Diese Arbeit wurde von der Französisch nationalen Forschungsagentur (ANR-14-CE02-0010) unterstützt. Wir danken Dr. Benjamin Field und Dr. Elodie Lanet für das kritische Lesen des Manuskripts. Wir danken Herrn Michel Terese für seine Hilfe bei Videobearbeitung.

1. Herstellung von 20 bis 50% (w / v) Saccharosegradienten Anmerkung: Die Gradienten sind aus 4 Schichten von Saccharose (50%, 35% und 2 Schichten aus 20%) in 13,2 ml Ultrazentrifugenröhrchen. Unserer Erfahrung nach in zwei getrennten Schichten, die die 20% Saccharose Gießen verbessert die Qualität der Polysoms Zubereitungen. <ol><li> Bereiten Sie die Stammlösungen. Stellen Sie sicher, dass alle Lösungen RNAse und DNAse frei. <ol><li> Bereiten 10X Salzlösung: 400 mM Tris-HCl pH 8,4, 200 mM KCl und 100 mM MgCl 2. </li><li> Bereiten Sie 2 M Saccharose-Lösung: Für 200 ml, lösen sich 137 g Saccharose in 1X Salzlösung. </li></ol></li><li> Verdünnen Sie die Saccharose-Lösung in Salzlösung, wie in Tabelle 1 beschrieben, um die Steigungen vorzubereiten. Die Volumina beziehen sich auf sechs Steigungen. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Endgültige Saccharosekonzentration </td><td> Sucrose 2M (ml) </td><td> Salzlösung 1X (ml) </td><td> Finale Vol. (ml) </td></tr><tr><td> 50% </td><td> 8.8 </td><td> 3.2 </td><td> 12 </td></tr><tr><td> 35% </td><td> 12.9 </td><td> 12.1 </td><td> 25 </td></tr><tr><td> 20% </td><td> 7.4 </td><td> 17.6 </td><td> 25 </td></tr><tr><td> 20% </td><td> 5.8 </td><td> 14.2 </td><td> 20 </td></tr></tbody></table> Tabelle 1. Verdünnungen der Saccharose - Lösung sechs Steigungen vorzubereiten. <ol start="3"><li> Gießen Sie die Schichten gemäß Tabelle 2. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Sucrose Schicht </td><td> 50% </td><td> 35% </td><td> 20% </td><td> 20% </td></tr><tr><td> Vol. (Ml) </td><td> 1,85 </td><td> 3,65 </td><td> 3.65 </td><td> 1,35 </td></tr></tbody></table> Tabelle 2. Volumen von Saccharoselösung pro Schicht. <ol start="4"><li> Nach jeder Schicht Gießen, halten Sie die Röhrchen in einem -40 ° C oder -80 ° C Gefrierschrank bis zur vollständigen Einfrieren, bevor die nächste Schicht Saccharose Zugabe. Das Einfrieren ist in der Regel nach 2 Stunden bei -40 ° C erreicht, sondern warten etwa 6 Stunden, bevor die nächste Schicht wird empfohlen, zu gießen. Die letzten 20% Schicht kann frisch des Experiments am Tag eingefroren oder hinzugefügt werden. Hinweis: jede Schicht Einfrieren bevor Sie die nächste Zugabe von jeder Vermischung oder Störung der Schichten verhindert. Gradienten können in einem -40 ° C oder -80 ° C Gefrierschrank für mindestens sechs Monate aufbewahrt werden. </li><li> Wenn sie nicht bereits geschehen ist, fügen Sie den Gradienten am Tag des Experiments die letzten 20% Schicht. Dann lassen Sie die Steigungen in einem kalten Raum auftauen oder einen Kühlschrank. </li></ol> 2. Herstellung von Cytosolextrakten Hinweis: Wir empfmit zwei Steigungen pro biologischen Probe zu beenden. 300 mg ist die optimale Menge an Pflanzenmaterial zwei Steigungen vorzubereiten , wenn sie mit 6 Tage alten Arabidopsis thaliana Keimlingen arbeiten. Wenn es mit weniger translational aktiven Geweben arbeiten, kann die Menge an Pflanzenmaterial erhöht werden, bis zu 600 mg. <ol><li> Ernte 6 Tage alten Sämlinge wachsen auf ½ Murashige und Skoog 8 - Medium mit 1% Saccharose oder gleichwertigen Medien ergänzt durch das Tiefgefrieren in flüssigem Stickstoff </li><li> Grind Pflanzenmaterial in einem vorgekühlten Mörser und Stößel mit flüssigem Stickstoff. </li><li> Man wiegt 300 mg pulverisiertes Material in einem vorgekühlten Waagschale. Führen Sie diesen Schritt schnell Auftauen der Probe zu vermeiden. Hinweis: Halten Sie die gefrorenen