Diese Arbeit wurde von der Cornell University Agricultural Experiment Station (CUAES) NYC-125433 Hatch Grant und Cornell unterstützt Start-Up zum OKV gewährten Zuschusses

Teil 1. Vorbereitung der festen Medium für den Anbau von Pflanzen. Wir typischerweise Kultur Pflanzen konzentriert Murasige und Skoog (MS)-Medium mit 1% Saccharose und 0,7% Agar. Es enthält alle notwendigen Zutaten, um die Pflanzen gesund. Für die Herstellung von 1L MS-, Medien-pH-Wert 5,7 <ol><li> Add 2,15 g MS-Pulver in 800 ml Wasser in 1,5-2.0L autoklavierbar Flasche. </li><li> Legen Sie einen Rührstab in die Flasche und lösen MS Pulver durch Rühren Medium auf eine mitreißende Platte. </li><li> Unter Rühren hinzufügen, Tropfen für Tropfen, bis 1N KOH pH-Wert von MS-Medium auf 5,7 einstellen. </li><li> Fügen Sie 10 g Saccharose, immer schön rühren. </li><li> Add 7 g Agar *. </li><li> Passen Sie das endgültige Volumen der vorbereiteten mittel-bis 1000 ml und durch Autoklavieren sterilisieren .* </li><li> Legen Sie autoklavierten Medium auf eine mitreißende Platte und cool it down Rühren, so dass Agar nicht Niederschlag am Boden der Flasche. Cool down mittel-bis ~ 60 ° C * </li><li> Gießen Sie 100 mm Petrischalen. Keep Platten bei 4 ° C. </li></ol> * Tipp 1: Versuchen Sie nicht, Agar aufzulösen. Es wird beim Autoklavieren zu lösen. * Tipp 2: Halten Sie das Rühren bar in eine Flasche beim Autoklavieren, du wirst es später brauchen. * Tipp 3: Wenn Sie Sie die Hände auf dem autoklavierten Flasche für 10 s halten kann, bedeutet dies, dass das Medium ausreichend abgekühlt hat, und Sie können beginnen Gießen Platten. Part 2. Pflanzenmaterial und Wachstumsbedingungen. <ol><li> Sterilisieren Samen von Arabidopsis thaliana <ol><li> Platz 100 mg Samen in Eppendorf Reaktionsgefäße und 1 ml 70% Ethanol. Gut mischen und inkubieren für 2 min. Spin Körnern, saugen Sie den Überstand. </li><li> 1 ml 1,8% Bleichmittel-Lösung * mit 0,1% Tween-20. Mix für 10 min. Spin down Samen und saugen Bleichlösung. </li><li> Dieser Schritt muss in einer Sterilbank durchgeführt werden. 1 ml sterilem Wasser, Samen, gut mischen. Spin down Samen, saugen Wasser. Wiederholen Sie diesen Schritt 5 mal. </li></ol></li><li> Spread-Samen auf den vorbereiteten in Schritt 1 Platten (100 Korn / Platte). </li><li> Halten gesetzten Platten für 24-48 h bei 4 ° C im Dunkeln für die Stratifizierung. </li><li> Wachsen Pflanzen für 14 Tage in der Klimakammer mit 8 h light/16 h dunkel Photoperiode (bei ​​einer photosynthetischen Photonenflussdichte von 250 mmol m -2 s -1) bei 23 / ​​19 ° C hell / dunkel Temperatur-Regime und 75% relative Feuchte. </li></ol> * Tipp 1: Bleach-Lösung wird-up durch Verdünnung eines Haushalts Clorox, mit 6% Natriumhypochlorit und Zugabe von Tween-20 bis zu einer Endkonzentration von 0,1%. Teil 3. Isolation von Protoplasten aus Arabidopsis-Keimlinge. <ol><li> Scheiben von 2 g 14-Tage alte Keimlinge mit einer frischen Rasierklinge in 15 ml Filter-sterilisiert TVL Solution. Wir hacken Pflanzenmaterial in sterile Einweg-Petrischalen. </li><li> Transfer gehackte Gewebe in 200 ml Becherglas mit 20 ml Filter-sterilisiert Enzymlösung, wirbeln den Becher zu lassen Gewebe mischen sich mit Enzymlösung, mit Parafilm und Alufolie abdecken. </li><li> Schütteln Sie Pflanzengewebe bei 35 Umdrehungen pro Minute im Dunkeln bei Raumtemperatur für 16-18 h. </li><li> Sammeln Sie die Freigabe Protoplasten in 50 ml Falcon-Röhrchen durch Sieben bis 8 Schichten der Gaze, pre-Nass-in W5-Lösung. </li><li> Sieve Protoplasten aus der Käse-Tuch noch einmal durch Waschen Sie das Tuch mit 15 ml W5-Lösung. </li><li> Sorgfältig Overlay Protoplasten mit 10 ml W5-Lösung, stören die Zucker-Gradienten; Zentrifuge für 7 min bei 100 g. </li><li> Sammeln Sie 10 ml des Protoplasten an der Schnittstelle von Enzymlösung und W5-Lösung (Abb. 1B) und an einem neuen 50 ml Falcon-Röhrchen. </li><li> 15 ml W5-Lösung, Zentrifuge für 5 min bei 60 g. Entfernen Sie den Überstand. </li><li> Wash Protoplasten frei von Enzymlösung durch Resuspendieren Protoplasten in 15 ml W5-Lösung, Zentrifuge 5 min bei 60 g </li><li> Entfernen Sie den Überstand resuspendieren pelletiert Protoplasten in 1-3ml W5-Lösung. </li><li> Bewerten Protoplasten Ausbeute durch Zellzählung mit einer Zählkammer. </li></ol> Teil 4. Reagenzien: TVL: 0,3 M Sorbit, 50 mM CaCl 2 Filter-sterilisiert und bei -20 ° C. Enzymlösung: 0,5 M Saccharose, 10 mM MES-KOH [pH 5,7], 20 mM CaCl 2, 40 mM KCl, 1% Cellulase (Onozuka R-10), 1% Macerozyme (R10). Filter-sterilisiert und frisch verwenden. W5-Lösung: 0,1% (w / v) Glucose, 0,08% (w / v) KCl, 0,9% (w / v) NaCl, 1,84% (w / v) CaCl 2, 2 mM MES-KOH pH 5,7. Filter-sterilisiert und bei Raumtemperatur lagern. Teil 5: Repräsentative Ergebnisse: Mit Arabidopsis Kulturbedingungen in den Teilen 1 und 2 beschrieben macht gesunde Setzlinge, geeignet für die Isolierung von Protoplasten (Abb. 1A). Protoplasten werden durch Saccharose Dichtegradient gereinigtHNO-Zentrifugation gesammelt und an der Schnittstelle von W5 und Enzym-Lösungen (Abb. 1B). Mit Protoplastenisolierung Verfahren in Teil 3 beschrieben wir in der Regel Ernte 05 bis 10 Oktober 6 von intakten Protoplasten (Abb. 1C) von einem Gramm frische Keimlinge. <img src="/files/ftp_upload/1149/Fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Isolation von Protoplasten aus Arabidopsis-Keimlinge. A, Gewebe aus 14-Tage alte Keimlinge von Arabidopsis gesammelt wurden, und konvertierte zum Protoplasten durch ein modifiziertes Verfahren (Chen und Halkier, 2000). B, wurden Protoplasten durch Saccharose-Dichtegradientenzentrifugation gereinigt und gesammelt an der Schnittstelle von Enzymlösung und W5 Uffer (weiße Pfeile). C, Hellfeld-Mikroskopie von Protoplasten. Mikrophotographien wurden mit Hilfe einer gekühlten CCD-Kamera eine Schnittstelle mit dem Zeiss Axioskop 2 plus Mikroskop.

