Name: measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers
Uploaded: 2014-08-22T14:00:00.000+00:00
Duration: 13 min 14 s
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 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>

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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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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><tbody><tr><td>Gamma counter</td><td>Perkin Elmer</td><td></td><td>Model: Wallac 1480 Wizard 3&quot;</td></tr><tr><td>Geiger-M&amp;uuml;ller counter</td><td>Ludlum Measurements Inc.</td><td></td><td>Model 3 survey meter</td></tr><tr><td>400 ml glass beakers</td><td>VWR</td><td>89000-206</td><td>For pre-absorption, absorption, and desorption solutions</td></tr><tr><td>Glass funnel</td><td>VWR</td><td>89000-466</td><td>For efflux funnel</td></tr><tr><td>Large tubing</td><td>VWR</td><td>529297</td><td>For efflux funnel</td></tr><tr><td>Medium tubing</td><td>VWR</td><td>684783</td><td>For bundling</td></tr><tr><td>Small tubing</td><td>VWR</td><td>63013-541</td><td>For aeration</td></tr><tr><td>Aeration manifold</td><td>Penn Plax Air Tech</td><td>vat 5.5</td><td>To control/distribute pressurized air into solutions</td></tr><tr><td>Glass scintillation vials</td><td>VWR</td><td>66022-128</td><td>For gamma counting</td></tr><tr><td>Glass centrifuge tubes</td><td>VWR</td><td>47729-576</td><td>For spin-drying root samples</td></tr><tr><td>Kimwipes</td><td>VWR</td><td>470173-504</td><td>For spin-drying root samples</td></tr><tr><td>Dissecting scissors</td><td>VWR</td><td>470001-828</td><td></td></tr><tr><td>Forceps</td><td>VWR</td><td>470005-496</td><td></td></tr><tr><td>Low-speed clinical centrifuge</td><td>International Equipment Co.</td><td>76466M-4</td><td>For spin-drying root samples</td></tr><tr><td>1 ml pipette</td><td>Gilson</td><td>F144493</td><td></td></tr><tr><td>10 ml pipette</td><td>Gilson</td><td>F144494</td><td></td></tr><tr><td>1 ml pipette tips</td><td>VWR</td><td>89079-470</td><td></td></tr><tr><td>10 ml pipette tips</td><td>VWR</td><td>89087-532</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler toledo</td><td>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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11.3K 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

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Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Biochemistry

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow injection analysis, has been developed. The concentrations of radioactive strontium were determined by flow scintillation counting, allowing for on-line and also on-site determination. The proposed system can determine radioactive strontium at industrial relevant levels without further modification using overall analysis time of less than 10 min per aqueous sample. The limit of the detection is 320 fg&#183;g-1 (1.7 Bq/g).

Automated 90Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

Here, we present the portable autoRAD platform for quick radiometric separation and determination of 90Sr, an important fission product highly pertinent to nuclear waste.

A quick, automated and portable system for the separation and determination of radiostrontium in aqueous samples, using Sr-resin and multi sequential flow ...

Chemistry

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

Bioengineering

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Biology

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology&#160;(herbaceous plant/root/soil system)&#160;and biomedical applications&#160;(rat femur and mouse lung).

This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system.

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron ...

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

Summary

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Chapters in this video

Measuring Carbon-based Contaminant Mineralization Using Combined CO₂ Flux and Radiocarbon Analyses

A Colorimetric Method for Measuring Iron Content in Plants

Microplot Design and Plant and Soil Sample Preparation for ¹⁵Nitrogen Analysis

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

Automated ⁹⁰Sr Separation and Preconcentration in a Lab-on-Valve System at Ppq Level

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor