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 e Preparação <ol><li> A produção de mudas de cevada hidropônico durante 7 dias em câmara de crescimento com clima controlado (para detalhes, ver 10). NOTA: É importante considerar examinando plantas em uma variedade de estágios de desenvolvimento, como as necessidades de nutrientes vai mudar com a idade. </li><li> Um dia antes da experimentação, agrupar várias mudas em conjunto para fazer uma única repetição (3 unidades por conjunto de DI, 6 plantas por pacote para CATE). Mudas Bundle envolvendo um pedaço de 2 cm de Tygon tubulação ao redor da porção basal dos brotos e proteger a tubulação com fita adesiva para criar um &quot;colar&quot;. NOTA: O número de plantas por pacote pode variar de acordo com as condições experimentais 10,13,14. Agrupamento é feito para melhorar as estatísticas e precisão de medição, particularmente quando a massa de raízes e / ou atividade específica são baixos. </li></ol> 2 Preparação das soluções experimentais / Materiais content &quot;&gt; NOTA: O seguinte é normalmente realizada um dia antes da experimentação. <ol><li> Para DI, reúna as seguintes: Pré-etiquetagem, rotulagem, e soluções de dessorção (para detalhes, ver 11), tubos de centrifugação (por spin-secagem de amostras de plantas) e frascos de amostra (para material vegetal e atividade específica [S o; Veja abaixo]). Arejar e misture todas as soluções. </li><li> Para CATE, reunir os seguintes: soluções, rotulagem aerado bem misturado e de eluição (para detalhes, ver 10), funis de efluxo, tubos de centrifugação (spin-para secagem de amostras de plantas) e frascos de amostra (para eluatos, amostras de plantas e determinação de S o fator de diluição e [D f, veja abaixo]). </li></ol> 3 Prepare radiofármaco ATENÇÃO: Os seguintes passos de segurança devem ser tomadas antes de trabalhar com radioatividade. <ol><li> Certifique-se de que os requisitos da Radioaclicença materiais tiva são entendidas e seguidas. Use equipamento de segurança adequado (ou seja, óculos de proteção, luvas, jaleco, chumbo colete / colar) e dosímetros (por exemplo, anel de TLD e crachá). Configure blindagem (ou seja, Plexiglas e tijolos de chumbo) e realizar o trabalho radioativo por trás dele. Certifique-se de que um contador de Geiger-Muller está presente, a fim de monitorizar por rotina para a contaminação. </li><li> Preparação de 42 K + <ol><li> Colocar num copo limpo e seco sobre o equilíbrio. Zero o equilíbrio. </li><li> Remover o frasco de marcador (20 mCi de 42 K 2 CO 3, em forma de pó), a partir de embalagens e despejar traçador para o copo. Tome nota da massa. </li><li> Pipeta 19,93 ml de dH 2 O, seguido por 0,07 mL de H 2 SO 4, para o copo. Isto irá conduzir a seguinte reação química: 42 K 2 CO 3 (S) + H 2 SO 4 (l) + H2O (l) 42 → </sup&gt; K 2 SO 4 (L) + CO2 (v) + 2H 2 O (l) </li><li> Calcula-se a concentração da solução de estoque radioactivo, determinada a massa e o peso molecular de K 2 CO 3, e o volume (20 ml). NOTA: Se estiver trabalhando com 13 NH 3/13 NH 4 + O marcador é produzido em um ciclotrão através do bombardeio de prótons do átomo de oxigênio da água (geralmente resultando em 100-200 atividade mCi, pois detalhes da produção, ver 12). Porque a quantidade de 14 NH 3/14 NH 4 + é extremamente baixo nessas soluções, a concentração de N na solução estoque é negligenciável. </li></ol></li></ol> 4. Influx direta (DI) Medição <ol><li> Para 42 K +, pipetar a quantidade de solução de estoque radioactivos necessária para alcançar a concentração final desejada de K + na solução de marcação. <ol><li> Para 13 NH 3/13 NH 4 +, pipeta uma pequena quantidade (&lt;0,5 ml) para a solução de etiquetagem. Permitir que a solução de etiquetagem para misturar bem (via aeração). </li></ol></li><li> Pipete 1 mL de uma sub-amostra de solução de etiquetagem para um frasco de amostra e repetir três vezes (quatro amostras no total). <ol><li> Medir a radioatividade em frascos (em &quot;contagens por minuto&quot;, CPM), utilizando um contador gama. Certifique-se de que o contador é programado de modo que as leituras de cpm são corrigidos para o decaimento de isótopos (isto é particularmente importante para estes marcadores de curta duração). </li><li> Calcule o S (expresso como cpm mol-1) pela média das contagens das quatro amostras (cpm ml -1) e dividindo pela concentração de substrato em solução (mol ml -1). </li></ol></li><li> Imergir raízes na solução de pré-marcação (não radioactivo) durante 5 minutos, para pré-equilibrar as plantas sob condições de ensaio (verpor exemplo, 10,13,14 a variações no tempo de pré-etiqueta). </li><li> Mergulhe raízes na solução de etiquetagem (radioativo) por 5 min. OBSERVAÇÃO: O tempo de inscrição podem variar de acordo com experiência 3,4,7-10. </li><li> Transferir a solução de dessorção raízes durante 5 segundos para remover a maior parte da radioactividade aderente superfície. Transferir raízes em um segundo recipiente de dessorção solução durante 5 min para as raízes mais claras do traçador extracelular. </li><li> Dissecar e tiros separados, brotos basais e raízes. </li><li> Coloque raízes em tubos de centrífuga e amostras de rotação durante 30 segundos em baixa velocidade, clínico-grade centrífuga (~ 5.000 xg) para remover água superficial e intersticial. </li><li> Pesar raízes (peso fresco, FW). </li><li> Contagem radioatividade em amostras de plantas (caule, basal e raiz; veja o passo 4.2.1). </li><li> Calcula-se o fluxo. Calcular o influxo para a planta por meio da fórmula Φ = Q * / S o peso L onde o fluxo é Φ(Mmol g -1 h -1), Q * é a quantidade de traçador acumulada no tecido (cpm, geralmente em raiz, caule e broto basal, combinado), o S é a atividade específica da solução de etiquetagem (cpm mol - 1), W é o peso fresco (g), e t é o tempo L rotulagem (hr). NOTA: cálculo mais sofisticado pode ser feito para dar conta de efluxo tracer simultânea das raízes durante a rotulagem e dessorção, com base em parâmetros obtidos a partir CATE (veja abaixo; para detalhes, ver 4). </li></ol> 5. compartimental Análise por Tracer efluxo (CATE) Medição <ol><li> Preparar a solução de rotulagem e medida S o (ver passos 4,1-4,2, acima). </li><li> Medir factor de diluição (D f). NOTA: Muitas vezes, a posição da amostra em relação ao detector, no contador de raios gama pode influenciar a quantidadeda radiação medida. Veja a discussão para mais detalhes. <ol><li> Depois de medir o S, adicionar 19 ml de H2O a cada amostra (de tal modo que o volume final = volume de eluato = 20 ml). Contagem radioatividade em cada amostra de 20 ml (veja o passo 4.2.1). </li><li> Calcular f D dividindo a cpm média das amostras de 1 ml da cpm média das amostras de 20 ml. </li></ol></li><li> Mergulhe raízes na solução de etiquetagem por 1 hora. </li><li> Retirar as plantas de solução de etiquetagem e de transferência de plantas para efluxo funil, garantindo todo o material de raiz está dentro do funil. Suavemente plantas seguras para o lado de efluxo de funil através da aplicação de uma pequena tira de fita adesiva sobre o colar de plástico. </li><li> Suavemente despeje o primeiro eluato de dentro do funil. Comece temporizador (contando). </li><li> Abra a torneira e recolher o fluido no frasco da amostra após 15 seg (note: tempo de eluição irá variar, veja abaixo). Feche a torneira. Delicadamente despeje a próxima fluido dentro do funil. </li><li> REPEAt passo 5.6 para o restante da série de eluição, o que se segue, a partir do primeiro para o eluato final: 15 seg (quatro vezes), 20 s (três vezes), 30 s (duas vezes), 40 sec (uma vez), 50 seg (uma vez), 1 min (25 vezes), para um período total de eluição de 29,5 minutos NOTA: Série A dessorção pode variar de acordo com as condições experimentais 7-10,13,14. </li><li> Uma vez que o protocolo de eluição é completa, as plantas de colheita (passos de 4,6-4,8, acima). </li><li> Contagem de radioactividade em eluatos e as amostras de plantas no contador gama (multiplicando a leitura para cada eluato por D f, ver 5.2). </li><li> Lote libertação do marcador (cpm g (FW raiz) -1 min -1) como uma função de tempo de eluição. Para condições de estado estacionário, realizar regressões lineares e cálculos de fluxos, meias-vidas de troca e tamanhos de piscina (para detalhes, ver 6-9). </li></ol>