Proben für nicht mehr als eine Woche in einem -80 ° C Gefrierschrank. </li><li> Hinzufügen 2,4 ml vorgekühltem Polysomen - Puffer (Salt Solution 4X, 5,26 mM EGTA, 0,5% (v / v) Octylphenoxy - poly (ethylenoxy) ethanol, verzweigt, 50 μg.ml -1 Cycloheximid, 50 μg.ml 1Chloramphenicol) und homogenisieren, indem sie mit der Pipettenspitze vermischt wird. Übertragen in zwei 1,5-ml-Röhrchen. Arbeiten Sie schnell die Proben, um zu verhindern Erwärmung. </li><li> Zentrifuge bei 16.000 xg für 15 min bei 4 ° C in einer Mikrozentrifuge zur Pelletierung Schutt. Hinweis: Um die RNA-Abbau zu verhindern, halten immer die Proben bei 4 ° C, verwenden RNase / DNase frei Lösungen und arbeiten in RNase / DNase freien Bedingungen. Heparin kann in die Polysomen - Puffer zugegeben werden, bis zu einer Endkonzentration von 300 μg.ml -1, RNA - Schutz zu verbessern. Da jedoch Heparin mit nachgeschalteter Analyse stören kann, wird es durch Ausführen einer Lithiumchloridfällung anstelle von Ethanolfällung (siehe Anmerkung 4.7) während der RNA-Fällungsschritt entfernt werden. </li></ol> 3. Polysomen Profilieren <ol><li> Sorgfältig pipettieren den Überstand, ohne das Pellet zu stören. Wenn Pflanzenfragmente pipettiert wurden, wiederholen Sie Schritt 2.5. Laden Sie den Überstand auf der Oberseite des Gradienten durch sanft pipetting auf die Seitenwand des Röhrchens in einem konstanten Strom. Verwenden Sie einen Gradienten für jede 1,5-ml-Röhrchen. </li><li> Transfer zum vorgekühlten Eimer und Zentrifuge bei 175.000 xg für 2 h 45 min bei 4 ° C, in einer Ultrazentrifuge (zB 32.000 Umdrehungen pro Minute bei einem SW41-Rotor verwendet wird). </li><li> Stellen Sie den Gradienten Sammelsystem auf , als in Figur 1 beschrieben ist . Die UV - Küvette hat einen 1 mm Weglänge. </li></ol><img alt="Abbildung 1" src="/files/ftp_upload/54231/54231fig1.jpg" /> Abbildung 1. Gradient Sammelsystem. Die UV - Küvette wird durch Polyvinylchlorid Schlauch auf eine Glaskapillare verbunden, die auf dem Boden des Gradienten abfällt. Die Steigung schreitet durch das System dank einer Schlauchpumpe. OD 260 wird kontinuierlich gelesen und 2 ml - Fraktionen werden gesammelt. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">eine größere Version zu sehen</a>diese Figur. <ol start="4"><li> Stellen Sie den Fraktionssammler auf RT und setzen Sie das Karussell mit einer Geschwindigkeit, die die Sammlung von 2 ml Fraktionen ermöglichen. Verwenden Sie vorgekühlten Sammelröhrchen. </li><li> Sammeln Sie die Fraktionen von unten eine Glaskapillare von Polyvinylchlorid Schlauch mit dem Gradienten Sammelsystem verbunden nach oben mit. Mit einem Kunststoffparaffinfilm die Verbindung zwischen dem Polyvinylchlorid-Rohr und der Glaskapillare zu sichern. </li><li> Die Extinktion bei 260 nm von der Unterseite zur Oberseite des Gradienten. Wenn der gesamte Gradienten gesammelt wird, legen Sie die Sammelröhrchen auf Eis vor der RNA-Extraktion. </li></ol> 4. RNA-Extraktion <ol><li> Pool der Fraktionen aus zwei Gradienten gesammelt, in 50 ml Zentrifugenröhrchen mit einer Kappe bedeckt, wie folgt: Polysomen: Fraktionen 1 bis 3 (12 ml) Monosomes: Fraktion 4 (4 ml) Überstand: Fraktionen 5 und 6 (8 ml) </li><li> Zu jeder Fraktion, fügen Sie 1 vol. 8M Guanidin-Hydrochlorid,50 &amp; mgr; g Acrylträger und 1,5 Vol. Isopropanol. Hinweis: Acryl Träger lineares Acrylamid, als Co-Fällmittel verwendet, um die Rückgewinnung von Nukleinsäuren während Alkoholfällung zu verbessern. </li><li> Mischen Sie durch die Rohre Umkehren und O / N bei -20 ° C auszufallen. </li><li> Zentrifuge bei 175.000 × g für 1 Stunde bei 4 ° C in einer Ultrazentrifuge (32.000 rpm in einem SW32 Rotor). Wenn das Fällungsschritt in einem Rohr durchgeführt