Die Arbeit in der Montgomery-Labor auf Phytochrom Reaktionen in Pflanzen wird durch die National Science Foundation (Grant No. MCB-0919100 bis BLM) und der Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of unterstützt Energy (Grant No. DE FG02 91ER20021 zu BLM). Wir danken Melissa Whitaker für die technische Unterstützung während der Dreharbeiten und kritische Durchsicht des Manuskripts, Stephanie Costigan für experimentelle Mitarbeit, Dr. Louis King für die Unterstützung bei der Entwicklung und Optimierung Fluorescence-Activated Cell Sorting Protokolle für Arabidopsis Protoplasten Sortierung und Dr. Melinda Rahmen für die Unterstützung bei der konfokalen Mikroskopie. Wir bedanken uns bei Marlene Cameron für die grafische Gestaltung Unterstützung und Karen Vogel für redaktionelle Unterstützung.

1. Plant Growth <ol><li> Bestätigt UAS-BVR X GAL4-GFP-Enhancer trap line isoliert als 4 beschrieben (Zusammenfassung siehe Abb.. 1) und Wildtyp-oder elterlichen Linien sind auf den Boden gesät, dh ~ 2000 sterilisierten Samen pro Zeile. </li><li> Pflanzen sind für 5 Wochen auf den Boden unter weißer Beleuchtung von 100 μmolm -2 s -1 gewachsen bei 22 ° C und 70% Luftfeuchtigkeit. </li></ol> 2. Blatt Protoplastenisolierung (angepasst von Denecke und Vitale 9) <ol><li> Zur Isolierung Protoplasten, bereiten TEX-Puffer. Für 1 Liter TEX-Puffer, wiegen Sie die folgenden Komponenten: 3,1 g Gamborg die B5-Salze, 0,5 g 2 - (N-Morpholino) ethansulfonsäure (MES, 2,56 mM), 0,75 g Calciumchlorid-Dihydrat (CaCl 2 .2 H 2 O, 6,75 mM), 0,25 g Ammoniumnitrat (NH 4 NO 3, 3,12 mM), auflösen 136,9 g Saccharose (0,4 Mio.). Bauteile komplett in ~ 900 ml entionisiertem, destilliertem Wasser (ddH 2 O) und bringen Sie den pH-Wert auf 5,7 mit 1 M KOH. Bringen Endvolumen von 1 Liter und Filter sterilisieren mit einem 0,2 &amp;mgr; Flaschenaufsatz-Filter angeschlossen an eine Vakuumpumpe. </li><li> Sammeln Sie grüne, gesunde Blätter von Pflanzen (~ 250 ml der Blätter lose in ein Becherglas verpackt) und spülen Sie mit ~ 40 ml ddH 2 O 4-mal, gefolgt von Spülen zweimal mit sterilem ddH 2 O. Mit einem Nr. 20 Skalpell schneiden Blätter in feine Streifen schneiden und spalten Gewebe zu gleichen Teilen in zwei 50 ml sterile Kunststoffröhrchen. Bereiten 1x Blatt Verdauung durch Zugabe von 5 ml Aliquot 10x Blatt Verdauung Stammlösung (10x Blatt Verdauung Stammlösung: Man löst 2% w / v Macerozyme R-10, 4% w / v Cellulase &quot;Onozuka&quot; R-10 in TEX-Puffer, Filter sterilisieren mit 0,2 um Filter und frieren 5 ml Aliquots bei -80 ° C) auf 45 ml frisches TEX-Puffer. Add ~ 25 ml 1x Blatt Verdauung Mischung pro 50 ml Tube für alle Blattgewebe zu decken. </li><li> Vacuum infiltrieren Blattgewebe in Verdaumischung in offenen Röhren für 1 Stunde bei Raumtemperatur mit einem Vakuum-Exsikkator an eine Wasserpumpe mit 3-hr Inkubation bei Raumtemperatur auf einer Wippe unter leichtem Schütteln folgte. Capped Rohre mit Blattgewebe in 1x Blatt Verdauung Mix sind in Alufolie aufbewahrt während dieses Prozesses umwickelt, um Lichteinfall zu verhindern. </li><li> Nach 3 Stunden, erhöhen Sie die Wippe Geschwindigkeit für ca. 2 Minuten bis Protoplasten freizugeben. Filter das rohe Protoplastensuspension durch zwei Schichten sterile Gaze, um Ablagerungen zu entfernen und das Filtrat in ein steriles Becherglas. Filter das Filtrat durch einen sterilen 100-um Nylon-Mesh in eine sterile Petrischale. Sammeln Sie die Durchströmung und übertragen sie auf eine neue sterile 50 ml-Tube. Verwenden Sie ca. 15-20 ml frischem TEX Puffer, um die sterile Petrischale waschen und sammeln keine Protoplasten an der Oberfläche haften. </li><li> Centrifuge die Durchströmung mit einem Swing-Eimer Rotor bei 100xg bei 10 ° C für 15 min (Acceleration 6, Verzögerung 0). </li><li> Nehmen Sie ~ 25 bis 30 ml der Flüssigkeit unterhalb der schwimmenden Protoplasten Schicht, die Rest-und pelletiert Schmutz enthält, mit einer sterilen 9 &quot;Glas Pasteur Pipette an einer Schlauchpumpe, ohne die schwimmenden Protoplasten