Journal of Experimental Botany, Vol. 54, No. 383, pp. 813-824,
February 1, 2003
© 2003 Oxford University Press
Electrophysiological responses of maize roots to low water potentials: relationship to growth and ABA accumulation
Received 29 April 2002; Accepted 23 September 2002
Department of Agronomy, Plant Sciences Unit, 1-87 Agriculture Building, University of Missouri, Columbia, MO 65211, USA
1 Present address and to whom correspondence should be sent: Broom’s Barn Experimental Station, Higham, Bury St Edmunds, Suffolk IP28 6NP, UK. Fax: +44 (0)1284 811191. E-mail: eric.ober{at}bbsrc.ac.uk
Abbreviations: 
w, water potential(s); FLU, fluridone; PEG, polyethylene glycol; Em, membrane potential; SHAM, salicylhydroxamic acid; CCCP, carbonyl cyanide m-chlorophenylhydrazone; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.
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Abstract |
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The maintenance of root elongation is an important adaptive response to low water potentials (
w), but little is known about its regulation. An important component may be changes in root cell electrophysiology, which both signal and maintain growth maintenance processes. As a first test of this hypothesis, membrane potentials (Em) were measured within the cell elongation zone of maize (Zea mays L.) primary roots. Seedlings were grown in oxygenated solution culture, and low w was imposed by the gradual addition of polyethylene glycol. Cells hyperpolarized approximately 25 mV in response to low w, and after 48 h resting potentials remained significantly hyperpolarized at w lower than –0.3 MPa compared with roots at high w. Inhibitor experiments showed that the hyperpolarization was dependent on plasma membrane H+-ATPase activity. Previous work showed that accumulation of abscisic acid (ABA) is required for the maintenance of maize primary root elongation at low w. To determine if the mechanism of action of ABA involves changes in root electrophysiology, Em measurements were made during long-term exposure to low w. Steady-state resting Em were measured in regions in which maintenance of cell elongation was dependent on ABA accumulation (2–3 mm from the apex), or in which elongation was inhibited regardless of ABA status (6–8 mm from the apex). Em was substantially more negative in ABA-deficient roots specifically in the 2–3 mm region. The results suggest that set-points for ion homeostasis shifted in association with the maintenance of root cell elongation at low w, and that ABA accumulation plays a role in regulating the ion transport processes involved in this response. Key words: Abscisic acid, maize, membrane potential, root growth, water deficit.
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Introduction |
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Water deficit is often the major factor restricting plant growth, and drought or insufficient irrigation water limits agricultural productivity worldwide. Therefore, it is important to understand the mechanisms that plants use to adapt to water-limited conditions. For example, root growth is much less sensitive to low water potentials (
w) than shoot growth (Sharp and Davies, 1989), and this adaptive strategy helps the plant to maintain a supply of water under conditions of soil drying. Previous studies have shown that in the elongation zone of the maize (Zea mays L.) primary root, morphological, biochemical, molecular, and biophysical changes occur in response to low w (Sharp et al., 1988; Ober and Sharp, 1994; Saab et al., 1995; Wu et al., 1996, 2001; Conley et al., 1997). Detailed growth kinematic analysis revealed that cell elongation at low w is maintained preferentially toward the root apex (Sharp et al., 1988); in the apical few millimetres, elongation is completely maintained even under severe water deficit (w of –1.6 MPa). Many of the observed changes occur specifically within this region, but not a few millimetres further from the apex where cell elongation is not maintained at low w. This is strong evidence that these changes are not symptoms of stress injury, but are adaptive responses specifically related in some manner to the maintenance of root elongation. Thus, it is critical to discover how these processes are regulated.
It is likely that cellular water deficit is perceived at the membrane level and effects changes in ion activities that transduce the environmental challenge to adaptive changes in cell biochemistry and gene expression (Trewavas, 1976; Hetherington and Quatrano, 1991). Electrophysiological changes are integral parts of stress-activated signalling mechanisms in organisms ranging from bacteria to humans (Chamberlin and Strange, 1989). In plants, it is well known how algal cells respond to osmotic challenges through changes in ion transport and inorganic and organic solute accumulation (Bisson and Kirst, 1995). Descriptions of higher plant responses are less definitive. For example, responses to low w include a depolarization of the membrane potential (Em) (Göring et al., 1979; Cortes, 1997), hyperpolarization (Kinraide and Wyse, 1986; Li and Delrot, 1987; Lew, 1996; Shabala and Lew, 2002), or no change in Em (Cocucci et al., 1976; Wegner and Zimmermann, 1998). The variability in reported responses may be attributable to differences in species, tissue or cell type or the mode of low w imposition; there is no clear understanding. In addition, most studies have examined short-term responses to rapid osmotic perturbation and, as far as is known, none have examined the electrophysiological effects of long-term exposure to low w or addressed issues related to growth, with the exception of work on root hairs (Lew, 1996).
