Journal of Experimental Botany

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Journal of Experimental Botany, Vol. 51, No. 350, pp. 1585-1594, September 2000
© 2000 Oxford University Press

The effects of ABA on channel-mediated K⁺ transport across higher plant roots

Stephen K. Roberts¹ and Benjamin N. Snowman²

The Plant Laboratory, Department of Biology, University of York, PO Box 373, York YO1 5YW, UK

Received 12 August 1999; Accepted 22 November 1999

	Abstract

Top Abstract Introduction K+ transport across higher... Regulation of K+ transport... References

 
The transport and accumulation of K⁺ in higher plant roots is regulated by ABA. Molecular and electrophysiological techniques have identified a number of discrete transporters which are involved in the translocation of K⁺ from the soil solution to the shoots of higher plants. Furthermore, recent reports have shown that ABA regulates K⁺ channel activity in maize and Arabidopsis roots which suggests that ABA regulation of K⁺ transport in roots is, at least in part, ion channel-mediated. The signalling processes which underlie the ABA regulation of K⁺ channels have been investigated. The effects of ABA on the membrane potential of intact maize root cells were also studied. It was found that ABA regulated the membrane potential of root cells and that this regulation is consistent with the hypothesis that ABA-induced K⁺ accumulation in roots is mediated by K⁺ channels.

Key words: ABA, K⁺ channels, roots, maize.

	Introduction

Top Abstract Introduction K+ transport across higher... Regulation of K+ transport... References

 
The roots of higher plants are responsible for the transport of ions from the soil solution to the xylem vessels and, via the transpiration stream, to the shoot. Although there are many specialized root cell types, with respect to ion transport they can be generalised to those that mediate the uptake of ions from the soil solution (i.e. epidermal and cortical cells) and those that mediate ion release into the transpiration stream (i.e. stelar cells). Separating the cortex from the stele is a single layer of endodermal cells. Associated with these cells is a unique structure which prevents ions from the soil solution being transported directly to the xylem vessels via the extracellular space of the root (i.e. aploplastic transport; see Fig. 1). The structure, called the Casparian strip, is located between the walls of the endodermal cells and forms a barrier to apoplastic movement of ions. In its mature form, the Casparian strip is composed of lignin and suberin which render it impermeant to ions (Robards et al., 1973). Thus the presence of the Casparian strip ensures that ions must enter the root symplasm via the plasma membrane of the cortical and epidermal cells or the outer face of the endodermis. Likewise, efflux from the root symplasm to the transpiration stream must cross the plasma membrane of cells within the stele of the root (Fig. 1).

View larger version (70K): [in this window] [in a new window]   Fig. 1. Diagrammatic representation of apoplastic and symplastic ion transport across higher plant roots. The apoplastic pathway (dashed arrows) conducts ion movement through the cell wall matrix as far as the outer cell walls of the endodermis, since the Casparian strip blocks movement of ions to the stele. Consequently, ions are taken up into the symplasm (shaded area) at the plasma membranes of the epidermis or cortex or the outer face of the endodermis. Symplastic movement of ion (solid arrows) to the stelar symplasm occurs via the plasmadesmata. Release of the ions into the stelar apoplasts (and ultimately the xylem vessels) occurs across the plasma membrane of the stelar cells.

 
K⁺ transport across the root has been widely studied. K⁺ is a major plant nutrient and is important in many cellular functions including turgor regulation, charge balance, regulation of the membrane potential, and the activity of cytosolic enzymes (Evans and Sorger, 1966). An overview of the transporters that facilitate the radial movement of K⁺ across the root is presented. Also discussed is recent evidence showing that some of these transporters are regulated by ABA and that this regulation probably reflects a mechanism to induce K⁺ accumulation in roots during water stress.

	K+ transport across higher plant roots

Top Abstract Introduction K+ transport across higher... Regulation of K+ transport... References

 
K⁺ uptake from the soil solution
Tracer flux studies investigating K⁺ uptake by roots identified two distinct systems (Epstein et al., 1963); a high affinity pathway operating at extracellular K⁺ concentration less than 1 mM (System 1) and a low affinity pathway predominant at concentrations greater than approximately 1 mM (System 2). Subsequent to these findings two types of mechanism have been identified at the molecular level in plant cells which mediate K⁺ uptake across the plasma membrane: (1) ion channel proteins which form pores in the plasma membrane (Doyle et al., 1998) and allow passive K⁺ transport down its electrochemical gradient, and (2) carrier proteins (Maathuis and Sanders, 1994) which couple the transport of K⁺ to the transport of another ion with an electrochemical gradient favouring its uptake across the membrane. The electrochemical gradient for an ion (e.g. K⁺) is determined by the electric potential difference across the membrane (V_m), the cytosolic K⁺ activity () and the extracellular K⁺ activity (). The Nernst equation indicates whether passive K⁺ uptake can be mediated by ion channels or if there is a need for carrier-mediated transport. If the membrane potential is more negative than the Nernst potential for K⁺ (E_K:=59 log[/]) then net K⁺ uptake can proceed passively through ion channels. When the membrane potential is positive of E_K then net passive K⁺ efflux is favoured and K⁺ uptake must occur via a carrier-type mechanism.

