Electrophysiological responses of maize roots to … 10.1093/jxb/erg060 RESEARCH PAPER...

DOI: 10.1093/jxb/erg060

RESEARCH PAPER

Electrophysiological responses of maize roots to lowwater potentials: relationship to growth and ABAaccumulation

Eric S. Ober1 and Robert E. Sharp

Department of Agronomy, Plant Sciences Unit, 1-87 Agriculture Building, University of Missouri, Columbia, MO65211, USA

Received 29 April 2002; Accepted 23 September 2002

Abstract

The maintenance of root elongation is an important

adaptive response to low water potentials (yw), but

little is known about its regulation. An important

component may be changes in root cell electrophy-

siology, which both signal and maintain growth main-

tenance processes. As a ®rst 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 yw was imposed by the gradual add-

ition of polyethylene glycol. Cells hyperpolarized

approximately 25 mV in response to low yw, and after

48 h resting potentials remained signi®cantly hyper-

polarized at yw lower than ±0.3 MPa compared with

roots at high yw. 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 yw. To determine if the mechanism of action of

ABA involves changes in root electrophysiology, Em

measurements were made during long-term exposure

to low yw. 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 regard-

less of ABA status (6±8 mm from the apex). Em was

substantially more negative in ABA-de®cient roots

speci®cally in the 2±3 mm region. The results sug-

gest that set-points for ion homeostasis shifted in

association with the maintenance of root cell elonga-

tion at low yw, 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 de®cit.

Introduction

Water de®cit is often the major factor restricting plantgrowth, and drought or insuf®cient irrigation water limitsagricultural productivity worldwide. Therefore, it isimportant to understand the mechanisms that plants useto adapt to water-limited conditions. For example, rootgrowth is much less sensitive to low water potentials (yw)than shoot growth (Sharp and Davies, 1989), and thisadaptive strategy helps the plant to maintain a supply ofwater under conditions of soil drying. Previous studieshave shown that in the elongation zone of the maize (Zeamays L.) primary root, morphological, biochemical, mol-ecular, and biophysical changes occur in response to lowyw (Sharp et al., 1988; Ober and Sharp, 1994; Saab et al.,1995; Wu et al., 1996, 2001; Conley et al., 1997). Detailedgrowth kinematic analysis revealed that cell elongation atlow yw is maintained preferentially toward the root apex(Sharp et al., 1988); in the apical few millimetres,elongation is completely maintained even under severewater de®cit (yw of ±1.6 MPa). Many of the observedchanges occur speci®cally within this region, but not a fewmillimetres further from the apex where cell elongation is

Journal of Experimental Botany, Vol. 54, No. 383, ã Society for Experimental Biology 2003; all rights reserved

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: [email protected]: yw, water potential(s); FLU, ¯uridone; PEG, polyethylene glycol; Em, membrane potential; SHAM, salicylhydroxamic acid; CCCP, carbonylcyanide m-chlorophenylhydrazone; FCCP, carbonyl cyanide p-tri¯uoromethoxyphenylhydrazone.

Journal of Experimental Botany, Vol. 54, No. 383, pp. 813±824, February 2003

at University of Missouri-Columbia on 18 May 2009 http://jxb.oxfordjournals.orgDownloaded from

http://jxb.oxfordjournals.org

not maintained at low yw. This is strong evidence thatthese changes are not symptoms of stress injury, but areadaptive responses speci®cally related in some manner tothe maintenance of root elongation. Thus, it is critical todiscover how these processes are regulated.

It is likely that cellular water de®cit is perceived at themembrane level and effects changes in ion activities thattransduce the environmental challenge to adaptive changesin cell biochemistry and gene expression (Trewavas, 1976;Hetherington and Quatrano, 1991). Electrophysiologicalchanges are integral parts of stress-activated signallingmechanisms in organisms ranging from bacteria to humans(Chamberlin and Strange, 1989). In plants, it is well knownhow algal cells respond to osmotic challenges throughchanges in ion transport and inorganic and organic soluteaccumulation (Bisson and Kirst, 1995). Descriptions ofhigher plant responses are less de®nitive. For example,responses to low yw include a depolarization of themembrane potential (Em) (GoÈring et al., 1979; Cortes,1997), hyperpolarization (Kinraide and Wyse, 1986; Liand Delrot, 1987; Lew, 1996; Shabala and Lew, 2002), orno change in Em (Cocucci et al., 1976; Wegner andZimmermann, 1998). The variability in reported responsesmay be attributable to differences in species, tissue or celltype or the mode of low yw imposition; there is no clearunderstanding. In addition, most studies have examinedshort-term responses to rapid osmotic perturbation and, asfar as is known, none have examined the electrophysio-logical effects of long-term exposure to low yw oraddressed issues related to growth, with the exception ofwork on root hairs (Lew, 1996).

An important regulator of ion ¯uxes that underliechanges in Em in response to low yw may be abscisic acid(ABA), which is synthesized by plant tissues under waterde®cit conditions. In stomatal guard cells, ABA activateschanges in ion transport leading to turgor loss and stomatalclosure (MacRobbie, 1997). ABA may play a role inregulating ion transport in other cell types as well, and mayfunction to shift tissues to `a new and different physio-logical state' in response to changing environmentalconditions (Hetherington and Quatrano, 1991). In roots,applied ABA can cause a range of effects on ion transportsuch as increased K+ ef¯ux, or in¯ux, depending on theconcentration of ABA, cell type, tissue K+ status, age ofthe tissue, temperature, etc. (van Steveninck and vanSteveninck, 1983; Roberts and Snowman, 2000). Similarfactors also determine whether applied ABA causes celldepolarization or hyperpolarization (Fromm et al., 1997;Zocchi and De Nisi, 1996).

