Journal of Experimental Botany

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Journal of Experimental Botany, Vol. 52, No. 359, pp. 1353-1359, June 1, 2001
© 2001 Oxford University Press

An improved Na⁺-selective microelectrode for intracellular measurements in plant cells

David E. Carden^1,4, Dermot Diamond² and Anthony J. Miller^1,3

¹ Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Hertfordshire, UK
² National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland

Received 15 September 2000; Accepted 12 February 2001

	Abstract

Top Abstract Introduction Materials and methods Results and discussion References

 
The high background K⁺ concentration in plant cells is a problem for intracellular measurements of Na⁺ using ion-selective microelectrodes. The discrimination between Na⁺ and K⁺ of the microelectrode ionophore molecule limits the usefulness of this technique. A new Na⁺-selective microelectrode with an ionophore incorporating a tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene has been developed. Microelectrodes made with this new sensor have superior selectivity for Na⁺ over K⁺ resulting in a lower limit of detection when compared with microelectrodes made using a commercially available ionophore (ETH227). Both types of microelectrodes were insensitive to changes in ionic strength and physiological ranges of pH, but only the calixarene-based electrodes showed no protein interference. To test the suitability of the calixarene-based microelectrodes for measurements in plants, they were used to measure Na⁺ in epidermal cells in the zone 10–20 mm from the root apex of barley (Hordeum vulgare L.). Seedlings were grown in a nutrient solution containing 200 mM NaCl for 1–6 d. The range of intracellular Na⁺ activity (a_Na) measured varied from 0.1 mM (limit of detection) to over 100 mM, and these values increased significantly with time. The membrane potential (E_m) of these cells was variable, but the values became significantly more negative with time, although there was no significant correlation between E_m and a_Na. These intracellular measurements could not be separated into distinct populations that might be representative of subcellular compartments.

Key words: Barley roots, calixarene, salt-stress, sodium activity, sodium-selective microelectrodes.

	Introduction

Top Abstract Introduction Materials and methods Results and discussion References

 
Agricultural productivity is adversely affected by salinity because most major crop species are glycophytes, which are sensitive to low salt concentrations (Greenway and Munns, 1980). A major part of the sensitivity of these plants to salinity may result from Na⁺ replacing K⁺ within the cell, particularly the cytosol (Maathuis and Amtmann, 1999). The importance of effluxing Na⁺ from the cytosol was indicated recently by the demonstration that plants overexpressing a vacuolar H⁺/Na⁺ exchanger had improved salt tolerance when compared with wild-type plants (Apse et al., 1999). Measurements of intracellular Na⁺ are, therefore, important for understanding the physiological basis of tolerance and ion-selective microelectrodes can be used to provide this information. Although commercially available electrodes have been successfully used to measure the Na⁺ concentrations in extracts from plant tissues (Rieger and Litvin, 1998), there is only one report of an intracellular measurement. Sodium-selective microelectrodes were used for intracellular measurements in the marine alga, Acetabularia (Amtmann and Gradmann, 1994). A limitation for the use of any ion-selective microelectrodes can be interference from other ions present in the cell, and in plant cells the relatively high background concentrations of K⁺ in the cytosol can limit the range of Na⁺ measurements.

One sodium ionophore, ETH227 (Fluka, Buchs, Switzerland) has been commonly used in poly(vinyl chloride) (PVC) membrane electrodes for clinical measurements (Anker et al., 1983). However, high concentrations of protein have been reported to interfere with electrodes made using ETH227 (Coombs et al., 1994). Sodium-selective electrodes based on calix[4]arene tetraesters have also been reported (Diamond, 1986), and the p-t-butyl calix[4]arene tetramethyl ester derivative was shown to be particularly effective (Cadogan et al., 1989). Like ETH227, this ligand is now also used extensively for blood-sodium measurements and is sold as sodium ionophore X (Fluka, Switzerland). Recently, the p-t-butyl calix[4]arene tetramethoxyethyl ester derivative was demonstrated to have better Na⁺ over K⁺ selectivity than both the tetramethyl ester derivative and ETH227, but this is not a major advantage for accurate blood sodium measurements, as the background K⁺ levels are relatively low (typically 2–4 mM) compared to Na⁺ (typically 120–140 mM) (Diamond and McKervey, 1996). However, the enhanced selectivity for Na⁺ over K⁺ offered by the tetramethoxyethyl ester derivative is important for measurements in plant cells, where the background K⁺ concentration can be much higher.