wird, die nicht kompatibel mit Ultrazentrifugation ist, zu einem geeigneten Rohr. </li><li> Überstand verwerfen und lösen Sie das Pellet in 200 &amp; mgr; l RNAse-freiem TE-Puffer (10 mM Tris-HCl pH 7,5-1 mM EDTA). Übertragen auf ein frisches 1,5-ml-Röhrchen. </li><li> Extrahieren RNA durch Zugabe von 1 vol. Wasser gesättigtem Phenol (pH 6,6) und 1 Vol. Chloroform: Isoamylalkohol (24: 1). Gründlich mischen. Zentrifuge bei 15.000 xg für 20 min bei 4 ° C. Übertragen Sie die wässrige Phase in ein neues 1,5-ml-Röhrchen. Anmerkung: Säure Phenol (pH 4.5) kann verwendet werden, um DNA-Kontamination zu minimieren. </li><li>Ausfällen RNA von 1/10 Vol Zugabe. 3 M Natriumacetat und 3 vol. 100% Ethanol. Lassen Sie Fällung bei -80 ° C für 20 min oder O / N bei -20 ° C. Zentrifuge bei 15.000 xg für 15 min bei 4 ° C. Hinweis: Wenn in der Polysomen - Puffer (vgl Note 2.5), führen ein Lithiumchlorid (LiCl) Fällung anstelle von Ethanolfällung unter Verwendung von Heparin richtig jede Spur von Heparin zu entfernen , die 9 mit nachgeschalteter Analyse stören könnten. Nach der Extraktion hinzufügen LiCl zu einer Endkonzentration von 2,5 M. Niederschlag Lassen Sie für 30 Minuten bei -20 ° C oder O / N bei 4 ° C. Zentrifuge bei 15.000 xg für 15 min bei 4 ° C. </li><li> Waschen Sie das Pellet mit 500 ul 75% Ethanol. Air das Pellet trocknen und in 30 ul TE-Puffer lösen. Beurteilen RNA Qualität durch Elektrophorese auf einem RNAse frei 1,2% Agarosegel (100 V, 15 min) oder durch Kapillarelektrophorese Methoden. </li></ol> 5. Datenanalyse <ol><li> Export-Rohdaten in eine Grafik und Datenanalyse-Software. Hinweis: Wie in 2A und B gezeigt, geben die Rohprofile qualitative Informationen: die Anzahl der Polysoms Spitzen, die die Höhe des monosome Spitze, und das Vorhandensein einer Schulter entsprechend den freien Ribosomen - Untereinheiten zu sehen ist. </li><li> Merge Profile Vergleich der Profile zwischen den Proben zu ermöglichen. indem zusätzliche Datenpunkte zu Beginn der Kurven Verwenden Sie den Bildschirmleser Werkzeug, um den X-Wert des monosome Spitze für jede Probe zu bestimmen und die monosome Gipfel auszurichten. Identifizieren Sie den tiefsten Punkt der Kurve und bestimmen dessen Y-Wert (mit dem Screenreader-Tool). Stellen Sie den tiefsten Punkt Y-Wert auf 0, indem Sie den &quot;Set Spalten Wert&quot; -Funktion. </li><li> Bestimmen Sie den Polysomen / monosomes Verhältnis , indem die Fläche unter der Kurve von Rohprofile (3C) zu integrieren. Verwenden Sie die Datenauswahlwerkzeug die Grenze des Gebiets zu definieren, zu integrieren. Dann die Integration verwenden Funktion (Analysis-Mathematik). </li><li> Normalisierendie Kurven an den monosome Spitze durch alle Datenpunkte einer Kurve von der Y-Wert des Gipfels des monosome Spitzenteilung (bestimmt den Screenreader-Tool). Wählen Sie die Spalte, dann unter Analysis-Mathematik, wählen Sie Normalisieren und wählen Sie die &quot;geteilt durch einen bestimmten Wert&quot; Methoden. Dieser Schritt ermöglicht einen Vergleich der relativen Pegel von Polysomen (Abbildung 3D). </li></ol><img alt="Figur 2" src="/files/ftp_upload/54231/54231fig2.jpg" /> Abbildung 2. Repräsentative Polysoms Profile. Arabidopsis thaliana Wildtyp (wt, Ökotyp Col-0) und mutierten Keimlinge wurden für sechs Tage auf ½ Murashige und Skoog - Medium unter langen Tag Photoperiode gezüchtet (16 Stunden Licht, 8 Stunden Dunkelheit). A: roh Polysoms Profile wt Sämlinge. B: roh Polysoms Profile aus dem mutierten Sämlinge. C: Die Bestimmung des Prozentsatzes von Polysomen und monosomes durch Integration der Fläche unter der Kurve D: Polysomen Profiles zum monosome Spitze normalisiert. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