High affinity transporters:
Using a yeast complementation method, a wheat cDNA (HKT1) was isolated which was (after heterologous expression in Xenopus oocytes) identified as a high affinity K⁺ symporter (Schachtman and Schroeder, 1994). Initially it was reported to be a K⁺ : H⁺ symporter (1 : 1 ratio), however, later studies concluded that HKT1 acted as a Na⁺ : K⁺ symporter in the presence of low concentrations of extracellular Na⁺ and as a Na⁺ : Na⁺ transporter (i.e. a low affinity Na⁺ transporter) when high (mM) Na⁺ was present (Rubio et al., 1995). Although in situ hybridization located expression to the cortex of the root, the activity of HKT1 in planta has yet to be confirmed either as a high affinity K⁺ transporter or as a low affinity Na⁺ transporter (Walker et al., 1996). More recently, other genes coding for high affinity K⁺ transporters have been identified. HvHAK1 was identified in barley by homology to HAK1 and the KUP family from S. occidentalis and E. coli, respectively (Santa-Maria et al., 1997). Heterologous expression studies using a yeast mutant (trk1trk2), which is defective in K⁺ uptake and requires high (mM) extracellular K⁺ to grow, revealed that HvHAK1 mediated K⁺ uptake (with a K_m of 27 µM) independently of (but was inhibited by) extracellular Na⁺. HvHAK1 is expressed in the roots and its expression is enhanced in K⁺-depleted conditions. In Arabidopsis, AtKUP genes were identified independently by three groups (Quintero and Blatt, 1997; Fu and Luan, 1998; Kim et al., 1998). They are highly homologous to the HvHAK1 and KUP genes in E. coli and are expressed in the roots and shoots. Heterologous expression in trk1trk2 yeast revealed that they mediate both high affinity (K_m approximately 20 µM) and low affinity K⁺ uptake and are also inhibited by extracellular Na⁺. The mechanism of K⁺ transport for the HAK and KUP proteins is not known.

Low affinity transporters:
Channel-mediated K⁺-dependent inward currents have been characterized in many plant cell types including those from the cortex of maize roots (Roberts and Tester, 1995) and the epidermis of wheat roots (Gassmann and Schroeder, 1994). The inward-rectifying (K_in) channels which underlie these currents were proposed to represent the pathway for low affinity K⁺ uptake (Kochian and Lucas, 1988). This type of channel only activates at potentials negative of E_K (in a voltage-dependent manner) and is K⁺ selective, showing lesser permeability for Rb⁺ and Na⁺ and channel blockage by extracellular Cs⁺. Complementation studies of the K⁺ uptake-defective yeast mutant identified two cDNAs (KAT1 and AKT1) coding for K⁺ channels from Arabidopsis (Sentenac et al., 1992; Anderson et al., 1992). Since their identification several homologues to AKT1 and KAT1 have been reported (Coa et al., 1995). However, the focus will exclusively be on AKT1 because it was found to be expressed predominantly in the cortex and epidermis of roots. Unlike expression of the AtKUP and HvHAK1, AKT1 expression is not regulated by K⁺ availability. Patch clamp analysis of AKT1 activity after heterologous expression in yeast (Bertl et al., 1997) revealed K_in channels which displayed similar biophysical properties (i.e. pharmacology, selectivity and activation kinetics) to the K_in channels observed in maize and wheat roots. However, AKT1 expression also conferred the ability of the K⁺ uptake-defective yeast mutant to grow at submillimolar K⁺ concentrations. These results suggested that the V_m of the yeast plasma membrane was either very negative (i.e. greater than -300 mV) or that AKT1 possessed a dual functionality that allowed K⁺ uptake which was thermodynamically uphill. Recently, an Arabidopsis T-DNA insertion AKT1-knockout mutant (akt1–1) has been identified (Hirsch et al., 1998). The absence of K_in channels in the cortex was confirmed using the patch clamp technique. The phenotype of akt1–1 was defective K⁺ uptake and growth in solutions as dilute as 10 µM K⁺; this suggested that AKT1 mediated K⁺ uptake at concentrations previously thought to be beyond the realms of K⁺ channels (i.e. high affinity K⁺ uptake). However, electrode impalement experiments with akt1–1 revealed that the V_m of the plasma membrane was more negative than -230 mV, indicating that channel-mediated K⁺ uptake from 10 µM K⁺-containing solutions was energetically possible. The akt1–1 phenotype was only apparent in the presence of extracellular , in the absence of the mutant and wild-type Arabidopsis grew similarly. The most likely explanation for this is that inhibits high affinity K⁺ transporters and maintains a sufficiently negative membrane potential to enable high affinity K⁺ uptake via K_in channels. Consistent with this, HvHAK1 is inhibited by (Santa-Maria et al., 1997). In summary, it appears that channels can mediate high affinity K⁺ uptake in some conditions. However, in most conditions, when high affinity K⁺ transporters are functional, channels are unlikely to contribute to K⁺ uptake from solutions which contain less than 100–200 µM K⁺ (Maathuis and Sanders, 1993)

