This work was supported by the Natural Sciences and Engineering Council of Canada (NSERC), the Canada Research Chair (CRC) program, and the Canadian Foundation for Innovation (CFI).

<html> 1. Pflanzen Kultur und Vorbereitung <ol><li> Wachsen Gerstenkeimlingen hydroponically für 7 Tage in einem klimatisierten Wachstumskammer (Details siehe 10). HINWEIS: Es ist wichtig zu prüfen, in Pflanzen bei einer Vielzahl von Entwicklungsstufen, wie Nährstoffbedarf wird mit dem Alter ändern. </li><li> Ein Tag vor dem Experiment, bündeln mehrere Sämlinge zusammen, um ein einzelnes Replikat (3 Pflanzen pro Bündel für DI, 6 Pflanzen pro Bündel für CATE) zu machen. Bundle Sämlinge, indem ein 2-cm-Stück Tygon-Schlauch um den basalen Teil der Triebe, und die Sicherung der Schlauch mit Klebeband, um einen &quot;Kragen&quot; zu schaffen. HINWEIS: Die Anzahl der Pflanzen pro Bündel kann von den experimentellen Bedingungen 10,13,14 variieren. Bündelung wird getan, um Statistiken und Messgenauigkeit zu verbessern, vor allem, wenn Wurzelmasse und / oder spezifische Aktivität sind gering. </li></ol> 2. Vorbereitung für Experimentelle Lösungen / Materialien Inhalt &quot;&gt; Hinweis: der folgende wird in der Regel vor dem Experiment 1 durchgeführt Tag. <ol><li> Für DI, sammeln die folgenden: Pre-Kennzeichnung, Etikettierung, und Desorption Lösungen (für Details siehe 11), Zentrifugenröhrchen (zum Trockenschleudern von Pflanzenproben) und Probenfläschchen (für Pflanzenmaterial und die spezifische Aktivität [S o; siehe unten]). Belüften und mischen alle Lösungen. </li><li> Cate, sammeln die folgenden: gut gemischten, Porenbeton Kennzeichnung und Elution Lösungen (für Details siehe 10), Auslauftrichter, Zentrifugenröhrchen (zum Trockenschleudern von Pflanzenproben) und Probenfläschchen (für Eluate, Pflanzenproben und Bestimmung von S o und Verdünnungsfaktor [D f, siehe unten]). </li></ol> 3. Bereiten Radiotracer ACHTUNG: Die folgenden Sicherheits Schritte sollten vor der Arbeit mit Radioaktivität eingenommen werden. <ol><li> Stellen Sie sicher, dass die Anforderungen der radiotive Materialien Lizenz verstanden und befolgt werden. Richtige Sicherheitsausrüstung (dh, Schutzbrille, Handschuhe, Kittel, Blei Weste / Kragen) und Dosimeter (zB TLD-Ring und Abzeichen) zu tragen. Bis Abschirmung gesetzt (dh, Plexiglas und Bleiziegel) und führen radioaktiven Arbeit dahinter. Stellen Sie sicher, dass ein Geiger-Müller-Zähler, um routinemäßig für die Kontaminationsmonitor vorhanden ist. </li><li> Herstellung von 42 K + <ol><li> Legen Sie ein sauberes, trockenes Becherglas auf die Waage. Null das Gleichgewicht. </li><li> Entfernen Fläschchen Tracer (20 mCi 42 K 2 CO 3, in Pulverform) aus der Verpackung und gießen Tracer in den Becher. Beachten Sie die Masse. </li><li> Pipette 