Name: catalytic scavenging of plant reactive oxygen species in vivo by anionic cerium oxide nanoparticles
Uploaded: 2018-08-26T15:00:00
Description: Watch this Scientific Journal Video about Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles at JoVE.com

Detta arbete fick stöd av University of California, Riverside och USDA nationella institutet för livsmedel och jordbruk, Hatch projektet 1009710 till J.P.G. Detta material bygger på arbete stöds av National Science Foundation under Grant nr 1817363 till J.P.G.

1. A. thaliana plantor
<ol><li>Så A. thaliana fröer i 5 x 5 cm disponibla krukor fyllda med standard jord mix. Sätta 32 av dessa krukor i en plastbricka fylld med vatten (~ 0,5 cm djup) och överför plastbehållaren med växter till en växt tillväxt kammare.<ol>
<li>Ange tillväxten kammare inställningar enligt följande: 200 µmol/ms fotosyntetiska aktiv strålning (PAR), 24 ± 1 ° C dag och 21 ± 1 ° C natt, 60% luftfuktighet och 14/10 h dag/natt lättreglering, respektive.</li>
	</ol>
</li...

<ol>
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I detta protokoll beskriver vi PNC syntes, karakterisering, fluorescerande färgämne märkning och confocal avbildning av nanopartiklarna inom mesophyll växtceller att ställa ut sin i vivo ROS rensning verksamhet. PNC syntetiseras från en blandning av cerium nitrat och PAA lösning i ammoniumhydroxid. PNC kännetecknas av absorption spectrophotomery och den koncentration som fastställs med hjälp av öl-Lamberts lag. Zeta potential mätningar bekräftade negativt laddade ytan av PNC för att förbättra lev...

Cerium oxide nanopartiklar (nanoceria) används allmänt i levande organismer, från grundforskning till bioteknik, på grund av deras distinkta katalytisk reaktiva syreradikaler (ROS) rensning förmåga1,2,3. Nanoceria har ROS rensning förmåga på grund av ett stort antal ytan syre vakanser som växlar mellan två oxidation (CE-3 + och Ce4 +) 4,5,6. CE-3 + dinglande obligationerna rensar effektivt ROS medan de galler stammarna på nanonivå främja regenerering av dessa defekt platser via redox cykling reaktioner7. Nanoceria har också nyligen använts för att studera och engineering plantera funktion8,9. Växter under abiotisk stress uppleva ansamling av ROS, orsakar oxidativa skador på lipider, proteiner och DNA10. I A. thaliana växter leder nanoceria katalytisk rensning av ROS i vivo till förbättrad växt fotosyntesen under hög ljus, värme och kylning betonar8. Tillämpa nanoceria att jord också ökar skjuta biomassa och korn avkastning av vete (Triticum aestivum)11. raps (Brassica napus) växter behandlas med nanoceria har högre växtbiomassa under salt stress12.
Nanoceria erbjuder bioengineers och plantera biologer ett nanoteknikbaserade verktyg för att förstå abiotisk stress svaren och förbättra anläggningen abiotisk stresstålighet. Nanocerias i vivo ROS rensning funktioner är oberoende av växtarter och lättköpt leverans till växt vävnader har potential att möjliggöra bred tillämpning utanför modellorganismer. Till skillnad från andra genetiskt-baserade metoder, nanoceria inte kräver radgenerering växt med överuttryck av antioxidant enzymer för högre ROS rensning förmåga13. Leaf lamina infiltration av nanoceria växter är ett praktiskt tillvägagångssätt för lab-baserad forskning.
Det övergripande målet med detta protokoll är att beskriva 1) syntesen och karakterisering av negativt laddade poly (akryl) sura nanoceria (PNC), 2) leverans och spårning av PNC hela blad celler, och 3) övervakningen av PNC-aktiverade ROS rensning i vivo. I detta protokoll är negativt laddade poly (akryl) sura nanoceria (PNC) syntetiseras och kännetecknas av deras Absorberingsspectrum, hydrodynamisk diameter och zeta potential. Vi beskriver en enkel leaf lamina infiltration metod för att leverera PNC in växten leaf vävnader. För in-vivo imaging av nanopartiklar distribution inom mesophyll celler användes ett fluorescerande färgämne (DiI) att märka PNC (DiI-PNC) och iaktta nanopartiklarna via confocal fluorescensmikroskopi. Slutligen förklarar vi hur du övervakar i vivo PNC ROS rensning genom konfokalmikroskopi.

