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

Este trabalho foi apoiado pela Universidade da Califórnia, Riverside e USDA National Institute of Food e agricultura, projeto Hatch 1009710 para J.P.G. Este material é baseado em trabalho, apoiado pela Fundação Nacional de ciência sob Grant no. 1817363 de J.P.G.

1. cultivo de plantas da . thaliana
<ol><li>A. thaliana semear em potes descartáveis 5 cm x 5 cm, preenchidos com mistura de solo padrão. Colocar 32 destes vasos em uma bandeja de plástico cheia de água (~ 0,5 cm de profundidade) e transferir a bandeja plástica com as plantas em uma câmara de crescimento de planta.<ol>
<li>Definir o crescimento câmara configurações como segue: 200 µmol/ms fotossinteticamente ativa radiação (PAR), 24 ± 1 ° C de dia e 21 ± 1 ° C à noite, 60% de umidade ...

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Neste protocolo, descrevemos PNC síntese, caracterização, tintura fluorescente rotulagem e imagem latente confocal das nanopartículas dentro de células do mesofilo de vegetais para expor seu na vivo ROS eliminação atividade. PNC são sintetizados a partir de uma mistura de PAA solução de hidróxido de amónio e nitrato de cério. PNC caracterizam-se por spectrophotomery de absorção e a concentração de determinada usando a lei da cerveja-Lambert. Medições de potencial zeta confirmaram a superfície...

Nanopartículas de óxido de cério (nanoceria) são amplamente utilizadas em organismos vivos, de pesquisa básica de bioengenharia, devido a suas espécies distintas catalítico reativas de oxigênio (ROS) eliminação de capacidade1,2,3. Nanoceria ter ROS eliminação habilidades devido a um grande número de vagas de superfície oxigênio que alternam entre dois oxidação Estados (Ce3 + e4 +Ce) 4,5,6. Os Ce3 + oscilação títulos efetivamente eliminar ROS enquanto as cepas de treliça em nanoescala promovem a regeneração destes sites de defeito através de redox reações7de ciclismo. Nanoceria também têm sido usados recentemente para estudar e engenharia planta função8,9. Plantas sob estresse abiótico experimentam acúmulo de ROS, causando danos oxidativos de lipídios, proteínas e DNA10. Em plantas da . thaliana , nanoceria catalítico eliminação de ROS na vivo leva a fotossíntese da planta melhorada sob alta luz, calor e refrigeração salienta8. Aplicação nanoceria para o solo também aumentos atirar rendimento da biomassa e o grão de trigo (Triticum aestivum)11; as plantas de canola (Brassica napus) tratadas com nanoceria têm maior biomassa de plantas sob estresse sal12.
Nanoceria oferecer bioengineers e biólogos da planta uma ferramenta baseada em nanotecnologia para compreender as respostas de estresse abiótico e aumentar a tolerância de estresse abiótico de planta. Na vivo ROS eliminação capacidades do Nanoceria são independentes das espécies de plantas, e a entrega fácil nos tecidos vegetais tem o potencial para permitir ampla aplicação fora de organismos modelo. Ao contrário de outros métodos baseados em geneticamente, nanoceria não exigem gerando linhas de planta com a superexpressão de enzimas antioxidantes para ROS maior eliminação de capacidade13. Infiltração de lâmina de folha de nanoceria de plantas é uma abordagem prática para a pesquisa em laboratório.
O objetivo geral do presente protocolo é para descrever 1) a síntese e caracterização de poli carregados negativamente (acrílico) ácido nanoceria (PNC), 2) a entrega e rastreamento de PNC em toda as células da folha e 3) a vigilância de eliminação ROS PNC-habilitado em vivo. Neste protocolo, poli carregados negativamente (acrílico) ácido nanoceria (PNC) são sintetizados e caracterizada pelo seu espectro de absorção, diâmetro hidrodinâmico e potencial zeta. Nós descrevemos um método de infiltração de lâmina de folha simples para lançar PNC para fábrica de tecidos da folha. Para imagens em vivo de nanopartículas distribuição dentro das células do mesofilo, uma tintura fluorescente (DiI) foi usada para rotular PNC (DiI-PNC) e observar as nanopartículas através de microscopia de fluorescência confocal. Finalmente, vamos explicar como monitorar na vivo PNC ROS eliminação através de microscopia confocal.

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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>
		</tr>
		<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>
		</tr>
		<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>
			<td></td>
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		<tr height="21">
			<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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		<tr height="21">
			<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>
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			<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>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
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			<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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		</tr>
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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>
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			<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>
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			<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>
			<td></td>
		</tr>
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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>
			<td></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

Acumulação de (ROS) de espécies reactivas de oxigénio é uma marca registrada da resposta ao estresse abiótico planta. ROS desempenham um papel duplo em plantas actuando como sinalização moléculas em níveis baixos e danificando moléculas em níveis elevados. Acumulação de ROS em plantas estressadas pode danificar metabolitos, enzimas, lipídeos e DNA, causando uma redução do crescimento das plantas e o rendimento. A capacidade de nanopartículas de óxido de cério (nanoceria) para eliminar cataliticamente ROS na vivo fornece uma única ferramenta para compreender e tolerância de estresse abiótico bioengineer planta. Aqui, apresentamos um protocolo para sintetizar e caracterizar nanoceria revestido poli (acrílico), ácido (PNC), interface as nanopartículas com plantas através de infiltração de lâmina de folha e monitorar sua distribuição e eliminação na vivo usando confocal de ROS microscopia. Atuais ferramentas moleculares para manipular a acumulação de ROS em plantas são limitadas a espécie de modelo e exigem métodos de transformação laborioso. Este protocolo para na vivo ROS eliminação tem potencial para ser aplicado ao tipo selvagem plantas com folhas largas e estrutura da folha como Arabidopsis thaliana.

Caracterização e síntese PNC . PNC foram sintetizados, purificada e caracterizada a seguir o método descrito na seção de protocolo 2. Figura 1 A mostra a coloração das soluções de nitrato de cério, PAA, a mistura de nitrato de cério e PAA e PNC. Uma mudança de cor do branco para amarelo claro é vista depois de PNC é sintetizado. Após a purificação com um filtro de 10 kDa, PNC for...

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

Limpeza catalítica de planta reativas de oxigênio espécies na Vivo por nanopartículas de óxido de cério aniônicos

Aqui, apresentamos um protocolo para a síntese e caracterização de nanopartículas de óxido de cério (nanoceria) de ROS (espécies reativas de oxigênio) eliminação na vivo, nanoceria de imagens em tecidos vegetais por microscopia confocal e na vivo monitoramento de nanoceria ROS eliminação pela microscopia confocal.

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.

Research

JoVE Journal

Bioengineering

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

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