In the maize primary root, it has been established thataccumulation of ABA helps to maintain root elongation atlow yw, chie¯y through the suppression of ethylenesynthesis (Saab et al., 1990; Sharp et al., 1994; Spollenet al., 2000; Sharp, 2002). However, it is not known howregulation of the ethylene biosynthesis pathway by

endogenous ABA is brought about. It is hypothesizedthat changes in ion transport and intracellular activity bothsignal (in the short-term) and maintain (in the long-term)root cellular responses to water de®cit, and that ABA mayfunction primarily in the regulation of these electrophy-siological changes. As a ®rst step to test the hypothesis,conventional electrophysiological techniques have beenused to measure changes in Em, which occur as a functionof changes in ion transport and accumulation. Thistechnique does not provide information on which ionspecies are affected, but it is much more sensitive todetecting intracellular changes than are measurements ofchanges in ion ¯ux or activity (Ullrich and Novacky,1991). The objectives of the study were to measure inintact, growing primary roots of maize the changes in Em

that occur within the elongation zone in response to thegradual imposition of low yw and relief from stress, howthese changes are associated with the spatial pattern of cellelongation during long-term exposure to low yw, and theextent to which the long-term changes are dependent onABA accumulation.

Materials and methods

Plant culture

Maize seeds (Zea mays L., cv. Fr273FrMo17) were sown ongermination paper moistened with 0.25 mM CaCl2, then germinatedin the dark at 2960.1 °C for 46 h. Seedlings with a primary rootlength of approximately 3 cm were transferred to solution culture(culture chambers described below) and grown under the sameconditions. When necessary, illumination was provided by a dimblue-green safelight (Saab et al., 1990) to minimize potential effectsof white light on root growth (Pilet and Ney, 1978). The medium,formulated to re¯ect the nutrient content of a typical soil solution(Barber, 1984), contained (in mM): 0.25 CaCl2, 0.3 MgSO4, 0.15K2HPO4, 0.4 NH4NO3, 5 MES; (in mM): 20 FeSO4, 6.0 H3BO3, 0.9MnSO4, 0.6 ZnSO4, 0.15 CuSO4, 0.1 NaMoO4, 0.01 CoCl2, 0.11NiCl2; adjusted to pH 6.0 with NaOH (approximately 2.75 mM ®nalNa+ concentration).

Even though maize does not normally grow with roots immersedin water, electrophysiological studies must usually be conducted ontissue bathed in an electrically conductive solution, which alsofacilitates the uptake of experimental compounds. Faced with thisrestriction, a system of gradually imposing low yw in oxygenatedsolution culture was developed using polyethylene glycol (PEG) asan osmoticum (Verslues et al., 1998). Supplemental oxygenationwas shown to be required for optimum root growth in solutions ofPEG, and to shorten the time to attain steady-state growth rates athigh and low yw. All solutions were oxygenated to achieve an O2

partial pressure of 45 kPa. This level of oxygenation at both high andlow yw restored tissue O2 partial pressure levels within the rootelongation zone to those found in roots in well-aerated vermiculite(Verslues et al., 1998). Solution O2 partial pressures were measuredwith 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 excludedfrom entering the root apoplast (Carpita et al., 1979), and thusremoves water from the cell and cell wall space. In this way itmimics the drying effects of a soil environment. In contrast,osmotica such as salts, mannitol, or sorbitol penetrate the cell wall

814 Ober and Sharp



and the cells themselves, and therefore may alter the normalresponse to low yw. There were no toxic effects of PEG apparentunder the conditions used (Verslues et al., 1998). Low yw wereimposed by gradually replacing the growth medium with solutions ofPEG dissolved in growth solution. Solution yw were determined byisopiestic thermocouple psychrometry (Boyer and Knipling, 1965).

ABA accumulation was decreased in some experiments bytreatment with ¯uridone (FLU), an inhibitor of carotenoidbiosynthesis (Moore and Smith, 1984; Saab et al., 1990; Sharpet al., 1994; Spollen et al., 2000). FLU was added at a ®nalconcentration of 1 mM during germination and in the growthmedium. To con®rm that effects of FLU on root growth and Em werea result of ABA de®ciency, additional experiments were conductedin which the ABA level in the growth zone of FLU-treated roots wasrestored by adding (R,S) 6ABA to the PEG solution.

In experiments using other inhibitors, stock solutions wereprepared as follows: KCN was dissolved in water to prepare a 100mM solution, and used at a concentration of 1 mM; salicylhy-droxamic acid (SHAM) was prepared as a 10 mM solution in 5 mMHEPES buffer, pH 8.0, and used at full strength; a stock solution of 5mM NaVO4 was prepared in 5 mM MES, pH 6.4, and used at aconcentration of 0.5 mM; a 10 mM solution of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was prepared in 95% ethanol, andused at a concentration of 10 mM.

