IEM möchte den dänischen Research Council (FTP und FNU) für die Finanzierung zu bestätigen. ERL erkennt die Unterstützung eines ARC DP gewähren. AB erkennt die Unterstützung des ARC Centre of Excellence in Plant Cell Walls Zuschuss.

Ein. Tissue Collection &amp; Vorbereitung <ol><li> Sammeln Sie 100 mg Frischgewicht Pflanzengewebe (ein Minimum von 10 mg Trockengewicht) in mindestens dreifacher Ausfertigung für jedes Gewebe von Interesse. Die folgenden Schritte beschreiben die Herstellung der Zellwand Material aus vegetativen Geweben. Im Falle der Lagerung Gewebe wird un-wanted Stärke enzymatisch, bevor mit der Extraktion von Zellwandpolymere wie zuvor beschrieben 13 entfernt. </li><li> Homogenisierung der Proben zu einem feinen Pulver mit flüssigem Stickstoff unter Verwendung eines Qiagen TissueLyser II, mit 24 Rohradapter Sets und 3 mm Wolframcarbid Kügelchen (30 Hz, 2 x 30 Sekunden). Alternativ, wenn nur wenige Proben verarbeitet werden, ist ein Mörser und Stößel genutzt. </li><li> Übertragen Sie die Homogenate in 10 ml Kunststoff-konische Röhrchen. </li><li> Planen Zellwandmaterial durch Waschen der Homogenate in 10 ml 80% v / v Ethanol bei RT und Vortexen kräftig für 2 min. </li><li> Zentrifuge Proben bei 3.500 xg und Überstand verwerfen. Wiederholen der 70% Ethanol wäscht mindestens dreimal oder bis der Überstand klar ist, insbesondere für Gewebe enthalten Chlorophyll. </li><li> Führen Sie eine letzte Wäsche mit 100% Aceton und lassen Sie die Pellets mit Alkohol unlöslichen Rückständen (AIR) an der Luft trocknen über Nacht. </li><li> Die Luftproben Sieb mit einer Maschenweite 0,4 mm 2, um ein feines, homogenes Pulver zu erreichen und zu entfernen größeren, schlecht gemahlenen Partikel, die irgendwann verbleiben im Homogenat. </li><li> 10 mg wiegen AIR Probe in Mikroröhrchen auf, jeweils mit einem 3 mm Glasperlen, um das Mischen der Proben zu erleichtern. </li></ol> 2. Extraktion von Zellwand Glykane <ol><li> Pektinen, und Polymere mit Pektine zugeordnet sind, aus den Proben durch Zugabe von 500 ul 50 uM CDTA (pH 7,5) zu jeder Probe extrahiert. </li><li> Vortexen die Rohre zu gewährleisten, ist das Lösungsmittel in Kontakt mit dem Probenmaterial und dann Mischen mit der TissueLyser 3 h bei 8 Hz. </li><li> Zentrifuge Proben bei 12.000 xg, sorgfältig reMove der Überstände und bei 4 ° C. </li><li> Waschen Pellets in 1 ml deionisiertem Wasser zu verdünnen und entfernen Sie alle verbleibenden Lösungsmittel, Wirbel und Zentrifuge bei 13.000 x g. Wiederholen Sie diesen Schritt, ohne das Pellet und entfernen Sie die gesamte Flüssigkeit aus den Rohren, bevor Sie den nächsten Schritt. </li><li> Vernetzen Glycane werden sequentiell von der verbleibenden Zellwand Pellet mit 500 ul 4 M NaOH extrahiert mit 0,1% w / v NaBH4, nach dem gleichen Verfahren in den Schritten 2.1 beschrieben - 2,4 für die Extraktion CDTA. NaBH 4 zugegeben, um den Aldehyd (oder Keto)-Gruppe am reduzierenden Ende der Polysaccharide zu einer Alkohol wodurch Basis Schälen von Polysacchariden zu reduzieren. </li><li> Nach Zentrifugation werden Überstände wieder entfernt, gelagert bei 4 ° C, und die Pellets zweimal in deionisiertem Wasser, bevor zum nächsten Extraktion. </li><li> Als optionaler Schritt werden restliche Polymere wie Cellulose mit 500 ul cadoxen (31% v / v 1,2-diamin extrahiertennoethane mit 0,78 M CdO) nach dem gleichen Verfahren wie in den Schritten 2.1 beschrieben - 2.4. Alternativ können absolute Zellulose Inhalt verbleibenden Pellets ermittelt Acetic / Nitric Assays (siehe Diskussion) werden. </li></ol> 3. Printing Microarrays <ol><li> Centrifuge Überstände, die Zellwandpolymere bei 13.000 xg gewonnen, um Partikel zu entfernen. </li><li> Legen Sie 50 ul jeder Probe in ein Polypropylen-384-Well Mikrotiterplatten mit einem pre-designed benutzerdefinierte Layout, wo Proben nach Gewebetyp und Extraktionstyp angeordnet sind. </li><li> Verdünnen Sie die Zellwandpolymer Probe in einer 0, 5 und 25 × seriellen Verdünnungsreihe mit destilliertem Wasser. </li><li> Parameter wie Pin