An important regulator of ion fluxes that underlie changes in Em in response to low w may be abscisic acid (ABA), which is synthesized by plant tissues under water deficit conditions. In stomatal guard cells, ABA activates changes in ion transport leading to turgor loss and stomatal closure (MacRobbie, 1997). ABA may play a role in regulating ion transport in other cell types as well, and may function to shift tissues to ‘a new and different physiological state’ in response to changing environmental conditions (Hetherington and Quatrano, 1991). In roots, applied ABA can cause a range of effects on ion transport such as increased K+ efflux, or influx, depending on the concentration of ABA, cell type, tissue K+ status, age of the tissue, temperature, etc. (van Steveninck and van Steveninck, 1983; Roberts and Snowman, 2000). Similar factors also determine whether applied ABA causes cell depolarization or hyperpolarization (Fromm et al., 1997; Zocchi and De Nisi, 1996).
In the maize primary root, it has been established that accumulation of ABA helps to maintain root elongation at low w, chiefly through the suppression of ethylene synthesis (Saab et al., 1990; Sharp et al., 1994; Spollen et al., 2000; Sharp, 2002). However, it is not known how regulation of the ethylene biosynthesis pathway by endogenous ABA is brought about. It is hypothesized that changes in ion transport and intracellular activity both signal (in the short-term) and maintain (in the long-term) root cellular responses to water deficit, and that ABA may function primarily in the regulation of these electrophysiological changes. As a first step to test the hypothesis, conventional electrophysiological techniques have been used to measure changes in Em, which occur as a function of changes in ion transport and accumulation. This technique does not provide information on which ion species are affected, but it is much more sensitive to detecting intracellular changes than are measurements of changes in ion flux or activity (Ullrich and Novacky, 1991). The objectives of the study were to measure in intact, growing primary roots of maize the changes in Em that occur within the elongation zone in response to the gradual imposition of low w and relief from stress, how these changes are associated with the spatial pattern of cell elongation during long-term exposure to low w, and the extent to which the long-term changes are dependent on ABA accumulation.
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Materials and methods |
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Plant culture
Maize seeds (Zea mays L., cv. Fr27xFrMo17) were sown on germination paper moistened with 0.25 mM CaCl2, then germinated in the dark at 29±0.1 °C for 46 h. Seedlings with a primary root length of approximately 3 cm were transferred to solution culture (culture chambers described below) and grown under the same conditions. When necessary, illumination was provided by a dim blue-green safelight (Saab et al., 1990) to minimize potential effects of white light on root growth (Pilet and Ney, 1978). The medium, formulated to reflect the nutrient content of a typical soil solution (Barber, 1984), contained (in mM): 0.25 CaCl2, 0.3 MgSO4, 0.15 K2HPO4, 0.4 NH4NO3, 5 MES; (in µM): 20 FeSO4, 6.0 H3BO3, 0.9 MnSO4, 0.6 ZnSO4, 0.15 CuSO4, 0.1 NaMoO4, 0.01 CoCl2, 0.11 NiCl2; adjusted to pH 6.0 with NaOH (approximately 2.75 mM final Na+ concentration).
Even though maize does not normally grow with roots immersed in water, electrophysiological studies must usually be conducted on tissue bathed in an electrically conductive solution, which also facilitates the uptake of experimental compounds. Faced with this restriction, a system of gradually imposing low w in oxygenated solution culture was developed using polyethylene glycol (PEG) as an osmoticum (Verslues et al., 1998). Supplemental oxygenation was shown to be required for optimum root growth in solutions of PEG, and to shorten the time to attain steady-state growth rates at high and low w. All solutions were oxygenated to achieve an O2 partial pressure of 45 kPa. This level of oxygenation at both high and low w restored tissue O2 partial pressure levels within the root elongation zone to those found in roots in well-aerated vermiculite (Verslues et al., 1998). Solution O2 partial pressures were measured with a commercial O2 sensor (ISO2, World Precision Instruments, Sarasota, Florida) or an O2 microsensor (Ober and Sharp, 1996).