K⁺ transport in the stele
After entering the root symplasm, K⁺ can diffuse (via plasmodesmata) to the cells within the stele. Radial diffusion across the root probably results from a K⁺ gradient and/or potential difference between the cytosol of the cortical and stelar cells. It was originally proposed that oxygen deficiency in the stele prompted a passive ‘leak’ of K⁺ into the stelar apoplast (Crafts and Broyer, 1938). However, it is now clear that ion release is highly regulated and likely to involve ion channels. Early studies in support of this notion showed that fusicoccin (which enhances H⁺ ATPase activity and hyperpolarizes the membrane potential) inhibited K⁺ release from the stele of Plantago (DeBoer and Prins, 1985) and onion (Clarkson and Hanson, 1986) roots, whereas depolarization of the membrane potential of stelar cells increased the release of K⁺ into the apoplast (Clarkson and Hanson, 1986). Recent technological advances have allowed access to the plasma membrane of the root stelar cells and several ion channels have now been identified using the patch clamp technique. Isolated root steles from barley and maize roots have been treated with cell wall-digesting enzymes to release protoplasts from the barley xylem parenchyma cells (XPCs; Wegner and Raschke, 1994) and maize stelar cells (Roberts and Tester, 1995). Also fluorescent microscopy has been used to identify stelar protoplasts isolated from transgenic Arabidopsis plants which have GFP expression restricted to the root stele (Maathius et al., 1998).

K_out channels:
Outwardly-rectifying K⁺-dependent currents displaying sigmoidal activation kinetics have been identified in stelar cells from Arabidopsis, maize and barley roots. The K_out channels that underlie these currents activate at potentials positive of E_K and are highly selective for K⁺ amongst the monovalent cations (Wegner and Raschke, 1994; Roberts and Tester, 1995). It is noteworthy that the absolute permeability of the K_out channel for Rb⁺ is a fraction of that for K⁺, indicating that flux studies, in which Rb⁺ is used as a tracer for K⁺, will yield an under-estimation of K⁺ transport through these channels (Roberts and Tester, 1997). K_out channels are also permeable to Ca²⁺ such that under physiological conditions, channel activation will mediate both a K⁺ efflux and a Ca²⁺ influx (Roberts and Tester, 1997). A new gene (SKOR) has been recently identified and shown to be expressed exclusively in the stele of Arabidopsis roots (Gaymard et al., 1998). Although it has high homology to AKT1 and KAT1, heterologous expression of SKOR in Xenopus oocytes showed that this gene coded for a K_out channel and that it shared many properties with K_out channels observed in maize and barley roots (e.g. similar activation kinetics, activation potentials and a capability to mediate Ca²⁺ influx). SKOR-knockout Arabidopsis mutants had a 50% reduction in the shoot K⁺ content, illustrating that K_out channels mediate K⁺ release to the xylem. Moreover, the mutants also exhibited increased shoot Ca²⁺ content indicating that SKOR-type channels are involved in Ca²⁺ re-absorption from the xylem.

NORC:
The XPCs from barley roots also possess a unique outward current with exponential activation kinetics (Wegner and Raschke, 1994). It appears to be non-selective amongst monovalent anions and cations and is activated at high cytosolic Ca²⁺ concentrations (see below for regulation of K_out channels). The physiological role of NORC is unknown, though it is likely that the activation of this channel would clamp the membrane to 0 mV and induce a net efflux of ions into the xylem (since the chemical gradient for most monovalent ions is outwardly-directed). It has been suggested (Wegner and De Boer, 1997a) that the NORC-mediated efflux of ions will drive water influx and turgor pressure changes in the xylem vessels which may be involved in long-distance signalling during wounding responses.