<ol>
	<li>Kronzucker, H. J., Coskun, D., Schulze, L. M., Wong, J. R., Britto, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+as+nutrient+and+toxicant.">Sodium as nutrient and toxicant.</a> Plant Soil. 369, 1-23 (2013).</li><li>Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NH4++toxicity+in+higher+plants:+a+critical+review.">NH4+ toxicity in higher plants: a critical review.</a> J. Plant Physiol. 159, 567-584 (2002).</li><li>Epstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+ion+absorption+by+roots.">Mechanism of ion absorption by roots.</a> Nature. 171, 83-84 (1953).</li><li>Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+unidirectional+influx+be+measured+in+higher+plants?+A+mathematical+approach+using+parameters+from+efflux+analysis.">Can unidirectional influx be measured in higher plants? A mathematical approach using parameters from efflux analysis.</a> New Phytol. 150, 37-47 (2001).</li><li>Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+potassium+transport+in+plants.">Cellular mechanisms of potassium transport in plants.</a> Physiol. Plant. 133, 637-650 (2008).</li><li>Walker, N. A., Pitman, M. G., L&#252;ttge, U., >Pitman, M. .. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+fluxes+across+membranes.">Measurement of fluxes across membranes.</a> Encyclopedia of plant physiology. 2 Part A, (1976).</li><li>Kronzucker, H. J., Siddiqi, M. Y., Glass, A. D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+13NH4++efflux+in+spruce+roots+-+A+test+case+for+phase+identification+in+compartmental+analysis.">Analysis of 13NH4+ efflux in spruce roots - A test case for phase identification in compartmental analysis.</a> Plant Physiol. 109, 481-490 (1995).</li><li>Siddiqi, M. Y., Glass, A. D. M., Ruth, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+uptake+of+nitrate+in+barley.+3.+Compartmentation+of+NO3-.">Studies of the uptake of nitrate in barley. 3. Compartmentation of NO3-.</a> J. Exp. Bot. 42, 1455-1463 (1991).</li><li>Lee, R. B., Clarkson, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen-13+studies+of+nitrate+fluxes+in+barley+roots.+1.+Compartmental+analysis+from+measurements+of+13N+efflux.">Nitrogen-13 studies of nitrate fluxes in barley roots. 1. Compartmental analysis from measurements of 13N efflux.</a> J. Exp. Bot. 37, 1753-1767 (1986).</li><li>Coskun, D., Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+mechanism+of+potassium+release+from+barley+roots:+an+in+planta+42K++analysis.">Regulation and mechanism of potassium release from barley roots: an in planta 42K+ analysis.</a> New Phytol. 188, 1028-1038 (2010).</li><li>Britto, D. T., Kronzucker, H. J., Maathuis, F. .. J. .. M. .. ,. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluxes+measurements+of+cations+using+radioactive+tracers.">Fluxes measurements of cations using radioactive tracers.</a> Plant Mineral Nutrients: Methods and Protocols, Methods in Molecular Biology. Volume 953, 161-170 (2013).</li><li>Meeks, J. C., Knowles, R. ,., Blackburn, T. .. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13N+techniques.">13N techniques.