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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • 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 prom....

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.<.......

Representative 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.......

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.......

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.

....

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

References

  1. Xu, C., Qu, X. Cerium oxide nanoparticle: A remarkably versatile rare earth nanomaterial for biological applications. NPG Asia Materials. 6 (3), 90-116 (2014).
  2. Nelson, B., Johnson, M., Walker, M., Riley, K., Sims, C.

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