The ability of cerium oxide nanoparticles to catalytically scavenge reactive oxygen species in vivo provides a unique tool to understand and engineer mechanisms of plant signaling and tolerance to stress. Reactive oxygen species play a key role in plants by acting as signal molecules at low levels and damaging molecules at high levels. The main advantage of this technique is the potential to be applied to diverse wild type plant species with broad leaves and leaf structure, like Arabidopsis thaliana.

After growing the Arabidopsis thaliana plants, weigh out 1.08 grams of Cerium nitrate and dissolve it in 2.5 milliliters of biology-grade water in a 50-milliliter conical tube. Weigh out 4.5 grams of Poly acid and dissolve it in five milliliters of biology-grade water in a different 50-milliliter conical tube. Combine these two solutions and then use a digital vortex mixer to mix them thoroughly at 2, 000 rpm for 15 minutes.

In a fume hood, transfer 15 milliliters of a 7.2-molar ammonium hydroxide solution to a 50-milliliter glass beaker. Begin stirring at 500 rpm and at room temperature. Add the Cerium nitrate and Poly acid mixture drop wise.

Cover the beaker with a piece of paper to prevent substantial loss of the solution and let it stir for 24 hours. After this, transfer the resulting solution to a 50-milliliter conical tube. Centrifuge at 3, 900 times g for one hour to remove any debris or large agglomerates.

Then, transfer the supernatant into three 50-milliliter, 10-kilodalton filters. Fill the remainder of each filter with molecular-grade water, resulting in a total dilution of 45 milliliters. Centrifuge at 3, 900 times g for 15 minutes.

Repeat this process at least six times. Using a UV-vis spectrophotometer, measure the absorbance of each eluent from 220 nanometers to 700 nanometers to ensure that no free polymers or other reagents are present in the final PNC solution. Use a five-milliliter syringe to take up the collected PNC solution.

Next, filter the solution against a 20-nanometer pore-sized syringe filter and collect the filtered solution in a 50-milliliter conical tube. Transfer a diluted final PNC solution into a plastic cuvette and measure its absorbance from 220 nanometers to 700 nanometers. Using a particle sizer, measure the hydrodynamic diameter of the synthesized PNC, then store the final PNC solution in a refrigerator at four degrees Celsius until ready to use.

After labeling the PNC with DiI fluorescent dye, transfer 0.9 milliliters of a solution containing 0.5 millimolar of either the PNC solution or the DiI-PNC solution to a tube. Add 0.1 milliliter of infiltration buffer and mix by vortexing. Prepare a 10-millimolar TES infiltration buffer as a negative control.

Next, transfer 0.2 milliliters of the sample and buffer mixture to a one-milliliter sterile needleless syringe. Tap to remove any possible air bubbles. Just before infiltration, retrieve the plant from the growth chamber.

Using a chlorophyll meter, measure the chlorophyll content in the A.thaliana leaves. Measure each leaf with three replicates as outlined in the text protocol. Choose leaves with similar chlorophyll content.

Gently press the tip of the needleless syringe against the bottom of the leaf lamina and depress the plunger to infiltrate the leaves. Using a delicate task wiper, gently wipe off any excess solution that remains on the surface of the leaf lamina, then label the plant. Keep the infiltrated plants on the bench to incubate for three hours.

First, place a pea-sized amount of observation gel onto a glass slide, roll the gel and spread it out until it is approximately two centimeters in diameter and one millimeter thick. Using a cork borer with a diameter of 0.3 centimeters, cut out a circular section in the center of the gel to create a well. Fill the well completely with PFD, which will provide a deeper and better confocal imaging resolution.

Then, use a cork borer with a diameter of 0.2 centimeters to collect leaf disks from the previously-infiltrated A.thaliana plants. Mount each leaf disk in a PFD-filled well with the infiltrated side facing up. After this, place a square cover slip on top of the leaf disk.

Press gently and evenly on the cover slip to seal with the well of observation gel and ensure that no air bubbles remain trapped. To begin, prepare 25 micromolar of H2DCFDA dye and 10 micromolar of DHE dye in TES infiltration buffer in separate 1.5-milliliter microcentrifuge tubes. Using a cork borer with a diameter of 0.2 centimeters, collect leaf disks from the adapted PNC-infiltrated A.thaliana plants.

Use the sharp tip of forceps to make three to four holes on each leaf disk to accelerate the dye-loading process. Next, load an equal amount of leaf disks into each dye tube. Incubate in darkness for 30 minutes.

After this, rinse the leaf disks with double-distilled water three times. Use observation gel to mount each leaf to a glass slide as previously described. Load the slide on a confocal microscope and manually focus to a region of leaf mesophyl cells as outlined in the text protocol, then expose the leaf disks to a UVA laser at 405 nanometers for three minutes to generate ROS.

Record the ROS signal intensity change in time series per leaf disk. Image the leaf disk using a 40X water objective and the confocal microscope settings shown here. Use a plant infiltrated with only infiltration buffer solution as the negative control.

After purification, the synthesized PNC is characterized using a UV-vis spectrophotometer and is seen to have a peak of absorbance at 271 nanometers. The purification process is confirmed by measuring the final eluent. PNC samples are then labeled with a fluorescent DiI dye to determine the distribution of the nanoparticles in vivo.

Three peaks are observed for the DiI-labeled PNC, while a measurement of the final eluent confirms that the non-reacted chemicals are washed out during purification. The distribution of DiI-PNC in leaf mesophyl cells is determined via confocal imaging. While no DiI dye signals are detected in the control samples the DiI-PNC is observed co-localized with chloroplasts in the infiltrated leaves.

In vivo ROS scavenging enabled by PNC is monitored in the leaf disk by measuring the fluorescence intensity changes in the DHE and DCF dyes. After three minutes of UV stress, the PNC-infiltrated leaves show significantly less intensity for both dye types when compared to the no-nanoparticle buffer control. A CAT mimetic activity assay reveals a decrease in the resorufin level in the mixture containing PNC.

This level is indicative of the hydrogen peroxide level, which therefore is also decreasing, and confirms the CAT mimetic activity of the synthesized PNC. This protocol for synthesizing nanoceria is a simple, stepwise procedure that generates cerium oxide nanoparticles with controlled size, charge, and ROS scavenging abilities. Plant genetic modification methods for ROS manipulation in vivo are currently limited to plant model species.

In contrast, nanoceria ROS scavenging has the potential to be applied to diverse wild type plant species both in the laboratory and in the field.