Schicht und verlassen ~ 10 bis 15 ml Volumen. </li><li> Add frischen TEX-Puffer auf ein Endvolumen von 40 ml, während sanft Resuspendieren des Protoplasten. </li><li> Wiederholen Sie die Schritte 2,5 bis 2,7 zweimal so viel Zelltrümmer wie möglich zu entfernen. Die Zentrifugation der Zeit ist, 10 Minuten in der ersten Wiederholung und 5 Minuten in der letzten Wiederholung reduziert. </li><li> Absaugen schwimmenden Protoplasten mit einem Schnitt, sterile 1 ml Transferpipette in ein neues 15 ml Tube. Gehen Sie direkt zu sortieren oder zu speichern in Dunkeln bis zu 2 Stunden bei 4 ° C bis zur Sortierung. </li></ol> 3. Protoplast Sortierung nach Fluorescence-Activated Cell Sorting (FACS) <ol><li> Vor dem Sortieren untersuchen Protoplasten durch konfokale Laser Scanning Mikroskopie (CLSM) mit einem 488-nm-Laser für die Anregung zu Protoplasten Integrität und die Anwesenheit von GFP-Fluoreszenz in den Protoplasten Pool zu bestätigen. Minimal Schutt ist notwendig, um Verstopfungen zu vermeiden die FACS-Sortierung Düse. </li><li> Sortieren isolierten Protoplasten in TEX-Puffer über FACS (BD FACSVantage SE, BD Biosciences) mit einer 200-um Düse auf der Makro-Art Kopf Ereignisraten zwischen 6.000 und 15.000, mit einem Systemdruck von etwa 9 psi nach einem adaptierten Protokoll 10. </li><li> Wild-Typ nicht-GFP Protoplasten werden verwendet, um die Autofluoreszenz Schwellenwerte zu ermitteln. </li><li> Um GFP-positive Protoplasten, Art-Zellen mit einem luftgekühlten Argonlaser (Spectra Physics Modell 177, Newport Corporation, Irvine, CA) betrieben bei 100 mW auf einer 488-nm Argon Linie GFP-Fluoreszenz zu identifizieren mit einem 530/30 Band sammeln Pass-Filter. </li><li> Nach der Sammlung von GFP-positive Protoplasten untersuchen sortiert Protoplasten durch CLSM wie in Schritt 3.1. </li><li> Auszug Gesamt-RNA aus sortierten Protoplasten unter Verwendung eines Qiagen RNeasy Plant Mini Kit. cDNA kann dann für die Hybridisierung hergestellt werden, wie in Affymetrix GeneChip Expr beschriebenITZUNG Analysis Technical Manual und dann hybridisiert ATH1 Arabidopsis Affymetrix Whole Genome-Arrays. </li></ol> Repräsentative Ergebnisse Wir erkannten eine beträchtliche Anzahl von GFP-positive Zellen im GFP-Kanal durch FACS (Abb. 2). In Optimierung Tests unter Verwendung von GFP-Enhancer-trap Eltern, die konstitutiv GFP-exprimierenden Linie, J0571, angezeigt ~ 17% auf 24% GFP-positive Protoplasten, während die Linie mit vaskulären und dermalen Ausdruck, J1071, hatte ~ 1,4% GFP-positive Protoplasten (Tabelle 1). Sortieren von 3 x 500 ul (~ 1,5 ml insgesamt Protoplastensuspension) von J0571 Protoplasten für 1 h gab ~ 100.000 GFP-positive Protoplasten. Sortieren von 3 x 500 ul (~ 1,5 ml insgesamt Protoplastensuspension) von J1071 Protoplasten für 1,5 h gab ~ 3.000 GFP-positive Protoplasten. Konfokale Bilder zeigte eine sehr hohe Ausbeute an Protoplasten für J0571 und J1071 Proben vor der Sortierung wurde (Abb. 3C und 3E) durchgeführt, und die sortierten Fraktionen enthielten nur helle GFP-Fluoreszenz-Protoplasten (Abb. 3G und Daten nicht gezeigt). Dieser Befund bestätigt, dass intakte GFP-positive Protoplasten via FACS sortiert werden können. Die nicht-GFP Protoplasten können auch sortiert und gesammelt in einem separaten Kanal. Die nicht-GFP Protoplasten dienen als ideales negative Kontrolle für die anschließende Microarray-Analysen auf die spezifischen Veränderungen der Genexpression bei der Reduktion von holophytochrome Ebenen zu erkennen. RNA-Extraktion aus isolierten Protoplasten (1 ml) ergibt RNA in ausreichender Menge für den Nachweis von fluorospectrometry (Abb. 4). Isolierte RNA wurde anhand einer NanoDrop Instrument (NanoDrop 1000, Thermo Scientific) und quantifiziert NanoDrop 3.7.1 RNA-Quantifizierung Software. RNA-Ausbeuten aus C24-Wildtyp-Protoplasten oder FACS-sortiert, überschritten GFP-positive enhancer trap Protoplasten die mindestens 20 ng für den Einsatz in brauchte vorsortiert RNA-Labeling-Assays für die Microarray-(Tabelle 2). <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Enhancer-Trap-Linie </td><td> % Der GFP-positive Protoplasten </td><td> Anzahl der sortierten GFP Protoplasten </td></tr><tr><td> J0571 </td><td> 17,18% ~ 24,06% </td><td> 26.400 ~ 36.000 </td></tr><tr><td> J1071 </td><td> ~ 1,43% </td><td> 1.000 ~ 1.300 </td></tr></tbody></table> Tabelle 1. Anteil der GFP-positive Protoplasten von zwei Enhancer-trap-Linien vor der Sortierung und der Anzahl der GFP-positive Protoplasten, indem Sie 500 ul der Protoplastensuspension durch die Fluorescence Activated Cell Sorter (FACSVantage, BD) gesammelt. