10 kDa filter and centrifuge at 3,900 x g for 15 min. Repeat this step at least six times.
  10. Measure the absorbance of the eluent in each cycle with a UV-VIS spectrophotometer from 220-700 nm to ensure no free polymers and other reagents are present in the final PNC solution.
  11. Take the collected PNC solution into the 5 mL syringe and filter it against a 20 nm pore size syringe filter. Collect the filtered PNC solution in a 50 mL conical tube.
  12. Take a diluted final PNC solution in a plastic cuvette and measure its absorbance with the UV-VIS spectrophotometer from 220-700 nm. PNC absorbance peak is at 271 nm.
  13. Calculate its concentration by using Beer-Lambert's law: A = εCL. A is the absorbance of the peak value for a given sample, ɛ is the molar absorption coefficient of PNC (cm-1 M-1), L is the optical path length (cuvette width, 1 cm in this method), and C is the molar concentration of measured nanoparticles.
  14. Measure the hydrodynamic diameter and zeta potential of the synthesized PNC using a particle size and zeta potential analyzer (Figure 1).
  15. Store the final PNC solution in a refrigerator (4 °C) until further use.
    NOTE: Please refer to Wu et al.8 for more protocol details about PNC characterization.

3. Labeling PNC with DiI Fluorescent Dye

  1. Mix 0.4 mL of 5 mM (58 mg/L) PNC with 3.6 mL of molecular biology grade water in a 20 mL glass vial and stir at 500 rpm.
  2. Add 24 µL 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate dye solution (DiI, 2.5 mg/mL; dilute in DMSO) into 176 µL of DMSO (dimethyl sulfoxide) to make the DiI dye solution.
  3. Add the DiI dye dropwise to the PNC solution, stirring at 1,000 rpm for 1 min at ambient temperature.
  4. Transfer this resulting mixture into a 15 mL 10 kDa filter and fill the tube to the top with molecular biology grade water to make the total dilution 15 mL.
  5. Purify the DiI labeled PNC (DiI-PNC) solution from DMSO and any possible free DiI dye by a benchtop centrifugation with the 15 mL 10 kDa filter at 3,900 x g for 5 min.
    1. Repeat Step 3.5 at least five times.
  6. Filter the final DiI-PNC solutions through a 20 nm pore size syringe filter.
  7. Measure the absorbance of final DiI-PNC by UV-VIS spectrophotometry and calculate its concentration according to Beer-Lambert's law (Figure 2). See Step 2.13 for more details.
  8. Store it in a refrigerator at 4 °C for further use.

4. Infiltration of Plant Leaves with PNC

  1. Add 0.1 mL of infiltration buffer (100 mM TES, 100 mM MgCl2, pH 7.5, adjusted by HCl) into 0.9 mL of 0.5 mM PNC or DiI-PNC solution and vortex it. Use a solution of 10 mM TES infiltration buffer as a negative control.
  2. Transfer 0.2 mL of the PNC or DiI-PNC infiltration solution to a 1 mL sterile needleless syringe. Tap to remove any possible air bubbles.
  3. Retrieve the plant from the growth chamber just before infiltration with nanoparticles to avoid possible stomata closure under room light conditions.
  4. Before infiltration, measure the chlorophyll content from A. thaliana leaves with similar size using a chlorophyll meter. Measure each leaf with three replicates (each replicate consisting of at least three measurements)14. Choose the A. thaliana leaves with similar chlorophyll content for the infiltration experiment.
  5. Infiltrate the leaves slowly with the recently prepared PNC or DiI-PNC solution by gently pressing the tip of the needleless syringe against the bottom of the leaf lamina (abaxial side) and depress the plunger (Figure 3A).
  6. Gently wipe off the excess solution that remains on the surface of leaf lamina (Figure 3B) using a delicate task wiper (Figure 3C) and label the plant. Use new delicate task wipes for each group of leaves.
  7. Keep the infiltrated A. thaliana plants on the bench for leaf adaptation and incubation with PNC or DiI-PNC for 3 h.
    NOTE: Infiltrated A. thaliana plants are then ready for further use (Figure 3D).

