Journal of Experimental Botany, Vol. 52, No. 356, pp. 541-549,
April 2001
© 2001 Oxford University Press
The use of microelectrodes to investigate compartmentation and the transport of metabolized inorganic ions in plants
Department of Biochemistry and Physiology, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK
Received 23 May 2000; Accepted 24 November 2000
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Abstract |
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 Top Abstract IntroductionMicroelectrode access to cellsAssaying membrane transport...Ion-selective microelectrode...Ammonium and nitrate...Phosphate and sulphate...Conclusions and future workReferences |
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Microelectrode measurements can be used to investigate both the intracellular pools of ions and membrane transport processes of single living cells. Microelectrodes can report these processes in the surface layers of root and leaf cells of intact plants. By careful manipulation of the plant, a minimum of disruption is produced and therefore the information obtained from these measurements most probably represents the ‘in vivo’ situation. Microelectrodes can be used to assay for the activity of particular transport systems in the plasma membrane of cells. Compartmental concentrations of inorganic metabolite ions have been measured by several different methods and the results obtained for the cytosol are compared. Ion-selective microelectrodes have been used to measure the activities of ions in the apoplast, cytosol and vacuole of single cells. New sensors for these microelectrodes are being produced which offer lower detection limits and the opportunity to measure other previously unmeasured ions. Measurements can be used to determine the intracellular steady-state activities or report the response of cells to environmental changes.
Key words: Compartmentation, cytoplasm, ion-selective microelectrodes, plasma membrane.
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Introduction |
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TopAbstractIntroductionMicroelectrode access to cellsAssaying membrane transport...Ion-selective microelectrode...Ammonium and nitrate...Phosphate and sulphate...Conclusions and future workReferences |
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Microelectrodes are small probes that can be inserted into plant tissues to measure the electrical potential difference between the probe tip and an external reference point, usually another electrode placed in the external solution bathing the plant tissue. The measuring microelectrode can be placed on the surface of plant tissues or directly inserted into single cells. The microelectrode is the interface between the circuitry, for measuring the potential difference, and the plant material. This electrical contact occurs at the surface of a metal and aqueous solution, with the actual measurement of a membrane potential requiring the movement of charge at the boundary. The chief requirement for a good electrical contact is that the properties of this interface are stable and do not change significantly during a recording. For intracellular measurements the probe is commonly a glass micropipette that has been back-filled with a salt-solution and has a tip diameter suitable for insertion into a plant cell. The tip diameter of this micropipette is selected to give a size that will not destroy the cell and allows the plasma membrane to seal around the tip once inserted into a cell to record a stable membrane potential. The glass micropipette tip is commonly called a microelectrode, this provides a salt solution bridge between the back-filling solution and the metal circuit contact in the base of the micropipette holder.
One of the benefits of microelectrode measurements is that they can be used to study metabolism in single cells. The way plants modify metabolism in response to changes in the environment is usually studied by molecular and biochemical analysis of whole tissue samples. Another layer of complexity in these responses has been revealed as methods for analysis of single cells have developed. For example, micropipette sampling of the vacuole of single cells has shown heterogeneity within and between tissues (Tomos and Leigh, 1999
). Microelectrode measurements can be used to report the response of a single cell to a particular treatment. A large component of the electrical potential difference between the inside of a cell and the external solution is determined by the activity of the plasma membrane proton pump. This activity depends on many extra- and intracellular factors such as cytosolic pH and ATP concentration (reviewed by Sze et al., 1999). The membrane potential can be an indicator of a cell's health and energy status, because when the plasma membrane is damaged or the cytosolic ATP pool is depleted there is a decrease in the magnitude of the membrane potential. For example, chemical inhibitor treatments such as cyanide, that reversibly deplete cytosolic ATP concentrations, also reduce the membrane potential difference (Blatt et al., 1990). However, the usefulness of this as a defining parameter is limited because the membrane potential is also dependent on other factors such as the ionic composition of the external solution (e.g. the potassium concentration, Hirsch et al., 1998; Pitman et al., 1970). Nutrient starvation has been reported to increase the resting membrane potential of cells (discussed by Meharg and Blatt, 1995), but some of these reports can be discounted because the external K+ concentration was increased for the measurements in replete cells.