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			<td height="21" style="height:21px;width:567px;">Cerium (III) nitrate hexahydrate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">238538-100G</td>
			<td style="width:265px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Molecular Biology Grade Water, Corning</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">45001-044&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Falcon 50 mL Conical Centrifuge Tubes</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">14-959-49A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Poly (acrylic acid) 1,800 Mw</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">323667-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Fisherbrand Digital Vortex Mixer</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">02-215-370</td>
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			<td height="21" style="height:21px;width:567px;">Fisherbrand Digital Vortex Mixer Accessory, Insert Retainer</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">02-215-391</td>
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			<td height="21" style="height:21px;width:567px;">Fisherbrand Digital Vortex Mixer Accessories: Foam Insert Set</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">02-215-395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Ammonium hydroxide solution</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">05002-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">PYREX Griffin Beakers, Graduated, Corning</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">13912-149&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">RCT basic</td>
			<td style="width:228px;">IKA</td>
			<td style="width:165px;">3810001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Eppendorf Microcentrifuge 5424</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">80094-126</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Amicon Ultra-15 Centrifugal Filter Units</td>
			<td style="width:228px;">Millipore-Sigma</td>
			<td style="width:165px;">UFC901024</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Allegra X-30 Series Benchtop Centrifuge</td>
			<td style="width:228px;">Beckman Coulter</td>
			<td style="width:165px;">B06314</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">UV-2600 Sptecrophotometer</td>
			<td style="width:228px;">Shimadzu</td>
			<td style="width:165px;">UV-2600 120V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Whatman Anotop 10 syringe filter</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">WHA68091102</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">BD Disposable Syringes with Luer-Lok Tips</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">14-829-45</td>
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			<td height="21" style="height:21px;width:567px;">Zetasizer Nano S</td>
			<td style="width:228px;">Malvern Panalytical</td>
			<td style="width:165px;">Zen 1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">42364-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Dimethyl Sulfoxide, ACS</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">BDH1115-1LP</td>
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			<td height="21" style="height:21px;width:567px;">Sunshine Mix #1 LC1</td>
			<td style="width:228px;">Green Island Distributors, Inc</td>
			<td style="width:165px;">5212601.CFL080P</td>
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			<td height="21" style="height:21px;width:567px;">Adaptis 1000</td>
			<td style="width:228px;">Conviron</td>
			<td style="width:165px;">A1000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">TES, &#62;99% (titration</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">T1375-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Magnesium chloride</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">M8266-1KG</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">Air-Tite All-Plastic Norm-Ject Syringe</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">14-817-25</td>
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			<td height="21" style="height:21px;width:567px;">Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">06-666A</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Carolina Observation Gel</td>
			<td style="width:228px;">Carolina</td>
			<td style="width:165px;">132700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Corning microscope slides, frosted one side, one end</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">CLS294875X25-72EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Cork Borer Sets with Handles</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">S50166A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Perfluorodecalin</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">P9900-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Micro Cover Glasses, Square, No. 1</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Leica Laser Scanning Confocal Microscope TCS SP5</td>
			<td style="width:228px;">Leica Microsystems</td>
			<td style="width:165px;">TCS SP5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">2&#8242;,7&#8242;-Dichlorofluorescin diacetate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">D6883-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Dihydroethidium</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">D7008-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Eppendorf Uvette cuvettes</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">Z605050-80EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Chlorophyll meter&#160;</td>
			<td style="width:228px;">Konica Minolta</td>
			<td style="width:165px;">SPAD-502</td>
			<td></td>
		</tr>
	</tbody>
</table>

catalytic scavenging of plant reactive oxygen species in vivo by anionic cerium oxide nanoparticles

Reaktivt syre arter (ROS) ackumulering är ett kännetecken för växt abiotisk stress svar. ROS spelar en dubbel roll i växter genom att agera som signalmolekyler på låga nivåer och skadliga molekyler på höga nivåer. Ansamling av ROS i stressade växter kan skada metaboliter, enzymer, lipider och DNA, orsakar en minskning av växters tillväxt och avkastning. Cerium oxide nanopartiklar (nanoceria) förmåga att katalytiskt rensa ROS i vivo ger en unikt verktyg att förstå och bioengineer växt abiotisk stresstålighet. Här presenterar vi ett protokoll för att syntetisera och karakterisera poly (akryl) syra belagda nanoceria (PNC), gränssnitt nanopartiklarna med växter via leaf lamina infiltration och övervaka sin distribution och ROS rensning i vivo med confocal mikroskopi. Aktuella molekylära verktyg för att manipulera ROS ackumulering i växter är begränsad till modell arter och kräver mödosamma transformation metoder. Detta protokoll för i vivo ROS rensning har potential att användas för vildtyp växter med breda blad och löv struktur som Arabidopsis thaliana.

PNC syntes och karakterisering . PNC var syntetiseras, renas och kännetecknas följande metoden beskrivs i protokollet avsnitt 2. Figur 1 A visar färgning av lösningar av cerium nitrat, PAA, blandningen av cerium nitrat och PAA och PNC. En färgförändring från vit till ljusgul ses efter PNC syntetiseras. Efter rening med 10 kDa filter karakteriserades PNC med en UV-VIS-spektrofotometer. En t...

Watch this Scientific Journal Video about Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles at JoVE.com

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

Katalytisk rensning av växten reaktivt syre arter In Vivo av anjon Cerium Oxide nanopartiklar

Här presenterar vi ett protokoll för syntes och karakterisering av cerium oxide nanopartiklar (nanoceria) för ROS (reaktiva syreradikaler) rensning i vivo, nanoceria imaging i växt vävnader av konfokalmikroskopi, och i vivo övervakning av nanoceria ROS rensning av konfokalmikroskopi.

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

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.

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Bioengineering

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

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