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Pflanzenlinie </td><td> RNA-Ausbeute (ng) </td></tr><tr><td> C24 WT </td><td> 12486,5 </td></tr><tr><td> J0571 </td><td> 60 </td></tr><tr><td> J1071 </td><td> 71,5 </td></tr></tbody></table> Tabelle 2. Von RNA-Isolierung aus Protoplasten Yield. RNA wurde aus vorsortierten C24-Wildtyp-oder sortiert GFP-positive Protoplasten von zwei enhancer trap Linien isoliert und quantifiziert. <img src="/files/ftp_upload/1925/1925fig1.jpg" alt="Abbildung 1" /> Abbildung 1. GAL4-Enhancer-trap-basierte Induktion von Biliverdin-Reduktase (BVR) die Expression in transgenen Arabidopsis thaliana Pflanzen. (A). Ein einzelner aus einer Bibliothek von GAL4-basierten Enhancer-trap-Linien, die eine GAL4-responsive GFP-Markergen enthält, ausgewählt werden kann mit einer Zeile mit einem GAL4-responsive Zielgen um die Expression des Zielgens in induzieren GAL4-enthaltenden Zellen markiert überquert werden von GFP-Fluoreszenz. Basierend auf einer Figur aus Dr. Jim Haseloff (http://www.plantsci.cam.ac.uk/Haseloff/geneControl/GAL4Frame.html). (B) Herstellung von Phytochrom-Chromophor, Phytochromobilin (PΦB) und holophytochrome in übergeordneten Linien. (C) links, Reduktion von Biliverdin IXα (BV IXα) und PΦB von Biliverdin-Reduktase (BVR) Aktivität BR und PΦR jeweils. BVR-Aktivität führt zu Erschöpfung der PΦB und führt zu einer Reduktion bei der Herstellung von photoaktiven holophytochrome. Richtig, wird die Reaktion durch BVR katalysierten gezeigt. <img src="/files/ftp_upload/1925/1925fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Fluorescence Activated Cell Sorting (FACS) Erwerb Dot-Plots. Vergleich von Protoplasten Zellsortierung für C24-Wildtyp (A), J0571 (B) und J1071 (C). (A) Erwerb Dot-Plot von nicht-fluoreszierenden GFP C24-Wildtyp-Protoplasten durch ein 488-nm Argon-Laser angeregt und zur Autofluoreszenz Schwelle zu bestimmen. (B) und (C) Erwerb Dot-Plots zeigen Anteile von Protoplasten, dass GFP positive Reaktion auf Anregung durch einen 488-nm-Laser sind. R3 Sortierweichen in B und C begrenzen die GFP-positive Ziele, die durch Fluorescence-Activated Cell Sorter sortiert wurden (FACSVantage, BD) und gesammelt. Red-Kanal zeigt verte für Chlorophyll-Autofluoreszenz von Protoplasten und grünen Kanal zeigt Werte für GFP-Fluoreszenz. <img src="/files/ftp_upload/1925/1925fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Konfokale Mikroskopie von pflanzlichen Protoplasten für die Zell-Sortierung eingesetzt. Konfokalen Laser-Scans von Protoplasten vor (A, C, E) und nach (G) Sortierung über Fluorescence-Activated Cell Sorter (FACS). B, D, F und H sind DIC Bilder. Protoplasten von C24-Wildtyp (A, B), J1071 (C, D) und J0571 (E, F, G, H) gezeigt. Und Autofluoreszenz (LP 650 nm) aus Anregung mit einem 488-nm-Laser erzielt - Bilder C, E und G sind Bilder von GFP-Fluoreszenz (575 nm BP 505 nm) zusammengeführt. A bis H sind durchschnittlich 4 Scans unter 63x Öl. Bar = 10 pm. <img src="/files/ftp_upload/1925/1925fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Quantifizierung von RNA aus Protoplasten durch fluorospectrometry isoliert. RNA von (A) C24 Wildtyp (B) J0571 und (C) J1071 extrahiert. A zeigt RNA für vorsortierte Wildtyp-Protoplasten. B und C zeigen RNA aus GFP-positive Protoplasten durch Fluorescence-Activated Cell Sorting (FACS) sortiert. RNA-Quantifizierung Software, NanoDrop 3.7.1, (NanoDrop 1000, Thermo Scientific).