Microelectrodes can be used to determine the membrane transport properties of the cell, and can also be modified to measure intracellular free ion activities. Ion-selective microelectrodes actually respond to changes in activity and this parameter is more relevant biologically than the more familiar concentration (Miller, 1995). Inorganic nutrient ions can be divided into two types according to whether or not they are substrates for metabolic assimilation within the cell. Ions such as Ca2+, K+, Na+, and Cl- are considered as essential for growth because they are cofactors for life processes, but are not directly incorporated into organic molecules. In contrast, the ‘metabolite ions’ inorganic phosphate (Pi), NH4-, NO3, and SO42- are assimilated by cells. The intracellular concentrations of these ions can be indicators of the metabolic activity of a cell. There are several methods to follow changes in the vacuolar activities of these ions in single cells but the cytosolic pools are more difficult to measure. Changes in the cytosolic pools of a primary substrate can indicate the metabolic activity of a cell. Furthermore, the transport of a primary substrate into a cell provides entry into the metabolic pool, so the presence of a particular type of membrane transporter can indicate the activity of a particular assimilatory pathway.
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Microelectrode access to cells |
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TopAbstractIntroductionMicroelectrode access to cellsAssaying membrane transport...Ion-selective microelectrode...Ammonium and nitrate...Phosphate and sulphate...Conclusions and future workReferences |
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The main limiting factor for any type of microelectrode measurements is obtaining access to the cells to be measured. The electrode must be inserted into an identified cell, with minimal damage to the surrounding tissue and the plant must be as intact as possible avoiding any tissue dissection that may stimulate wound responses. The roots of hydroponically grown plants can be easily prepared for electrophysiological measurements without any major disruption to the plant. For similar reasons the leaves of aquatic plants can be used conveniently for measurements. More recently methods have been developed for inserting microelectrodes into the cells of intact leaves. However, these techniques require electrical contact to be made through the apoplast of the leaf tissue (Lauver et al., 1992
), so that the edge of the leaf is in contact with an aqueous solution into which the bath reference electrode can be inserted (Fig. 1). At the leaf surface, epidermal cells can be directly accessed while mesophyll cells can be measured through the stomatal aperture. The tough waxy leaf cuticle may be problematic for impaling the epidermal cells of some plants, but this difficulty can be overcome by optimizing the shape of the electrode tip. A short robust electrode tip is less likely to break or become blocked during the impalement of leaf cells.
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After the microelectrode impalement, identifying the compartmental location of an electrode tip can be difficult. The electrode tip pushes through the cell wall tending to jump quickly across the plasma membrane into either the cytoplasm or the vacuole. The intracellular compartmental location of the tip cannot be easily identified unless the electrode can report a parameter that is a defining characteristic. This is the principle exploited by triple-barrelled electrodes that include a pH-selective tip to identify the compartment (Walker et al., 1995
). When the electrode tip is located in the vacuole, the electrical potential difference is that across two membranes, the plasma membrane and the tonoplast. This information has been used to define the electrode tip location by measuring differences in the electrical resistance (Bates et al., 1982), but this is technically difficult and symplastic connections between cells complicate the measurement. Just before entering each cell, a small electrical potential difference can be measured and this has been defined as the apoplast (Felle, 1998). Vibrating ion-selective probes have also been used for extracellular measurements of ion fluxes at the surface of hydroponically grown roots (Henriksen et al., 1990; Kochian et al., 1992).
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Assaying membrane transport using microelectrodes |
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TopAbstractIntroductionMicroelectrode access to cellsAssaying membrane transport...Ion-selective microelectrode...Ammonium and nitrate...Phosphate and sulphate...Conclusions and future workReferences |
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When the transmembrane transport of any ion or molecule includes the movement of charge, the electrical response produced can be reported by a single microelectrode inserted into the cell. For example, nitrate and ammonium transporter activity has been assayed using the changes in membrane potential that are elicited when these substrates are applied externally to cells (McClure et al., 1990
; Glass et al., 1992; Ayling, 1993; Wang et al., 1994). The extent of these nitrogen-elicited changes in membrane potential depended on the nitrogen status of the plant. For ammonium and rice root cells, a biphasic response could be detected and the ammonium-elicited changes in the membrane potential could be fitted with Michaelis-Menten kinetics (Wang et al., 1994). For rice, the ammonium-elicited changes in membrane potential were significantly larger for plants previously grown in 2 µM when compared to those in 100 µM ammonium. The nitrate-elicited changes in membrane potential are present in root cells of seedlings grown without any nitrogen source, but the magnitude of the response increases after treatment with nitrate.
