Katalytisk Scavenging plante reaktive ilt arter In Vivo af anioniske Cerium oxid nanopartikler
Summary
Vi præsenterer her, en protokol til syntese og karakterisering af cerium oxid nanopartikler (nanoceria) for ROS (reaktiv ilt arter) Gaderenovation i vivo, nanoceria imaging i plantevæv ved Konfokal mikroskopi og i vivo overvågning af nanoceria ROS scavenging af Konfokal mikroskopi.
Abstract
Reaktive ilt arter (ROS) akkumulering er kendetegnende for anlægget abiotisk stressrespons. ROS spiller en dobbelt rolle i planter ved at fungere som signalering molekyler på lavt niveau og beskadige molekyler på højt niveau. Ophobning af ROS i stressede planter kan beskadige metabolitter, enzymer, lipider og DNA, forårsager en reduktion af planternes vækst og udbytte. Evnen af cerium oxid nanopartikler (nanoceria) til katalytisk skyllepumpetab ROS i vivo giver et unikt værktøj til at forstå og bioengineer plante abiotisk stresstolerance. Vi præsenterer her, en protokol for at syntetisere og karakterisere poly (akryl) syre coated nanoceria (PNC), interface nanopartikler med planter via blad lamina infiltration og overvåger deres distribution og ROS Gaderenovation i vivo bruger Konfokal mikroskopi. Nuværende molekylære værktøjer til at manipulere ROS ophobning i planter er begrænset til model arter og kræve omstændelig transformation metoder. Denne protokol for i vivo ROS scavenging har potentiale til at blive anvendt til vildtype planter med brede blade og blade struktur som Arabidopsis thaliana.
Introduction
Cerium oxid nanopartikler (nanoceria) er udbredt i levende organismer, fra grundforskning til bioteknologi, på grund af deres forskellige katalytisk reaktive ilt arter (ROS) skylleluften evne1,2,3. Nanoceria har ROS scavenging evner på grund af et stort antal overflade ilt ledige stillinger, der veksler mellem to oxidation stater (CE-3 + og CE-4 +) 4,5,6. CE-3 + dinglende obligationer skyllepumpetab effektivt ROS mens gitter stammer på nanoplan fremme regenerering af disse defekt websteder via redox cykling reaktioner7. Nanoceria har også været brugt for nylig for at studere og engineering plant funktion8,9. Planter under abiotisk stress opleve ophobning af ROS, forårsager oxidative skader på lipider, proteiner og DNA10. I A. thaliana planter fører nanoceria katalytisk scavenging ROS i vivo til forbedret plante fotosyntese under høj lys, varme og nedkøling understreger8. Anvende nanoceria til jord også øger skyde biomasse, og korn udbytte af hvede (Triticum aestivum)11; raps (Brassica napus) planter behandlet med nanoceria har højere plantebiomasse under salt stress12.
Nanoceria tilbyder bioengineers og plante biologer en nanoteknologi-baseret værktøj til at forstå abiotisk stress svar og forbedre plante abiotisk stresstolerance. Nanocerias i vivo ROS skylleluften kapaciteter er uafhængige af plantearter, og den facile levering i plantevæv har potentiale til at aktiverer bred anvendelse uden for modelorganismer. Nanoceria kræver i modsætning til andre genetisk baserede metoder, ikke generere plante linjer med overekspression af antioxidant enzymer til højere ROS skylleluften evne13. Leaf lamina infiltration af nanoceria planter er en praktisk tilgang til lab-baseret forskning.
Det overordnede mål med denne protokol er at beskrive 1) syntese og karakterisering af negativt ladede poly (akryl) syre nanoceria (PNC), 2) for levering og sporing af PNC hele blad celler, og 3) overvågningen af PNC-aktiveret ROS skylleluften i vivo. I denne protokol, er negativt ladede poly (akryl) syre nanoceria (PNC) syntetiseret og karakteriseret ved deres absorptionsspektrum, hydrodynamiske diameter og zeta potentiale. Vi beskriver et simpelt blad lamina infiltration metode til at levere PNC til plante blad væv. For i vivo billeddannelse af nanopartikel fordeling inden for mesofyllet celler, blev et fluorescerende farvestof (DiI) brugt til at mærke PNC (DiI-PNC) og observere nanopartikler via Konfokal Fluorescens mikroskopi. Endelig, vi forklare hvordan man kan overvåge i vivo PNC ROS scavenging gennem Konfokal mikroskopi.
Protocol
Representative Results
Discussion
I denne protokol beskriver vi PNC syntese, karakterisering, fluorescerende farvestof mærkning og konfokal billeddannelse af nanopartikler i mesofyllet planteceller til at udstille deres i vivo ROS skylleluften aktivitet. PNC er syntetiseret fra en blanding af cerium nitrat og PAA løsning i ammoniumhydroxid. PNC er karakteriseret ved absorption spectrophotomery og koncentrationen bestemmes ved hjælp af øl-Lamberts lov. Zeta potentielle målinger bekræftet PNC negativt ladede overflade for at øge levering ti…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af University of California, Riverside og USDA National Institute for fødevarer og landbrug, Hatch projektet 1009710 til J.P.G. Dette materiale er baseret på arbejde støttet af National Science Foundation under Grant nr. 1817363 til J.P.G.
Materials
| 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 |
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