Growth studies

After germination, seedlings were grown in a Plexiglas chamber(600 ml) housing 21 seedlings (Verslues et al., 1998). Caryopseswere suspended on a holder above the solution and the primary rootsgrew downward through perforated transparent root guides fash-ioned from plastic drinking straws (i.d. 6 mm). A Plexiglas coverwas used to enclose the shoots within a humid atmosphere, whichminimized transpirational water loss. The air/O2 mix, suppliedthrough a perforated plastic tube extending along the bottom of thebox, vigorously aerated and stirred the solution. Root elongation ratewas monitored by marking the position of the root apex along theside of the box at various times.

Spatial growth analysis

Spatial patterns of longitudinal strain rate (local relative elongationrate, % h±1) were determined using procedures modi®ed from Silket al. (1984) and Sharp et al. (1988). Seedlings were removed fromsolution and the primary root tip was marked at approximately 1 mmintervals with water-insoluble ink (Pelican, 17 Black) using a ®nepaint brush. Seedlings were returned to solution, and afterelongation rates recovered (usually within 1±2 h), roots werephotographed every 15 (high yw) or 30 min (low yw). Enlargedphotocopies were made from the negatives using a micro®cheviewer so that the displacement of marks from the root apex could bedigitized to obtain longitudinal velocities, which were then inter-polated to 0.5 mm intervals using cubic splines. The velocity datawere then differentiated with respect to position using a 5-pointformula (Erickson, 1976) to give the spatial distribution oflongitudinal strain rate.

ABA determinations

To assay tissues for ABA content, seedlings were removed fromsolution, roots were rinsed brie¯y in distilled water, and then blotteddry. The apical 10 mm of the primary roots were excised, placed inmicrocentrifuge tubes and immediately frozen at ±80 °C. Sampleswere weighed, freeze-dried, then reweighed to obtain the mass oftissue water. Distilled water (500 ml) was added to the tubes andABA was extracted overnight at 4 °C. Aliquots were assayed forABA 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 rootwas transferred from germination paper to a small (15 ml) Plexiglaschamber (Fig. 1). The chamber was designed such that the root grewdown through a guide tube, which provided physical support for theroot during impalements, yet permitted enough movement to allowunrestricted growth. Roots were measured in a vertical orientation inorder to minimize the confounding effects of gravistimulation on Em,ion and hormone distributions within the elongation zone (Pilet andRivier, 1981; Ishikawa and Evans, 1990). Microelectrodes weremounted in a rubber septum, positioned through a port drilled into amovable slide on the side of the chamber, and accessed the rootthrough openings in the guide tube. The seal at the port was madewatertight 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 aeratedin a 15 ml tube adjacent to the chamber, then circulated through theroot chamber at 3.0 ml min±1 using a peristaltic pump. In someexperiments, solutions in the root chamber were oxygenated directlyand the bubbles were separated from the root by a baf¯e.

Root elongation rates in this chamber were comparable to ratesobtained in the larger chamber used for the growth studies. Em

measurements were made on roots elongating at approximately themean rate for each treatment. Impalements were viewed through amicroscope (®tted with long-working-distance objectives) mountedhorizontally in front of the chamber, which was illuminated frombehind with a ®bre optic light source ®tted with a blue/green ®lter.The set-up was enclosed within a Faraday cage on a vibration-isolated table. Preliminary tests showed that illuminating seedlingswith the blue/green light did not cause any transient electrophysio-logical changes within the root elongation zone.

Em measurements were made with standard glass microelectrodes®lled with 3 M KCl and connected to the head stage of a high inputimpedance electrometer (FD223, World Precision Instruments,Sarasota, Florida) via Ag/AgCl wires. Voltage output from theelectrometer was sent to a chart recorder and collected on computerdisk via a data acquisition system. Measuring electrodes weremounted 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 wereobtained using electrodes ®lled with 3 M KCl or 100 mM KCl (pH2.0), but the latter tended to be associated with more noise. Using 3M KCl, stable Em were obtained immediately upon impalement andwere held for as long as 50 min; therefore, there was no apparentleakage of electrolyte from the electrode into the cell that affectedthe measurements.

Em were measured in epidermal and cortical cells within the rootelongation zone. Epidermal cells had slightly less negative potentialsthan cells in underlying layers. The differences between successivelayers were small (on average 2 mV) and variable; for treatmentcomparisons, therefore, values from all measured layers (epidermisthrough the ®fth cortical cell layer) were combined. Mature roottissues have typically been used in studies of root Em, and fewstudies have attempted measurements of cells near the apex of intact,growing roots (see, for example, Papernik and Kochian, 1997). Theprimary dif®culty is that, as the root grows, the impaled target cellmoves away from the electrode; thus, to keep the electrode tip withinthe cell, the electrode must be moved to `track' root movement. Themicroelectrodes were pulled to produce long, thin shanks, whichincreased their ¯exibility and the duration of successful impale-ments. During an impalement the tip bent slightly as the root grew,and the microelectrode was frequently repositioned using themicromanipulator. The greased ®ttings in the chamber port aroundthe microelectrode barrel helped to dampen any abrupt movements.Preliminary tests showed that merely bending the microelectrode tip

