Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

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 ability^1,2,3. 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.<.......

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.......

Materials

Name	Company	Catalog Number	Comments
Cerium (III) nitrate hexahydrate	Sigma-Aldrich	238538-100G
Molecular Biology Grade Water, Corning	VWR	45001-044
Falcon 50 mL Conical Centrifuge Tubes	VWR	14-959-49A
Poly (acrylic acid) 1,800 Mw	Sigma-Aldrich	323667-100G
Fisherbrand Digital Vortex Mixer	Fisher Scientific	02-215-370
Fisherbrand Digital Vortex Mixer Accessory, Insert Retainer	Fisher Scientific	02-215-391
Fisherbrand Digital Vortex Mixer Accessories: Foam Insert Set	Fisher Scientific	02-215-395
Ammonium hydroxide solution	Sigma-Aldrich	05002-1L
PYREX Griffin Beakers, Graduated, Corning	VWR	13912-149
RCT basic	IKA	3810001
Eppendorf Microcentrifuge 5424	VWR	80094-126
Amicon Ultra-15 Centrifugal Filter Units	Millipore-Sigma	UFC901024
Allegra X-30 Series Benchtop Centrifuge	Beckman Coulter	B06314
UV-2600 Sptecrophotometer	Shimadzu	UV-2600 120V
Whatman Anotop 10 syringe filter	Sigma-Aldrich	WHA68091102
BD Disposable Syringes with Luer-Lok Tips	Fisher Scientific	14-829-45
Zetasizer Nano S	Malvern Panalytical	Zen 1600
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate	Sigma-Aldrich	42364-100MG
Dimethyl Sulfoxide, ACS	VWR	BDH1115-1LP
Sunshine Mix #1 LC1	Green Island Distributors, Inc	5212601.CFL080P
Adaptis 1000	Conviron	A1000
TES, >99% (titration	Sigma-Aldrich	T1375-100G
Magnesium chloride	Sigma-Aldrich	M8266-1KG
Air-Tite All-Plastic Norm-Ject Syringe	Fisher Scientific	14-817-25
Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers	Fisher Scientific	06-666A
Carolina Observation Gel	Carolina	132700
Corning microscope slides, frosted one side, one end	Sigma-Aldrich	CLS294875X25-72EA
Cork Borer Sets with Handles	Fisher Scientific	S50166A
Perfluorodecalin	Sigma-Aldrich	P9900-25G
Micro Cover Glasses, Square, No. 1	VWR	48366-045
Leica Laser Scanning Confocal Microscope TCS SP5	Leica Microsystems	TCS SP5
2′,7′-Dichlorofluorescin diacetate	Sigma-Aldrich	D6883-250MG
Dihydroethidium	Sigma-Aldrich	D7008-10MG
Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL	Fisher Scientific	05-408-129
Eppendorf Uvette cuvettes	Sigma-Aldrich	Z605050-80EA
Chlorophyll meter	Konica Minolta	SPAD-502