This paper reports a comparison of ion-selective microelectrodes made using the calix[4]arene tetramethoxyethyl ester derivative with the commercially available sensor, ETH227. To demonstrate the utility of the calixarene Na⁺-microelectrodes, they were used to make measurements in barley root epidermal cells which had been treated with high concentrations of NaCl.

	Materials and methods

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Preparation of single- and double-barrelled Na⁺-selective microelectrodes
Single- and double-barrelled microelectrodes were prepared using filamented borosilicate glass as described previously (Zhen et al., 1992; Miller, 1996). The ion-selective barrel was silanized by the addition of one or two drops of 2% (v/v) dimethyldichlorosilane in chloroform (BDH, Poole, UK) to the blunt end of the micropipette barrel using a glass pipette. All chemicals were analytical grade and unless stated otherwise, were obtained from Fluka. Two different ionophores were tested, the commercially-available Na⁺-selective ionophore ETH227 (Fluka) was compared to p-t-butyl calix[4]arene tetramethoxyethyl ester derivative. The Na⁺ sensor cocktail contained by weight, the ionophore 10% (ETH227 or calixarene), the plasticizer 89.5% (2-nitrophenyl octyl ether) and lipophilic anion (sodium tetraphenylborate) 0.5%. To this cocktail, 10% PVC was added (by wt) and then dissolved in approximately 6 vol of tetrahydrofuran after vigorous shaking for 10 min. This final cocktail could be stored for 2–3 months without any loss of activity.

Backfilling and calibration of the microelectrodes to test for the effects of ionic strength, K⁺ and H⁺
The microelectrode was backfilled behind the membrane with a 100 mM NaCl solution using a 70 mm long Microfil needle (World Precision Instruments Inc., Stevenage, UK). The tip was also stored ‘wet’ in a solution of 100 mM NaCl for approximately 30 min in order to condition the microelectrode. The microelectrodes were calibrated as described previously using a modified glass funnel that incorporated a U-bend (Zhen et al., 1992; Miller, 1996). Calibration solutions were prepared (Table 1) with varying Na⁺ activity (a_Na) and some of these contained a fixed background activity of 70 mM K⁺ (a_K). This value of a_K was chosen because of the cytosolic homeostatic a_K reported over a wide range of external K⁺ concentrations (Walker et al., 1996). The calibration solutions were made from chloride salts of Na⁺ and K⁺, and the pH was buffered at pH 7.0 with 5 mM 2-[N-morpholino]ethanesulphonic acid and Tris[hydroxymethyl]aminomethane (Sigma-Aldrich, Poole, UK). Both types of Na⁺-selective microelectrodes were also tested for interference by H⁺ using calibration solutions adjusted to a range of different pH values from 4.0 to 8.0. Each of these solutions contained a constant 100 mM NaCl and was buffered to the appropriate pH with 5 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane/HCl. All electrodes were calibrated in Na⁺ calibration solutions before and after the pH interference test to ensure proper functioning of the electrode being tested. To test if the electrode output was modified by changes in the ionic strength some of the calibration solutions had extra magnesium chloride added (Table 1). Calibration curves were all fitted with the Nicolsky–Eisenman relationship (Walker et al., 1995) using SigmaPlot software (Jandel Scientific, Ekrath, Germany). The divalent cation Mg²⁺ was chosen to vary the ionic strength because the interference on the response of Na⁺-selective electrodes was minimal (data not shown).

View this table: [in this window] [in a new window]   Table 1. Composition of the solutions used to calibrate the two types of Na+-selective microelectrodes These solutions all had a background concentration of 70 mM K+ and to change the total ionic strength of the solution MgCl2 was added to some. The ion activities (a) for Na+, K+ and Mg2+ were calculated using activity coefficients obtained from Debeye–Hückel equation (Dean, 1973).

