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. Planta cultura y Preparación <ol><li> Crezca las plantas de semillero de cebada hidropónico durante 7 días en una cámara de crecimiento con temperatura controlada (para más detalles, véase 10). NOTA: Es importante tener en cuenta el examen de plantas en diversas etapas de desarrollo, como las necesidades de nutrientes van a cambiar con la edad. </li><li> Un día antes de la experimentación, agrupar varias plantas juntas para hacer una sola réplica (3 plantas por paquete para DI, 6 plantas por paquete para CATE). Plántulas Bundle por envolver un trozo de 2 cm de tubo Tygon alrededor de la parte basal de los brotes, y asegurar el tubo con cinta adhesiva para crear un &quot;collar&quot;. NOTA: El número de plantas por paquete puede variar según las condiciones experimentales 10,13,14. La agrupación se realiza para mejorar las estadísticas y la precisión de la medición, sobre todo cuando la masa de raíces y / o actividad específica son bajos. </li></ol> 2 Preparación de Soluciones Experimental / Materiales contenido &quot;&gt; NOTA: La siguiente se realiza típicamente 1 día antes de la experimentación. <ol><li> Por DI, reunir los siguientes: Pre-etiquetado, etiquetado y soluciones de desorción (para más detalles, véase 11), tubos de centrifugación (por centrifugado de las muestras de las plantas), y los viales de muestra (por material vegetal y la actividad específica [S o; vea abajo]). Airear y mezclar todas las soluciones. </li><li> Para CATE, reunir los siguientes: soluciones, etiquetado aireado bien mezclado y elución (para más detalles, véase 10), embudos, tubos de centrifugación de flujo de salida (por centrifugado de las muestras de plantas), y los viales de muestra (por eluidos, muestras de plantas, y determinación de S O y factor de dilución [D f, véase más adelante]). </li></ol> 3. Preparar radiotrazadores ATENCIÓN: Las siguientes medidas de seguridad se deben tomar antes de trabajar con la radiactividad. <ol><li> Asegúrese de que los requisitos de la radioactiva licencia materiales se entienden y lo siguió. Utilice el equipo de seguridad adecuado (es decir, gafas, guantes, bata de laboratorio, plomo chaleco / cuello) y dosímetros (por ejemplo, un anillo de TLD y placa). Configure blindaje (es decir, plexiglás y ladrillos de plomo) y realizar el trabajo radiactiva detrás de él. Asegúrese de que un contador Geiger-Müller está presente con el fin de monitorear rutinariamente para la contaminación. </li><li> Preparación de 42 K + <ol><li> Coloque un vaso de precipitados limpio y seco en el equilibrio. Cero la balanza. </li><li> Eliminar vial de trazador (20 mCi de 42 K 2 CO 3, en forma de polvo) de los envases y verter trazador en el vaso de precipitados. Tome nota de la masa. </li><li> Pipeta 19,93 ml de dH 2 O, seguido de 0,07 ml de H 2 SO 4, en el vaso de precipitados. Esto conducirá a la siguiente reacción química: 42 K 2 CO 3 (S) + H 2 SO 4 (l) + H2O (l) → 42 </sarriba&gt; K 2 SO 4 (L) + CO 2 (v) + 2H 2 O (l) </li><li> Calcular la concentración de la solución madre radiactivo, dada la masa y el peso molecular de K 2 CO 3, y el volumen (20 ml). NOTA: Si se trabaja con 13 NH 3/13 NH 4 + El marcador se produjo en un ciclotrón mediante el bombardeo de protones del átomo de oxígeno del agua (por lo general resulta en la actividad 100-200 mCi; para los detalles de producción, ver 12). Debido a que la cantidad de NH 14 3/14 NH 4 + es extremadamente baja en estas soluciones, la concentración de N de la solución madre es insignificante. </li></ol></li></ol> 4. afluencia directa (DI) Medición <ol><li> Para 42 K +, pipetear la cantidad de solución de stock radiactivo requerida para alcanzar la concentración final deseada de K + en la solución de etiquetado. <ol><li> Para NH 13 3/13 NH 4 +, pipeta una pequeña cantidad (&lt;0,5 ml) en la solución de etiquetado. Deje que la solución de etiquetado para mezclar bien (a través de la aireación). </li></ol></li><li> Pipetear una sub-muestra de 1 ml de solución de etiquetado en un vial de la muestra y repetir tres veces (4 muestras en total). <ol><li> Medir la radiactividad en viales (en &quot;cuentas por minuto, cpm)&quot;, usando un contador gamma. Asegúrese de que el contador está programado de tal modo que las lecturas de CPM se corrigieron para la desintegración isotópica (esto es particularmente