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The authors have nothing to disclose

The protocol we present here is an easy and cheap method for generating polysome profiles and isolating mRNAs associated with polysomes, single ribosomes or free of ribosomes. A wide range of different polysome fractionation methods is described in the literature. The method we have described here has been optimized to keep only the necessary compounds and has been adapted for plant material. In particular, we reduced the amount of detergent11 and added chloramphenicol to the buffer to fix the chloroplastic ribosomes to the mRNA (as cycloheximide does for the cytosolic ribosomes)12 . We have also reduced the total ultracentrifugation time7.
To obtain high quality polysome profiles, it is essential to use freshly collected plant material and to perform all steps at 4&#176;C. When using tissues that are poorly translationally active, more plant material can be loaded on the gradient (up to 600 mg).
Analysis of polysome profiles can provide insights into both the overall translational status of cells13 and the translational status of a specific mRNA. We have used RNAs isolated by this method for different applications. Using the RNAs for microarrays allowed us to identify a class of cadmium stress response genes for which transcription and translation are uncoupled 14. We also used this method to identify small RNAs associated with polysomal fractions. This identification was made by northern blotting15 of RNA extracted from polysomal fractions, and provided biochemical evidence for a translational component in the miRNA pathway in plants. In another study, a cis-NAT RNA was identified by quantitative RT-PCR. This cis-NAT is associated to the phosphate homeostasis and promote translation of the PHO1;2 transcript 16. 
The main limits of the polysome profiling approach are the lack of information concerning both the position of the ribosome on the mRNA and its progression along the mRNA. Ribosome profiling has emerged to address these limitations 17 and has been successfully used on plant tissues18. Ribosome profiling provides a global measurement of translation by taking advantage of the advances in sequencing technology. Nevertheless, as for any sequencing based assays, the quality of the results depends on the mapping of the sequences to the genome, therefore focusing on the small ribosome-protected fragments makes it difficult to deconvolute repetitive sequences. Moreover, the digestion of RNA not protected by the ribosomes leads to the loss of regulatory information contained in the 3' and 5' UTRs. It is then impossible to distinguish transcript variants that have different 3' or 5' UTRs and show different levels of translation19. The polysome profiling method described here is rapid and does not require specific technical skills. Altogether, these two methods represent complementary approaches to study translation regulation.