</a> Nitrogen isotope techniques. , 273-303 (1993).</li><li>Coskun, D., Britto, D. T., Li, M., Becker, A., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+ammonia+gas+transport+accounts+for+futile+transmembrane+cycling+under+NH3/NH4++toxicity+in+plant+roots.">Rapid ammonia gas transport accounts for futile transmembrane cycling under NH3/NH4+ toxicity in plant roots.</a> Plant Physiol. 163, 1859-1867 (2013).</li><li>Coskun, D., Britto, D. T., Li, M., Oh, S., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+and+plasticity+of+potassium+channels+and+high-affinity+transporters+in+roots+of+barley+and+Arabidopsis.">Capacity and plasticity of potassium channels and high-affinity transporters in roots of barley and Arabidopsis.</a> Plant Physiol. 162, 496-511 (2013).</li><li>Johansson, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+K++modulates+the+activity+of+the+Arabidopsis+potassium+channel+SKOR+via+an+unusual+mechanism.">External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism.</a> Plant J. 46, 269-281 (2006).</li><li>Nocito, F. F., Sacchi, G. A., Cocucci, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+depolarization+induces+K++efflux+from+subapical+maize+root+segments.">Membrane depolarization induces K+ efflux from subapical maize root segments.</a> New Phytol. 154, 45-51 (2002).</li><li>Wang, M. Y., Glass, A. D. M., Shaff, J. E., Kochian, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium+uptake+by+rice+roots.+3.+Electrophysiology.">Ammonium uptake by rice roots. 3. Electrophysiology.</a> Plant Physiol. 104, 899-906 (1994).</li><li>Walker, D. J., Leigh, R. A., Miller, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+homeostasis+in+vacuolate+plant+cells.">Potassium homeostasis in vacuolate plant cells.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 10510-10514 (1996).</li><li>Holm, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NH3+and+NH4++permeability+in+aquaporin-expressing+Xenopus+oocytes.">NH3 and NH4+ permeability in aquaporin-expressing Xenopus oocytes.</a> Pflugers Archiv. Eur. J. Physiol. 450, 415-428 (2005).</li><li>Britto, D. T., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-stimulation+of+13NH4++efflux+provides+evidence+for+the+cytosolic+origin+of+tracer+in+the+compartmental+analysis+of+barley+roots.">Trans-stimulation of 13NH4+ efflux provides evidence for the cytosolic origin of tracer in the compartmental analysis of barley roots.</a> Funct. Plant Biol. 30, 1233-1238 (2003).</li><li>Malagoli, P., Britto, D. T., Schulze, L. M., Kronzucker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Futile+Na++cycling+at+the+root+plasma+membrane+in+rice+(Oryza+sativa+L.):+kinetics,+energetics,+and+relationship+to+salinity+tolerance.">Futile Na+ cycling at the root plasma membrane in rice (Oryza sativa L.): kinetics, energetics, and relationship to salinity tolerance.</a> J. Exp. Bot. 59, 4109-4117 (2008).</li><li>Kronzucker, H. J., Britto, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+transport+in+plants:+a+critical+review.">Sodium transport in plants: a critical review.</a> New Phytol. 189, 54-81 (2011).</li></ol>