The usual nitrate effect on membrane potential is to give a transient depolarization (the potential becomes less negative) followed by a relatively slower hyperpolarization (the potential becomes more negative). The magnitude of the nitrate-elicited depolarization increases with duration of exposure of the root to nitrate (Glass et al., 1992). In maize the size of the hyperpolarization was shown to depend on the concentration of nitrate (McClure et al., 1990), while in barley and Arabidopsis root hairs it was the initial depolarization that depended on the nitrate concentration (Glass et al., 1992; Meharg and Blatt, 1995). Michaelis-Menten kinetics could be fitted to the plots of nitrate concentration and both parts of the electrical changes (depolarization and hyperpolarization) suggesting that both changes must be related to the activity of the nitrate transport system. From these types of measurements it was assumed that the initial depolarization involves the actual proton/ nitrate cotransport step. The later hyperpolarization results from stimulation of the plasma membrane proton pump due to the cytosolic acidification associated with the activity of the nitrate transporter. More recently, the characterization of nitrate transporter genes expressed in Xenopus oocytes has provided more support for this idea because nitrate cotransport with protons produces a depolarization that does not recover (Zhou et al., 1998, 2000). The oocyte lacks the proton pump activity that, in plant cells, restores the membrane potential. Changes in membrane potential that are associated with nitrate transport have been used to assay transporter activity in Arabidopsis mutants (Wang and Crawford, 1996). The presence of particular transporter activity in a cell may be an indicator of the cell's nutritional and metabolic activity.
The addition of sulphate outside plant cells has also been shown to give changes in the membrane potential that depend on the sulphur-status of the plant. Sulphate-starved plants showed larger depolarizations of membrane potential when compared with replete plants (Lass and Ullrich-Eberius, 1984). The addition of phosphate to phosphate-starved root cells can give both a depolarization and an acidification of the cytoplasm (Ullrich and Novacky, 1990). The P-status of the plants also influences the magnitude of the response, and like sulphate, the depolarization was largest in starved plants (Dunlop and Gardiner, 1993). The plasma membrane transport of other metabolites such as amino acids and sugars can also be assayed by the changes in membrane potential (Felle and Johannes, 1990; Novacky et al., 1978).
One of the possible difficulties for this type of measurement is the heterogeneity of response of cells to a particular treatment. The best way to assay the activity of a transporter is to apply differing concentrations of the substrate to the same cell and then measure the electrical response. In practice, it is difficult to maintain an electrical recording from a single cell for sufficient time to gather enough data. Another problem is that the activity of the transporter being assayed can depend on the resting membrane potential of the cell. The cloning and characterization of single genes in heterologous expression systems, like Xenopus oocytes, has identified how the membrane potential of a cell can influence the activity of the transporter protein (Zhou and Miller, 2000). In addition to being a component of the driving force for transport, the affinity of the carrier protein for a substrate (Km) can change with alterations in the membrane voltage. For example, amino acid and sucrose carriers all showed a voltage-sensitive Km for these substrates with the value decreasing at more negative voltages (Boorer et al., 1996; Zhou et al., 1997). In plant cells, the substrate-elicited changes in membrane potential may not always be a direct indicator of the substrate's membrane transporter activity. When the addition of a particular substrate outside a cell elicits a change in membrane potential this could arise through effects on transport systems unrelated to those transporting the ions replaced in the bathing solution. For example, the transport of an added ion could be electrically neutral, resulting in no change in membrane potential, but the accumulation of the ion or a cotransported species that then stimulates an electrogenic transport system.
The two-electrode voltage clamp of a single cell can be used to assay transport, but this can only be applied to certain cell types of intact plants. Root hairs can be used and these measurements involve the insertion of two microelectrodes or a double-barrelled electrode into a single cell. These measurements include passing current to maintain the membrane potential at a fixed value during the application of the substrate, to do this the cells must be electrically isolated, so root hairs or single cells are best for this type of assay (Meharg et al., 1994). This is a powerful way to analyse the activity of a transporter and it can be used to generate kinetic models (Meharg and Blatt, 1995). For most types of transporter, the activity depends in some way on the availability of the substrate. For example, ammonium and nitrate transporter activity may be used to indicate the nitrogen status of a cell. This is likely to be a useful tool for the characterization of genetically modified plants.