Electrophysiology, root growth and ABA during water de®cit 815



did not affect the voltage signal. The depth of impalement wasdetermined using the micromanipulator scale, and reaching a celllayer was determined by observing the impalement of cells, detectedas a signi®cant increase in potential above the Donnan potential ofthe cell wall (approximately ±40 mV). For Em recordings lastingseveral minutes or more, cells within the third to sixth cortical cell

layer were impaled, since the deeper impalements helped to anchorthe microelectrode tip in place. Checks on microelectrode resistancewere routinely performed during measurements and if a microelec-trode became plugged (indicated by an increased input resistance), itwas replaced with a fresh microelectrode. Under `steady-state'conditions (i.e. when solution yw and root elongation rates were

Fig. 1. A schematic drawing of the chamber used to measure primary root elongation rate and root tip Em in vertically-oriented maize seedlingsgrowing in solution culture. Solutions were oxygenated (oxygen partial pressure of 45 kPa) before ¯owing into the root chamber. In someexperiments, solutions in the root chamber were oxygenated directly and the bubbles were separated from the root by a baf¯e. The solution wasallowed to drip freely into and out of the system in order to electrically isolate the system from the pumps and lines outside the Faraday cage.

816 Ober and Sharp



constant), an Em value was recorded if the potential remained steadyfor at least 1 min, electrode resistance had not changed, and theimpalement showed signs of maintaining a good seal (indicated bythe 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 andtrough voltages.

The possibility of measuring voltage artefacts during changes inyw was examined. A gradual improvement in the seal around themicroelectrode could have resulted in an apparent increase inpotential over time as cells lost turgor. This appears unlikely sinceelectrode resistance did not change over the course of measurementsas yw decreased. When sudden increases in resistance wereobserved, often due to a plugged microelectrode tip, the data werediscarded. 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-yw solutions had thesame ionic composition). However, there was no change inmeasured potential across the electrodes when the microelectrodewas positioned in the bath solution at the root surface during the shiftin yw.

Results

Em responses during imposition of and recovery fromlow yw

To study dynamic responses of root cell Em to changes inyw, measurements were made in cortical cells between 6±8mm from the apex, within the zone of elongation at highyw (see Fig. 5). At high yw, values of Em were generallybetween ±120 to ±132 mV (Fig. 2); the scatter in valueswas due in part to an oscillation of Em that had anamplitude of 5±10 mV and a frequency of 5±10 min.Detailed analyses of Em oscillations in roots have beenreported previously (Souda et al., 1990).

Upon imposition of a decrease in yw to ±0.28 MPa, Em

began to hyperpolarize (Fig. 2) and reached a new levelafter the shift in yw was completed (after 80 min; Fig. 2,inset). The mean hyperpolarization at 60±80 min after lowyw imposition was 2463 mV (n=11). Root elongation wasinhibited after a change in yw to ±0.28 MPa, but eventuallyrecovered 48 h later (Verslues et al., 1998). Whenmeasurements were made in roots growing under thesenew steady-state conditions, Em was ±13663 mV (n=8),which was not signi®cantly different from the value at highyw (±13063 mV, n=8; see Table 3). Thus, the initialhyperpolarization in response to a decrease in yw of ±0.28MPa was transient and Em returned to near normal levels asthe roots acclimated to the new condition.

When ±0.8 MPa PEG was used to impose low yw, thepattern of hyperpolarization was similar to the experimentsusing ±0.28 MPa PEG during the ®rst 80 min because therate of decline in yw was similar. Beyond 80 min, as yw

declined further to ±0.8 MPa, there was no furtherhyperpolarization (DEm=2566 mV, n=6, after the shift inyw was completed). In contrast to the ±0.28 MPatreatment, Em values remained hyperpolarized (±14565mV, n=13) under steady-state conditions at ±0.8 MPa (48 h

after stress imposition). Similar results were obtained at ayw of ±1.6 MPa (see Table 3). In summary, imposition oflow yw always caused a hyperpolarization, but a new,more negative resting Em was maintained under steady-state conditions only when solution yw was less than ±0.3MPa.

Roots growing at low yw depolarized when the yw wasincreased by gradually replacing the PEG solution withsolution of high yw (Fig. 3). Root Em depolarized beyondthe normal resting Em observed at high yw, but not furtherthan 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 thenormal resting value at high yw (data not shown).Depolarizations in response to hypo-osmotic treatmentshave been observed before, e.g. in Chara (Bisson andKirst, 1995) and Arabidopsis root hairs (Lew, 1996).Following a hypo-osmotic shift, the hyperpolarized statecould be recovered by re-imposition of the PEG solution(Fig. 3). Long-term recordings from single cells weredif®cult to obtain routinely in growing roots, but thisexample demonstrates the dynamic nature of an individualcell's response to changing yw.