 

Protein interference
The effect of protein on the response of Na⁺-selective microelectrodes was tested by calibrating with solutions containing 70 mM a_K and supplemented with 100 mg ml^-1 albumin (BDH, Poole, Dorset, UK). This protein concentration represented the upper range of expected cytosolic concentration (Coombs et al., 1994). The electrodes were calibrated both before and after immersion in the protein solutions to test if there was any hysteresis in the effect of the albumin on the output. Also, solid albumin was checked for Na⁺ contamination by inductively-coupled plasma spectrometry (ICP) after ashing at 550 °C for 24 h. In addition, two desalting techniques were used to remove free Na⁺ from the albumin, and the desalted albumin was reanalysed by ICP. Desalted albumin samples were also used in calibration solutions to determine whether the reduction in Na⁺ resulted in a reduction in observed protein effect on the Na⁺-selective microelectrodes. The first method involved centrifuging an aqueous albumin sample at 5000 g for 2 h through a Centricon-10 ultrafilter (Amicon, Beverly, MA, USA). The second method desalted an aqueous albumin sample by passing a sample either once or twice through a Sephadex PD-10 gel filtration column (Pharmacia, Uppsala, Sweden).

Plant material
Seeds of barley (Hordeum vulgare L. cv. Triumph) were germinated and grown in a modified Hoagland's nutrient solution (Walker et al., 1996), and after 7 d the plants were transferred to a solution containing an additional 200 mM NaCl. A single-step increase in NaCl concentration was used because previous root elongation measurements had indicated no difference between growth at either 50 or 200 mM NaCl (data not shown). For the electrode impalements the root was placed into a Perspex chamber and held in position by a pair of Perspex blocks and Apiezon grease (M&I Material, Manchester, UK). Only epidermal cell measurements are reported here and these are defined as the first layer of cells encountered by the approaching microelectrode tip.

	Results and discussion

Top Abstract Introduction Materials and methods Results and discussion References

 
Na calibration and K interference
Figure 1 shows a typical calibration of the calixarene-based Na⁺-selective microelectrode using two different calibration series. The first was a pure NaCl solution with no added interfering ions and the electrode responded beyond -log a_Na 4 (0.1 mM). The slope of this calibration was 63.8 mV and the limit of detection was -log a_Na 4.03 (Table 2), these two parameters define the response curve of any ion-selective microelectrode (IUPAC, 1994). It is not known why this slope value is higher than the theoretical value of 59 mV, but super-Nernstian responses have been explained by effects at the electrode surface (Pungor, 1998). The ETH227-based Na⁺-selective microelectrode calibrated with a slope of 59.4 mV, but the limit of detection was higher at –log a_Na 3.87. When potassium was added at a constant background a_K of 70 mM (fixed interference method), both types of Na⁺-selective microelectrodes calibrated although the slopes were lower and the detection limits were higher in both (Fig. 1; Table 2). The calixarene-based electrode maintained its greater range of response, with a higher slope (58.4 mV) and a lower limit of detection of –log a_Na 3.47 (equivalent to 0.3 mM a_Na) compared to that of the ETH227-based electrode. The selectivity coefficient values, k_NaK, showed that the calixarene-based microelectrode was more selective for Na⁺ against K⁺ (Table 2). The value obtained log(k_NaK)=–2.32±0.14 is very similar (–2.4) to that obtained with conventional macro (8.0 mm tip) PVC membrane electrodes based on the same calix[4]arene derivative (Diamond and Nolan, 2000).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Calibration curves for the calixarene-based Na-selective microelectrode in NaCl solutions with and without added 70 mM aK. The lines drawn show the fitted Nicolsky–Eisenman relationship for each data set (Walker et al., 1995). Plots for three different types of solution are shown: NaCl only (•); Na with constant 70 mM aK (); isotonic series containing Na with constant 70 mM aK and with Mg to maintain a constant background ionic strength ().

 

View this table: [in this window] [in a new window]   Table 2. Comparison of the electrode responses of Na+-selective microelectrodes made using the sensors Limits of detection, slopes, and selectivity coefficients of the two Na+-selective electrodes based on either ETH227 or the calixarene ionophore. These parameters were calculated by fitting the Nicolsky–Eisenman equation (Walker et al., 1995). Data are means (±SE) of four replications.

 

Interference by pH, nitrate, ionic strength and protein
Both the calixarene- and ETH227-based Na⁺-selective microelectrodes were insensitive to changes in pH over a physiological range of pH 4.0 to 8.0 (data not shown). Figure 1 shows the response of the calixarene-based microelectrode to changes in a_K and ionic strength of the calibration solutions. One calibration series was in pure NaCl solution, while the second had a constant 70 mM a_K background, with some of these values obtained from solutions that had added MgCl₂ to maintain the overall ionic strength. The electrode response to the constant a_K interference was insensitive to changes in total ionic strength for both calixarene- (Fig. 1) and ETH227-based microelectrodes (data not shown). Recently, high concentrations of nitrate were found to interfere with the response of K⁺-selective microelectrodes (Cuin et al., 1999), but the calixarene-based electrodes did not show a similar sensitivity to nitrate (data not shown).

