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. Plante Culture et préparation <ol><li> Pousser les semis d&#39;orge hydroponique pendant 7 jours dans une chambre de croissance à température contrôlée (pour plus de détails, voir 10). NOTE: Il est important de considérer l&#39;examen des plantes à divers stades de développement, comme les besoins en nutriments vont changer avec l&#39;âge. </li><li> Un jour avant l&#39;expérimentation, regrouper plusieurs plants ensemble pour faire une seule répétition (3 plants par paquet de DI, 6 plantes par paquet pour CATE). Semis Bundle en enroulant un morceau de tube Tygon 2 cm autour de la partie basale des pousses, et la fixation du tube avec du ruban adhésif pour créer un «collier». REMARQUE: Le nombre de plants par faisceau peut varier selon les conditions expérimentales 10,13,14. Le regroupement est fait pour améliorer les statistiques et la précision de mesure, en particulier lorsque la masse racinaire et / ou une activité spécifique sont faibles. </li></ol> 2 Préparation des solutions expérimentales / Matériaux contenu &quot;&gt; REMARQUE: Ce qui suit est généralement effectuée un jour avant l&#39;expérimentation. <ol><li> Pour DI, rassemblez les informations suivantes: pré-étiquetage, l&#39;étiquetage et des solutions de désorption (pour plus de détails, voir 11), tubes de centrifugation (pour l&#39;essorage des échantillons de plantes), et flacons d&#39;échantillon (du matériel végétal et l&#39;activité spécifique [S o; voir ci-dessous]). Aérer et mélanger toutes les solutions. </li><li> Pour CATE, recueillir les suivants: solutions, d&#39;étiquetage et d&#39;élution aéré bien mélangé (pour plus de détails, voir 10), entonnoirs efflux, tubes de centrifugation (pour l&#39;essorage des échantillons de plantes), et flacons d&#39;échantillon (pour éluats, des échantillons de plantes, et détermination de S o et le facteur de dilution [D f; voir ci-dessous]). </li></ol> 3 Préparer radiotraceurs ATTENTION: Les étapes de sécurité suivantes doivent être prises avant de travailler avec la radioactivité. <ol><li> Veiller à ce que les exigences de la radioactive matériaux licence sont comprises et respectées. Porter un équipement de sécurité approprié (c.-à-lunettes, gants, blouse de laboratoire, le plomb gilet / collier) et dosimètres (par exemple, l&#39;anneau de TLD et insigne). Mettre en place de protection (c.-à-plexiglas et briques de plomb) et effectuer des travaux radioactifs derrière elle. Assurez-vous que d&#39;un compteur Geiger-Müller est présent afin de surveiller régulièrement la contamination. </li><li> Préparation de 42 K + <ol><li> Placez un récipient propre et sec sur la balance. La balance à zéro. </li><li> Retirer le flacon de traceur (20 mCi de 42 K 2 CO 3, sous forme de poudre) de l&#39;emballage et versez traceur dans le bécher. Prenez note de la masse. </li><li> Pipette 19,93 ml de dH 2 O, suivi par 0,07 ml de H 2 SO 4, dans le bécher. Cela conduira à la réaction chimique suivante: 42 K 2 CO 3 (S) + H 2 SO 4 (l) + H 2 O (l) → 42 </sjusqu&#39;à&gt; K 2 SO 4 (L) + CO 2 (v) + 2H 2 O (l) </li><li> Calculer la concentration de la solution mère radioactive, compte tenu de la masse et le poids moléculaire de K 2 CO 3, et le volume (20 ml). NOTE: Si vous travaillez avec 13 NH 3/13 NH 4 + Le traceur est produit dans un cyclotron par le bombardement de protons de l&#39;atome d&#39;oxygène de l&#39;eau (qui entraîne généralement 100-200 activité mCi pour des détails de production, voir 12). Parce que la quantité de NH 14 3/14 NH 4 + est extrêmement faible dans ces solutions, la concentration en N de la solution mère est négligeable. </li></ol></li></ol> 4 Afflux directe (DI) Mesure <ol><li> Pour 42 K +, pipette la quantité de solution mère radioactif nécessaire pour atteindre la concentration finale souhaitée de K + dans la solution d&#39;étiquetage. <ol><li> Pour 13 NH 3/13 NH 4 +, pipette une petite quantité (&lt;0,5 ml) dans la solution d&#39;étiquetage. Laisser la solution d&#39;étiquetage à mélanger soigneusement (par l&#39;intermédiaire d&#39;aération). </li></ol></li><li> Introduire à la pipette un sous-échantillon de 1 ml de solution d&#39;étiquetage dans un flacon échantillon et répéter trois fois (4 échantillons au total). <ol><li> Mesurer la radioactivité dans des flacons (en &quot;coups par minute&quot;, cpm), à l&#39;aide d&#39;un compteur gamma. Assurez-vous que le compteur est programmé de telle sorte que les lectures cpm sont corrigées pour la décroissance isotopique (ceci est particulièrement important pour ces traceurs de courte durée). </li><li> Calculer S o (exprimée en cpm mol -1) par la moyenne des comptages des quatre échantillons (cpm ml -1) et en divisant par la concentration du substrat dans la solution (mol ml -1). </li></ol></li><li> Plongez dans les racines pré-étiquetage (non radioactif) solution pendant 5 min, d&#39;effectuer une pré-équilibrer les plantes dans des conditions d&#39;essai (voirpar exemple, 10,13,14 des variations de temps de pré-étiquette). </li><li> Plongez racines dans l&#39;étiquetage (radioactifs) solution pendant 5 min. NOTE: Les temps d&#39;étiquetage peuvent varier en fonction expérience 3,4,7-10. </li><li> Transfert des racines de solution de désorption de 5 sec pour éliminer la plus grande partie de la radioactivité adhérant en surface. Transfert des racines dans un deuxième bécher de solution de désorption pendant 5 min à racines plus claires de traceur extracellulaire. </li><li> Disséquer et pousses séparées, pousses basales, et les racines. </li><li> Passer racines dans des tubes à centrifuger et des échantillons de rotation pendant 30 s à une faible vitesse, de qualité clinique centrifugeuse (~ 5 000 x g) pour éliminer l&#39;eau interstitielle et superficielle. </li><li> Peser racines (poids frais, FW). </li><li> Compter la radioactivité dans des échantillons de plantes (tournage, tournage de base, et la racine; voir l&#39;étape 4.2.1). </li><li> Calculer le flux. Calculer afflux dans la plante en utilisant la formule Φ = Q * / S o poids L où Φ est le flux(Pmol g -1 h -1), Q * est la quantité de traceur accumulée dans les tissus (cpm, généralement dans la racine, tirer, et tirer de base, combiné), S o est l&#39;activité spécifique de la solution de marquage (cpm pmol - 1), w est le poids frais des racines (g), et L t est le temps d&#39;étiquetage (h). REMARQUE: calcul plus sophistiqué peut être faite pour tenir compte simultanée traceur efflux de racines pendant l&#39;étiquetage et la désorption, en fonction des paramètres obtenus à partir de CATE (voir ci-dessous pour des détails, voir 4). </li></ol> 5. parcellaire Analyse par Tracer efflux (CATE) Mesure <ol><li> Préparer une solution d&#39;étiquetage et mesure S o (voir les étapes 04.01 à 04.02, ci-dessus). </li><li> Mesurer le facteur de dilution (D f). REMARQUE: Souvent, la position de l&#39;échantillon par rapport au détecteur dans le compteur gamma peut influencer la quantitéde rayonnement mesuré. Voir discussion pour plus de détails. <ol><li> Après la mesure de S o, ajouter 19 ml de H 2 O pour chaque échantillon (tel que le volume final = volume d&#39;éluat = 20 ml). Nombre de radioactivité dans chaque échantillon de 20 ml (voir l&#39;étape 4.2.1). </li><li> Calculer D f en divisant les cpm moyenne des échantillons de 1 ml par le cpm moyenne des échantillons de 20 ml. </li></ol></li><li> Plongez racines dans une solution d&#39;étiquetage pendant 1 heure. </li><li> Retirez les plantes de solutions d&#39;étiquetage et de transfert des plantes à efflux entonnoir, tout en s&#39;assurant que le système racinaire est dans l&#39;entonnoir. Doucement plantes sécurisés à côté de l&#39;efflux entonnoir en appliquant une petite bande de ruban adhésif sur le collier en plastique. </li><li> Verser doucement le premier éluat dans l&#39;entonnoir. Lancement d&#39;une temporisation (comptage). </li><li> Ouvrez le robinet et recueillir l&#39;éluat dans le flacon d&#39;échantillon après 15 secondes (note: le temps d&#39;élution varie; voir ci-dessous). Fermez le robinet. Verser délicatement la prochaine éluat dans l&#39;entonnoir. </li><li> REPEAt étape 5.6 pour le reste de la série d&#39;élution, qui suit, de la première à l&#39;éluat final: 15 sec (quatre fois), 20 s (trois fois), 30 sec (deux fois), 40 s (une fois), 50 sec (une fois), 1 min (25 fois), pour une période d&#39;élution total de 29,5 min REMARQUE: série de désorption peut varier en fonction des conditions expérimentales 7-10,13,14. </li><li> Une fois que le protocole d&#39;élution est terminée, les plantes de la récolte (étapes 4.6 à 4.8 ci-dessus). </li><li> Compter de la radioactivité dans les éluats et des échantillons de plantes dans le compteur gamma (multipliant la lecture pour chaque éluat par D f, voir 5.2). </li><li> Terrain de presse de traceur (cpm g (FW racine) -1 min -1) en fonction du temps d&#39;élution. Pour l&#39;état d&#39;équilibre, effectuer des régressions linéaires et des calculs de flux, la demi-vie de change, et les tailles de la piscine (pour plus de détails, voir 6-9). </li></ol>