The initial low yw-induced hyperpolarization couldhave been caused by energized ion transport processes(requiring ATP), or by passive movement of ions down

Fig. 2. The effect of imposing low yw on the Em of cortical cellslocated 6±8 mm from the primary root apex (within the elongationzone at high yw; see Fig. 5). Low yw was imposed by slowlyintroducing oxygenated PEG solution (yw of ±0.28 MPa) into thechamber (beginning at the arrow). The time-course of bulk solutionyw decline is shown in the inset. Each root was continuouslymonitored and Em values were recorded every 5 min during successfulimpalements. Roots of the high yw control (open circles) weremeasured over the same time course but without the addition of PEG.Data from 17 (low yw) and 22 (high yw) individual roots wereanalysed by repeated measures analysis of variance (Greenhouse andGeisser, 1959) using Genstat 5 (VSN International Ltd, Oxford, UK).The error bar indicates the maximum LSD (Greenhouse-Geissere=0.0769, 10 d.f.) for comparison between treatments at a samplingtime. Curves were ®tted to the data using a third-order regressionprocedure using SigmaPlot software (SPSS UK Ltd, Woking, UK).




their electrochemical gradient. To differentiate betweenthese possibilities, roots were pretreated with metabolicinhibitors before the imposition of low yw. Mitochondrialelectron transfer is completely blocked by the combinationof CN± and SHAM, which effectively inhibits the H+-ATPase and energized ion transport. This treatment causedan immediate depolarization, and after imposition of lowyw (±0.28 MPa) there was no hyperpolarization within 60min (Table 1), the usual time frame for the full response(Fig. 2). Vanadate, a speci®c inhibitor of the plasmamembrane H+-ATPase, also prevented the hyperpolariza-tion (Table 1). The protonophore CCCP dissipates H+

gradients, and thereby inhibits ion transport processes thatare dependent on the gradient for H+-coupled transport orfor ATP production and H+-ATPase activity. CCCP alsocaused a depolarization followed by a lack of hyperpolar-ization after low yw imposition (Table 1). Similar resultswere also obtained by treating roots with FCCP orerythrosin B (data not shown). These results indicate thatthe hyperpolarization response to low yw required anactive plasma membrane H+-ATPase.

Preliminary experiments were conducted to examinewhether, in addition to H+, other ions were required for thehyperpolarization. To indicate whether ¯uxes of K+ wereessential, roots were grown for 48 h in K+-free (replaced byNa+) media, or pre-treated with Ba2+, which blocks inward-and outward-rectifying K+ channels (MacRobbie, 1997).

Both treatments were effective in preventing the hyperpo-larization normally observed after the addition of ±0.3MPa PEG (data not shown). When roots were grown for 48h in a Cl±-free medium (replaced by SO4

2±), hyperpolar-ization was similarly blocked. Further experimentation isneeded to describe accurately the role of speci®c ion¯uxes, but these preliminary results suggest a role for K+

and Cl± in the hyperpolarization response to low yw, whichis consistent with other studies (Lew, 1996; Teodoro et al.,1998).

ABA and root growth during long-term exposure to lowyw

Previous studies using vermiculite as a growth mediumhave demonstrated that ABA accumulation is required forthe maintenance of maize primary root elongation at lowyw (Saab et al., 1990; Sharp et al., 1994; Spollen et al.,2000). For the present study it was necessary to show thatsimilar results could be obtained in solution culture. Inseedlings exposed to a yw of ±1.6 MPa, the rate of rootelongation 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 theaddition of 0.5 mM ABA to the PEG solution (Fig. 4). Thisconcentration of applied ABA raised the ABA content ofthe root apical centimetre (encompassing the elongationzone; see Fig. 5) to the normal level at ±1.6 MPa (Table 2).By contrast, the addition of 1.0 mM ABA to FLU-treatedroots more than doubled the normal internal level (71617ng g ±1 H2O, n=4) and did not restore the elongation rate(data not shown). Similar results were obtained withvermiculite-grown roots (Sharp et al., 1994), and illustratethat non-physiological levels of ABA can inhibit rootgrowth. These ®ndings emphasize the importance ofstudying hormone levels in an appropriate range for theparticular condition (Jacobs, 1959; Sharp, 2002). At highyw, as in the previous vermiculite studies, treatment withFLU had minimal effects on root ABA content (Table 2) orelongation rate (data not shown).

Relationship of Em changes to steady-state growthpatterns and ABA accumulation

Determining the relationship of electrophysiologicalchanges to the response of root growth during impositionof low yw is complicated by the dynamics of rapidlychanging patterns of cell elongation. By contrast, thepatterns of cell elongation under steady-state conditions athigh yw and low yw (±1.6 MPa) were de®ned by kinematicanalysis. Figure 5 shows which regions were elongating,which regions had ceased growth at low yw, and whichregions were dependent on ABA for growth maintenanceat low yw. As with the responses of overall root elongationrate, the growth patterns were similar to those obtained

Fig. 3. Response of cortical cell Em to changes in solution yw,measured at 4±6 mm from the apex. The root was initially growingunder steady-state conditions at low yw (approximately ±0.8 MPa).The solution yw was then increased by replacing the PEG (arrow, ±PEG) with high yw growth solution (±0.03 MPa). After the initialdepolarization, solution yw was again decreased (arrow, +PEG). Smalltriangles indicate measurements of solution yw (values are in MPa).Similar traces were obtained in four independent experiments.

818 Ober and Sharp



previously in vermiculite-grown roots (Sharp et al., 1988;Saab et al., 1992). The elongation zone extended to 12 mmfrom the apex at high yw and was shortened to approxi-mately 7 mm in PEG solution at ±1.6 MPa. Maximallongitudinal strain rates occurred between 3 mm and 6 mmfrom the apex at high yw, but at low yw rates began to slowbeyond 3 mm. Treatment with FLU at low yw decreasedstrain rates at all positions beyond the ®rst millimetre,whereas restoration of the ABA level largely restored thenormal growth pattern. Two regions are particularlyimportant to note: at low yw, strain rates were almostfully maintained at 2±3 mm from the apex, but only ifABA accumulation was not blocked. In the region 6±8 mmfrom the apex, strain rates were almost completelyinhibited at low yw regardless of the ABA status of thetissue. Thus, cells 2±3 mm from the apex were dependent

on ABA accumulation for growth maintenance at low yw,whereas later, when cells had been displaced 6±8 mm fromthe apex, ABA accumulation was no longer suf®cient toprevent growth inhibition. These regions were selected forcomparative electrophysiological measurements.