Sodium-selective microelectrodes made with both sensors showed a large shift in the calibration curve when 100 mg ml^-1 albumin was added to the standard solutions. Figure 2 shows a typical example of a protein test on calixarene- and ETH227-based electrodes. The slope of the linear portion of the calibration curve was unaffected, but the detection limit was raised by almost 1 log unit in both types of electrodes. The calibration without albumin before and after indicated that the result was not due to electrode hysteresis, and was entirely reversible (not shown). However, using ICP analysis the albumin showed significant Na⁺ contamination (3450 ppm), this gives a background 15 mM Na⁺ in all the protein calibration solutions. Assuming that all this Na⁺ was free in solution, then each calibration solution contained a contaminating a_Na of 11.25 mM, using an activity coefficient of =0.75. The calibration curve without albumin was recalculated to correct for this Na⁺ contamination. This correction showed that all of the observed protein effect on the calixarene-based Na⁺-selective microelectrode could be explained by the Na⁺ contamination of the albumin added to the calibration solutions (Fig. 2). In contrast, the ETH227-based microelectrode showed a residual protein effect that could not be accounted for by this contaminating Na⁺ (Fig. 2). In addition, various desalting methods were used to attempt to remove the Na⁺ contaminating the albumin (Table 3). Centrifuging an aqueous albumin solution through a Centricon-10 ultrafilter removed 23% of the contaminating Na⁺. Passing an aqueous albumin solution down a Sephadex column removed more (49%), and by passing it twice down the filter 72% was removed. When the desalted albumin was added to the Na⁺ calibration solutions the apparent protein effect was reduced by approximately the same magnitude as the decreases in Na⁺ content shown in Table 3. However, the ETH227 electrodes still showed a shift in the calibration curve that could not be wholly explained by the remaining Na⁺ contamination.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Test for protein effect on ETH227- and calixarene-based Na+-selective microelectrodes. (A) The ETH227 microelectrode was calibrated with () and without (before •, after ) albumin at 100 mg ml-1 added to the calibration solutions containing 70 mM aK. The calibration plot () was calculated by subtracting the Na+ contaminating the albumin measured by ICP and this assumes that all of the Na+ was free in solution and uses an activity coefficient of 0.75. The arrow on the graph indicates the shift obtained after this correction and is the remaining effect of protein on the electrode response. (B) The calixarene-based electrode was also calibrated with () and without (before •, after ) albumin at 100 mg ml-1, but after the correction for contaminating Na+ there was no residual protein effect (not shown). For both types of electrode the lines drawn show the fitted Nicolsky–Eisenman relationship for each data set (Walker et al., 1995).

 

View this table: [in this window] [in a new window]   Table 3. Reduction of Na in albumin due to various desalting methods compared to untreated albumin sample

 
Both calixarene- and ETH227-based Na⁺-selective microelectrodes can function as satisfactory Na⁺-selective microelectrodes, but there were some important differences in their response to K⁺ and protein interference. The calixarene-based electrode had a higher k_NaK resulting in lower detection limits than the ETH227-based electrode, and this result agrees qualitatively with that of macroelectrodes (Cadogan et al., 1989). The results reported here used the recommended fixed interference method (IUPAC, 1994), while the earlier quoted results used the separate solutions method (Cadogan et al., 1989), emphasizing the need to give details of the calibration procedure when quoting selectivity factors (IUPAC, 1994). More recent results using the fixed interference method report similar quantitative values for k_NaK (Diamond and Nolan, 2001). The higher k_NaK of the calixarene-based electrode is an important difference when making intracellular measurements in plants due to the high a_K in the cytosol (Walker et al., 1996). Both electrodes were also insensitive to pH, ensuring no complications of subcellular location (cytoplasm or vacuole) for plant cell measurements. The lack of sensitivity to total ionic strength enables calibration solutions to be made without first measuring the intracellular ionic strength.