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The authors have nothing to disclose.

Comme le montrent les exemples ci-dessus, la méthode de traceur radioactif est un puissant moyen de mesurer les flux unidirectionnels de nutriments et de substances toxiques in planta. Figure 1 montre que le NH 3 afflux peut atteindre plus de 225 pmol g -1 h -1, ce qui est peut-être le plus de bonne foi flux transmembranaire jamais enregistré dans un système de centrale 13, mais l&#39;ampleur de ce flux ne serait pas visible si seulement ...

L&#39;absorption et la distribution des éléments nutritifs et les substances toxiques influencent fortement la croissance des plantes. En conséquence, l&#39;étude des processus de transport sous-jacents constitue un domaine de recherche important en biologie végétale et en sciences agricoles de 1,2, en particulier dans les contextes d&#39;optimisation nutritionnelle et les contraintes environnementales (par exemple, le stress salin, la toxicité de l&#39;ammonium). Au premier rang des méthodes de mesure des flux dans les plantes est l&#39;utilisation de traceurs radio-isotopiques, qui a été développé de manière significative dans les années 1950 (voir, par exemple, 3) et continue à être largement utilisé aujourd&#39;hui. D&#39;autres méthodes, telles que la mesure de l&#39;épuisement des nutriments à partir du milieu de la racine et / ou l&#39;accumulation dans les tissus, l&#39;utilisation de micro-électrodes vibrants ions sélectifs tels que MIFE (microélectrode ion estimation du flux) et SIET (balayage de la technique de l&#39;électrode sélective d&#39;ions), et l&#39;utilisation de colorants fluorescents ioniques spécifiques, sont également largement utilisés, mais ils sont limités dans leur capacité à détecter la grippe netxes (c&#39;est à dire, la différence entre les flux et l&#39;efflux). L&#39;utilisation de radio-isotopes, d&#39;autre part, permet au chercheur la capacité unique d&#39;isoler et de quantifier les flux unidirectionnels, qui peut être utilisé pour régler les paramètres cinétiques (par exemple, K M et V max), et donner un aperçu de la capacité, énergétique, mécanismes et la régulation, des systèmes de transport. Mesures de flux unidirectionnels à base de radiotraceurs sont particulièrement utiles dans des conditions où le flux dans la direction opposée est élevé, et le chiffre d&#39;affaires de piscines intracellulaires est rapide 4. En outre, les méthodes radiotraceurs permettent des mesures à effectuer en vertu des concentrations assez élevées de substrat, contrairement à beaucoup d&#39;autres techniques (voir «Discussion», ci-dessous), parce que l&#39;isotope tracé est observé sur un fond d&#39;un autre isotope du même élément. Ici, nous fournissons des instructions détaillées pour la mesure radio-isotopique de unidirectionnelle et nET flux de nutriments minéraux et des substances toxiques dans les plantes intactes. L&#39;accent sera mis sur la mesure du flux de potassium (K +), un macro plante 5, et de l&#39;ammoniac / ammonium (NH 3 / NH 4 +), un autre macronutriments qui est, cependant, toxique lorsqu&#39;il est présent à des concentrations élevées (par exemple, 1 10 mM) 2. Nous allons utiliser les radio-isotopes 42 K + (t 1/2 = 12,36 h) et 13 NH 3/13 NH 4 + (t 1/2 = 9,98 min), respectivement, pour les plants intacts de l&#39;orge du système de modèle (Hordeum vulgare L .), dans la description des deux principaux protocoles: afflux directe (DI) et analyse parcellaire par