Die Proteinsynthese ist ein wesentlicher und energetisch kostspieliger Prozess in allen 1 - Zellen. Zuallererst Zellen müssen Energie in der Herstellung der Translationsmaschinerie, die Ribosomen investieren. Zum Beispiel erzeugt eine teilungsaktiven Hefezelle so viel wie 2000 Ribosomen pro Minute. Eine solche Produktion erfordert bis 60% der Gesamttranskriptionsaktivität und bis zu 90% der Gesamt Spleißen Aktivität der Zelle 2. Zusätzlich wird Energie für die Synthese von Aminosäuren, Aminoacyl-tRNA und Peptidbindungen erforderlich. In Pflanzen, das Hinzufügen einer Aminosäure einer Peptidkette Kosten 4,5-5,9 Moleküle ATP 3. Daher ist es nicht verwunderlich, dass die Translation von mRNA zu Protein ein wichtiger Ort der Regulierung, vor allem, wenn es mit wechselnden Umweltbedingungen fertig zu werden. Die Einleitung Schritt der Übersetzung, dass die Assoziation einer mRNA mit dem Ribosom ist, ist das Hauptziel der RegulierungÜbersetzung 4. Als Folge der Regulation der Translation sowie andere post-transkriptionale regulatorische Schritte können nur 40% der Variationen in Proteinkonzentration von mRNA Menge 5,6 erläutert. So gibt die Studie der Gesamt-mRNA relativ schlechte Informationen über Protein Hülle und Fülle. Auf der anderen Seite gibt der Verband der mRNA mit Ribosomen besseren Einblick in die Proteinmenge durch den Zugang zu diesen in Translation beteiligt mRNAs zu geben. Aktiv werden übersetzt mRNAs mit mehreren Ribosomen in Strukturen genannt Polysomen assoziiert. Im Gegensatz dazu wird schlecht übersetzt mRNAs mit nur einem Ribosom (monosome) zugeordnet werden. Folglich kann die Translations Status einer mRNA durch Überwachung seiner Verbindung mit Ribosomen 7 ausgewertet werden. Dieses Protokoll beschreibt die Isolierung von Polysomen aus 6 Tage alten Arabidopsis thaliana Keimlinge, die anschließende Isolierung von RNA und die Analyse der Ergebnisse. Polysomes und monosomes werden durch einen Saccharose-Dichtegradienten getrennt. Gradienten werden in sechs Fraktionen gesammelt. Einige der Fraktionen werden vereinigt drei zu erhalten gut getrennten Fraktionen: Polysomen, monosomes und die Leichtfraktion (im Folgenden als Überstand), die enthält die freien 60S und 40S ribosomalen Untereinheiten und mRNAs, die nicht mit Ribosomen assoziiert. Globale Translationsaktivität kann durch Erzeugen eines Polysomen / monosome Verhältnis geschätzt werden, die durch Integration der Fläche unter der Kurve bestimmt wird, und durch die Polysomen Profilen verglichen wird. mRNAs und Proteine ​​werden dann aus den verschiedenen Fraktionen und zur Analyse mittels RT-PCR, qRT-PCR, Northern Blot, Microarray, Western-Blot oder Proteomik extrahiert. Dieses Protokoll wurde für andere Pflanzen und Geweben validiert. Das Gerät benötigt, um dieses Protokoll zu erfüllen sind häufig in den meisten Labors gefunden: Es besteht keine Notwendigkeit für einen Gradienten ausgestattet. Einfrieren jede Schicht bevor die nächste Zugabe verhinderns aus einem Mix oder Störung der Schichten. Kein Schlauch piercer für Gradienten Sammlung verwendet, die durch Eintauchen eines Glaskapillarrohres in dem Gradienten erreicht werden kann. Daher bleiben die teuren Ultrazentrifugenröhrchen unbeschädigt und wiederverwendet oft werden kann. Zusammen macht dies das vorliegende Protokoll eine einfache und billige Methode für Polysoms Profilierung.