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root-localized+phytochrome+chromophore+synthesis+is+required+for+tissue-specific+photoregulation+of+root+elongation+and+impacts+sensitivity+to+jasmonic+acid+in+Arabidopsis+thaliana.">Root-localized phytochrome chromophore synthesis is required for tissue-specific photoregulation of root elongation and impacts sensitivity to jasmonic acid in Arabidopsis thaliana.</a> , .</li><li>Lagarias, D. M., Crepeau, M. W., Maines, M. D., Lagarias, J. 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Gene Expression Profiling durch Mikroarrays (1) hat angedeutet, dass mehr als 30% der Gene in Arabidopsis-Keimlinge Licht reguliert 11 sind und (2) hat eine große Gruppe von Genen, Licht Signaltransduktionsproteine ​​in der Phytochrom Signalkaskade 12, 13 beteiligten identifiziert . Solche Experimente deuten darauf hin, dass Licht eine schnelle und langfristige Veränderungen in der Genexpression induziert. Jeder Pool von Phytochrome kann die Kontrolle nur eine Teilmenge der Entwicklungs-und A...

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<td>Anti-BVR antibody</td>
<td>&nbsp;</td>
<td>QED Bioscience Inc.</td>
<td>56257-100</td>
<td>&nbsp;</td>
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<td>Cellulase &#x201C;Onozuka&#x201D; R-10</td>
<td>&nbsp;</td>
<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
<td>MSPC 0930</td>
<td>&nbsp;</td>
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<td>Gamborg&#x2019;s B5 basal salt mixture</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5768</td>
<td>&nbsp;</td>
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<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
<td>PTC 001</td>
<td>&nbsp;</td>
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<td>MES, low moisture content</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M3671</td>
<td>&nbsp;</td>
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<td>Murashige and Skoog salts</td>
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<td>Caisson Laboratories</td>
<td>74904</td>
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<td>Phytablend</td>
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<td>Caisson Laboratories</td>
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<td>RNeasy Plant Minikit</td>
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investigating tissue- and organ-specific phytochrome responses using facs-assisted cell-type specific expression profiling in arabidopsis thaliana