High molecular weight PEG (PEG 8000, Sigma-Aldrich, St Louis, Missouri) was used as an osmoticum because it is virtually excluded from entering the root apoplast (Carpita et al., 1979), and thus removes water from the cell and cell wall space. In this way it mimics the drying effects of a soil environment. In contrast, osmotica such as salts, mannitol, or sorbitol penetrate the cell wall and the cells themselves, and therefore may alter the normal response to low w. There were no toxic effects of PEG apparent under the conditions used (Verslues et al., 1998). Low w were imposed by gradually replacing the growth medium with solutions of PEG dissolved in growth solution. Solution w were determined by isopiestic thermocouple psychrometry (Boyer and Knipling, 1965).
ABA accumulation was decreased in some experiments by treatment with fluridone (FLU), an inhibitor of carotenoid biosynthesis (Moore and Smith, 1984; Saab et al., 1990; Sharp et al., 1994; Spollen et al., 2000). FLU was added at a final concentration of 1 µM during germination and in the growth medium. To confirm that effects of FLU on root growth and Em were a result of ABA deficiency, additional experiments were conducted in which the ABA level in the growth zone of FLU-treated roots was restored by adding (R,S) ±ABA to the PEG solution.
In experiments using other inhibitors, stock solutions were prepared as follows: KCN was dissolved in water to prepare a 100 mM solution, and used at a concentration of 1 mM; salicylhydroxamic acid (SHAM) was prepared as a 10 mM solution in 5 mM HEPES buffer, pH 8.0, and used at full strength; a stock solution of 5 mM NaVO4 was prepared in 5 mM MES, pH 6.4, and used at a concentration of 0.5 mM; a 10 mM solution of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was prepared in 95% ethanol, and used at a concentration of 10 µM.
Growth studies
After germination, seedlings were grown in a Plexiglas chamber (600 ml) housing 21 seedlings (Verslues et al., 1998). Caryopses were suspended on a holder above the solution and the primary roots grew downward through perforated transparent root guides fashioned from plastic drinking straws (i.d. 6 mm). A Plexiglas cover was used to enclose the shoots within a humid atmosphere, which minimized transpirational water loss. The air/O2 mix, supplied through a perforated plastic tube extending along the bottom of the box, vigorously aerated and stirred the solution. Root elongation rate was monitored by marking the position of the root apex along the side of the box at various times.
Spatial growth analysis
Spatial patterns of longitudinal strain rate (local relative elongation rate, % h–1) were determined using procedures modified from Silk et al. (1984) and Sharp et al. (1988). Seedlings were removed from solution and the primary root tip was marked at approximately 1 mm intervals with water-insoluble ink (Pelican, 17 Black) using a fine paint brush. Seedlings were returned to solution, and after elongation rates recovered (usually within 1–2 h), roots were photographed every 15 (high w) or 30 min (low w). Enlarged photocopies were made from the negatives using a microfiche viewer so that the displacement of marks from the root apex could be digitized to obtain longitudinal velocities, which were then interpolated to 0.5 mm intervals using cubic splines. The velocity data were then differentiated with respect to position using a 5-point formula (Erickson, 1976) to give the spatial distribution of longitudinal strain rate.
ABA determinations
To assay tissues for ABA content, seedlings were removed from solution, roots were rinsed briefly in distilled water, and then blotted dry. The apical 10 mm of the primary roots were excised, placed in microcentrifuge tubes and immediately frozen at –80 °C. Samples were weighed, freeze-dried, then reweighed to obtain the mass of tissue water. Distilled water (500 µl) was added to the tubes and ABA was extracted overnight at 4 °C. Aliquots were assayed for ABA content by a monoclonal antibody-based radioimmunoassay (Quarrie et al., 1988; Saab et al., 1990).
Electrophysiology
For Em measurements, one seedling with a 3 cm long primary root was transferred from germination paper to a small (15 ml) Plexiglas chamber (Fig. 1). The chamber was designed such that the root grew down through a guide tube, which provided physical support for the root during impalements, yet permitted enough movement to allow unrestricted growth. Roots were measured in a vertical orientation in order to minimize the confounding effects of gravistimulation on Em, ion and hormone distributions within the elongation zone (Pilet and Rivier, 1981; Ishikawa and Evans, 1990). Microelectrodes were mounted in a rubber septum, positioned through a port drilled into a movable slide on the side of the chamber, and accessed the root through openings in the guide tube. The seal at the port was made watertight by applying high vacuum silicone grease (Dow Corning, Midland, Michigan) around the pipette barrel and the rubber septum. To minimize vibrations in the root chamber, solutions were aerated in a 15 ml tube adjacent to the chamber, then circulated through the root chamber at 3.0 ml min–1 using a peristaltic pump. In some experiments, solutions in the root chamber were oxygenated directly and the bubbles were separated from the root by a baffle.
