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

The overall goal of the following experiment is to measure the unidirectional fluxes of potassium and ammonia into and out of roots of intact barley seedlings, and to characterize the functioning of key nutrient transport systems in plant membranes. This is achieved by first growing seedlings for one week in hydroponic solutions of specific chemical composition to ensure that the plants are at a nutritional steady state. Hydroponic culture allows roots to be accessible for experimental manipulation.

As a second step, roots of intact plants are immersed for variable periods of time in experimental solutions, including uptake solutions, which have the substrate of interest spiked with its radioactive isotope. This step will be used to determine the rates of transport into and out of seedlings. Next, plants are either dissected immediately after a short uptake period for unidirectional influx experiments or transferred to an FLX funnel after a longer uptake for measurement of tracer release.

Using compartmental analysis by tracer, flx or Kate, results are obtained that can reveal key aspects of the capacity, energetics, mechanisms, and regulation of transport systems. This method can help answer key questions related to plant nutritional physiology, such as how are mineral nutrients and toxicants transported in and out of plants? How do such fluxes respond to changing environments and how do they affect substrate cellular and tissue compartmentation?

And lastly, how do abiotic stresses that compromise ecological environments in agriculture such as salinity, drought, and heavy metal toxicity affect plant nutrient fluxes and dynamics. The main advantage of this technique over existing methods such as substrate depletion or accumulation assays, or iron selective vibrating electrode measurements is that we're able to measure unidirectional fluxes as opposed to net flexes, which is a difference between influx and eFlex. By doing so, we are able to gain valuable insight into the capacity energetics, mechanisms and regulation of transport systems for plant nutrients and intoxicants.

The model species barley will be used in this experiment, grow the barley seedlings hydroponically for seven days in a climate controlled growth chamber one day prior to experimentation bundle several seedlings together to make a single replicate. Wrap a two centimeter piece of tigon tubing around the basal portion of the chutes and secure the tubing with tape to create a collar. Use three plants per bundle for the direct influx or DI assay and six plants per bundle for the compartmental analysis by tracer, flx, or Kate assay one day prior to the experiment.

Prepare the following materials and solutions for DI gather pre labeling, labeling and DESORPTION solutions, centrifugation tubes and sample vials, aerate and mix all solutions for Kate. Gather the following well. Mixed aerated labeling and elution solutions, efflux funnels, centrifugation tubes, and sample vials.

Prepare the radio tracers on the day of the experiment following all the requirements of the radioactive materials license of the institution. Wear proper safety equipment and dosimeters and use appropriate shielding for preparation of the radioactive potassium isotope. Potassium 42.

Place a clean, dry beaker on the balance and zero the balance. Remove a vial of the tracer from its packaging and pour the powder into the beaker. Take note of the mass pipette, 19.93 milliliters of distilled water into the beaker, followed by 0.07 milliliters of sulfuric acid.

Subsequently, the concentration of the radioactive stock solution is calculated. Given the mass and molecular weight of potassium carbonate and the volume of the solution, use a Geiger Mueller counter to routinely monitor for contamination. The radioactive nitrogen 13 isotope is produced in a cyclotron and arrives as a liquid for DI measurements.

Using potassium 42 pipette the amount of radioactive stock solution required to reach the desired final concentration of potassium into the labeling solution For DI measurements using nitrogen 13 pipette a small amount less than 0.5 milliliters of the radio tracer into the labeling solution. Allow the labeling solution to mix thoroughly via aeration. Next, pipette a one milliliter sub-sample of labeling solution into each of four sample vials.

Measure the radio activity in the vials using a gamma counter. Ensure that the counter is programmed such that the counts per minute or CPM readings are corrected. For isotopic decay, which is particularly important for short-lived tracers, calculate the specific activity of the labeling solution S not expressed as counts per minute per micromole by averaging the counts of the four samples and dividing by the concentration of substrate in solution, immerse the barley roots in a non-radioactive pre labeling solution for five minutes to pre equilibrate the plants under test conditions.

After that, immerse the roots in the radioactive labeling solution for five minutes. Transfer the roots to a DESORPTION solution for five seconds to remove the bulk of surface adhering radio activity. Then transfer the roots into a second beaker of Desorption solution for five minutes.

To further clear the roots of extracellular tracer, dissect and separate the shoots basal shoots and roots. Place the roots in centrifuge tubes and spin the samples for 30 seconds in a low speed clinical grade centrifuge. To remove surface and interstitial water, weigh the roots to obtain the fresh weight.

Measure the radioactivity in the plant samples using a gamma counter, calculate the influx into the plant using this formula. Begin this procedure by preparing the labeling solution and measuring S knot as shown earlier. After measuring s, add 19 milliliters of water to each sample such that the final volume is equal to the EIT volume of 20 milliliters.

Count the radio activity in each 20 milliliter sample. Immerse the roots in the labeling solution for one hour. After one hour, remove the plants from the labeling solution and transfer them to the FLX funnel, ensuring that all root material is within the funnel.

Gently secure the plants to the side of the efflux funnel by applying a small strip of tape over the plastic collar Gently pour the first elu into the funnel. Start a timer to count up in seconds, and after 15 seconds, open the spigot and collect the EIT in the sample vial. Close the spigot gently pour the next EIT into the funnel.

In this manner, collect the EIT for the remainder of the Elucian series for a total EU period of 29.5 minutes. Once the EU protocol is complete, harvest plants as shown earlier, count the radio activity in the EITs and the plant samples using the gamma counter, multiplying the reading for each EIT by the dilution factor plot tracer release as a function of elution time for steady state conditions, perform linear regressions and calculations of fluxes. Half lies of exchange and pool sizes.

Shown here are representative isotherms for ammonia influx as a function of varying external concentrations of ammonia. In intact roots of barley seedlings grown at high ammonia or ammonium, and either low or high potassium ammonia fluxes were significantly higher at low potassium McKayla's. Menin analyses of the isotherms reveal that high potassium has relatively little effect on the substrate affinity of ammonia uptake transporters, but significantly reduces the transport capacity.

This next result highlights the rapid plasticity of the potassium uptake system. In roots of intact barley seedlings grown at moderate potassium and high ammonium. An almost 350%increase in potassium influx was observed within five minutes of ammonium withdrawal from the external solution.

This ammonium withdrawal effect was sensitive to the potassium channel blockers, tetraethyl ammonium barium, an cesium. These plots show the steady state potassium 42 efl in roots of intact barley seedlings grown at low potassium and moderate nitrate, and the immediate effects of 10 millimolar caesium chloride, five millimolar potassium sulfate, and five millimolar ammonium sulfate on flx potassium flx was inhibited by either caesium or potassium, but stimulated by ammonium. Cate can also be used to estimate concentrations and turnover times of the substrate in subcellular compartments.

Regression analysis of the slowly exchanging phase of tracer release along with tracer retention in plant tissues can reveal important information on pool size and half lives of exchange of subcellular components such as the cell wall, cytoplasm and va. This table shows cape parameters extracted from measurements of steady state potassium 42 flx in barley seedlings grown either with one millimolar nitrate or 10 millimolar ammonium. The latter representing a toxic scenario.

High ammonium causes suppression of all potassium fluxes and a significant decline in pool size. Once mastered, the efficiency of the DI methodology can be improved by staggering treatments 30 seconds apart. In doing so, we can examine up to 10 conditions in a single experiment.

Similarly, several Kate runs can be conducted simultaneously given sufficient time between runs. After watching this video, you should have a good understanding of how to measure fluxes of nutrients and intoxicants in intact plants by use of radioactive tracers.