Licht vermittelt eine Reihe von Entwicklungs-und adaptive Prozesse über den gesamten Lebenszyklus einer Anlage. Pflanzen nutzen Licht absorbierende Moleküle, so genannte Photorezeptoren zu spüren und sich an Licht. Die rot / Dunkelrot-Licht-absorbierenden Photorezeptoren Phytochrom wurden umfassend untersucht. Phytochrome existieren als eine Familie von Proteinen mit unterschiedlichen und sich überschneidenden Funktionen in allen höheren Pflanzen, in denen sie studiert haben,<sup&gt; 1</sup&gt;. Phytochrom-vermittelten Reaktionen Licht, das von Keimung der Samen durch blühende und Seneszenz-Bereich, sind oft auf bestimmte pflanzliche Gewebe oder Organen lokalisiert<sup&gt; 2</sup&gt;. Trotz der Entdeckung und Aufklärung der individuellen und redundant Phytochrom Funktionen durch Mutationsanalysen, aussagekräftige Berichte auf unterschiedlichen Seiten des photoperception und die molekularen Mechanismen der lokalen Pools von Phytochrome, die vermitteln, räumlich-spezifische Phytochrom Reaktionen begrenzt sind. Wir haben Experimente auf der Hypothese, dass bestimmte Websites von Phytochrom photoperception regulieren Gewebe-und Organ-spezifische Aspekte der Photomorphogenese und die lokalisierte Phytochrom Pools engagieren verschiedene Teilmengen von nachgeschalteten Zielgene in Zell-Zell-Signal basiert. Wir entwickelten ein biochemischer Ansatz zur selektiven Reduktion funktioneller Phytochrome in einem Organ-oder Gewebe-spezifische Weise in transgenen Pflanzen. Unsere Untersuchungen basieren auf einem bipartiten Enhancer-trap Ansatz, der in Transaktivierung der Expression eines Gens unter der Kontrolle des Upstream Activation Sequence (UAS) Element Ergebnisse der Transkriptionsaktivator GAL4 Basis<sup&gt; 3</sup&gt;. Das Biliverdin-Reduktase (<em&gt; BVR</em&gt;)-Gen unter die Kontrolle der UAS wird stillschweigend in der Abwesenheit von GAL4 Transaktivierung in der FH-BVR Elternteil aufrechterhalten<sup&gt; 4</sup&gt;. Genetische Kreuzungen zwischen einem UAS-BVR transgene Linie und ein GAL4-GFP-Enhancer trap line führen zu spezifischen Ausdruck der<em&gt; BVR</em&gt; Gen in Zellen gekennzeichnet durch<em&gt; GFP</em&gt; Ausdruck<sup&gt; 4</sup&gt;. BVR Akkumulation in Arabidopsis-Pflanzen führt Phytochrom-Chromophor-Mangel<em&gt; In planta</em<sup&gt; 5-7</sup&gt;. So sind transgene Pflanzen, die wir haben, zeigen GAL4-abhängige Aktivierung der produzierten<em&gt; BVR</em&gt; Gen, was in der biochemischen Inaktivierung von Phytochrom sowie GAL4-abhängigen<em&gt; GFP</em&gt; Ausdruck. Photobiologische und molekulargenetische Analysen<em&gt; BVR</em&gt; Transgenen Linien sind nachgiebig Einblick in Gewebe-und Organ-spezifische Phytochrom-vermittelten Reaktionen, die mit entsprechenden Stellen des photoperception in Verbindung gebracht wurden<sup&gt; 4, 7, 8</sup&gt;. Fluorescence Activated Cell Sorting (FACS) von GFP-positive, Enhancer-trap-induzierte<em&gt; BVR</em&gt;-Exprimierenden Pflanzenprotoplasten mit Zelltyp-spezifische Gene Expression Profiling durch Microarray-Analyse gekoppelt wird verwendet, um vermeintliche nachgeschaltete Zielgene bei der Vermittlung von räumlich-spezifische Phytochrom Reaktionen beteiligt sind. Diese Forschung baut unser Verständnis von Seiten der Lichtwahrnehmung, die Mechanismen, durch die verschiedenen Gewebe oder Organe in Licht-regulierte Pflanzenwachstums und der Entwicklung zusammenarbeiten und die Weiterentwicklung der molekularen Zergliederung komplexer Phytochrom-vermittelten Zell-Zell-Signalkaskaden.