importante para tales trazadores de corta duración). </li><li> Calcular S o (expresada como cpm -1 mol) por el promedio de los recuentos de las cuatro muestras (cpm ml -1) y dividiendo por la concentración de sustrato en solución (mol ml -1). </li></ol></li><li> Sumergir raíces en solución de pre-etiquetado (no radiactivo) durante 5 min, para pre-equilibrar las plantas bajo condiciones de prueba (véasepor ejemplo, 10,13,14 para variaciones en el tiempo de pre-label). </li><li> Sumergir las raíces en el etiquetado de solución (radiactivo) durante 5 min. NOTA: Los tiempos de etiquetado pueden variar en función del experimento 3,4,7-10. </li><li> Transferencia raíces a la solución de desorción durante 5 segundos para eliminar la mayor parte de la radiactividad-adhesión superficie. Transferencia raíces en un segundo vaso de precipitados de la solución de desorción durante 5 min a las raíces más claras de trazador extracelular. </li><li> Diseccionar y brotes separados, brotes basales y raíces. </li><li> Coloque raíces en tubos de centrifugación y las muestras de centrifugado durante 30 segundos en una baja velocidad, centrífuga clínica-grado (~ 5.000 xg) para eliminar de la superficie y el agua intersticial. </li><li> Pesar raíces (peso fresco, FW). </li><li> Contar la radiactividad en muestras vegetales (brotes, brotes basales, y la raíz; vea el paso 4.2.1). </li><li> Calcular el flujo. Calcular afluencia en la planta usando la fórmula Φ = Q * / S o en peso de L donde Φ es el flujo(Mol -1 h -1 g), Q * es la cantidad de trazador acumulada en el tejido (cpm, por lo general en la raíz, disparar y disparar basal, combinado), S o es la actividad específica de la solución de etiquetado (mol cpm - 1), w es el peso fresco de la raíz (g), y L t es el tiempo de etiquetado (hr). NOTA: cálculo más sofisticado se puede hacer para dar cuenta de flujo de salida de trazador simultánea de las raíces durante el etiquetado y desorción, en base a los parámetros obtenidos de CATE (véase más adelante; para más detalles, ver 4). </li></ol> 5. compartimental Análisis Tracer Eflujo (CATE) Medición <ol><li> Prepare la solución de etiquetado y medida S o (consulte los pasos 4.1 a 4.2, más arriba). </li><li> Mida factor de dilución (D f). NOTA: A menudo, la posición de la muestra en relación con el detector en el contador gamma puede influir en la cantidadde radiación medida. Véase la discusión para más detalles. <ol><li> Después de medir o S, añadir 19 ml de H 2 O a cada muestra (de manera que el volumen final = volumen eluido = 20 ml). Conteo de radiactividad en cada muestra de 20 ml (ver paso 4.2.1). </li><li> Calcular D f dividiendo el cpm promedio de las muestras de 1 ml por la cpm media de las muestras de 20 ml. </li></ol></li><li> Sumerja sus raíces en la solución de etiquetado durante 1 hora. </li><li> Retire las plantas de las plantas de soluciones de etiquetado y de transferencia efluir embudo, asegurando todo el material de la raíz está dentro del embudo. Gently plantas seguras al lado del embudo de flujo de salida mediante la aplicación de una pequeña tira de cinta adhesiva sobre el anillo de plástico. </li><li> Vierta suavemente el primer eluato en el embudo. Iniciar temporizador (contando). </li><li> Abra el grifo y recoger el eluido en el vial de la muestra después de 15 segundos (nota: tiempo de elución variará, véase más adelante). Cierre el grifo. Vierta suavemente la próxima eluato en el embudo. </li><li> REPEAt paso 5.6 para el resto de la serie de elución, que sigue, desde el primero al eluato final: 15 seg (cuatro veces), 20 seg (tres veces), 30 seg (dos veces), 40 seg (una vez), 50 sec (una vez), 1 min (25 veces), durante un período de elución total de 29,5 min NOTA: Serie de desorción puede variar según las condiciones experimentales 7-10,13,14. </li><li> Una vez que el protocolo de elución es completa, las plantas de cosecha (pasos 4.6 a 4.8, más arriba). </li><li> Contar la radiactividad en los eluidos y muestras de plantas en el contador gamma (multiplicando la lectura para cada eluato por D f, ver 5.2). </li><li> Parcela liberación trazador (cpm g (FW raíz) -1 min -1) como una función de tiempo de elución. Para condiciones de estado estacionario, realizar regresiones lineales y los cálculos de los flujos, la vida media de intercambio y tamaños de piscina (para más detalles, consulte 6-9). </li></ol>