19.93 ml dH 2 O, gefolgt von 0,07 ml H 2 SO 4, in das Becherglas. Dies wird die folgende chemische Reaktion zu fahren: 42 K 2 CO 3 (S) + H 2 SO 4 (l) + H 2 O (l) → 42 </sbis&gt; K 2 SO 4 (L) + CO 2 (v) + 2H 2 O (l) </li><li> Berechnen der Konzentration des radioaktiven Stammlösung angesichts der Masse und einem Molekulargewicht von K 2 CO 3, und das Volumen (20 ml). HINWEIS: Wenn Sie mit 13 NH 13.03 NH 4 + Der Tracer wird in einem Zyklotron über die Protonenbeschuss von dem Sauerstoffatom des Wassers erzeugt (was in der Regel 100-200 mCi Aktivität; Produktions Details siehe 12). Da die Menge der 14 NH 14.03 NH 4 + ist extrem niedrig in diesen Lösungen ist die N-Konzentration der Stammlösung zu vernachlässigen. </li></ol></li></ol> 4. Direkte Influx (DI) Mess <ol><li> Für 42 K +, Pipette die Menge der radioaktiven Stammlösung erforderlich, um die gewünschte endgültige Konzentration von K + in die Etikettierungslösung zu erreichen. <ol><li> 13 NH 13.03 NH 4 +, Pipette eine kleine Menge (&lt;0,5 ml) in die Markierungslösung. Lassen Sie die Markierungslösung gründlich mischen (über Belüftung). </li></ol></li><li> Pipettieren 1 ml Teilstichprobe von Kennzeichnungslösung in ein Probengefäß und wiederholen dreimal (4 Proben insgesamt). <ol><li> Messen Radioaktivität in Fläschchen (in &quot;counts per minute&quot;, cpm), mit einem Gamma-Zähler. Sicherzustellen, daß der Zähler derart programmiert ist, dass cpm Ablesungen für die Isotopen Zerfall korrigiert (dies ist besonders wichtig für solche kurzlebige Tracer). </li><li> Berechnen S o (als cpm ausgedrückt mmol -1) durch Mitteln der Zählungen der vier Proben (cpm ml -1) und dividiert durch die Konzentration des Substrats in Lösung (mmol ml -1). </li></ol></li><li> Tauchen Wurzeln in vor-Kennzeichnung (nicht-radioaktive)-Lösung für 5 min vor, ins Gleichgewicht Pflanzen unter Testbedingungen (siehezB 10,13,14 für Variationen in der Vor-Label-Zeit). </li><li> Tauchen Wurzeln in der Kennzeichnung (radioaktiv)-Lösung für 5 min. HINWEIS: Labeling Zeiten können auf Experiment 3,4,7-10 variieren. </li><li> Übertragen Wurzeln Desorptionslösung 5 Sekunden, um den Großteil der Oberfläche haftende Radioaktivität zu entfernen. Übertragen Wurzeln in einem zweiten Becherglas der Desorption Lösung für 5 min auf weitere deutliche Wurzeln der extrazellulären Tracer. </li><li> Sezieren und separater Sprossen, basale Triebe und Wurzeln. </li><li> Zeigen Wurzeln in Zentrifugenröhrchen und Spin Proben für 30 Sekunden in einem Low-Speed-, klinische-Grade-Zentrifuge (~ 5.000 xg), um Oberfläche und Porenwasser zu entfernen. </li><li> Wiegen Wurzeln (Frischgewicht, FW). </li><li> Graf Radioaktivität in Pflanzenproben (shoot, basal-Shooting, und die Wurzel, siehe Schritt 4.2.1). </li><li> Berechnen