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Ion-selective microelectrode measurements of compartmentation |
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TopAbstractIntroductionMicroelectrode access to cellsAssaying membrane transport...Ion-selective microelectrode...Ammonium and nitrate...Phosphate and sulphate...Conclusions and future workReferences |
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Intracellular measurements using ion-selective microelectrodes have been used to study the compartmentation of nutrients, intracellular signalling and transport mechanisms (Miller, 1995
). The theory and preparation of these ion-selective microelectrodes has been described in earlier reviews (Ammann, 1986; Felle, 1993; Miller, 1995). One of the advantages is that the method is non-destructive so that measurements are made on living cells enabling direct measurement of ion activities. Intact plants are used and experimental treatments can be applied during the electrode measurements. The measurements are quantitative and allow simultaneous measurement of the complete electrical and chemical gradients across membranes. The intracellular compartmental location of the tip can be identified by the incorporation of a second ion-selective barrel, such as pH (Walker et al., 1995). Other criteria can be used to define the compartmental location, for example, measurements of intracellular ion activity can be divided into two (cytosolic and vacuolar) populations (Miller and Zhen, 1991). The possibility of a tip location within other intracellular organelles is discounted because these compartments are so much smaller than the vacuole and cytoplasm.
Nitrate-selective microelectrodes have revealed vacuolar tissue differences in barley roots between epidermal and cortical cells (Zhen et al., 1991), with lower activities accumulated in the epidermis. Later measurements also showed that these two vacuolar pools could be remobilized at different rates (van der Leij et al., 1998) and these differences can be explained by the relative pool sizes. The cells of the root cortex are much larger than those in the epidermis and so represent a bigger nitrate pool (van der Leij et al., 1998). However, this explanation does not apply to vacuolar filling; for this process the vacuole of cortical cells appears to fill faster than epidermal cells (Zhen et al., 1991). These tissue disparities between cortical and epidermal cells of barley roots may be explained by differences in the activity of the tonoplast nitrate transporter (Miller and Smith, 1992) and/or nitrate reductase (assimilation) in the two types of cell (Rufty et al., 1986). The development of techniques to determine the pattern of gene expression in single cells should give some information on these tissue differences (Karrer et al., 1995; Brandt et al., 1999). However, if these techniques involve pooling cell samples the differences between individual cells of the same tissue will not be revealed.
The information provided by these intracellular ion measurements is needed to determine transport mechanisms and for the experimental design of other techniques. For example, in vitro biochemical enzyme assays can make use of this in vivo information to use realistic concentrations of substrates. Also for patch-clamping (Ward, 1997) the intracellular activities of ions are needed for the design of pipette and bath solutions. Although the ion selectivity of a channel can be determined in patch clamp experiments, the function of the channel in vivo is determined by the prevailing intracellular chemical and electrical gradients and ion-selective microelectrode measurements provide this information. Furthermore, these measurements can also report the electrical gradients across the plasma membrane and tonoplast in vivo providing complete pictures of the electrochemical gradients that exist in a living cell.
The trans-tonoplast potential difference is usually small and these values are calculated from the differences between measurements obtained when the compartmental location of the tip is known (e.g. pH electrodes, Miller and Smith, 1992). During a few cell impalements the tip location changes compartments during a recording and the reference barrel during this transition (Fig. 2) reports the in vivo trans-tonoplast potential. Figure 2 shows a recording obtained from a wild type Arabidopsis leaf cell during which the electrode tip spontaneously moves from the cytosol to the more acidic vacuole. During this compartmental transition the pH changes from 7.3 to 5.8 and the membrane potential from –150 to –120 mV, indicating a trans-tonoplast potential difference of 30 mV (Fig. 2). This value for the trans-tonoplast potential is larger than that measured in Kalanchoë leaf slices (25 mV; Rona et al., 1980) and that calculated from pH microelectrode measurements in barley root cells (12 mV; Miller and Smith, 1992). Measurements of the trans-tonoplast potential in the leaf cells are important, for example, changes in the potential may provide the driving force for the diurnal accumulations of organic anions in the vacuole during crassulacean acid metabolism.
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The main difficulty in the use of ion-selective microelectrodes is the reliability of their manufacture. Electrode tip geometry is a compromise between obtaining microelectrode tips that are small enough to impale cells successfully and yet large enough to provide a surface area of ion-selective membrane that allows sufficient response to changes in ion-concentration at the tip. At each development stage during the fabrication of ion-selective microelectrodes a proportion are unusable. Almost a quarter of the double-barrelled nitrate microelectrodes originally prepared successfully yield intracellular measurements, while the figure falls to 9% for triple-barrelled electrodes (Table 1
). Some types of electrode are sensitive to interference by proteins (Coombs et al., 1994) and other ions (Cuin et al., 1999). During the development of new types of ion-selective microelectrodes it is important to calibrate in solutions containing a range of ions that might be present in the cell.