The authors have nothing to disclose.

Tal como demonstrado nos exemplos acima, o método de traçador é um meio poderoso para medir fluxos unidireccionais de nutrientes e de agentes tóxicos in planta. Figura 1 mostra que o influxo de NH3 pode chegar a mais de 225 g mol -1 hora -1, o que é, talvez, o maior bona fide fluxo transmembrana já registrado em um sistema de planta 13, mas a magnitude deste fluxo não seria visível se apenas fluxos líquidos foram medidos. Isto aconte...

A absorção e distribuição dos nutrientes e toxinas influenciar fortemente o crescimento da planta. Assim, a investigação de processos de transporte subjacentes constitui uma importante área de pesquisa em biologia de plantas e ciências agrárias 1,2, especialmente nos contextos de otimização nutricional e estresse ambiental (por exemplo, estresse salino, a toxicidade de amônio). O chefe entre os métodos para a medição de fluxos em plantas é o uso de traçadores radioisótopos, que foi desenvolvido de forma significativa na década de 1950 (ver, por exemplo, 3) e continua a ser amplamente utilizado hoje. Outros métodos, como a medição de esgotamento dos nutrientes do meio de raiz e / ou acumulação nos tecidos, o uso de microeletrodos vibrando íon-seletivo, tais como Mife (microeletrodos ion estimativa de fluxo) e SIET (varredura técnica do eletrodo íon-seletivo), e uso de corantes fluorescentes íon-seletivo, também são amplamente aplicadas, mas são limitados em sua capacidade de detectar gripe netxes (isto é, a diferença entre o influxo e de efluxo). A utilização de radioisótopos, por outro lado, permite que o investigador a capacidade única para isolar e quantificação dos fluxos unidireccionais, que pode ser usada para resolver os parâmetros cinéticos (por exemplo, K M e V max), e fornecer informações sobre a capacidade, energética, mecanismos e regulação, dos sistemas de transporte. Medições de fluxos unidirecionais feitos com radiofármacos são particularmente úteis em condições onde o fluxo na direção oposta é alta, eo volume de negócios de pools intracelulares é rápida 4. Além disso, métodos de radiofármaco permite que as medições sejam realizadas sob concentrações bastante elevadas de substrato, ao contrário de muitas outras técnicas (ver &quot;Discussão&quot;, abaixo), porque o isótopo traçado observa-se contra um fundo de outro isótopo do mesmo elemento. Aqui, nós fornecemos instruções detalhadas para a medição de radioisótopos unidirecional e net fluxos de nutrientes minerais e substâncias tóxicas em plantas intactas. A ênfase será feita na medição de fluxo de potássio (K +), um macronutriente planta 5, e de amoníaco / amónio (NH 3 / NH 4 +), que é um outro macronutriente, no entanto, tóxicos quando presente em concentrações elevadas (por exemplo, 1 10 mM) 2. Vamos usar os radioisótopos 42 K + (t = 12,36 meia hr) e 13 NH 3/13 NH 4 + (t 1/2 = 9,98 min), respectivamente, em plântulas intactas do sistema modelo de cevada (Hordeum vulgare L .), na descrição de dois protocolos principais: influxo directa (DI) e análise compartimental por efluxo do marcador (CATE). Devemos notar desde o início que este artigo simplesmente descreve as etapas necessárias para realizar cada protocolo. Sempre que necessário, breves explicações sobre cálculos e teoria são fornecidos, mas detalhadas exposições de cada técnica&#39;S de fundo e teoria pode ser encontrada em vários artigos importantes sobre o assunto 4,6-9. É importante ressaltar que estes protocolos são amplamente transferíveis ao fluxo análise de outros nutrientes / tóxicos (por exemplo, 24 de Na +, Na + 22, 86 Rb +, 13 NO 3 -) e outras espécies de plantas, embora com algumas ressalvas (veja abaixo) . Ressaltamos também a importância de que todos os pesquisadores que trabalham com materiais radioativos devem trabalhar sob uma licença organizadas através ionizante regulador de segurança de radiação de sua instituição.

<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.

A Figura 1 mostra isotérmicas encontrados utilizando a técnica DI (com 13 N), para o influxo de NH 3 em raízes de plântulas de cevada intactos cultivados em alta (10 mM), NH 4 +, e qualquer baixa (0,02 mM) ou alta (5 mM ) K +. As isotérmicas de apresentar uma cinética de Michaelis-Menten, quando NH 3 fluxos são representados como uma função da concentração externa de NH 3 ([NH3] ext; ajustados por alteraç...

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.

Medindo Fluxos de nutrientes minerais e substâncias tóxicas em plantas com Traçadores Radioativos

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

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11.2K visualizações.  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.

Vídeo: 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.

Expressão da proteína recombinante a partir da celulase Cel5A<em&gt; Trichoderma reesei</em&gt; Em Plantas de tabaco

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

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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.

Medindo Taxas de Herbicida Metabolismo em Dicot Weeds com um ensaio extirpado Folha

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.

A Bolsa Benthic de O<sub&gt; 2</sub&gt;, N<sub&gt; 2</sub&gt; e nutrientes dissolvidos Usando pequeno núcleo incubações

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.

Monitoramento Contínuo de Nutrientes e Sedimentos em Bacias 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.

Avaliação de metano e óxido nitroso fluxos de arrozal por meio estático fechado Chambers manter plantas dentro Headspace

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 a produção de vídeo infravermelho tridimensional de congelamento em 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.

Um protocolo otimizado de Rhizobox para visualizar o crescimento da raiz e receptividade aos 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.

Medindo a forma e o tamanho das partículas de lodo ativado imobilizada em ágar com um Pipeline de Software de código aberto

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.

Distribuição da água de xylem em plantas lenhosas visualizadas com um microscópio de elétron da Cryo-exploração

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.

Usando Caenorhabditis elegans para estudar trans-e multi-Generational efeitos de toxicants

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