Höhe, Erhebung und Verweilzeit und Waschschritte sind auf der Software-Steuerung des Microarrayer gesetzt. </li><li> Die Feuchtigkeit der Druckkammer wird mit 60% gesteuert wird, um Verdunstung zu verhindern Probe. </li><li> Der Druckauftrag wird gestartet mit LabNEXT software und ein Programm, das auf dem Mikroarray Layout entspricht. </li><li> Der Roboter verwendet Kapillarkanal Stifte, um Lösungen aus der Probe Platte auf 20 x 20 cm Nitrozellulosemembran, die zu einer flachen Platte in der Maschine befestigt ist gedruckt. Jeder Punkt auf dem Array enthält 15 nl einer Lösung und ist in dreifacher Ausfertigung gedruckt. </li><li> Identische Microarrays werden nebeneinander auf der Membran und Schnitt in einzelnen Arrays gedruckt, nachdem der Druckauftrag abgeschlossen ist. </li><li> In jedem Versuch die Arrays können modifiziert werden, um mehr oder weniger Proben, Verdünnungen oder repliziert unterzubringen. </li></ol> 4. Sondierung Glycan Microarrays <ol><li> Nach dem Drucken, blockieren die einzelnen Mikroarrays in 5% w / v Magermilchpulver in Phosphat-gepufferter Kochsalzlösung (MPBS) bei Raumtemperatur für 2 Stunden, um die unspezifische Bindung zu reduzieren. </li><li> Sonde Mikroarrays mit monoklonalen Antikörpern, die für Zellwand-Glykan Epitope für 2 Stunden in MPBS. Die Mehrzahl der monoklonalen antibOdies gegen Zellwand Glycane sind kommerziell von drei Unternehmen; Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ), Carbosource Services ( <a href="http://www.carbosource.net" target="_blank">www.carbosource.net</a> ) und PlantProbes ( <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ). </li><li> Einschließen Negativkontrolle, ein Mikroarray mit nur MPBS und keine primären Antikörper inkubiert. </li><li> Waschen der Mikroarrays 3mal in phosphatgepufferter Kochsalzlösung (PBS) für 5 min auf unspezifische Bindung zu entfernen. </li><li> Sonde der Mikroarrays mit sekundärem Antikörper, konjugiert an Meerrettichperoxidase (HRP) in MPBS für 2 Stunden. Die meisten monoklonalen Antikörper gegen Zellwand Glykane erfordern anti-Maus oder anti-Ratte Sekundärantikörper. </li><li> Wiederholen Waschschritte 3 mal mit PBS-Puffer für 5 min auf unspezifische Bindung zu entfernen. </li><li> Entwickeln Sie die Microarrays mit chromogenen (3,3-Diaminobenzidin) oder chemiluminecent (Luminol) Substraten. </li></ol> 5. Quantifizierung <ol><li> Nach der Entwicklung, scannen die einzelnen Mikroarrays mit einem hochauflösenden (1.200 dpi) Desktop-Scanner und speichern Sie die Bilder als negativ, 16-Bit-TIFF-Dateien (Abbildung 3). </li><li> Berechnen Sie die integrale Intensität jedes Spots mit Xplore Image Processing Software (LabNEXT) mit einem automatisierten Raster-Werkzeug ausgestattet. Das Integral Spotintensität wird aus der Summe der Pixel in der Umgebung jeder Rasterfläche Spot stammt. </li><li> Die Grid-Daten für jeden Mikroarray als txt-Datei exportiert und kann manuell in eine Excel-Tabelle zur Analyse importiert werden. Eine Online-Werkzeug ( <a href="http://arraytemplates.charliehart.com.au/" target="_blank">http://microarray.plantcell.unimelb.edu.au/</a> ) wurde entwickelt, um automatisch zu übersetzen und verarbeiten Daten einzelner txt Dateien. </li><li> Der integrierte Spotintensität wird über Druck repliziert gemittelt und Verdünnungen, eine &quot;Kassamittelkurs erhaltenIntensität &quot;Wert für jede Probe (Abbildung 2). Alternativ werden spot Signale entsprechend nur einem Verdünnungswert auf dem Array verwendet werden, um die relative Abundanz Glycan Epitop für jede Probe zu quantifizieren. </li><li> Die relative mittlere Spotintensitäten zwischen verschiedenen Proben werden als Heatmap (Abbildung 4) mithilfe der bedingten Formatierung in Excel oder online HeatMapper Werkzeuge (präsentiert <a href="http://bar.utoronto.ca/welcome.htm" target="_blank">http://bar.utoronto.ca/welcome.htm</a> ). Die Daten für jeden Antikörperart wird auf 100 korrigiert und eine 5% Cutoff-Wert enthalten sind die Hintergrundsignals und Fehlalarme zu entfernen. </li></ol>

