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 tubing around the basal portion of the shoots, and securing the tubing with tape to create a &#8220;collar&#8221;. 
	NOTE: The number of plants per bundle may vary based on experimental conditions 10,13,14. Bundling is done to improve statistics and measuring accuracy, particularly when root mass and/or specific activity are low.</li>
</ol>
2. Preparation of Experimental Solutions/Materials
NOTE: The following is typically performed 1 day prior to experimentation.
<ol>
	<li>For DI, gather the following: Pre-labeling, labeling, and desorption solutions (for details, see 11), centrifugation tubes (for spin-drying of plant samples), and sample vials (for plant material and specific activity [So; see below]). Aerate and mix all solutions.</li>
	<li>For CATE, gather the following: Well-mixed, aerated labeling and elution solutions (for details, see 10), efflux funnels, centrifugation tubes (for spin-drying of plant samples), and sample vials (for eluates, plant samples, and determination of So and dilution factor [Df; see below]).</li>
</ol>
3. Prepare Radiotracer
CAUTION: The following safety steps should be taken prior to working with radioactivity.
<ol>
	<li>Ensure that the requirements of the radioactive materials license are understood and followed. Wear proper safety equipment (i.e., goggles, gloves, lab coat, lead vest/collar) and dosimeters (e.g., TLD ring and badge). Set up shielding (i.e., Plexiglas and lead bricks) and perform radioactive work behind it. Ensure that a Geiger-M&#252;ller counter is present in order to routinely monitor for contamination.</li>
	<li>Preparation of 42K+
	<ol>
		<li>Place a clean, dry beaker on the balance. Zero the balance.</li>
		<li>Remove vial of tracer (20 mCi of 42K2CO3, in powder form) from packaging and pour tracer into the beaker. Take note of the mass.</li>
		<li>Pipette 19.93 ml of dH2O, followed by 0.07 ml of H2SO4, into the beaker. This will drive the following chemical reaction: 
		42K2CO3 (s) + H2SO4 (l) + H2O (l)&#160;&#8594; 42K2SO4 (l) + CO2 (v) + 2H2O (l)</li>
		<li>Calculate the concentration of the radioactive stock solution, given the mass and molecular weight of K2CO3, and the volume (20 ml). 
		NOTE: If working with 13NH3/13NH4+ The tracer is produced in a cyclotron via the proton bombardment of the oxygen atom of water (typically resulting in 100-200 mCi activity; for production details, see 12). Because the quantity of 14NH3/14NH4+ is extremely low in these solutions, the N concentration of the stock solution is negligible.</li>
	</ol>
	</li>
</ol>
4. Direct Influx (DI) Measurement
<ol>
	<li>For 42K+, pipette the amount of radioactive stock solution required to reach the desired final concentration of K+ into the labeling solution.
	<ol>
		<li>For 13NH3/13NH4+, pipette a small amount (&#60;0.5 ml) into the labeling solution. Allow the labeling solution to mix thoroughly (via aeration).</li>
	</ol>
	</li>
	<li>Pipette a 1 ml sub-sample of labeling solution into a sample vial and repeat three times (4 samples in total).
	<ol>
		<li>Measure radioactivity in vials (in &#8220;counts per minute&#8221;, cpm), using a gamma counter. Ensure that the counter is programmed such that cpm readings are corrected for isotopic decay (this is particularly important for such short-lived tracers).</li>
		<li>Calculate So (expressed as cpm &#181;mol-1) by averaging the counts of the four samples (cpm ml-1) and dividing by the concentration of substrate in solution (&#181;mol ml-1).</li>
	</ol>
	</li>
	<li>Immerse roots in pre-labeling (non-radioactive) solution for 5 min, to pre-equilibrate plants under test conditions (see e.g., 10,13,14 for variations in pre-label time).</li>
	<li>Immerse roots in labeling (radioactive) solution for 5 min. 
	NOTE: Labeling times can vary based on experiment 3,4,7-10.</li>
	<li>Transfer roots to desorption solution for 5 sec to remove the bulk of surface-adhering radioactivity. Transfer roots into a second beaker of desorption solution for 5 min to further clear roots of extracellular tracer.</li>
	<li>Dissect and separate shoots, basal shoots, and roots.</li>
	<li>Place roots in centrifuge tubes and spin samples for 30 sec in a low-speed, clinical-grade centrifuge (~5,000 x g) to remove surface and interstitial water.</li>
	<li>Weigh roots (fresh weight, FW).</li>
	<li>Count radioactivity in plant samples (shoot, basal shoot, and root; see step 4.2.1).</li>
	<li>Calculate the flux. Calculate influx into the plant using the formula 
	&#934; = Q*/SowtL 
	where &#934; is the flux (&#181;mol g-1 hr-1), Q* is the quantity of tracer accumulated in tissue (cpm, usually in root, shoot, and basal shoot, combined), So is the specific activity of the labeling solution (cpm &#181;mol-1), w is the root fresh weight (g), and tL is the labeling time (hr). 
	NOTE: More sophisticated calculation can be made to account for simultaneous tracer efflux from roots during labeling and desorption, based on parameters obtained from CATE (see below; for details, see 4).</li>
</ol>
5. Compartmental Analysis by Tracer Efflux (CATE) Measurement
<ol>
	<li>Prepare labeling solution and measure So (see steps 4.1 - 4.2, above).</li>
	<li>Measure dilution factor (Df). 
	NOTE: Often, the position of the sample relative to the detector in the gamma counter can influence the quantity of radiation measured. See discussion for details.
	<ol>
		<li>After measuring So, add 19 ml of H2O to each sample (such that final volume = eluate volume = 20 ml). Count radioactivity in each 20-ml sample (see step 4.2.1).</li>
		<li>Calculate Df by dividing the average cpm of the 1-ml samples by the average cpm of the 20-ml samples.</li>
	</ol>
	</li>
	<li>Immerse roots in labeling solution for 1 hr.</li>
	<li>Remove plants from labeling solution and transfer plants to efflux funnel, ensuring all root material is within the funnel. Gently secure plants to side of efflux funnel by applying a small strip of tape over the plastic collar.</li>
	<li>Gently pour the first eluate into the funnel. Start timer (counting up).</li>
	<li>Open the spigot and collect the eluate in the sample vial after 15 sec (note: elution time will vary; see below). Close the spigot. Gently pour the next eluate into the funnel.</li>
	<li>Repeat step 5.6 for the remainder of the elution series, which follows, from the first to the final eluate: 15 sec (four times), 20 sec (three times), 30 sec (twice), 40 sec (once), 50 sec (once), 1 min (25 times), for a total elution period of 29.5 min 
	NOTE: Desorption series can vary based on experimental conditions 7-10,13,14.</li>
	<li>Once elution protocol is complete, harvest plants (steps 4.6 - 4.8, above).</li>
	<li>Count radioactivity in eluates and plant samples in the gamma counter (multiplying the reading for each eluate by Df, see 5.2).</li>
	<li>Plot tracer release (cpm g (root FW)-1 min-1) as a function of elution time. For steady-state conditions, perform linear regressions and calculations of fluxes, half-lives of exchange, and pool sizes (for details, see 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. .. 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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. 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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. 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The authors have nothing to disclose.

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...

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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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11.2K Views.  University of Toronto. In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Video: Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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