A second important difference between the electrodes based on two different ionophores was the difference in sensitivity to protein. Effects of protein and macromolecules on Na⁺-selective microelectrode performance have been reported before (Coombs et al., 1994) and the protein effect on the ETH227-based electrode can only be partly explained by the presence of contaminating Na⁺. The residual protein effect occurs by some unknown mechanism, but this was not observed for the calixarene-based microelectrodes. The Na⁺-selective microelectrode made using the calixarene sensor was superior in its performance over ETH227, and was chosen as the electrode to use for intracellular plant measurements.

Intracellular measurements
Figure 3 shows an example of a double-barrelled intracellular recording obtained from a barley root epidermal cell that had been growing in 200 mM NaCl for approximately 10 d. The upper two recordings show the output from the reference barrel of the microelectrode and a differential output obtained by subtracting the reference from the Na⁺-sensing barrel voltage. The lower trace shows the resulting intracellular measurement of a_Nacalculated using the calibration curve (Fig. 3). The microelectrode was advanced to touch the root surface at the start of the recording, then penetrating the cell membrane after 20 s, and after 2 min it was deliberately withdrawn from the cell. The membrane potential (E_m) was slightly positive on the root surface but then quickly jumped to -85 mV and remained stable throughout the impalement, with the Na⁺-selective electrode recalibrated after the recording, to confirm this measurement. The Na⁺-electrodes showed differing success rates depending on the complexity of construction, 95% of single barrels tips showed good calibration response, but 30% of double-barrelled electrodes gave measurements in cells. One double-barrelled electrode could be used for between 2–6 intracellular measurements.

View larger version (21K): [in this window] [in a new window]   Fig. 3. An intracellular recording obtained by impaling a barley root epidermal cell with a double-barrelled Na+-selective microelectrode. The upper trace (A) is the voltage reported by the reference barrel, and the middle trace (B) is the difference trace obtained by subtracting the reference from the Na+-selective barrel output. The lower trace (C) is the aNa in the cell obtained using the VISER software (Walker et al., 1995) to convert the differential output and the calibration curve for the electrode (not shown). The electrode tip was advanced to touch the root surface at time zero, with the cell impalement occurring after 20 s then at 2.5 min the tip was deliberately withdrawn.

 
Double-barrelled Na⁺-selective microelectrodes were successfully used to measure intracellular root epidermal a_Na and E_m at high external concentrations of NaCl. The measurements showed that there was a wide range of values for both a_Na and E_m (Fig. 4) over the time-course measured, however there were significant changes in both parameters, with E_m becoming more negative (P<0.05), and a_Na increasing (P<0.05) with time in NaCl (Fig. 4). Although both parameters increased with time there was no significant correlation between E_m and a_Na. After 2 d in 200 mM NaCl the change in the E_m values may represent the recovery of the cells as they adjusted to the salt stress. The salinity was applied to the plants in a single step addition of 200 mM NaCl and this may also have imposed an osmotic shock. Approximately 30% of epidermal cells did not register an E_m upon impalement although apparently remaining turgid, and a cell vitality test using Evan's blue stain did not indicate an equivalent cell death rate (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4. The pooled intracellular electrode measurements obtained after treating barley roots with 200 mM NaCl. (A) Em measurements of epidermal root cells of the barley growing in a full nutrient solution containing 200 mM NaCl obtained by double-barrelled microelectrodes. Data are means (±SE) of between 5 and 8 measurements. (B) Intracellular Na+ measurements of epidermal root cells of the barley growing in a full nutrient solution containing 200 mM NaCl obtained by double-barrelled microelectrodes.

 
By deliberately growing the barley plants in high external concentrations of NaCl, it was hoped that the subsequent a_Na measurements would separate into two distinct populations, that of the cytosol and of the vacuole. This would enable the assignment of the data to subcellular compartments as was possible for nitrate (Zhen et al., 1991). Although the range of values measured presumably included both cytosolic and vacuolar measurements, neither E_m nor a_Na separated into two distinct populations that could be assigned to these two main intracellular compartments. To identify cytosolic measurements of a_Na it will be necessary to use calixarene Na⁺ sensor in triple-barrelled microelectrodes (Walker et al., 1995), that include a third barrel selective for pH.

	Acknowledgments

 
IACR is grant-aided by the Biotechnology and Biological Sciences Council (BBSRC) of the UK. The authors also wish to thank David Walker and Susan Smith for their helpful discussion and the BBSRC for the studentship award, which supported DEC during this work.