Sie den Fluss. Berechnen Zustrom in die Anlage mit Hilfe der Formel Φ = Q * / S o Gew L wobei Φ die Fluss(Mmol g -1 h -1), Q * die Menge des Tracers im Gewebe angesammelt (cpm, in der Regel in Wurzel, schießen, schießen und basalen, kombiniert), die spezifische Aktivität des Markierungslösung (cpm mmol S o - 1) ist, W die Wurzel-Frischgewicht (g) und t L die Kennzeichnung Zeit (hr). HINWEIS: Weitere anspruchsvolle Berechnung kann gemacht werden, um für die gleichzeitige Tracer Efflux aus Wurzeln beim Etikettieren und Desorption ausmachen, basierend auf von CATE erhaltenen Parameter (siehe unten; Details siehe 4). </li></ol> 5. Compartmental Analyse von Tracer Efflux (CATE) Mess <ol><li> Bereiten Etikettierungslösung und Maßnahme S o (siehe Schritte 4,1-4,2, oben). </li><li> Messen Verdünnungsfaktor (D f). HINWEIS: Häufig kann die Position der Probe relativ zu dem Detektor im Gamma-Zähler die Menge beeinflussender Strahlung gemessen. Siehe Diskussion für Details. <ol><li> Nach Messung S o, fügen 19 ml H 2 O zu jeder Probe (wie das Endvolumen = Eluat Volumen = 20 ml). Graf Radioaktivität in jeder 20-ml-Probe (siehe Schritt 4.2.1). </li><li> Berechnen D f durch Division der durchschnittlichen cpm der 1-ml-Proben durch den Mittelwert cpm der 20-ml-Proben. </li></ol></li><li> Tauchen Wurzeln in Etikettierungslösung für 1 Stunde. </li><li> Entfernen Sie Pflanzen aus Etikettierungslösung und Übertragungsanlagen zu Trichter Auslauf, muss, im Trichter alle Wurzelmaterial ist. Sanft sicheren Pflanzen seitlich Auslauftrichter durch Anlegen einer kleinen Bandstreifen über die Kunststoffkragen. </li><li> Gießen Sie vorsichtig das erste Eluat in den Trichter. Timer starten (Hochzählen). </li><li> Öffnen Sie die Zapfen zu sammeln und das Eluat in das Probengefäß nach 15 sec (Hinweis: Elutionszeit variieren, siehe unten). Schließen Sie den Hahn. Gießen Sie vorsichtig die nächste Eluat in den Trichter. </li><li> Repeat Schritt 5.6 für den Rest der Elution Serie, die folgt von der ersten zu der letzten Eluat: 15 sec (vier Mal), 20 s (dreimal), 30 s (zweimal), 40 s (einmal), 50 sec (einmal), 1 min (25-mal), für eine Gesamtdauer von 29,5 Elution min HINWEIS: Desorption Serie kann von den experimentellen Bedingungen 7-10,13,14 variieren. </li><li> Sobald Elutionsprotokoll abgeschlossen ist, Ernte Pflanzen (Schritte von 4,6 bis 4,8, oben). </li><li> Graf Radioaktivität in Eluaten und Pflanzenproben in der Gamma-Zähler (Multiplikation der Lektüre für jeden Eluat durch D f, siehe 5.2). </li><li> Grundstück Tracer Release (cpm g (root FW) -1 min -1) als Funktion der Elutionszeit. Für stationären Bedingungen, führen lineare Regressionen und Berechnungen von Flussmitteln, Halbwertszeiten von Austausch und Poolgrößen (Details siehe 6-9). </li></ol>