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Kompp ist eine schnelle und sensitive Methode zur Profilierung der Glycan Zusammensetzung von Hunderten pflanzlichen Proben in einer Angelegenheit von Tagen. Diese Methode ergänzt die bereits vorhandenen bakteriellen oder Säugetier-Glykan Array-Plattformen für das Hochdurchsatz-Screening von Kohlenhydrat-Wechselwirkungen mit Glycan-bindende Proteine ​​wie Lektine, Rezeptoren, Antikörper und 16. Mit einer großen Vielfalt von Sonden zur Erkennung von Zellwand Glykane, ist es möglich, detaillierte Info...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> 3 mm Hartmetall Perlen </td><td> Qiagen </td><td> 69997 </td><td></td></tr><tr><td> Sammlung Mikroröhrchen (1,2 mm) </td><td> Qiagen </td><td> 19560 </td><td> 1,5 ml Mikrozentrifugenröhrchen können ebenfalls verwendet werden </td></tr><tr><td> Qiagen TissueLyser II </td><td> Qiagen </td><td> 85300 </td><td></td></tr><tr><td> 3 mm Glaskugeln </td><td> Sigma Aldrich </td><td> Z143928 </td><td></td></tr><tr><td> CDTA </td><td> Sigma Aldrich </td><td> 34588 </td><td></td></tr><tr><td> Cadmiumoxid </td><td> Sigma Aldrich </td><td> 202894 </td><td></td></tr><tr><td> 1,2-Diaminoethan </td><td> Sigma Aldrich </td><td> 03550 </td><td></td></<html> tr&gt; <tr><td> Nitrozellulosemembran (0,22 um Porengröße) </td><td> GE-Water &amp; Process Technologies </td><td> EP2HY00010 </td><td> unterschiedlichen Porengröße Membranen sind für verschiedene Pin-Typen geeignet </td></tr><tr><td> Xact II Microarrayer Roboter </td><td> Labnext </td><td> 001A </td><td> die Xact II Roboter wurde mit einem benutzerdefinierten 20 x 20 cm Keramikplatte auf die die Nitrozellulosemembran gebunden ist eingepasst </td></tr><tr><td> Xtend RM Microarray Stifte </td><td> Labnext </td><td> 0037-350 </td><td> Pins müssen für das Auffinden von Membranen geeignet </td></tr><tr><td> 384-Well-Mikrotiterplatten (Polypropylen) </td><td> Greiner </td><td> 781207 </td><td></td></tr><tr><td> Anti-Glycan monoklonalen Antikörpern </td><td> Pflanzen Probes / CarboSource / Biosupplies </td><td></td><td> Websites; PlantProbes ( <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ), Carbosource (ww.carbosource.net &quot;target =&quot; _blank &quot;&gt; www.carbosource.net) und Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ). </td></tr><tr><td> Anti-Ratten-IgG (ganze Molekül) - Peroxidase-Antikörper in Ziegen hergestellt. </td><td> Sigma </td><td> A9037 </td><td> die Art der Sekundärantikörper hängt von der primären Antikörper verwendet (z. B. in Ratten-, Mäuse, Ziegen usw. erhöht). </td></tr><tr><td> SIGMA FAST 3,3 &#39;-Diaminobenzidin Tabletten </td><td> Sigma </td><td> D4293 </td><td> die Art des Entwicklungsreagens hängt von den sekundären Antikörper verwendet und das Nachweisverfahren (farbmetrischen oder chemiluminecent). </td></tr><tr><td> SuperSignal West Pico Chemilumineszenz Substrat </td><td> Thermoscientific </td><td> 34080 </td><td> siehe oben </td></tr><tr><td> Xplore Image Processing Software </td><td> LabNext </td><td> 008 </td><td> viele Software mit automatischer Gridding Werkzeuge stehen zur Verfügungum Pixelwert von Microarray-Spots zu messen. </td></tr><tr><td> Pflanzliche Polysaccharide </td><td> Sigma / Megazyme </td><td></td><td></td></tr></tbody></table>

