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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 sciences^1,2, 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., K_M and V_max), 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 ‘Discussion’, 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⁺ (t_1/2 = 12.36 hr) and ¹³NH₃/¹³NH₄⁺ (t_1/2 = 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’s background and theory can be found in several key articles on the subject^4,6-9. 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’s ionizing radiation safety regulator.

Protokół

1. Plant Culture and Preparation

1. 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.
2. 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 “collar”.
   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.

2. Preparation of Experimental Solutions/Materials

NOTE: The following is typically performed 1 day prior to experimentation.

1. 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 [S_o; see below]). Aerate and mix all solutions.
2. 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 S_o and dilution factor [D_f; see below]).

3. Prepare Radiotracer

CAUTION: The following safety steps should be taken prior to working with radioactivity.

1. 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-Müller counter is present in order to routinely monitor for contamination.
2. Preparation of ⁴²K⁺
   1. Place a clean, dry beaker on the balance. Zero the balance.
   2. Remove vial of tracer (20 mCi of ⁴²K₂CO₃, in powder form) from packaging and pour tracer into the beaker. Take note of the mass.
   3. 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) → ⁴²K₂SO₄ (l) + CO₂ (v) + 2H₂O (l)
   4. 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.

4. Direct Influx (DI) Measurement

1. For ⁴²K⁺, pipette the amount of radioactive stock solution required to reach the desired final concentration of K⁺ into the labeling solution.
   1. For ¹³NH₃/¹³NH₄⁺, pipette a small amount (<0.5 ml) into the labeling solution. Allow the labeling solution to mix thoroughly (via aeration).
2. Pipette a 1 ml sub-sample of labeling solution into a sample vial and repeat three times (4 samples in total).
   1. Measure radioactivity in vials (in “counts per minute”, 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).
   2. Calculate S_o (expressed as cpm µmol^-1) by averaging the counts of the four samples (cpm ml^-1) and dividing by the concentration of substrate in solution (µmol ml^-1).
3. 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).
4. Immerse roots in labeling (radioactive) solution for 5 min.
   NOTE: Labeling times can vary based on experiment ^3,4,7-10.
5. 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.
6. Dissect and separate shoots, basal shoots, and roots.
7. 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.
8. Weigh roots (fresh weight, FW).
9. Count radioactivity in plant samples (shoot, basal shoot, and root; see step 4.2.1).
10. Calculate the flux. Calculate influx into the plant using the formula
    Φ = Q*/S_owt_L
    where Φ is the flux (µmol g^-1 hr^-1), Q* is the quantity of tracer accumulated in tissue (cpm, usually in root, shoot, and basal shoot, combined), S_o is the specific activity of the labeling solution (cpm µmol^-1), w is the root fresh weight (g), and t_L 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).

5. Compartmental Analysis by Tracer Efflux (CATE) Measurement

1. Prepare labeling solution and measure S_o (see steps 4.1 - 4.2, above).
2. Measure dilution factor (D_f).
   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.
   1. After measuring S_o, 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).
   2. Calculate D_f by dividing the average cpm of the 1-ml samples by the average cpm of the 20-ml samples.
3. Immerse roots in labeling solution for 1 hr.
4. 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.
5. Gently pour the first eluate into the funnel. Start timer (counting up).
6. 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.
7. 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.
8. Once elution protocol is complete, harvest plants (steps 4.6 - 4.8, above).
9. Count radioactivity in eluates and plant samples in the gamma counter (multiplying the reading for each eluate by D_f, see 5.2).
10. 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).

Wyniki

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...

Dyskusje

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 µmol 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...

Materiały

Name	Company	Catalog Number	Comments
Gamma counter	Perkin Elmer		Model: Wallac 1480 Wizard 3"
Geiger-Müller counter	Ludlum Measurements Inc.		Model 3 survey meter
400 ml glass beakers	VWR	89000-206	For pre-absorption, absorption, and desorption solutions
Glass funnel	VWR	89000-466	For efflux funnel
Large tubing	VWR	529297	For efflux funnel
Medium tubing	VWR	684783	For bundling
Small tubing	VWR	63013-541	For aeration
Aeration manifold	Penn Plax Air Tech	vat 5.5	To control/distribute pressurized air into solutions
Glass scintillation vials	VWR	66022-128	For gamma counting
Glass centrifuge tubes	VWR	47729-576	For spin-drying root samples
Kimwipes	VWR	470173-504	For spin-drying root samples
Dissecting scissors	VWR	470001-828
Forceps	VWR	470005-496
Low-speed clinical centrifuge	International Equipment Co.	76466M-4	For spin-drying root samples
1 ml pipette	Gilson	F144493
10 ml pipette	Gilson	F144494
1 ml pipette tips	VWR	89079-470
10 ml pipette tips	VWR	89087-532
Analytical balance	Mettler toledo	PB403-S/FACT