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The authors have nothing to disclose.

Wie in den obigen Beispielen gezeigt, ist der Radiotracer-Methode ein mächtiges Mittel zur Messung der unidirektionalen Flüsse der Nährstoffe und Giftstoffe in der Pflanze. Abbildung 1 zeigt, dass NH 3 Zustrom in über 225 mmol g -1 h -1, das ist vielleicht das ist zu erreichen höchste bona fide Transmembranfluss je in einem Anlagensystem 13 berichtet, aber das Ausmaß dieser Fluss nicht sichtbar sein, wenn nur die Nettoflüsse gemessen w...

Die Aufnahme und Verteilung von Nährstoffen und Schadstoffen stark beeinflussen das Pflanzenwachstum. Dementsprechend ist die Untersuchung der zugrunde liegenden Transportprozesse stellt einen wichtigen Bereich der Forschung in der Pflanzenbiologie und Agrarwissenschaften 1,2, vor allem in den Kontexten der Ernährungsoptimierung und Umweltbelastungen (zB Salzstress, Ammoniumtoxizität). Das wichtigste Verfahren zur Messung von Flussmitteln in Pflanzen ist die Verwendung von Radioisotopen-Tracer, die sich in den 1950er Jahren entwickelt wurde (siehe Beispiel 3) und ist bis heute weit verbreitet. Andere Verfahren, wie die Messung von Nährstoffverarmung aus dem Wurzelmedium und / oder Akkumulation in Geweben, die Verwendung von Ionen-selektiven Mikroschwing wie Mife (Mikro Ionenfluß Schätzung) und SIET (Rasterionenselektive Elektrode Technik), und die Verwendung von Ionen-selektiven Fluoreszenzfarbstoffe, werden ebenfalls häufig verwendet, sind aber in ihrer Fähigkeit, Netz Grippe erkennen begrenztxes (dh die Differenz zwischen Zufluss und Abfluss). Die Verwendung von Radioisotopen, auf der anderen Seite ermöglicht es dem Forscher die einzigartige Fähigkeit, zu isolieren und zu quantifizieren unidirektionalen Flüsse, die verwendet werden können, um die kinetischen Parameter zu lösen (zB K M und V max) und geben einen Einblick in die Fähigkeit, Energetik, Mechanismen und die Regulation von Transportsystemen. Gleichfluss-Messungen mit Radiotracer sind besonders unter Bedingungen, wo der Fluss in die entgegengesetzte Richtung ist hoch nützlich, und der Umsatz von intrazellulären Pools ist schnell 4. Außerdem ermöglichen Radiotracer Methoden Messungen unter relativ hohen Substratkonzentrationen im Gegensatz zu vielen anderen Verfahren durchgeführt werden (siehe &quot;Discussion&quot;, unten), weil die zurück Isotop vor dem Hintergrund der anderen Isotop des gleichen Elements beobachtet. Hier bieten wir detaillierte Schritte für die Radioisotopenmessung von unidirektionalen und net Flüsse von Mineralstoffen und Giftstoffen in intakten Pflanzen. Der Schwerpunkt wird auf die Flussmessung von Kalium (K +), einer Pflanze macronutrient 5 und Ammonium / Ammoniak hergestellt werden (NH 3 / NH 4 +), ein anderes Makronährstoffe, die, wenn sie bei hohen Konzentrationen (beispielsweise vorhanden ist, jedoch giftig, 1- 10 mM) 2. Wir werden die Radioisotope 42 K + (t 1/2 = 12,36 h) und 13 NH 13.03 NH 4 + (t 1/2 = 9,98 min), jeweils in intakten Keimlingen der Modellsystem Gerste (Hordeum vulgare L verwenden .), in der Beschreibung der beiden wichtigsten Protokolle: direkte Zustrom (DI) und Kompartment-Analyse durch Tracer-Efflux (CATE). Wir sollten von Anfang an, die in diesem Artikel beschrieben einfach die erforderlich sind, um jedes Protokoll führen Sie die Schritte beachten. Gegebenenfalls werden kurze Erklärungen zu den Berechnungen und Theorie zur Verfügung gestellt, aber detaillierte Darstellungen der einzelnen Verfahren&#39;S Hintergrund und Theorie kann in mehreren wichtigen Artikel über das Thema 4,6-9 gefunden werden. Wichtig ist, dass diese Protokolle allgemein übertragbar Analyse anderer Nährstoffe / Giftstoffe Strom (zB 24 Na +, Na + 22, 86 Rb +, 13 NO 3 -) und anderen Pflanzenspezies, wenn auch mit einigen Einschränkungen (siehe unten) . Wir betonen auch die Bedeutung, dass alle Forscher, die mit radioaktiven Stoffen muss unter einer Lizenz durch ionisierende Strahlung Sicherheitsregler ihrer Institution angeordnet zu arbeiten.

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gamma counter</td>
			<td style="width:103px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td>Model: Wallac 1480 Wizard 3&#34;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Geiger-M&#252;ller counter</td>
			<td style="width:103px;">Ludlum Measurements Inc.</td>
			<td style="width:119px;"></td>
			<td>Model 3 survey meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">400-mL glass beakers</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89000-206</td>
			<td>For pre-absorption, absorption, and desorption solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass funnel</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89000-466</td>
			<td>For efflux funnel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large tubing</td>
			<td style="width:103px;">VWR</td>
			<td align="right" style="width:119px;">529297</td>
			<td>For efflux funnel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medium tubing</td>
			<td style="width:103px;">VWR</td>
			<td align="right" style="width:119px;">684783</td>
			<td>For bundling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small tubing</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">63013-541</td>
			<td>For aeration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aeration manifold</td>
			<td style="width:103px;">Penn Plax Air Tech</td>
			<td style="width:119px;">vat 5.5</td>
			<td>To control/distribute pressurized air into solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass scintillation vials</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">66022-128</td>
			<td>For gamma counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass centrifuge tubes</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">47729-576</td>
			<td>For spin-drying root samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kimwipes</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470173-504</td>
			<td>For spin-drying root samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting scissors</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470001-828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470005-496</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Low-speed clinical centrifuge</td>
			<td style="width:103px;">International Equipment Co.</td>
			<td style="width:119px;">76466M-4</td>
			<td>For spin-drying root samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-mL pipette tips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89079-470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10-mL pipette tips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89087-532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analytical balance</td>
			<td style="width:103px;">Mettler toledo</td>
			<td style="width:119px;">PB403-S/FACT</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