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Root elongation rates in this chamber were comparable to rates obtained in the larger chamber used for the growth studies. Em measurements were made on roots elongating at approximately the mean rate for each treatment. Impalements were viewed through a microscope (fitted with long-working-distance objectives) mounted horizontally in front of the chamber, which was illuminated from behind with a fibre optic light source fitted with a blue/green filter. The set-up was enclosed within a Faraday cage on a vibration-isolated table. Preliminary tests showed that illuminating seedlings with the blue/green light did not cause any transient electrophysiological changes within the root elongation zone.
Em measurements were made with standard glass microelectrodes filled with 3 M KCl and connected to the head stage of a high input impedance electrometer (FD223, World Precision Instruments, Sarasota, Florida) via Ag/AgCl wires. Voltage output from the electrometer was sent to a chart recorder and collected on computer disk via a data acquisition system. Measuring electrodes were mounted on a three-dimensional micromanipulator (model MO-5, Narishige). The reference electrode (Ag/AgCl/3 M KCl in 2% agar) was located downstream from the chamber. Similar Em values were obtained using electrodes filled with 3 M KCl or 100 mM KCl (pH 2.0), but the latter tended to be associated with more noise. Using 3 M KCl, stable Em were obtained immediately upon impalement and were held for as long as 50 min; therefore, there was no apparent leakage of electrolyte from the electrode into the cell that affected the measurements.
Em were measured in epidermal and cortical cells within the root elongation zone. Epidermal cells had slightly less negative potentials than cells in underlying layers. The differences between successive layers were small (on average 2 mV) and variable; for treatment comparisons, therefore, values from all measured layers (epidermis through the fifth cortical cell layer) were combined. Mature root tissues have typically been used in studies of root Em, and few studies have attempted measurements of cells near the apex of intact, growing roots (see, for example, Papernik and Kochian, 1997). The primary difficulty is that, as the root grows, the impaled target cell moves away from the electrode; thus, to keep the electrode tip within the cell, the electrode must be moved to ‘track’ root movement. The microelectrodes were pulled to produce long, thin shanks, which increased their flexibility and the duration of successful impalements. During an impalement the tip bent slightly as the root grew, and the microelectrode was frequently repositioned using the micromanipulator. The greased fittings in the chamber port around the microelectrode barrel helped to dampen any abrupt movements. Preliminary tests showed that merely bending the microelectrode tip did not affect the voltage signal. The depth of impalement was determined using the micromanipulator scale, and reaching a cell layer was determined by observing the impalement of cells, detected as a significant increase in potential above the Donnan potential of the cell wall (approximately –40 mV). For Em recordings lasting several minutes or more, cells within the third to sixth cortical cell layer were impaled, since the deeper impalements helped to anchor the microelectrode tip in place. Checks on microelectrode resistance were routinely performed during measurements and if a microelectrode became plugged (indicated by an increased input resistance), it was replaced with a fresh microelectrode. Under ‘steady-state’ conditions (i.e. when solution w and root elongation rates were constant), an Em value was recorded if the potential remained steady for at least 1 min, electrode resistance had not changed, and the impalement showed signs of maintaining a good seal (indicated by the rapid attainment of a steady value without subsequent decay). When Em oscillated during longer measurements of a single cell, values for resting potentials were determined by averaging peak and trough voltages.
The possibility of measuring voltage artefacts during changes in w was examined. A gradual improvement in the seal around the microelectrode could have resulted in an apparent increase in potential over time as cells lost turgor. This appears unlikely since electrode resistance did not change over the course of measurements as w decreased. When sudden increases in resistance were observed, often due to a plugged microelectrode tip, the data were discarded. Another possible artefact is a change in junction potential, which is produced in some instances by changing solutions (although in this study both high- and low-w solutions had the same ionic composition). However, there was no change in measured potential across the electrodes when the microelectrode was positioned in the bath solution at the root surface during the shift in w.
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Em responses during imposition of and recovery from low
wTo study dynamic responses of root cell Em to changes in
w, measurements were made in cortical cells between 6–8 mm from the apex, within the zone of elongation at high w (see Fig. 5). At high w, values of Em were generally between –120 to –132 mV (Fig. 2); the scatter in values was due in part to an oscillation of Em that had an amplitude of 5–10 mV and a frequency of 5–10 min. Detailed analyses of Em oscillations in roots have been reported previously (Souda et al., 1990).