Under conditions of steady-state growth and yw, Em wasalso at steady state. Em measured at 4±8 mm from the apexchanged by only +4.864 mV (n=4) between 24 h and 48 hafter transfer to solution culture in roots growing at highyw. Furthermore, Em oscillations were negligible in rootsgrowing at a yw of ±1.6 MPa. The measurements revealedthat Em were increasingly more negative with increasingdistance from the root apex (Table 3). Values at high yw

ranged from approximately ±120 mV at 1 mm from theapex to approximately ±140 mV at 10 mm from the apex.This longitudinal gradient in Em may re¯ect developmen-tal changes in the number or activity of ion transportcomponents as cells grow and mature. Similar gradientshave 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 rootsat high yw at both the 2±3 mm and 6±8 mm locations(Table 3).

At high yw, Em was not measurably affected bytreatment with FLU at either 2±3 or 6±8 mm from theapex (Table 3). At ±1.6 MPa, in contrast, cells in the 2±3mm region of FLU-treated roots were signi®cantly morehyperpolarized compared with non-treated roots. When

Table 1. The effect of metabolic inhibitors on the response of cortical cell Em to the imposition of low yw

The same procedure was used as in Fig. 2 except that roots were treated with inhibitors prior to the addition of PEG. Em values were recordedapproximately 60 min after PEG addition. Values are means 6se from (n) independent experiments. na=not applicable

Inhibitor Em before inhibitor (mV) Em after inhibitor (mV) Em after PEG (mV)

Vanadate ±12961.4 (3) ±10264.3 ±9967.5CCCP ±12364.2 (2) ±81617 ±85615CN±/SHAM ±12263.5 (2) ±91623 ±96628None ±12462.4 (17) na ±14962.9

Table 2. Effect of 1 mM ¯uridone (FLU) with or without 0.5mM exogenous (6)-ABA on the ABA content of the apicalcentimetre of roots growing at high (±0.03 MPa) or low (±1.6MPa) yw

Measurements were made 48 h (high yw) or 72 h (low yw) aftertransfer to solution (see Fig. 4). Data are means 6se (n=4±7),combined from at least two independent experiments. nd=notdetermined.

Treatment ABA content (ng g±1 H2O)

±0.03 MPa ±1.6 MPa

±FLU 1364 3362+FLU 862 1964+FLU, +ABA nd 3266

Fig. 4. Effect of treatment with 1 mM ¯uridone (FLU) with or without0.5 mM exogenous (6)-ABA on the elongation of roots exposed to thedevelopment of low yw in solution culture. Low yw was imposed byintroducing oxygenated PEG solution (yw of ±1.6 MPa) at the arrow.The time-course of bulk solution yw decline is shown in the inset. Theelongation of control roots maintained at high yw is also shown. Dataare means 6se (n=21). Experiments were repeated at least twice withsimilar results.




FLU-treated roots were supplied with ABA to restore thenormal ABA content (Table 2) and spatial growth pattern(Fig. 5), potentials 2±3 mm from the apex were returnedclose to the normal values at low yw (Table 3). In the 6±8mm region, where growth was inhibited regardless of ABAstatus, the hyperpolarization was similar in the control andFLU-treated roots, and was unaffected by the addition ofABA. The steady-state data show that ABA de®ciencyresulted in a greater than normal hyperpolarization at lowyw, speci®cally in the region in which ABA was requiredfor maintenance of cell elongation.

Discussion

Early electrophysiological responses to low yw

As soil begins to dry during the initial stages of drought,the responses of roots are critical to the survival of theplant, yet little is understood about how roots perceive lowyw or how adaptive responses are regulated. Furthermore,it is mainly the tissues at the root tips comprising themeristem and cell elongation zone that are essential forcontinued root elongation during dry conditions and afterstress is relieved. Therefore, this study focused on cellswithin the elongation zone, and the results show that anearly response to the imposition of low yw is Em

hyperpolarization, involving activation of the plasmamembrane H+-ATPase. This response may be triggeredby turgor-sensitive stretch-activated membrane channelsor by other osmo-sensing elements (Lew, 1996).

A more negative Em would increase the driving force forion uptake. An accumulation of inorganic ions, which arethen replaced by synthesis or uptake of organic osmolytesduring long-term exposure to low yw, appears to be aresponse to osmotic stress that is conserved throughoutnature: human brain glioma cells accumulate principallyNa+ and inositol (Chamberlin and Strange, 1989); in E. colithe response can involve K+ and proline (Yim andVillarejo, 1994); some algal cells accumulate Na+, thenreplace it with glycerol (Bisson and Kirst, 1995).Surprisingly, there is no de®nitive description of similarevents in higher plants. During long-term water de®citthere is signi®cant osmotic adjustment within the elonga-tion zone of the maize primary root (Sharp et al., 1990),and accumulation of proline accounts for up to half of thedecrease in osmotic potential within the apical fewmillimetres (Voetberg and Sharp, 1991). It is possiblethat early accumulation of inorganic ions, driven by ahyperpolarized Em, is the signal that activates theaccumulation of proline. Interestingly, the deposition ofproline within the growth zone also depends on theaccumulation of ABA (Ober and Sharp, 1994).