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There are many different techniques for studying intracellular ion concentrations, but a major prerequisite for any method should be that the methodology involves the minimum disturbance to the cells being measured. A previous review (Miller and Smith, 1996
) has discussed the methods for measuring intracellular nitrate and many of these methods can be used for other assimilated ions. With the exception of direct measurement of extracted cell sap, these techniques provide an average value for the whole tissue or organ, but comparing these values with single cell analysis can provide a test of the various methods. The tissue averaging techniques employed fall into three groups, compartmental efflux analyses, cell fractionation studies, and nuclear magnetic reasonance (NMR) studies. In some cases the diverse methods have produced a wide range of different values for the cellular concentrations of metabolized inorganic ions.
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Ammonium and nitrate compartmentation |
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TopAbstractIntroductionMicroelectrode access to cellsAssaying membrane transport...Ion-selective microelectrode...Ammonium and nitrate...Phosphate and sulphate...Conclusions and future workReferences |
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The mechanism of NH4+ transport across the plasma membrane and tonoplast requires knowledge of the concentrations of NH4+ in the cytoplasm and vacuole as well as in the external medium. When the pH of the compartment is known, information on the concentration of NH4+ in subcellular compartments can be used to calculate the proportion present as ammonia (NH3). Published values for intracellular concentrations of NH4+ are relatively few and vary widely, depending on the methodology used (Table 2
). Of the techniques listed previously, only NMR allows the direct in vivo measurement of [NH4+]. Extraction of vacuolar sap is only suitable for large cells (such as the internodal cells of the giant alga Chara) and gives no information as to cytoplasmic concentrations. Efflux analyses rely on estimations of cytoplasmic and vacuolar volume. A small error in estimation of compartmental volumes (which may vary with position along a root) will lead to a relatively large error in estimated [NH4+]. For example, estimated concentration will be doubled if the assumed cytoplasmic volume is altered from 10% to 5% of total cell volume (Miller and Smith, 1996). NMR compartmentation studies rely on the fact that properties of the 14N-ammonium signal vary according to pH (Lee and Ratcliffe, 1991). Aarnes et al. studied [NH4+] in spruce using 14N NMR, but was unable to perform a detailed analysis of the 14N-ammonium signal (Aarnes et al., 1995).
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The difficulties encountered using the techniques described above make the development of a simple technique for direct in vivo measurement of NH4+ concentrations in subcellular compartments desirable. Ammonium-selective microelectrodes offer such a method and these have been used for extracellular measurements (Henriksen et al., 1990
; Hanstein and Felle, 1999). The main problem for intracellular measurements is interference by K+, cytosolic potassium concentration is around 72 mM (Walker et al., 1996) and at this level of interference the effective limit of detection is 1 mM. Nonetheless, these electrodes were used in Chara cells growing in 1 mM ammonium nutrient solution to give cytosolic and vacuolar activities of 7.3 and 30.8 mM, respectively (Wells and Miller, 2000). The ammonium selectivity of these electrodes is provided by a macrotetralide antibiotic, nonactin. However, new types of ammonium sensor have been reported that offer improved selectivity for NH4+ over K+ (Siswanta et al., 1994) and microelectrodes made using these molecules offer a better prospect for measurements in plant cells.
A large range of values have been reported for cytosolic nitrate activities, but microelectrode measurements suggest that in mature root cells this parameter is regulated at a value that is independent of changes in the external concentration (Miller and Smith, 1996). The vacuolar nitrate pools change with the external supply and this store is remobilized to maintain the cytosolic concentration of nitrate (van der Leij et al., 1998). Compartmental tracer efflux analysis has yielded different results suggesting that the cytosolic pools change with the external concentration (Siddiqi et al., 1991).
