Name: measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers
Uploaded: 2014-08-22T14:00:00
Description: Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

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).

1. Plant Culture and Preparation
<ol>
	<li>Grow barley seedlings hydroponically for 7 days in a climate-controlled growth chamber (for details, see 10). 
	NOTE: It is important to consider examining plants at a variety of developmental stages, as nutrient requirements will change with age.</li>
	<li>One day prior to experimentation, bundle several seedlings together to make a single replicate (3 plants per bundle for DI, 6 plants per bundle for CATE). Bundle seedlings by wrapping a 2-cm piece of Tygon t...

<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>

As demonstrated in the examples above, the radiotracer method is a powerful means of measuring unidirectional fluxes of nutrients and toxicants in planta. Figure 1 shows that NH3 influx can reach in excess of 225 &#181;mol g-1 hr-1, which is perhaps the highest bona fide transmembrane flux ever reported in a plant system13, but the magnitude of this flux would not be visible if only net fluxes were measured. This is because a large efflux of N...

The uptake and distribution of nutrients and toxicants strongly influence plant growth. Accordingly, the investigation of underlying transport processes constitutes a major area of research in plant biology and agricultural sciences1,2, especially in the contexts of nutritional optimization and environmental stresses (e.g., salt stress, ammonium toxicity). Chief among methods for the measurement of fluxes in plants is the use of radioisotopic tracers, which was developed significantly in the 1950s (see e.g., 3) and continues to be widely used today. Other methods, such as measurement of nutrient depletion from the root medium and/or accumulation in tissues, use of ion-selective vibrating microelectrodes such as MIFE (microelectrode ion flux estimation) and SIET (scanning ion-selective electrode technique), and use of ion-selective fluorescent dyes, are also widely applied, but are limited in their ability to detect net fluxes (i.e., the difference between influx and efflux). The use of radioisotopes, on the other hand, allows the researcher the unique ability to isolate and quantify unidirectional fluxes, which can be used to resolve kinetic parameters (e.g., KM and Vmax), and provide insight into the capacity, energetics, mechanisms, and regulation, of transport systems. Unidirectional flux measurements made with radiotracers are particularly useful under conditions where the flux in the opposite direction is high, and the turnover of intracellular pools is rapid4. Moreover, radiotracer methods allow measurements to be conducted under fairly high substrate concentrations, unlike many other techniques (see &#8216;Discussion&#8217;, below), because the traced isotope is observed against a background of another isotope of the same element.
Here, we provide detailed steps for the radioisotopic measurement of unidirectional and net fluxes of mineral nutrients and toxicants in intact plants. Emphasis will be made on flux measurement of potassium (K+), a plant macronutrient5, and ammonia/ammonium (NH3/NH4+), another macronutrient which is, however, toxic when present at high concentrations (e.g., 1-10 mM)2. We will use the radioisotopes 42K+ (t1/2 = 12.36 hr) and 13NH3/13NH4+ (t1/2 = 9.98 min), respectively, in intact seedlings of the model system barley (Hordeum vulgare L.), in the description of two key protocols: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We should note from the outset that this article simply describes the steps necessary to perform each protocol. Where appropriate, brief explanations of calculations and theory are provided, but detailed expositions of each technique&#8217;s background and theory can be found in several key articles on the subject4,6-9. Importantly, these protocols are broadly transferable to flux analysis of other nutrients/toxicants (e.g., 24Na+, 22Na+, 86Rb+, 13NO3-) and to other plant species, albeit with a few caveats (see below). We also stress the importance that all researchers working with radioactive materials must work under a license arranged through their institution&#8217;s ionizing radiation safety regulator.

<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.

Figure 1 shows isotherms found using the DI technique (with 13N), for the influx of NH3 into roots of intact barley seedlings grown at high (10 mM) NH4+, and either low (0.02 mM) or high (5 mM) K+. The isotherms display Michaelis-Menten kinetics when NH3 fluxes are plotted as a function of external NH3 concentration ([NH3]ext; adjusted by changes in solution pH13). NH3 fluxes were sign...

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.

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

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