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			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 13.2 ml</td>
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			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 38.5 ml</td>
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			<td style="width:132px;">326823</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Glass capillary tube</td>
			<td style="width:109px;">Drummond Scientific</td>
			<td style="width:132px;">1-000-1000</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge&#160;</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">Optima series</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW41</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">331362</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW32</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">369650</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Peristaltic pump</td>
			<td style="width:109px;">Any</td>
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			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Tygon R3607 polyvinyl chloride tubing&#160;</td>
			<td>Fisher Scientific</td>
			<td>070534-22</td>
			<td>Polyvinyl chloride tubing, 2.29 mm&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Fraction collector Model 2110</td>
			<td style="width:109px;">Bio-Rad</td>
			<td style="width:132px;">731-8120</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV cuvette</td>
			<td style="width:109px;">Hellma</td>
			<td style="width:132px;">170.700-QS</td>
			<td>Quartz flow-through cuvette</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV Spectrophotometer</td>
			<td style="width:109px;">Varian</td>
			<td style="width:132px;">Cary50</td>
			<td>Read every 0.0125 sec</td>
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			<td height="40" style="height:40px;width:233px;">All chemicals</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td>Use only Molecular Biology Grade</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Murashige and Skoog Basal Salt Mixture (MS)</td>
			<td>Sigma-Aldrich</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Rnase-Free water</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>10083251&#160;</td>
			<td></td>
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			<td height="63" style="height:63px;width:233px;">Octylphenoxy poly(ethyleneoxy)ethanol, branched (Nonidet P40)</td>
			<td style="width:109px;">Euromedex</td>
			<td style="width:132px;">UN3500</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Linear acrylamide (acryl carrier)</td>
			<td style="width:109px;">ThermoFischer scientific</td>
			<td style="width:132px;">AM9520</td>
			<td>RNA precipitation carrier</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:233px;">OriginPro 8</td>
			<td style="width:109px;">OriginLab</td>
			<td style="width:132px;"></td>
			<td>Analysis software</td>
		</tr>
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an easy method for plant polysome profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