traceur efflux (CATE). Il faut noter d&#39;emblée que cet article décrit simplement les étapes nécessaires pour effectuer chaque protocole. Le cas échéant, une brève explication des calculs et la théorie sont fournis, mais détaillées expositions de chaque techniqueLe contexte et la théorie de &#39;peuvent être trouvés dans plusieurs articles de fond sur le sujet 4,6-9. Fait important, ces protocoles sont largement transférables au flux analyse d&#39;autres éléments nutritifs / substances toxiques (par exemple, 24 Na +, 22 Na +, 86 Rb +, 13 NO 3 -) et à d&#39;autres espèces de plantes, mais avec quelques mises en garde (voir ci-dessous) . Nous soulignons également l&#39;importance que tous les chercheurs travaillant avec des matières radioactives doivent travailler sous une licence arrangé par ionisants la réglementation de la sécurité de rayonnement de leur institution.

<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 figure 1 montre les isothermes trouvés en utilisant la technique de DI (à 13 N), pour l&#39;arrivée de NH 3 dans les racines de plants d&#39;orge intactes cultivées à élevée (10 mM), NH 4 +, et soit faible (0,02 mM) ou élevée (5 mM ) K +. Les isothermes montrent une cinétique de Michaelis-Menten NH 3 lorsque les flux sont tracés en fonction de la concentration externe NH 3 ([NH 3] de poste; ajustés par...

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.

Mesurer les flux d&#39;éléments nutritifs et des substances toxiques minéraux dans les usines avec Traceurs radioactifs

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 vues.  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.

Vidéo: 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 de recombinant cellulase Cel5A de<em&gt; Trichoderma reesei</em&gt; Dans les usines de tabac

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

Recherche

Environnement

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.

Mesurer les taux de métabolisme dans l&#39;herbicide Mauvaises herbes dicotylédones avec un dosage excisés Feuille

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.

Le benthique Échange de O<sub&gt; 2</sub&gt;, N<sub&gt; 2</sub&gt; et nutriments dissous par petit noyau incubations

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.

Surveillance de Instream continu de nutriments et de sédiments dans les bassins hydrographiques agricoles

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.

Évaluation de méthane et de protoxyde d’azote flux de rizière au moyen de la statique fermé Chambers maintenir les plantes dans l’espace de tête

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.

Protocole de fabrication en trois dimensions vidéo infrarouge de gel dans les plantes

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 protocole de Rhizobox optimisé pour visualiser la croissance racinaire et la réceptivité aux nutriments localisées

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.

Mesurer la forme et la taille des particules de boues activées immobilisées dans la gélose avec un Pipeline de logiciels Open Source

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.

La distribution d'eau de Xylem dans les usines ligneuses visualisée avec un microscope électronique cryo-scanné

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.

Utilisation de Caenorhabditis elegans pour étudier les effets trans et multigénérationnels des substances toxiques

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