<ol>
	<li>Cocking, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+the+Isolation+of+Plant+Protoplasts+and+Vacuoles.">A Method for the Isolation of Plant Protoplasts and Vacuoles.</a> Nature. 187, 962-963 (1960).</li><li>Chen, S., Halkier, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+glucosinolate+uptake+by+leaf+protoplasts+of+Brassica+napus.">Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.</a> J Biol Chem. 275, 22955-22960 (2000).</li><li>Robert, S., Zouhar, J., Carter, C., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+vacuoles+from+Arabidopsis+rosette+leaf-derived+protoplasts.">Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts.</a> Nat. Protocols. 2, 259-262 (2007).</li><li>Yoo, S. D., Cho, Y. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2, 1565-1572 (2007).</li><li>Zhai, Z., Sooksa-nguan, T., Vatamaniuk, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+RNAi+as+a+reverse+genetic+approach+for+gene+functional+analysis+in+protoplasts.">Establishing RNAi as a reverse genetic approach for gene functional analysis in protoplasts.</a> Plant Phys. 149, 642-652 (2009).</li></ol>

Die vorläufige Patentanmeldung beschreibt dieses Verfahren für den Einsatz in funktionellen Analysen von Pflanzen-Genen wurden mit Cornell Center For Technology Enterprise und Vermarktung (CCTEC), Juni 2008 eingereicht worden. Docket No 50341/015001: Vatamaniuk, OK, und Zhai, Z. Methode zur schnellen funktionellen Analysen von Pflanzen-Genen.