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The authors have nothing to disclose.

Como se demuestra en los ejemplos anteriores, el método radiotrazador es un poderoso medio de la medición de los flujos unidireccionales de nutrientes y sustancias tóxicas en planta. Figura 1 muestra que NH 3 afluencia puede llegar a más de 225 mmol g -1 h -1, que es tal vez el más alto bona fide transmembrana flujo siempre informó en un sistema planta 13, pero la magnitud de este flujo no sería visible si sólo se midieron los flujos ...

La absorción y distribución de los nutrientes y las sustancias tóxicas influyen fuertemente en el crecimiento vegetal. En consecuencia, la investigación de los procesos de transporte subyacentes constituye un área importante de la investigación en biología de las plantas y de las ciencias agrícolas 1,2, especialmente en el contexto de la optimización nutricional y el estrés ambiental (por ejemplo, estrés por salinidad, toxicidad de amonio). El principal de los métodos para la medición de los flujos en las plantas es el uso de trazadores radioisotópicos, que fue desarrollado significativamente en los años 1950 (véase, por ejemplo, 3) y sigue siendo ampliamente utilizado hoy. Otros métodos, como la medición de agotamiento de los nutrientes del medio de la raíz y / o la acumulación en los tejidos, el uso de microelectrodos vibrantes ion-selectivos tales como MIFE (microelectrodos ión estimación de flujo) y SIET (escanear técnica electrodo selectivo de iones), y el uso de colorantes fluorescentes selectivos de iones, también se aplican ampliamente, pero están limitados en su capacidad para detectar la gripe netaxes (es decir, la diferencia entre la afluencia y de flujo de salida). El uso de radioisótopos, por otro lado, permite al investigador la capacidad única de aislar y cuantificar los flujos unidireccionales, que se puede utilizar para resolver los parámetros cinéticos (por ejemplo, K M y V max), y proporcionar una idea de la capacidad, energética, mecanismos y regulación, de los sistemas de transporte. Las mediciones de flujo unidireccionales hechas con radiotrazadores son particularmente útiles en condiciones donde el flujo en la dirección opuesta es alta, y el volumen de piscinas intracelular es rápida 4. Por otra parte, los métodos de radiotrazadores permiten mediciones que se llevan a cabo bajo concentraciones bastante altas de sustrato, a diferencia de muchas otras técnicas (ver &quot;Discusión&quot;, más abajo), ya que el isótopo trazado se observa contra un fondo de otro isótopo del mismo elemento. Aquí, ofrecemos los pasos detallados para la medición radioisotópica de unidireccional y net flujos de nutrientes minerales y las sustancias tóxicas en plantas intactas. Se hará énfasis en la medición de flujo de potasio (K +), un macronutriente planta 5, y amoniaco / amonio (NH 3 / NH 4 +), otro de macronutrientes que es, sin embargo, tóxico cuando está presente en altas concentraciones (por ejemplo, de 1 10 mM) 2. Usaremos los radioisótopos 42 K + (t 1/2 = 12.36 h) y 13 NH 3/13 NH 4 + (t 1/2 = 9,98 min), respectivamente, en las plántulas intactas de la cebada sistema modelo (Hordeum vulgare L .), en la descripción de los dos protocolos clave: afluencia directa (DI) y el análisis compartimental por flujo de salida de trazador (CATE). Debemos tener en cuenta desde el principio que este artículo sólo se describen los pasos necesarios para llevar a cabo cada protocolo. En su caso, se proporcionan breves explicaciones de los cálculos y la teoría, pero detalladas exposiciones de cada técnica&#39;S fondo y la teoría se pueden encontrar en varios artículos sobre el tema clave 4,6-9. Es importante destacar que estos protocolos son ampliamente transferibles al flujo análisis de otros nutrientes / sustancias tóxicas (por ejemplo, 24 Na +, 22 Na +, Rb + 86, 13 NO 3 -) y para otras especies de plantas, aunque con algunas salvedades (véase más adelante) . También hacemos hincapié en la importancia de que todos los investigadores que trabajan con materiales radiactivos deben trabajar bajo una licencia organizado a través ionizante regulador de la seguridad radiológica de su institución.

<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.

La Figura 1 muestra isotermas encontrados utilizando la técnica de DI (con 13 N), para la afluencia de NH 3 en las raíces de las plántulas de cebada cultivadas intactas a alta (10 mM) NH 4 +, y, o bien bajo (0,02 mM) o alta (5 mM ) K +. Las isotermas muestran la cinética de Michaelis-Menten cuando los flujos de NH 3 se representan gráficamente como una función de la concentración externa NH 3 ([NH 3] ext; ajus...

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.

Flujos de nutrientes minerales y sustancias tóxicas de medición en plantas con Trazadores Radiactivos

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 vistas.  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.

Video: 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.

Expresión recombinante de celulasa de Cel5A<em&gt; Trichoderma reesei</em&gt; En plantas de tabaco

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

Investigación

Medio ambiente

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.

Medición de Precios de los herbicidas Metabolismo en Dicot malas hierbas con un ensayo extirpados Leaf

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.

El intercambio de O bentónica<sub&gt; 2</sub&gt;, N<sub&gt; 2</sub&gt; y nutrientes disueltos usando pequeños incubaciones Core

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.

Caudal continuo monitoreo de nutrientes y sedimentos en las cuencas agrícolas

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.

Evaluación de metano y óxido nitroso flujos de arrozal mediante estática cerrado cámaras de mantenimiento de plantas en espacios vacíos

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.

Protocolo para la producción de Video infrarroja tridimensional de congelación en las plantas

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.

Un protocolo de Rhizobox optimizada para visualizar el crecimiento de las raíces y la capacidad de respuesta a nutrientes localizadas

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.

Medición de la forma y el tamaño de las partículas de lodo activado inmovilizado en Agar con una tubería de Software de código abierto

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.

Distribución de agua de Xylem en plantas leñosas visualizada con un microscopio electrónico de barrido crio-

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.

Uso de Caenorhabditis elegans para el estudio de los efectos trans y multigeneracionales de los tóxicos

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