In der Literatur sind Polysomen Profile oft aus der leichten Fraktion in die Schwerfraktion als Ergebnis der Art und Weise gezeigt sind, die Gradienten gesammelt, also von oben nach unten. Da in dem beschriebenen Protokoll hier die Gradienten von unten nach oben gesammelt werden, mit der schweren Fraktion (die Polysomen) und gehen die Profile zeigen wir auf die leichte Fraktion (freie Ribosomen - Untereinheiten und RNAs) (2A) beginnen. Wir sammeln dann jeden Gradienten in sechs 2-ml-Fraktionen, aber auch kleinere Fraktionen können gesammelt werden, wenn eine detailliertere Analyse der Polysoms Inhalt durchgeführt werden muss. Zusammenführen und die Normierung der Kurven auf die monosome Peak (2D) ermöglicht den Vergleich von Profilen aus verschiedenen Linien oder Wachstumsbedingungen. Sie liefert Informationen über die relative Menge von Polysomen unabhängig von der Höhe der Initiation. Eine weitere Möglichkeit, eineAlysieren die Profile ist die Fläche unter der Kurve zu berechnen, so kann man die relative Menge an mRNA bestimmen mit zugehörigem entweder den monosomes oder den Polysomen (Abbildung 2C). Dieses Verhältnis ist spezifisch für eine Pflanze und Wachstumsbedingungen. Jedoch kann dieser Ansatz nicht in dem Fall von schlecht translatorisch aktiven Geweben relevant sein. Wir haben diese Methode für A verwendet thaliana ganze Sämlinge, junge und alte Rosetten sowie für N. benthamiana (3C), S. lycopersicum (Abbildung 3E) und O. sativa Blätter (3D). Die Profilform hängt von den Wachstumsbedingungen, Pflanzenalter und den analysierten Geweben. Hier haben wir A. thaliana 6 Tage alten Keimlingen. In diesem Stadium ist die Translationsaktivität hoch ist , und das Profil zeigt , gut geformte Spitzen (2A und B). Dies ist auch der Fall,wenn A. thaliana Blumen verwendet (Abbildung 3A). Bei der Verwendung von 4 Wochen alt A. thaliana Rosetten (3B), enthalten die Proben meist voll entwickelten Erwachsenen lässt , wo Zellen teilen sich nicht. Daher ist die Gesamtmenge an Polysomen und monosomes niedriger. Das Profil zeigt weniger, aber immer noch gut Polysoms Spitzen geformt. Neben dem monosome Spitze zeigt die Schulter, um die große Menge an freiem 60S ribosomalen Untereinheiten. Mit anderen Art von Pflanzen oder Geweben können die Polysoms Peaks können kaum sichtbar. Dies ist der Fall , wenn sie 30 Tage alt O. Verwendung sativa Blätter (3D). Selbst wenn die Menge an mRNAs in Translation beteiligt ist sehr niedrig (kein Polysomen peak kann auf das Profil zu sehen), das Vorhandensein des monosome peak zeigt an, dass die Fraktionierung richtig durchgeführt wurde und daß mRNAs kann weiter aus der Fraktion für eine weitere Analyse entnommen werden . Die Qualität der extrahierten RNA-Qualität wird durch Agarose-Gelelektrophorese beurteilt ( <str<html> Ong&gt; Abbildung 4). Die 25S und 18S ribosomale RNA cytosolischen sollte auf dem Gel deutlich sichtbar sein. Untere Bänder Chloroplasten ribosomale RNA entsprechend sollte auch sichtbar sein , wenn die RNA aus grünen Geweben 10 extrahiert werden. <img alt="Figur 3" src="/files/ftp_upload/54231/54231fig3.jpg" /> Abbildung 3. Polysomen Profile aus verschiedenen Pflanzenmaterial und Arten. (A) Arabidopsis thaliana Blumen (300 mg), (B) Arabidopsis thaliana 4 Wochen alt Rosetten (600 mg), (C) Nicotiana benthamiana (junge Blätter von 40 Tage alt kurzen Tageswachsene Pflanzen - 300mg), (D) Oryza sativa ( Blätter 30 Tage alten Pflanzen - 300 mg), (E) Solanum lycopersicum (junge Blätter von 35 Tage alt kurzen Tageswachsene Pflanzen - 300 mg). Die monosome Peaks sind durch Pfeile angedeutet ist.iles / ftp_upload / 54231 / 54231fig3large.jpg &quot;target =&quot; _ blank &quot;&gt; Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen. <img alt="Abbildung 4" src="/files/ftp_upload/54231/54231fig4.jpg" /> . (- 15 min 100V) Abbildung 4. Beurteilung der RNA - Qualität RNA aus den angegebenen Fraktionen wurden geladen auf einem 1,2% Agarosegel und durch Elektrophorese (500 ng von Sprossen von 6 Tage alten Arabidopsis thaliana Keimlingen extrahiert). Cytosolic (25S und 18S) rRNA angegeben werden durch Pfeilspitzen und Chloroplasten (23S und 16S) rRNA durch eine Klammer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig4large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig4large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

Watch this Scientific Journal Video about An Easy Method for Plant Polysome Profiling at JoVE.com

An Easy Method for Plant Polysome Profiling

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Eine einfache Methode für Pflanzen Polysomen Profilieren

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

an-easy-method-for-plant-polysome-profiling

Nanyang Technological University

Research

JoVE Journal

Biology

12.4K Aufrufe.  Aix-Marseille Université. This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Serie: An Easy Method for Plant Polysome Profiling

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

Oligo Messe Profiling (OLIMP) der extrazellulären Polysacchariden

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Forschung

Biologie

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Profiling Thiolredox Proteome Mit Isotopen-Massenspektrometrie Tagging

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Glycan Profiling of Plant Cell Wall Polymers mit Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Analyse der Translation Initiation Während Stress-Bedingungen durch Polysomprofile Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Eine effiziente Methode zur quantitativen, Einzel-Zell-Analyse von Chromatin Modifikation und Reaktor Architektur in Whole-Mount-Samenanlagen in<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Bewertung der Selective mRNA Translation in Säugerzellen durch Polysomen Profilieren

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

Lasergestützte Mikrodissektion (LAM) als Werkzeug für die Transkriptionsprofilierung einzelner Zelltypen

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Ein verbesserter Schnittverfahren für widerspenstige Pflanzengewebeproben

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Standardisierte Methode für Hochdurchsatz-Sterilisation von Arabidopsis Samen

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.