Um die hohe Ausbeute an intakten Protoplasten ist es sehr wichtig, um Start-up mit gesunden Pflanzen. Verwenden Sie Filter-sterilisiert Lösungen für die Isolierung von Protoplasten. Beachten Sie, dass Protoplasten zerbrechlich sind. Deshalb, wenn Sie Handling Protoplasten, nicht zu verwechseln, Pipette oder Wirbel zu heftig, da es bremst sie. Stattdessen mischen Protoplasten durch langsam drehende oder Abkleben der Zentrifugenröhrchen. Die beschriebene Vorgehensweise ergeben intakte Protoplasten, die lebensfähig sin...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Murashige and Skoog Basal Salt Mixture (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5524</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1296</td>
<td>&nbsp;</td>
<tr>
<td>Petri Dishes (100x15mm)</td>
<td>&nbsp;</td>
<td>Kreckeler Scientific</td>
<td>82-4001</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase (Onozuka R&#x2010;10)</td>
<td>&nbsp;</td>
<td>RPI Corp</td>
<td>C32200</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme (R10)</td>
<td>&nbsp;</td>
<td>RPI Corp</td>
<td>M22010</td>
<td>&nbsp;</td>
<tr>
<td>Cheesecloth wipes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>06-665-29</td>
<td>&nbsp;</td>
<tr>
<td>Lab Rotator</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1167152Q</td>
<td>&nbsp;</td>
<tr>
<td>Allegra X&#x2010;15R Centrifuge</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>392932</td>
<td>&nbsp;</td>
<tr>
<td>Axioskop2 Plus Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana

Protoplasten sind Pflanzenzellen, die hatten ihre Zellwände enzymatisch entfernt haben. Isolation von Protoplasten aus verschiedenen pflanzlichen Geweben wurde erstmals berichtet, mehr als 40 Jahren<sup&gt; 1</sup&gt; Und hat sich seitdem so angepasst, dass eine Vielzahl von zellulären Prozessen, wie subzelluläre Lokalisierung von Proteinen, die Isolierung von intakten Organellen und gezielte Gen-Inaktivierung durch doppelsträngige RNA-Interferenz (RNAi)-Studie<sup&gt; 2-5</sup&gt;. Die meisten der Protoplastenisolierung Protokolle verwenden Blattgewebe von reifen Arabidopsis (zB 35-Tage alte Pflanzen)<sup&gt; 2-4</sup&gt;. Wir modifizierten bestehenden Protokollen durch den Einsatz von 14-Tage alten Arabidopsis-Keimlinge. In diesem Verfahren ergab ein Gramm 14-Tage alte Keimlinge 5 10<sup&gt; 6</sup&gt; -10<sup&gt; 7</sup&gt; Protoplasten, dass intakte mindestens 96 Stunden bleiben. Die Ausbeute an Protoplasten aus Sämlingen ist vergleichbar mit Zubereitungen aus Blättern von Arabidopsis reifen, sondern von 35-36 Tagen, Isolierung von Protoplasten wird in 15 Tagen abgeschlossen. Dies ermöglicht die Verringerung der Zeit und das Wachstum münden, die für die Isolierung von Protoplasten, wenn reifen Pflanzen verwendet werden, erforderlich sind, und beschleunigt den Downstream Studien, dass intakte Protoplasten erfordern.

Watch this Scientific Journal Video about Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana at JoVE.com

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Dieses Video zeigt ein Verfahren zur Isolierung von intakten Protoplasten aus Geweben des 14-Tage alte Keimlinge von Arabidopsis. Da die isolierten Protoplasten intakt bleiben für mindestens 96h und sind aus Sämlingen statt einer Monate alten reifen Pflanzen isoliert, beschleunigt dieses Verfahren Assays erfordert intakte Protoplasten.

Isolation von Protoplasten aus Geweben von 14-Tage alte Keimlinge von Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

isolation-protoplasts-from-tissues-14-day-old-seedlings-arabidopsis

Research

JoVE Journal

Biology

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

35.7K Aufrufe.  Cornell University. Dieses Video zeigt ein Verfahren zur Isolierung von intakten Protoplasten aus Geweben des 14-Tage alte Keimlinge von Arabidopsis. Da die isolierten Protoplasten intakt bleiben für mindestens 96h und sind aus Sämlingen statt einer Monate alten reifen Pflanzen isoliert, beschleunigt dieses Verfahren Assays erfordert intakte Protoplasten.

Serie: Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

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 ...

Forschung

Biologie

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 ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Schnelle Analyse von Circadian Phänotypen in Arabidopsis Protoplasten Transfizierte mit einem lumineszenten Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

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 ...

Raum-zeitliche Untersuchung der ERL1-Dynamik mittels konfokaler Bildgebung

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Autor im Rampenlicht: Bildbasierte Methoden zur Untersuchung von Membrantransportereignissen in Zellen der Stomata-Linie  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Bildbasierte Methoden zur Untersuchung von Membrantransportereignissen in stomatalen Zellen

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

Eine verbesserte Methode zur Messung von Pollenhydratationsprofilen in Arabidopsis thaliana

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Autor im Rampenlicht: Ein hochauflösender, in vivo Pollenhydratations-Bioassay mit einem einzigen Korn für Arabidopsis thaliana  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Ein hochauflösender Einzelkorn-In-vivo-Pollenhydratations-Bioassay für Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...