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Phosphate and sulphate compartmentation |
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TopAbstractIntroductionMicroelectrode access to cellsAssaying membrane transport...Ion-selective microelectrode...Ammonium and nitrate...Phosphate and sulphate...Conclusions and future workReferences |
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The concentration of cytosolic phosphate is maintained constant by resupply from the vacuole (Mimura, 1995
), this information is mostly based on measurements made with NMR of whole tissues (reviewed by Schachtman et al., 1998). Other measurements in yeast cells provide evidence for a similar situation in which the cytosolic pool is maintained by remobilization of vacuolar stores (Shirahama et al., 1996). In contrast, when radiolabelled 32P-phosphate was used for compartmental tracer efflux studies in root cells of the grass, Agrostis, the cytosolic concentration was found to depend on the external concentration (Macklon et al., 1996). There was a 10-fold increase in cytosolic phosphate concentration as the external concentration was increased from 10 to 100 µM. Compartmental tracer efflux analysis has also been used to estimate cytosolic concentrations of sulphate. Using 35S-labelled sulphate the cytosolic concentrations in excised tissues of a tropical legume were estimated as 83 nmol g-1 in roots, and 95 nmol g-1 and 147 nmol g-1 in epidermal and mesophyll leaf cells, respectively (Bell et al., 1994). The same technique gave a cytosolic concentration of 0.7 mM in carrot root cells (Cram, 1983). The prospects for making new types of ion-selective microelectrode suitable for measuring these anions are good. For example, a new sensor has been developed for the measurement of phosphate, as HPO42- (Carey and Riggan, 1994). Sulphate-selective macroelectrodes have also been reported (Li et al., 1999), but the pH sensitivity of these electrodes may be a problem for intracellular measurements. These may therefore be best used in triple-barrelled format including parallel pH measurements. |
Conclusions and future work |
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TopAbstractIntroductionMicroelectrode access to cellsAssaying membrane transport...Ion-selective microelectrode...Ammonium and nitrate...Phosphate and sulphate...Conclusions and future workReferences |
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Vacuolar heterogeneity of ion concentrations is well established in plants but the situation in the cytosol is still under some debate. The range of values measured in the cytosol seems to depend on the methodology used. The cell averaging nature of the techniques employed may explain some of this variation for the cytosolic concentrations. For example, perhaps there may be differences in compartmentation along the length of the root. Recent measurements in roots of several different species have shown that the regulation of cytosolic nitrate is different in expanding cells closer to the root tip (AJ Miller and SJ Smith, unpublished work). Very few papers report using more than one method to measure the intracellular concentrations, but vacuolar nitrate activities in barley roots were found to be similar using single cell sap sampling and nitrate microelectrodes (Zhen et al., 1991
). More measurements comparing results obtained using different techniques on the same tissue are needed to provide some explanations for the range of values reported for cytosolic ion concentrations. For nitrate, ammonium and phosphate, compartmental tracer efflux studies have shown that the cytosolic concentrations reflect changes in the external concentrations, whereas homeostasis of cytosolic nitrate and phosphate has been demonstrated by NMR and ion-selective microelectrode measurements. One of the major consequences of the regulation of these cytosolic pools is that the cytosolic activities are not representative of the changes in the external supply. These observations raise difficult questions about how a plant cell senses its nutrient status. For example, the extent of induction of nitrate influx into barley root cells increased with increasing external nitrate (Siddiqi et al., 1990); the nitrate flux to the xylem changes reflecting the external supply (Andrews, 1986). These observations require complicated explanations, invoking sensors of nitrate fluxes or concentrations in the vacuole or at the cell surface. Testing these models requires more single cell measurements to determine when homeostasis of cytosolic nitrate breaks down.
Microelectrode measurements are most appropriate for cells at the surface of tissues or in single cells because in these situations impaling does not involve any damage to cells overlying the cell of interest. Cells that are found deeper in tissues can only be accessed by first damaging outer layers of cells or in leaves through the stomatal aperture. Ion-selective microelectrodes are the only technique for measuring cytosolic ion activities in single cells. Microelectrode access to other intracellular compartments may be feasible by using special tissues that have large organelles. For example, the arc6 Arabidopsis mutant has a few large chloroplasts (Pyke et al., 1994), so leaf mesophyll microelectrode measurements may record from this compartment. Nitrate-, and now also ammonium-selective microelectrodes have been successfully used to measure intracellular concentrations in plant cells. The measurements made with these microelectrodes have confirmed the vacuolar heterogeneity reported by other techniques, but for nitrate they suggest homeostasis of cytosolic activity. New types of ion-selective microelectrode are being developed that are suitable for measuring cytosolic concentrations of other ions so the prospects for using this technique to measure other types of metabolites are feasible.
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Acknowledgments |
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IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. The authors also receive funding from EU BIOTECH grant numbers BIO4CT972231 and BIO4CT972310.
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Notes |
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1 To whom correspondence should be addressed. Fax: +44 1582 763010. E-mail: tony.miller{at}bbsrc.ac.uk
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TopAbstractIntroductionMicroelectrode access to cellsAssaying membrane transport...Ion-selective microelectrode...Ammonium and nitrate...Phosphate and sulphate...Conclusions and future workReferences |
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