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=0.0769, 10 d.f.) for comparison between treatments at a sampling time. Curves were fitted to the data using a third-order regression procedure using SigmaPlot software (SPSS UK Ltd, Woking, UK).Upon imposition of a decrease in
w to –0.28 MPa, Em began to hyperpolarize (Fig. 2) and reached a new level after the shift in w was completed (after 80 min; Fig. 2, inset). The mean hyperpolarization at 60–80 min after low w imposition was 24±3 mV (n=11). Root elongation was inhibited after a change in w to –0.28 MPa, but eventually recovered 48 h later (Verslues et al., 1998). When measurements were made in roots growing under these new steady-state conditions, Em was –136±3 mV (n=8), which was not significantly different from the value at high w (–130±3 mV, n=8; see Table 3). Thus, the initial hyperpolarization in response to a decrease in w of –0.28 MPa was transient and Em returned to near normal levels as the roots acclimated to the new condition.
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When –0.8 MPa PEG was used to impose low
w, the pattern of hyperpolarization was similar to the experiments using –0.28 MPa PEG during the first 80 min because the rate of decline in w was similar. Beyond 80 min, as w declined further to –0.8 MPa, there was no further hyperpolarization (
Em=25±6 mV, n=6, after the shift in w was completed). In contrast to the –0.28 MPa treatment, Em values remained hyperpolarized (–145±5 mV, n=13) under steady-state conditions at –0.8 MPa (48 h after stress imposition). Similar results were obtained at a w of –1.6 MPa (see Table 3). In summary, imposition of low w always caused a hyperpolarization, but a new, more negative resting Em was maintained under steady-state conditions only when solution w was less than –0.3 MPa.
Roots growing at low w depolarized when the w was increased by gradually replacing the PEG solution with solution of high w (Fig. 3). Root Em depolarized beyond the normal resting Em observed at high w, but not further than the diffusion potential (approximately –91 mV, determined after treating cells with CN–; Table 1). Within 1 h after the removal of PEG, Em returned to the normal resting value at high w (data not shown). Depolarizations in response to hypo-osmotic treatments have been observed before, e.g. in Chara (Bisson and Kirst, 1995) and Arabidopsis root hairs (Lew, 1996). Following a hypo-osmotic shift, the hyperpolarized state could be recovered by re-imposition of the PEG solution (Fig. 3). Long-term recordings from single cells were difficult to obtain routinely in growing roots, but this example demonstrates the dynamic nature of an individual cell’s response to changing w.
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The initial low
w-induced hyperpolarization could have been caused by energized ion transport processes (requiring ATP), or by passive movement of ions down their electrochemical gradient. To differentiate between these possibilities, roots were pretreated with metabolic inhibitors before the imposition of low w. Mitochondrial electron transfer is completely blocked by the combination of CN– and SHAM, which effectively inhibits the H+-ATPase and energized ion transport. This treatment caused an immediate depolarization, and after imposition of low w (–0.28 MPa) there was no hyperpolarization within 60 min (Table 1), the usual time frame for the full response (Fig. 2). Vanadate, a specific inhibitor of the plasma membrane H+-ATPase, also prevented the hyperpolarization (Table 1). The protonophore CCCP dissipates H+ gradients, and thereby inhibits ion transport processes that are dependent on the gradient for H+-coupled transport or for ATP production and H+-ATPase activity. CCCP also caused a depolarization followed by a lack of hyperpolarization after low w imposition (Table 1). Similar results were also obtained by treating roots with FCCP or erythrosin B (data not shown). These results indicate that the hyperpolarization response to low w required an active plasma membrane H+-ATPase.
Preliminary experiments were conducted to examine whether, in addition to H+, other ions were required for the hyperpolarization. To indicate whether fluxes of K+ were essential, roots were grown for 48 h in K+-free (replaced by Na+) media, or pre-treated with Ba2+, which blocks inward- and outward-rectifying K+ channels (MacRobbie, 1997). Both treatments were effective in preventing the hyperpolarization normally observed after the addition of –0.3 MPa PEG (data not shown). When roots were grown for 48 h in a Cl–-free medium (replaced by SO42–), hyperpolarization was similarly blocked. Further experimentation is needed to describe accurately the role of specific ion fluxes, but these preliminary results suggest a role for K+ and Cl– in the hyperpolarization response to low w, which is consistent with other studies (Lew, 1996; Teodoro et al., 1998).