Figur 1 zeigt Isothermen gefunden unter Verwendung der DI-Technik (13 N) für den Zustrom von NH 3 in die Wurzeln von intakten Gerstenkeimlingen bei hohen gezüchtet (10 mM), NH 4 +, und entweder niedrig (0,02 mm) oder hoch (5 mM ) K +. Die Isothermen angezeigt Michaelis-Menten-Kinetik bei der NH 3 Flüsse in Abhängigkeit von der externen NH 3-Konzentration ([NH 3] ext, durch Änderungen in der Lösung auf pH 1...

Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Mess Flüsse von Mineralstoffen und Giftstoffe in Pflanzen, die mit radioaktiver Tracer

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

measuring-fluxes-mineral-nutrients-toxicants-plants-with-radioactive

Research

JoVE Journal

Environment

11.2K Aufrufe.  University of Toronto. In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Serie: Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Expression von rekombinanten Cellulase aus Cel5A<em&gt; Trichoderma reesei</em&gt; In Tabakpflanzen

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Forschung

Umwelt

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

Messraten von Herbizid Metabolism in DICOT Weeds mit einem herausgeschnittenen Blatt Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

Die Benthic Austausch von O<sub&gt; 2</sub&gt;, N<sub&gt; 2</sub&gt; Und gelöste Nährstoffe mit kleinen Kern Inkubationen

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in water resources is a prerequisite for understanding the drivers of the pollutant loads and for making informed water resource management decisions. The commonly used &#34;grab sampling&#34; method provides the concentrations of pollutants at the time of sampling (i.e., a snapshot concentration) and may under- or overpredict the pollutant concentrations and loads. Continuous monitoring of nutrients and sediment has recently received more attention due to advances in computing, sensing technology, and storage devices. This protocol demonstrates the use of sensors, sondes, and instrumentation to continuously monitor in situ nitrate, ammonium, turbidity, pH, conductivity, temperature, and dissolved oxygen (DO) and to calculate the loads from two streams (ditches) in two agricultural watersheds. With the proper calibration, maintenance, and operation of sensors and sondes, good water quality data can be obtained by overcoming challenging conditions such as fouling and debris buildup. The method can also be used in watersheds of various sizes and characterized by agricultural, forested, and/or urban land.

With the advancement of technology and the rise in end-user expectations, the need and use of higher temporal resolution data for pollutant load estimation has increased. This protocol describes a method for continuous in situ water quality monitoring to obtain higher temporal resolution data for informed water resource management decisions.

Kontinuierliche Instream Überwachung von Nährstoffen und Sediment in landwirtschaftlichen Wasserscheiden

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

Bewertung von Methan und Lachgas Wärmeflüsse vom Reisfeld mittels statischer geschlossen Kammern, die Pflege der Pflanzen im Gasraum

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

Protokoll zur Herstellung von dreidimensionalen Infrarot-Video des Einfrierens in Pflanzen

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Eine optimierte Rhizobox-Protokoll zum Wurzelwachstum und Reaktionsfähigkeit auf lokalisierte Nährstoffe zu visualisieren

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

Messung von Form und Größe der Belebtschlamm Partikel unbeweglich in Agar mit einer Open-Source-Software-Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

Xylem-Wasserverteilung in Holzpflanzen visualisiert mit einem Kryo-Scanning-Elektronenmikroskop

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Verwendung von Caenorhabditis elegans zur Untersuchung der trans- und mehrgenerationenlichen Wirkungen von Toxischen Stoffen

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...