Fig. 5. The spatial distribution of longitudinal strain rate (relativeelongation rate) within the apical 12 mm of maize primary rootsgrowing at high yw (±0.03 MPa) or at low yw (±1.6 MPa PEG) withor without the addition of ¯uridone (FLU, 1 mM) or FLU plus 0.5 mMABA. Measurements were made 48 h (high yw) or 72 h (low yw)after transfer to solution culture (see Fig. 4). Strain rates werecalculated from the averaged velocity distributions of 3±5 roots. Barson the abscissa denote the region 2±3 mm from the apex in whichmaintenance of cell elongation at low yw was dependent on ABAaccumulation, and the region 6±8 mm from the apex where elongationwas inhibited at low yw regardless of the level of ABA in the tissue(see text for further details).

Table 3. Steady-state resting Em of cells located either 2±3 mm or 6±8 mm from the apex of roots growing at high (±0.03 MPa)or low (±1.6 MPa) yw

Roots were grown with or without the addition of ¯uridone (FLU) or FLU plus 0.5 mM (6)-ABA. Measurements were made 48 h (high yw) or72 h (low yw) after transfer to solution (see Fig. 4), and were included in the analysis only if roots were elongating within 10% of the mean ratefor each particular treatment. Values represent the mean 6se (n=3±12), and include measurements from epidermal and cortical cell layerscombined from at least three separate roots from at least two independent experiments. Data were analysed by ANOVA and Fisher's LSD.Values followed by the same letter are not signi®cantly different (P=0.05). nd=not determined.

Treatment Em (mV)

±0.03 MPa ±1.6 MPa

2±3 mm 6±8 mm 2±3 mm 6±8 mm

±FLU ±12163 a ±13063 b ±13364 bc ±14062 c+FLU ±12363 a ±13065 ab ±15863 d ±13665 bc+FLU, +ABA nd nd ±14163 c ±13762 bc

820 Ober and Sharp



Furthermore, recent measurements of low yw-induced ion¯uxes at the root surface con®rm that increases in K+, Cl±

and Na+ in¯ux contribute to short-term osmotic adjustmentand the restoration of turgor (Shabala and Lew, 2002).

Few other studies have used growing cells to examineelectrophysiological responses to a decrease in yw. One, astudy of root hairs in Arabidopsis, also showed ahyperpolarization (Lew, 1996). In contrast, another studyinferred an inhibition of the proton pump based onmeasurements of ion ¯uxes at the root surface near theapex of the maize primary root (Shabala and Newman,1998). Hyperpolarizations in response to decreased yw

were also observed in mature root tissue of beet (Kinraideand Wyse, 1986) and Arabidopsis (Shabala and Lew,2002), and in carrot suspension cells (Reuveni et al.,1987). Also, algal species that regulate turgor or cellvolume generally hyperpolarize when the yw decreases(Bisson and Kirst, 1995). In contrast to these studies, Em

depolarizations in response to low yw were measured insun¯ower root (Cortes, 1997), maize coleoptile (GoÈringet al., 1979) and Chara in¯ata cells (Kourie and Findlay,1990). Most previous studies have used a very rapidimposition of low yw to evoke electrophysiologicalresponses. In this study, when low yw was imposed at amuch faster rate than usual, cells transiently depolarizedbefore the hyperpolarization (data not shown). Thus, oneexplanation for the varying effects of low yw on Em maybe the rapidity and severity of the stress imposition. It canbe argued that higher plant cells, and roots in particular, arerarely exposed to hyperosmotic shock; instead, the yw ofthe rhizosphere slowly declines as water evaporates or isextracted from the soil. Therefore, a relatively slow declinein yw may be necessary to observe a cellular response thatclosely relates to that which occurs naturally in ®eld-grownroots subjected to drought. Conversely, roots in drying soilthat have accumulated solutes for osmotic adjustment riskinjury when subjected to a sudden in¯ux of water afterrainfall. Depolarization-induced solute ef¯ux may be animportant adaptive response to avoid excessive turgorduring such hypo-osmotic shock.

A shift in steady-state root electrophysiology at low yw

Typically, electrophysiological responses to environmen-tal stimuli are studied in a single cell, are transient, andoccur on the order of seconds or milliseconds. Much lessattention has been focused on electrophysiology understeady-state conditions. Under widely varying conditions,the steady-state cytoplasmic activities of ions normally arecontrolled within certain set limits (Cram, 1980; Walkeret al., 1996). The idea of the `set-point' is that perturbationof ion activity in response to environmental challengesgenerates an `error' signal that triggers other processes,and then the ion activity is brought back to the set-pointvalue (Cram, 1980). In this sense it is likely of importancethat 48 h after imposition of low yw (<±0.3 MPa), the