ABA and root growth during long-term exposure to low w
Previous studies using vermiculite as a growth medium have demonstrated that ABA accumulation is required for the maintenance of maize primary root elongation at low w (Saab et al., 1990; Sharp et al., 1994; Spollen et al., 2000). For the present study it was necessary to show that similar results could be obtained in solution culture. In seedlings exposed to a w of –1.6 MPa, the rate of root elongation was similar in PEG solution and vermiculite (Verslues et al., 1998). As in the vermiculite studies, treatment with FLU decreased root ABA accumulation (Table 2) and severely inhibited root elongation (Fig. 4). Root elongation was almost completely restored by the addition of 0.5 µM ABA to the PEG solution (Fig. 4). This concentration of applied ABA raised the ABA content of the root apical centimetre (encompassing the elongation zone; see Fig. 5) to the normal level at –1.6 MPa (Table 2). By contrast, the addition of 1.0 µM ABA to FLU-treated roots more than doubled the normal internal level (71±17 ng g –1 H2O, n=4) and did not restore the elongation rate (data not shown). Similar results were obtained with vermiculite-grown roots (Sharp et al., 1994), and illustrate that non-physiological levels of ABA can inhibit root growth. These findings emphasize the importance of studying hormone levels in an appropriate range for the particular condition (Jacobs, 1959; Sharp, 2002). At high w, as in the previous vermiculite studies, treatment with FLU had minimal effects on root ABA content (Table 2) or elongation rate (data not shown).
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Relationship of Em changes to steady-state growth patterns and ABA accumulation
Determining the relationship of electrophysiological changes to the response of root growth during imposition of low
w is complicated by the dynamics of rapidly changing patterns of cell elongation. By contrast, the patterns of cell elongation under steady-state conditions at high w and low w (–1.6 MPa) were defined by kinematic analysis. Figure 5 shows which regions were elongating, which regions had ceased growth at low w, and which regions were dependent on ABA for growth maintenance at low w. As with the responses of overall root elongation rate, the growth patterns were similar to those obtained previously in vermiculite-grown roots (Sharp et al., 1988; Saab et al., 1992). The elongation zone extended to 12 mm from the apex at high w and was shortened to approximately 7 mm in PEG solution at –1.6 MPa. Maximal longitudinal strain rates occurred between 3 mm and 6 mm from the apex at high w, but at low w rates began to slow beyond 3 mm. Treatment with FLU at low w decreased strain rates at all positions beyond the first millimetre, whereas restoration of the ABA level largely restored the normal growth pattern. Two regions are particularly important to note: at low w, strain rates were almost fully maintained at 2–3 mm from the apex, but only if ABA accumulation was not blocked. In the region 6–8 mm from the apex, strain rates were almost completely inhibited at low w regardless of the ABA status of the tissue. Thus, cells 2–3 mm from the apex were dependent on ABA accumulation for growth maintenance at low w, whereas later, when cells had been displaced 6–8 mm from the apex, ABA accumulation was no longer sufficient to prevent growth inhibition. These regions were selected for comparative electrophysiological measurements.
Under conditions of steady-state growth and w, Em was also at steady state. Em measured at 4–8 mm from the apex changed by only +4.8±4 mV (n=4) between 24 h and 48 h after transfer to solution culture in roots growing at high w. Furthermore, Em oscillations were negligible in roots growing at a w of –1.6 MPa. The measurements revealed that Em were increasingly more negative with increasing distance from the root apex (Table 3). Values at high w ranged from approximately –120 mV at 1 mm from the apex to approximately –140 mV at 10 mm from the apex. This longitudinal gradient in Em may reflect developmental changes in the number or activity of ion transport components as cells grow and mature. Similar gradients have been reported previously (Mertz and Higinbotham, 1976; Papernik and Kochian, 1997). In the roots growing at –1.6 MPa, cells were hyperpolarized compared to the roots at high w at both the 2–3 mm and 6–8 mm locations (Table 3).
At high w, Em was not measurably affected by treatment with FLU at either 2–3 or 6–8 mm from the apex (Table 3). At –1.6 MPa, in contrast, cells in the 2–3 mm region of FLU-treated roots were significantly more hyperpolarized compared with non-treated roots. When FLU-treated roots were supplied with ABA to restore the normal ABA content (Table 2) and spatial growth pattern (Fig. 5), potentials 2–3 mm from the apex were returned close to the normal values at low w (Table 3). In the 6–8 mm region, where growth was inhibited regardless of ABA status, the hyperpolarization was similar in the control and FLU-treated roots, and was unaffected by the addition of ABA. The steady-state data show that ABA deficiency resulted in a greater than normal hyperpolarization at low w, specifically in the region in which ABA was required for maintenance of cell elongation.
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Early electrophysiological responses to low
wAs soil begins to dry during the initial stages of drought, the responses of roots are critical to the survival of the plant, yet little is understood about how roots perceive low
w or how adaptive responses are regulated. Furthermore, it is mainly the tissues at the root tips comprising the meristem and cell elongation zone that are essential for continued root elongation during dry conditions and after stress is relieved. Therefore, this study focused on cells within the elongation zone, and the results show that an early response to the imposition of low w is Em hyperpolarization, involving activation of the plasma membrane H+-ATPase. This response may be triggered by turgor-sensitive stretch-activated membrane channels or by other osmo-sensing elements (Lew, 1996).
