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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</ol>

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><tbody><tr><td>Gamma counter</td><td>Perkin Elmer</td><td></td><td>Model: Wallac 1480 Wizard 3&quot;</td></tr><tr><td>Geiger-M&amp;uuml;ller counter</td><td>Ludlum Measurements Inc.</td><td></td><td>Model 3 survey meter</td></tr><tr><td>400 ml glass beakers</td><td>VWR</td><td>89000-206</td><td>For pre-absorption, absorption, and desorption solutions</td></tr><tr><td>Glass funnel</td><td>VWR</td><td>89000-466</td><td>For efflux funnel</td></tr><tr><td>Large tubing</td><td>VWR</td><td>529297</td><td>For efflux funnel</td></tr><tr><td>Medium tubing</td><td>VWR</td><td>684783</td><td>For bundling</td></tr><tr><td>Small tubing</td><td>VWR</td><td>63013-541</td><td>For aeration</td></tr><tr><td>Aeration manifold</td><td>Penn Plax Air Tech</td><td>vat 5.5</td><td>To control/distribute pressurized air into solutions</td></tr><tr><td>Glass scintillation vials</td><td>VWR</td><td>66022-128</td><td>For gamma counting</td></tr><tr><td>Glass centrifuge tubes</td><td>VWR</td><td>47729-576</td><td>For spin-drying root samples</td></tr><tr><td>Kimwipes</td><td>VWR</td><td>470173-504</td><td>For spin-drying root samples</td></tr><tr><td>Dissecting scissors</td><td>VWR</td><td>470001-828</td><td></td></tr><tr><td>Forceps</td><td>VWR</td><td>470005-496</td><td></td></tr><tr><td>Low-speed clinical centrifuge</td><td>International Equipment Co.</td><td>76466M-4</td><td>For spin-drying root samples</td></tr><tr><td>1 ml pipette</td><td>Gilson</td><td>F144493</td><td></td></tr><tr><td>10 ml pipette</td><td>Gilson</td><td>F144494</td><td></td></tr><tr><td>1 ml pipette tips</td><td>VWR</td><td>89079-470</td><td></td></tr><tr><td>10 ml pipette tips</td><td>VWR</td><td>89087-532</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler toledo</td><td>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

Universidad San Sebastian

Research

JoVE Journal

Environment

11.3K 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

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

Messen auf Kohlenstoffbasis Contaminant Mineralisation mit kombinierten CO<sub&gt; 2</sub&gt; Flux und Radiokarbon-Analysen

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

Forschung

Umwelt

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Ein kolorimetrischen Verfahren zur Messung der Eisengehalt in Pflanzen

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Biochemie

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

Mikroplot-Design und Pflanzen- und Bodenprobenvorbereitung für 15Stickstoffanalysen

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.

Zweidimensionale Visualisierung und Quantifizierung von labilen, anorganischen Pflanzennährstoffen und Verunreinigungen im Boden

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow injection analysis, has been developed. The concentrations of radioactive strontium were determined by flow scintillation counting, allowing for on-line and also on-site determination. The proposed system can determine radioactive strontium at industrial relevant levels without further modification using overall analysis time of less than 10 min per aqueous sample. The limit of the detection is 320 fg&#183;g-1 (1.7 Bq/g).

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Here, we present the portable autoRAD platform for quick radiometric separation and determination of 90Sr, an important fission product highly pertinent to nuclear waste.

Automatisierte 90Sr Trennung und Anreicherung in einem Labor-Valve System auf Ppq-Ebene

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow ...

Chemie

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

Eine Methode zur Erhaltung von Feuchtgebietswurzeln und Rhizosphären für die Elementarbildgebung

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

Workflow basiert auf der Kombination der isotopischen Tracer Experimente zu untersuchen, mikrobiellen Stoffwechsel von mehreren Nährstoffquellen

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

Biotechnik

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Ein Cell-to-cell Makromolekulare Transport Assay In Planta Verwendung Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Biologie

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hydroponics: ein vielseitiges System Nährstoff Allocation und Pflanzen Reaktionen auf die Nährstoffverfügbarkeit und Belastung durch toxische Elemente zu studieren

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology&#160;(herbaceous plant/root/soil system)&#160;and biomedical applications&#160;(rat femur and mouse lung).

This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system.

Neutronenradiographie und Computertomographie biologischer Systeme am Hochflussisotopenreaktor des Oak Ridge National Laboratory

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron ...

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

Zusammenfassung

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Messen auf Kohlenstoffbasis Contaminant Mineralisation mit kombinierten CO