	Notes

 
³ To whom correspondence should be addressed. Fax: +44 1582 763010. E-mail: tony.miller{at}bbsrc.ac.uk

⁴ Present address: Botanisches Institut, Universität Giessen, Senckenbergstrasse 17–21, D-35390 Giessen, Germany.

	Abbreviations

 
E_m, membrane potential difference; ICP, inductively-coupled plasma spectrometry; PVC, poly(vinyl chloride); a_K, potassium activity; a_Na, sodium activity.

	References

Top Abstract Introduction Materials and methods Results and discussion References

 
Anker P, Jenny H-B, Wuthier U, Asper R, Ammann D, Simon W. 1983. Potentiometry of Na⁺ in undiluted serum and urine with use of an improved neutral carrier-based solvent polymeric membrane-electrode. Clinical Chemistry 29, 1508–1512.[Abstract/Free Full Text]

Amtmann A, Gradmann D. 1994. Na⁺ transport in Acetabularia bypasses conductance of plasmalemma. Journal of Membrane Biology 139, 117–125.[Web of Science][Medline]

Apse MP, Aharon GS, Snedden WA, Blumwald E. 1999. Salt tolerance conferred by overexpression of a vacuolar Na⁺/H⁺ antiport in Arabidopsis. Science 285, 1256–1258.[Abstract/Free Full Text]

Cadogan AM, Diamond D, Smyth MR, Deasy M, McKervey MA, Harris SJ. 1989. Sodium-selective polymeric membrane electrodes based on calix[4]arene ionophores. Analyst 114, 1551–1554.

Coombs HV, Miller AJ, Sanders D. 1994. Disruptive effects of protein on performance of liquid membrane-based ion-selective microelectrodes. American Journal of Physiology 267, C1027-C1035.[Abstract/Free Full Text]

Cuin TA, Miller AJ, Laurie SA, Leigh RA. 1999. Nitrate interference with potassium-selective microelectrodes. Journal of Experimental Botany 50, 1709–1712.[Abstract/Free Full Text]

Dean JA. 1973. Lange's handbook of chemistry. New York: McGraw-Hill Book Company.

Diamond D. 1986. Neutral Carrier Based Ion-Selective Electrodes. Analytical Chemistry Symposium Series 25, 155.

Diamond D, McKervey MA. 1996. Calixarene-based sensing agents. Chemical Society Reviews 25, 15–24.

Diamond D, Nolan K. 2001. Designer ligands for chemical sensors. Analytical Chemistry 73, 22A–29A.[Medline]

Greenway H, Munns R. 1980. Mechanisms of salt tolerance in non-halophytes. Annual Review of Plant Physiology 31, 149–190.[Web of Science]

IUPAC. 1994. Recommendations for nomenclature of ion-selective electrodes (IUPAC recommendations 1994). Pure and Applied Chemistry 66, 2528–2536.

Maathuis FJM, Amtmann A. 1999. K⁺ nutrition and Na⁺ toxicity: the basis of cellular toxicity K⁺/Na⁺ ratios. Annals of Botany 84, 123–133.[Abstract/Free Full Text]

Miller AJ. 1996. Ion-selective microelectrodes for measurement of intracellular ion concentrations. Methods in Cell Biology 49A, 275–291.

Pungor E. 1998. The theory of ion-selective electrodes. Analytical Sciences 14, 249–256.

Rieger M, Litvin P. 1998. Ion selective electrodes for measurement of sodium and chloride in salinity experiments. Journal of Plant Nutrition 21, 205–215.

Walker DJ, Leigh RA, Miller AJ. 1996. Potassium homeostasis in vacuolate plant cells. Proceedings of the National Academy of Sciences, USA 93, 10510–10514.[Abstract/Free Full Text]

Walker DJ, Smith SJ, Miller AJ. 1995. Simultaneous measurement of intracellular pH and K⁺ or NO₃^- in barley root cells using triple-barreled, ion-selective microelectrodes. Plant Physiology 108, 743–751.[Abstract]

Zhen R-G, Koyro H-W, Leigh RA, Tomos AD, Miller AJ. 1991. Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling. Planta 185, 356–361.[Web of Science]

Zhen R-G, Smith SJ, Miller AJ. 1992. A comparison of nitrate-selective microelectrodes made with different nitrate sensors and the measurement of intracellular nitrate activities in cells of excised barley roots. Journal of Experimental Botany 43, 131–138.[Abstract/Free Full Text]

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