glycan profiling of plant cell wall polymers using microarrays

Pflanzliche Zellwände sind komplexe Matrizen von heterogenen Glykane, die eine wichtige Rolle in der Physiologie und Entwicklung von Pflanzen spielen und die Rohstoffe für menschliche Gesellschaften (zB Holz, Papier, Textil-und Biokraftstoff-Industrie) 1,2. Das Verständnis der Biosynthese und Funktion dieser Komponenten bleibt eine Herausforderung. Zellwand Glycane sind chemisch und konformativ vielfältigen aufgrund der Komplexität ihrer Bausteine, die Glycosylreste. Diese Form Bindungen an mehreren Positionen unterscheiden sich in Ringstruktur, isomerer oder anomeren Konfiguration, und darüber hinaus werden mit einer Anordnung von nicht-substituierten Zuckerreste. Glycan Zusammensetzung variiert in verschiedenen Zell-und / oder Gewebetypen oder sogar Sub-Domänen eines einzelnen Zellwand 3. Außerdem ist deren Zusammensetzung auch während der Entwicklung ein, oder in Reaktion auf Umweltreize 4 modifiziert. In exProzess von 2.000 Gene Pflanzliche Zellwände sind komplexe Matrizen von heterogenen Glykane wird vorausgesagt, dass in der Zellwand Glykan Biosynthese und Modifikation in Arabidopsis 5 einbezogen werden. Jedoch haben relativ wenige der biosynthetischen Gene funktionell charakterisiert 4,5. Reversen Genetik Ansätze sind schwierig, da die Gene oft differentiell exprimiert werden, oft in geringen Konzentrationen, zwischen Zelltypen 6. Außerdem sind mutierte Studien oft durch Gen-Redundanz oder kompensatorische Mechanismen behindert zur Gewährleistung einer angemessenen Zellwand Funktion 7 wird beibehalten. So neuartige Ansätze sind nötig, um schnell prägen die vielfältige Palette von Glykanstrukturen und funktionelle Genomik Ansätze zum Verständnis der Zellwandbiosynthese und Modifikation zu erleichtern. Monoklonale Antikörper (mAbs) 8,9 haben als ein wichtiges Instrument zur Bestimmung Glykan Struktur und Verteilung in Pflanzen entstanden. Diese erkennen distINCT Epitope in die Hauptklassen der pflanzlichen Zellwand Glykane, darunter Pektine, Xyloglucanen, Xylane, Mannane, Glucane und Arabinogalactane. Kürzlich ist ihre Verwendung im großen Maßstab Screening-Experimenten wurde erweitert, um die relative Häufigkeit von Glykanen in einer breiten Palette von Pflanzen-und Gewebetypen gleichzeitig 9,10,11 bestimmen. Hier präsentieren wir eine Microarray-basierte Glykan Screening-Methode aufgerufen Umfassende Microarray Polymer Profiling (Kompp) (Abbildungen 1 und 2) 10,11, die mehrere Proben ermöglicht (100 Sek.) gescreent unter Verwendung einer miniaturisierten Microarray-Plattform mit reduzierter Reagenz-und Probenmengen werden. Die Spot-Signale auf dem Mikroarray kann formal quantifiziert werden, um semi-quantitative Daten über Glykan Epitop Auftreten zu geben. Dieser Ansatz wird auch zur Verfolgung Glykan Veränderungen in komplexen biologischen Systemen 12 und bietet einen umfassenden Überblick der Zellwand Zusammensetzung geeignet vor allem, wenn Vorkenntnisse of ist nicht verfügbar.