A more negative Em would increase the driving force for ion uptake. An accumulation of inorganic ions, which are then replaced by synthesis or uptake of organic osmolytes during long-term exposure to low w, appears to be a response to osmotic stress that is conserved throughout nature: human brain glioma cells accumulate principally Na+ and inositol (Chamberlin and Strange, 1989); in E. coli the response can involve K+ and proline (Yim and Villarejo, 1994); some algal cells accumulate Na+, then replace it with glycerol (Bisson and Kirst, 1995). Surprisingly, there is no definitive description of similar events in higher plants. During long-term water deficit there is significant osmotic adjustment within the elongation zone of the maize primary root (Sharp et al., 1990), and accumulation of proline accounts for up to half of the decrease in osmotic potential within the apical few millimetres (Voetberg and Sharp, 1991). It is possible that early accumulation of inorganic ions, driven by a hyperpolarized Em, is the signal that activates the accumulation of proline. Interestingly, the deposition of proline within the growth zone also depends on the accumulation of ABA (Ober and Sharp, 1994). Furthermore, recent measurements of low w-induced ion fluxes at the root surface confirm that increases in K+, Cl– and Na+ influx contribute to short-term osmotic adjustment and the restoration of turgor (Shabala and Lew, 2002).
Few other studies have used growing cells to examine electrophysiological responses to a decrease in w. One, a study of root hairs in Arabidopsis, also showed a hyperpolarization (Lew, 1996). In contrast, another study inferred an inhibition of the proton pump based on measurements of ion fluxes at the root surface near the apex of the maize primary root (Shabala and Newman, 1998). Hyperpolarizations in response to decreased w were also observed in mature root tissue of beet (Kinraide and Wyse, 1986) and Arabidopsis (Shabala and Lew, 2002), and in carrot suspension cells (Reuveni et al., 1987). Also, algal species that regulate turgor or cell volume generally hyperpolarize when the w decreases (Bisson and Kirst, 1995). In contrast to these studies, Em depolarizations in response to low w were measured in sunflower root (Cortes, 1997), maize coleoptile (Göring et al., 1979) and Chara inflata cells (Kourie and Findlay, 1990). Most previous studies have used a very rapid imposition of low w to evoke electrophysiological responses. In this study, when low w was imposed at a much faster rate than usual, cells transiently depolarized before the hyperpolarization (data not shown). Thus, one explanation for the varying effects of low w on Em may be the rapidity and severity of the stress imposition. It can be argued that higher plant cells, and roots in particular, are rarely exposed to hyperosmotic shock; instead, the w of the rhizosphere slowly declines as water evaporates or is extracted from the soil. Therefore, a relatively slow decline in w may be necessary to observe a cellular response that closely relates to that which occurs naturally in field-grown roots subjected to drought. Conversely, roots in drying soil that have accumulated solutes for osmotic adjustment risk injury when subjected to a sudden influx of water after rainfall. Depolarization-induced solute efflux may be an important adaptive response to avoid excessive turgor during such hypo-osmotic shock.
A shift in steady-state root electrophysiology at low w
Typically, electrophysiological responses to environmental stimuli are studied in a single cell, are transient, and occur on the order of seconds or milliseconds. Much less attention has been focused on electrophysiology under steady-state conditions. Under widely varying conditions, the steady-state cytoplasmic activities of ions normally are controlled within certain set limits (Cram, 1980; Walker et al., 1996). The idea of the ‘set-point’ is that perturbation of ion activity in response to environmental challenges generates an ‘error’ signal that triggers other processes, and then the ion activity is brought back to the set-point value (Cram, 1980). In this sense it is likely of importance that 48 h after imposition of low w (<–0.3 MPa), the resting Em of cells remained slightly but significantly hyperpolarized compared with roots of the same age at high w. There are other examples of small but physiologically significant shifts in Em. Differences up to 4 mV were important indicators of auxin sensitivity in tobacco protoplasts treated with auxin (LeBlanc et al., 1999) and, in animals, neurons of the visual cortex adapt over time to a repeated stimulus by shifting Em to a new resting potential 5–15 mV more negative (Carandini and Ferster, 1997). In roots under steady-state conditions at low w, cells within the elongation zone had just recently been formed in the meristem, and had never experienced a high w environment (compared with the now mature root cells displaced 20–50 mm from the apex). Therefore, factors establishing the altered resting Em in the apical region were active without the original stimulus (the change from high to low w). Since ion activities outside the root should not have changed after switching to low w, the steady-state change in Em indicates that programmed set-points for proton pump activity or intracellular ion activities governing Em (principally K+; Roberts and Snowman, 2000) had been shifted to a new value.