5. Preparation of Leaf Samples for Confocal Microscopy

  1. Roll a pea-size amount of observation gel to about a 1 cm radius (Figure 4A) and then spread it out until it is 1 mm thin on a glass slide (Figure 4B).
  2. Use a cork borer (diameter 0.3 cm) to cut out a circular section at the center of the observation gel on the glass slide (Figure 4C).
  3. Fill the cut well entirely with perfluorodecalin (PFD) for deeper and better confocal imaging resolution in leaf tissues.
  4. Use a cork borer (diameter 0.2 cm) to collect leaf discs from the adapted DiI-PNC infiltrated A. thaliana plants (Figure 4D).
  5. Mount the leaf disc in the PFD filled well; face the infiltrated (abaxial) side of the leaf up.
  6. Put a square coverslip on top of the leaf disc and gently press on the slide coverslip evenly to seal it with the well of observation gel and ensure no air bubbles remain trapped (Figure 4E).

6. Imaging DiI-PNC in Leaf Tissues by Confocal Microscopy

  1. Use a 40X objective lens in an inverted laser scanning confocal microscope.
  2. Drop two to three drops of ddH2O on the top of the 40X objective lens.
  3. Place the prepared DiI-PNC infiltrated leaf sample slide on top of the inverted 40X objective lens.
    1. Make sure the coverslip side but not the glass slide contact directly with the ddH2O on the lens.
  4. Find a region of interest in the sample under the microscope with either laser light or bright field.
  5. Start the microscope software and turn on the Argon laser (set at 20%).
  6. Set the pinhole to collect an optical slice less than 2 µm and a line average of 4.
  7. Image the sample with confocal microscope settings: 514 nm laser excitation (30 %); Z-Stack section thickness: 2 µm; PMT1: 550-615 nm (for DiI-PNC imaging); PMT2: 700-800 nm (for chloroplast imaging).
  8. Take representative confocal images of leaf samples from different individuals, a minimum of three biological replicates.

7. Imaging PNC in vivo ROS Scavenging by Confocal Microscopy

  1. Prepare 25 µM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, a dye for indicating a general ROS) and 10 µM dihydroethidium (DHE, a dye for indicating superoxide anion) dyes in TES infiltration buffer (pH 7.5) in 1.5 mL microcentrifuge tubes, separately.
  2. Use a cork borer (diameter 0.2 cm) to collect leaf discs from the adapted PNC infiltrated A. thaliana plants.
    1. Use the sharp tip of the forceps to make three to four holes on the leaf discs to accelerate dye loading process.
  3. Transfer the leaf discs to microcentrifuge tubes with H2DCFDA and DHE separately and incubate for 30 min under darkness.
  4. After incubation, rinse the leaf discs with ddH2O three times and mount it into the glass slide with observation gel (see Protocol Section 5).
  5. Put the slide on the confocal microscope and manually focus to a region of leaf mesophyll cells. See Protocol Section 6 for details.
  6. Expose the leaf discs to the UV-A (405 nm) laser for 3 min to generate ROS and record the ROS signal intensity change in time-series ("xyt") per leaf disc.
  7. Image the leaf disc with confocal microscope settings: 40X water objective; 496 nm laser excitation; PMT1: 500-600 nm (for DHE and DCFDA dye detection); PMT2: 700-800 nm (for chloroplasts detection). Use a plant infiltrated with only infiltration buffer solution as the negative control.

8. PNC Scavenging of H2O2 in vitro

  1. Conduct the CAT (catalase) mimetic activity of the synthesized PNC in vitro by following the methods in previous publications3,8,15
  2. Add 45.4 µL of 1x TES infiltration buffer (10 mM TES, 10 mM MgCl2, pH 7.5, adjusted by HCl), PNC (60 nM, 3 µL), and H2O2 (2 µM, 1 µL) into a well (white round bottom 96 well plate), and gently mix it by pipetting.
  3. Add 10-acetyl-3,7-dihydroxyphenoxazine (working concentration 100 µM, 0.5 µL) and horseradish peroxidase (HRP; working concentration 0.2 U/mL, 0.1 µL) into the well, gently mix it by pipetting, and incubate it for 30 min. 10-acetyl-3,7-dihydroxyphenoxazine reacts with H2O2 and is converted into resorufin in the presence of HRP.
    1. Wrap the plate with aluminum foil to avoid light during the incubation.
    2. Prepare a negative control by using reaction buffer or water to replace H2O2.
    3. Except for the stock solution, prepare all other solutions at ambient temperature.
  4. After the incubation, with a plate reader, monitor the absorbance at 560 nm to use resorufin for indicating the level of H2O2. Set time regime at 0, 2, 5, 10, 20, and 30 min.