resting Em of cells remained slightly but signi®cantlyhyperpolarized compared with roots of the same age athigh yw. There are other examples of small but physio-logically signi®cant shifts in Em. Differences up to 4 mVwere important indicators of auxin sensitivity in tobaccoprotoplasts treated with auxin (LeBlanc et al., 1999) and,in animals, neurons of the visual cortex adapt over time toa repeated stimulus by shifting Em to a new restingpotential 5±15 mV more negative (Carandini and Ferster,1997). In roots under steady-state conditions at low yw,cells within the elongation zone had just recently beenformed in the meristem, and had never experienced a highyw environment (compared with the now mature root cellsdisplaced 20±50 mm from the apex). Therefore, factorsestablishing the altered resting Em in the apical region wereactive without the original stimulus (the change from highto low yw). Since ion activities outside the root should nothave changed after switching to low yw, the steady-statechange in Em indicates that programmed set-points forproton pump activity or intracellular ion activities gov-erning 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 theinitial perception of water de®cit, maintenance of rootelongation during long-term exposure to low yw isequally critical for plant survival during drought. SinceABA is required for this process, and if the primarysite of action of ABA is at the membrane level, thenABA accumulation could regulate the maintenance ofresting Em under steady-state conditions at low yw.This hypothesis is supported by the observation thatABA-de®cient cells within the region 2±3 mm fromthe apex had signi®cantly different resting Em thanABA-suf®cient cells. ABA may play a role in ionhomeostasis within this region of the growth zone, butwith insuf®cient ABA this control is lost. Severalreports in the literature are consistent with this idea. Ithas been suggested that ABA shifts homeostatic set-points in guard cells of closed stomata (MacRobbie,1997). In leaf cells of ABA-de®cient tomato plants, theresting potential was signi®cantly more negative com-pared with wild-type plants (Herde et al., 1998). Inanother example, in yeast cells adapted to grow insaline media, over-expression of the Hal1 gene causedincreased levels of cellular K+

, which was associatedwith 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 theaddition of ABA. It was suggested that Hal1 is part ofthe K+-homeostatic mechanism, regulated by ABA.

An alternative to the control of ion homeostasis by ABAis that the greater hyperpolarization at 2±3 mm from theapex in the FLU-treated roots at low yw was merely a




consequence of growth inhibition, such that slowergrowing cells were relatively hyperpolarized comparedwith the faster growing cells at the same distance from theapex in the ±FLU roots. This relationship, however, breaksdown with other comparisons: for example, in the ±FLUtreatment longitudinal strain rates were quite similar at 2±3mm from the apex at high and low yw, but Em wassigni®cantly hyperpolarized at low yw. In the 6±8 mmregion, longitudinal strain rates differed greatly betweenhigh yw (±FLU) and low yw (+FLU), but resting Em werenot signi®cantly different. Overall, there was no signi®cantcorrelation (R2=0.01) between elongation rates and Em

values at 2±3 mm from the apex of individual rootsgrowing at high yw. Thus, the more negative Em at low yw

compared with high yw, or at 2±3 mm in +FLU comparedwith ±FLU roots at low yw, does not appear to beattributable to differences in the elongation rates of thecells. Experiments speci®cally designed to determinewhether or not ABA-modulated changes in Em bringabout changes in growth, or vice versa, are required toresolve this issue fully.

It is intriguing that the FLU-induced differences in Em at2±3 mm from the apex of roots at low yw did not occur at6±8 mm (Table 3). An explanation of the greaterhyperpolarization in ABA-de®cient roots in the apicalregion, but not the basal region, may be that it is related toa decrease in the net in¯ux of cations that occursspeci®cally near the apex of growing roots. Studies usingextracellular vibrating probes have shown that a net ¯ux ofcurrent, carried mostly by H+, enters the root near the apexand exits the root near the base of the elongation zone(Miller and Gow, 1989; Fromm et al., 1997). A smallportion of current is also carried by Ca2+ and K+ (Kochian,1995; Kiegle et al., 2000). The extent and density of H+

in¯ux are greater in faster- compared with slower-growingroots (Miller and Gow, 1989). This H+ shunt, or leakcurrent, would have a depolarizing effect on the electro-genic activity of the plasma membrane H+-ATPase. Thus,growing roots normally exhibit relatively depolarizedpotentials near the root apex compared with more distalcells, but it is not yet clear how these ion ¯uxes are relatedto growth.

Conclusion

It has been shown that there are early electrophysiologicalresponses of cells within the root elongation zone to theimposition of low yw, and steady-state changes in restingpotential during long-term exposure to low yw. Theseresponses may be part of the primary signals that induceother processes necessary for growth maintenance. Thecombination of electrophysiological techniques with kine-matic growth analysis and manipulation of endogenousABA levels revealed that ABA plays a role in regulatingthe steady-state Em at low yw in regions where cell

elongation is dependent on ABA accumulation. The resultssuggest that ABA controls homeostatic set-points for iontransport processes that shift when new environmentalconditions are encountered. Future studies will addresswhich ion species are involved in these responses, and howthey relate to the maintenance of root elongation at lowyw.

Acknowledgements

We thank Dr Anton Novacky for his help and advice during thecourse of this study, Dr Gary Krause and Alan Todd for help withstatistical analyses, and Dr Tony Miller for his advice and commentson the manuscript. This research was supported by National ScienceFoundation grant No. IBN-9306935 to RES and ESO, University ofMissouri Research Board grant No. RBN7-148 to RES, and theMissouri Agricultural Experiment Station project number MO-PSFCO355.

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