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Measuring net ion fluxes from plant roots using ion-selective microelectrodes

Protocol

 

Authors

Sergey Shabala, Lana Shabala and Ian Newman

Overview

This protocol outlines how to quantify kinetics of net ion fluxes from plant roots using non-invasive ion-selective microelectrodes, following the MIFE system.

Background

If an ion is taken up by root cells, its concentration in the proximity of the root surface will be lower than that further away. Vice versa, if the ion is extruded across epidermal root cells, there will be a pronounced electrochemical potential gradient directed away from the root surface. The principle of the method is to measure this electrochemical potential gradient by slow, square-wave movement of ion-selective microelectrode probes between two positions, close to (position 1), and distant from (position 2) the sample surface (Fig. 1). At each position, electrode voltage is recorded and then converted into approximate concentration using the calibrated Nernst slope of the electrode. It is assumed that convection and water uptake are negligibly small and unstirred layer conditions are met. More details on the theory of non-invasive MIFE ion flux measurements are available in Newman (2001) and Shabala et al (2006).


Fig. 1. The principle of the MIFE ion flux measurements (modified from Wherrett 2006)

Materials/Equipment

MIFETM hardware consists of (Fig. 2):

  1. MIFE main amplifier, preamplifier and controller
  2. Multi-manipulator providing 3- dimensional positioning (e.g. MMT-5, Narishige, Japan)
  3. One- or three-dimensional hydraulic micromanipulator (e.g. Narishige MHW-1 or MHW-3 models)
  4. Stepper motor and power supply to drive hydraulic manipulator
  5. Mechanical micromanipulator (e.g. Narishige MX-1) for positioning assembled MMT-5/MHW-3 ensemble.
  6. PC for system control and data acquisition
  7. CIO-DAS08 card for analogue to digital conversion


Fig.2

Units, terms, definitions

LIX – liquid ion exchanger;

OD– outer diameter

Procedure

Preparing electrodes

Step 1- pulling out electrode blanks

Step 2 – baking and salinising the blanks

Step 3 – filling the electrodes with an appropriate liquid ion exchanger

 

Calibrating electrodes

 

Conducting measurements

 

Data analysis

Literature references

Newman, I. A. (2001). Ion transport in roots: measurements of fluxes using ion-selective microelectrodes to characterize transporter function. Plant, Cell and Environment 24, 1-14

Shabala, L., Ross, T., McMeekin, T., and Shabala, S. (2006). Non-invasive microelectrode ion flux measurements to study adaptive responses of microorganisms to the environment. FEMS Microbiology Reviews 30, 472-486

Shabala, S. N., Newman, I. A., and Morris, J. (1997). Oscillations in H+ and Ca2+ ion fluxes around the elongation region of corn roots and effects of external pH. Plant Physiology 113, 111-118

Shabala, S. (2006) Non-invasive microelectrode ion flux measurements in plant stress physiology. In: Plant Electrophysiology – Theory and Methods (ed. A. Volkov). Springer, Heidelberg. pp. 35-71

Health, safety & hazardous waste disposal considerations

The silane used to make the electrodes hydrophobic is a toxic chemical, so the silaning should be done in a fume cabinet.

Notes and troubleshooting tips

MIFE Calibration Routine

To make a calibration, you will need to measure a range of standard solutions for each ion used. The range should cover the concentrations you expect to find. Optimum accuracy in linear fitting is obtained from equal factors change in standard concentrations chosen. Put electrodes into your first standard solution, close the Faraday cage and start data acquisition. Press the function key F7. In the window that opens, correct the temperature, type the names of all the ions to be used and press <ENTER>. When the data for the ion of the first solution is stable and in range, press F7 again and enter the concentration for that ion. After you press <ENTER> the data recorded will be used in the calibration calculation until a third press of F7, after about 20 seconds, allows you to terminates that set, either keeping it or rejecting it. Details are given under F7 in the Handbook file THECHART.DOC.

Repeat the process for the other concentrations and the other ions. When all needed data have been recorded type ALT-H to stop data acquisition. From the <E>lectrometer menu, choose the <A>verage option and the <C>alibration average. CHART will do the least squares fit to the data for each ion, create the .AVC file and display in a window the parameters of the electrodes. If you are unhappy with the calibrations, you can start data acquisition again to repeat as many ions as you wish. No need to quit CHART. Eventually, in running MIFEFLUX, you will have the option of selecting just those calibrations you want from the .AVC file(s) you have made.

Data averaging routine

You are required to specify a “Valid Time” at the end of each stage of manipulator movement, for which the actual “Stage Time” is provided. Pressing ENTER moves the highlight to the next stage. Pressing UPARROW moves up to the previous one to allow you to correct an error. Finally you must press ENTER to accept the order of the curve (“Kind of <F>it”) to fit the data (typing F to cycle through the orders). Linear is probably what you need. As the averaging is done, the chirp and the Line Count indicate progress. An AVM file (for “AVerage Manipulator”) is created with the same name as the DAT and LOG files of the data. The AVM file is read by the MIFEFLUX program to calculate the fluxes, but you can import it into a spreadsheet or view it in a text editor.

MIFEFLUX routine

The MIFEFLUX program is run under DOS in your current directory, which contains the AVC and AVM files for the flux calculations. The batch file MFLUX.BAT, which is in the C:MIFE directory, will do this, just by typing MFLUX in your current directory. The program first asks you for the AVC file(s) you want, then for the AVM file of your averaged flux data, and finally for the kind of analysis. After calculation is done you can calculate more fluxes using the same calibrations. The most often used defaults are indicated by and are obtained just by pressing <ENTER>.

 

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