Die relative Häufigkeit von Glykanen in sechs Gewebetypen (Anthere Filamente, Pollen, Ovarien, Petalen, Sepalen und Stigmatisierung) von Nicotiana alata Blüten wurde mit Kompp. 3A zeigt eine repräsentative Mikroarrays mit mAb JIM5 spezifisch für teilweise (niedrig) methylesterified Homogalacturonan (HG), ein Epitop, das auf pektischen Polysacchariden 14 auftritt wurde sondiert. Die JIM5 Epitop in CDTA Extrakten aller Blütengeweben detektiert jedoch ist am höchsten in Pollen und niedrigsten in s...

Watch this Scientific Journal Video about Glycan Profiling of Plant Cell Wall Polymers using Microarrays at JoVE.com

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Eine Technik namens<strong&gt; C</strong&gt; Mfassende<strong&gt; M</strong&gt; Icroarray<strong&gt; P</strong&gt; Olymer Profiling (Kompp) zur Charakterisierung von Pflanzenzellwand Glycane beschrieben. Dieses Verfahren kombiniert die Spezifität von monoklonalen Antikörpern, die auf definierte Glycan-Epitope mit einer Miniatur-Mikroarray analytische Plattform ermöglicht Screening Glycan Auftretens in einem breiten Bereich von biologischen Zusammenhänge.

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 ...

glycan-profiling-of-plant-cell-wall-polymers-using-microarrays

Research

JoVE Journal

Biology

14.5K Aufrufe.  University of Melbourne. Eine Technik namens C Mfassende M Icroarray P Olymer Profiling (Kompp) zur Charakterisierung von Pflanzenzellwand Glycane beschrieben. Dieses Verfahren kombiniert die Spezifität von monoklonalen Antikörpern, die auf definierte Glycan-Epitope mit einer Miniatur-Mikroarray analytische Plattform ermöglicht Screening Glycan Auftretens in einem breiten Bereich von biologischen Zusammenhänge.

Serie: Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Bacterial Gene Expression Analysis Using Microarrays

Bakterielle Gene Expression Analysis Using Microarrays

Forschung

Biologie

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Messen pflanzlichen Zellwand Extension (Creep) durch saure pH-Wert und von Alpha-Expansin Induced

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

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

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

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

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

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 ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

Genomweite Analyse der Aminoacylierung (Charging) Ebenen der tRNA mittels Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

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 ...

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 ...

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 ...

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 ...

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.

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 ...