The role of ABA
In addition to the rapid, early events involved in the initial perception of water deficit, maintenance of root elongation during long-term exposure to low w is equally critical for plant survival during drought. Since ABA is required for this process, and if the primary site of action of ABA is at the membrane level, then ABA accumulation could regulate the maintenance of resting Em under steady-state conditions at low w. This hypothesis is supported by the observation that ABA-deficient cells within the region 2–3 mm from the apex had significantly different resting Em than ABA-sufficient cells. ABA may play a role in ion homeostasis within this region of the growth zone, but with insufficient ABA this control is lost. Several reports in the literature are consistent with this idea. It has been suggested that ABA shifts homeostatic set-points in guard cells of closed stomata (MacRobbie, 1997). In leaf cells of ABA-deficient tomato plants, the resting potential was significantly more negative compared with wild-type plants (Herde et al., 1998). In another example, in yeast cells adapted to grow in saline media, over-expression of the Hal1 gene caused increased levels of cellular K+, which was associated with improved growth compared with the wild type (Gaxiola et al., 1992). Interestingly, in the same study, a Hal1 homologue in maize roots was induced by the addition of ABA. It was suggested that Hal1 is part of the K+-homeostatic mechanism, regulated by ABA.
An alternative to the control of ion homeostasis by ABA is that the greater hyperpolarization at 2–3 mm from the apex in the FLU-treated roots at low w was merely a consequence of growth inhibition, such that slower growing cells were relatively hyperpolarized compared with the faster growing cells at the same distance from the apex in the –FLU roots. This relationship, however, breaks down with other comparisons: for example, in the –FLU treatment longitudinal strain rates were quite similar at 2–3 mm from the apex at high and low w, but Em was significantly hyperpolarized at low w. In the 6–8 mm region, longitudinal strain rates differed greatly between high w (–FLU) and low w (+FLU), but resting Em were not significantly different. Overall, there was no significant correlation (R2=0.01) between elongation rates and Em values at 2–3 mm from the apex of individual roots growing at high w. Thus, the more negative Em at low w compared with high w, or at 2–3 mm in +FLU compared with –FLU roots at low w, does not appear to be attributable to differences in the elongation rates of the cells. Experiments specifically designed to determine whether or not ABA-modulated changes in Em bring about changes in growth, or vice versa, are required to resolve this issue fully.
It is intriguing that the FLU-induced differences in Em at 2–3 mm from the apex of roots at low w did not occur at 6–8 mm (Table 3). An explanation of the greater hyperpolarization in ABA-deficient roots in the apical region, but not the basal region, may be that it is related to a decrease in the net influx of cations that occurs specifically near the apex of growing roots. Studies using extracellular vibrating probes have shown that a net flux of current, carried mostly by H+, enters the root near the apex and exits the root near the base of the elongation zone (Miller and Gow, 1989; Fromm et al., 1997). A small portion of current is also carried by Ca2+ and K+ (Kochian, 1995; Kiegle et al., 2000). The extent and density of H+ influx are greater in faster- compared with slower-growing roots (Miller and Gow, 1989). This H+ shunt, or leak current, would have a depolarizing effect on the electrogenic activity of the plasma membrane H+-ATPase. Thus, growing roots normally exhibit relatively depolarized potentials near the root apex compared with more distal cells, but it is not yet clear how these ion fluxes are related to growth.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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It has been shown that there are early electrophysiological responses of cells within the root elongation zone to the imposition of low
w, and steady-state changes in resting potential during long-term exposure to low w. These responses may be part of the primary signals that induce other processes necessary for growth maintenance. The combination of electrophysiological techniques with kinematic growth analysis and manipulation of endogenous ABA levels revealed that ABA plays a role in regulating the steady-state Em at low w in regions where cell elongation is dependent on ABA accumulation. The results suggest that ABA controls homeostatic set-points for ion transport processes that shift when new environmental conditions are encountered. Future studies will address which ion species are involved in these responses, and how they relate to the maintenance of root elongation at low w. |
Acknowledgements |
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We thank Dr Anton Novacky for his help and advice during the course of this study, Dr Gary Krause and Alan Todd for help with statistical analyses, and Dr Tony Miller for his advice and comments on the manuscript. This research was supported by National Science Foundation grant No. IBN-9306935 to RES and ESO, University of Missouri Research Board grant No. RBN7-148 to RES, and the Missouri Agricultural Experiment Station project number MO-PSFCO355.
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