Watch this Scientific Journal Video about Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana at JoVE.com

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Die molekulare Basis der räumlich-spezifische Phytochrom Reaktionen wird untersucht mit Hilfe von transgenen Pflanzen, die Gewebe-und Organ-spezifische Phytochrom Mängel aufweisen. Die Isolierung von spezifischen Zellen mit induzierter Phytochrom-Chromophor Abreicherung durch Fluorescence-Activated Cell Sorting gefolgt von Microarray-Analysen wird genutzt, um Gene in räumlich-spezifische Phytochrom Reaktionen beteiligt sind.

Untersuchung Tissue-und Organ-spezifische Phytochrom Antworten mit FACS-gestützte zelltypspezifisch Expression Profiling in Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

investigating-tissue-organ-specific-phytochrome-responses-using-facs

Research

JoVE Journal

Biology

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

15.7K Aufrufe.  Michigan State University (MSU). Die molekulare Basis der räumlich-spezifische Phytochrom Reaktionen wird untersucht mit Hilfe von transgenen Pflanzen, die Gewebe-und Organ-spezifische Phytochrom Mängel aufweisen. Die Isolierung von spezifischen Zellen mit induzierter Phytochrom-Chromophor Abreicherung durch Fluorescence-Activated Cell Sorting gefolgt von Microarray-Analysen wird genutzt, um Gene in räumlich-spezifische Phytochrom Reaktionen beteiligt sind.

Serie: Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

Floral-dip Transformation von Arabidopsis thaliana Zum Untersuchen PTSO2: β-Glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Forschung

Biologie

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

Heterokaryon Technik zur Analyse von Zelltyp-spezifischen Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Arabidopsis thaliana Polar Glycerolipid Profiling durch Dünnschichtchromatographie (TLC) mit Gas-Flüssigkeits-Chromatographie (GLC) Coupled

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Zellspezifische Analyse<em&gt; Arabidopsis</em&gt; Blätter mit Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Effiziente und schnelle Isolierung von Frühphasen-Embryonen aus<em&gt; Arabidopsis thaliana</em&gt; Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Histochemische Färbung von<em&gt; Arabidopsis thaliana</em&gt; Sekundärzellwand-Elemente

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Hohe Auflösung Quantifizierung kristalliner Cellulose Akkumulation in<em&gt; Arabidopsis</em&gt; Roots Gewebespezifische Zellwand Änderungen überwachen

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Analyse von Arabidopsis Thaliana Wuchsverhalten in verschiedenen Licht-Qualitäten

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Cell-Type-Specific Epigenom Profilierung

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Zelltypspezifische Gene Expression Profiling in der Mausleber

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...