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Results

PNC synthesis and characterization.
PNC were synthesized, purified and characterized following the method described in Protocol Section 2. Figure 1A shows the coloration of the solutions of cerium nitrate, PAA, the mixture of cerium nitrate and PAA, and PNC. A color change from white to light yellow is seen after PNC is synthesized. After purification with a 10 kDa filter, PNC were characterize...

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Discussion

In this protocol, we describe PNC synthesis, characterization, fluorescent dye labeling, and confocal imaging of the nanoparticles within plant mesophyll cells to exhibit their in vivo ROS scavenging activity. PNC are synthesized from a mixture of cerium nitrate and PAA solution in ammonium hydroxide. PNC are characterized by absorption spectrophotomery and the concentration determined using Beer-Lamberts law. Zeta potential measurements confirmed the negatively charged surface of PNC for enhancing delivery to c...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the University of California, Riverside and USDA National Institute of Food and Agriculture, Hatch project 1009710 to J.P.G. This material is based upon work supported by the National Science Foundation under Grant No. 1817363 to J.P.G.

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Materials

NameCompanyCatalog NumberComments
Cerium (III) nitrate hexahydrateSigma-Aldrich238538-100G
Molecular Biology Grade Water, CorningVWR45001-044 
Falcon 50 mL Conical Centrifuge TubesVWR14-959-49A
Poly (acrylic acid) 1,800 MwSigma-Aldrich323667-100G
Fisherbrand Digital Vortex MixerFisher Scientific02-215-370
Fisherbrand Digital Vortex Mixer Accessory, Insert RetainerFisher Scientific02-215-391
Fisherbrand Digital Vortex Mixer Accessories: Foam Insert SetFisher Scientific02-215-395
Ammonium hydroxide solutionSigma-Aldrich05002-1L
PYREX Griffin Beakers, Graduated, CorningVWR13912-149 
RCT basicIKA3810001
Eppendorf Microcentrifuge 5424VWR80094-126
Amicon Ultra-15 Centrifugal Filter UnitsMillipore-SigmaUFC901024
Allegra X-30 Series Benchtop CentrifugeBeckman CoulterB06314
UV-2600 SptecrophotometerShimadzuUV-2600 120V
Whatman Anotop 10 syringe filterSigma-AldrichWHA68091102
BD Disposable Syringes with Luer-Lok TipsFisher Scientific14-829-45
Zetasizer Nano SMalvern PanalyticalZen 1600
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorateSigma-Aldrich42364-100MG
Dimethyl Sulfoxide, ACSVWRBDH1115-1LP
Sunshine Mix #1 LC1Green Island Distributors, Inc5212601.CFL080P
Adaptis 1000ConvironA1000
TES, >99% (titrationSigma-AldrichT1375-100G
Magnesium chlorideSigma-AldrichM8266-1KG
Air-Tite All-Plastic Norm-Ject SyringeFisher Scientific14-817-25
Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task WipersFisher Scientific06-666A
Carolina Observation GelCarolina132700
Corning microscope slides, frosted one side, one endSigma-AldrichCLS294875X25-72EA
Cork Borer Sets with HandlesFisher ScientificS50166A
PerfluorodecalinSigma-AldrichP9900-25G
Micro Cover Glasses, Square, No. 1VWR48366-045
Leica Laser Scanning Confocal Microscope TCS SP5Leica MicrosystemsTCS SP5
2′,7′-Dichlorofluorescin diacetateSigma-AldrichD6883-250MG
DihydroethidiumSigma-AldrichD7008-10MG
Fisherbrand Premium Microcentrifuge Tubes: 1.5 mLFisher Scientific05-408-129
Eppendorf Uvette cuvettesSigma-AldrichZ605050-80EA
Chlorophyll meter Konica MinoltaSPAD-502

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