K_in channels:
K_in channels have been observed in root stelar cells (Wegner and Raschke, 1994; Roberts and Tester, 1995; Maathuis et al., 1998) but are most extensively characterized in XPCs from barley (Wegner et al., 1994). K_in channels in the root stele are proposed to mediate K⁺ uptake from the xylem apoplast and hence may play a role in K⁺ circulation between the root and shoot. Although the K_in channels from XPCs share many properties with their counterparts in the root cortex and epidermis, they possess two distinguishing features. First, their activation potential is fixed at -110 mV and second, K_in channels from XPCs are not blocked by Cs⁺. The physiological relevance of these differences is unclear. Also, it remains to be seen if these characteristics are specific to barley or if the K_in channels of maize and Arabidopsis root stelar cells have similar properties.

	Regulation of K+ transport by ABA

Top Abstract Introduction K+ transport across higher... Regulation of K+ transport... References

 
Cram and Pitman were the first to report ABA regulation of ion transport in higher plant roots (Cram and Pitman, 1972). In this study, radiotracers were used to show that ABA applied to excised barley roots decreased the net efflux of K⁺ (and Cl^-) from the stelar cells to the xylem vessels, but was without effect on the net uptake of these ions in the root cortex. Since this study, there have been many concurring and contradicting reports of the effects of ABA on K⁺ transport across higher plant roots. Most reports on barley and maize roots show that ABA reduces K⁺ efflux into the xylem vessels (Cram and Pitman, 1972; Schaefer et al., 1975; Pitman and Wellfare, 1978; Behl and Jenschke, 1981; Bassirirad and Radin, 1992); the exception is a report that showed that ABA increased K⁺ efflux into the xylem vessels of maize roots (Collins and Kerrigan, 1974). However, it seems likely that this discrepancy results from the fact that the effects of ABA on ion transport in the root are sensitive to many factors including nutrient status of the roots, temperature, the availability of extracellular glucose, and aeration (Pitman et al., 1974; Fournier et al., 1987; Quintero et al., 1998). Also, it appears that the response to ABA is species dependent (e.g. only an ABA-induced promotion of K⁺ efflux into the xylem vessels has been reported for sunflower using a variety of experimental and growth condition; Fournier et al., 1987).

The molecular mechanisms which underlie ABA-regulated ion transport in root cells will be discussed. However, ABA regulation of ion transport has been extensively studied in guard cells—thus it will be useful to present a brief overview of current understanding of ABA regulation of ion transport in this cell type. For more detail on the regulation of ion transport in guard cells by ABA, the reader is referred to other reviews (Grabov and Blatt, 1998; Pei et al., 1998).

Ion channel regulation
Guard cells:
ABA causes a rapid depolarization of the guard cell plasma membrane (Thiel et al., 1992). This is thought to result from anion efflux following activation of S-type (for slow-activating) anion channels (Pei et al., 1997). However, inhibition of the plasma membrane H⁺ ATPase may also contribute to the ABA-induced depolarization (Goh et al., 1996). There are two dominant K⁺ channels in the plasma membrane of guard cells; K_in channels which are inhibited by ABA and K_out channels which are activated by ABA. ABA-induced membrane depolarization (to potentials positive of E_K) coupled with the up-regulation of K_out channel activity induces net K⁺ efflux from guard cells; the consequence of which is loss of cell turgor and stomatal pore closure.

There is evidence indicating that ABA regulation of ion channels in guard cells involves both Ca²⁺-dependent and Ca²⁺-independent pathways. ABA induces elevations of cytosolic Ca²⁺ in guard cells which may result from either a release from intracellular stores (Gilroy et al., 1990; Lee et al., 1996) or from an influx of Ca²⁺ across the plasma membrane (Schroeder and Hagiwara, 1989). Moreover, experimental elevation of cytosolic Ca²⁺ mimics some of the effects of ABA on ion channels (i.e. activation of anion channels and inhibition of the K_in channels). However, there are numerous reports which indicate that Ca²⁺-independent ABA signalling also occurs. For example, ABA-induced stomatal pore closure is not always associated with measurable elevations in cytosolic Ca²⁺ (McAinsh et al., 1992; though this could reflect technical problems which prevent detection of very small localized elevation of cytosolic Ca²⁺; Roberts et al., 1994). In addition, K_out channel activity is up-regulated by ABA, but insensitive to cytosolic Ca²⁺ (Lemtiri-Chlieh and MacRobbie, 1994). Several second messengers have been proposed to form part of the Ca²⁺-independent ABA signalling pathway. ABA increases cytosolic pH which may account for the up-regulation of K_out channels (Blatt and Armstrong, 1993) and there are reports which indicate a role for G-proteins in the regulation of ion channels, both as part of a Ca²⁺-dependent pathway (Fairley-Grenot and Assmann, 1991) and a Ca²⁺-independent membrane-delimited pathway (Wu and Assmann, 1994; Armstrong and Blatt, 1995). Also, there is good evidence showing that protein phosphorylation is a central component in ABA signal transduction. Stomatal closure is enhanced by phosphatase PP1 and PP2A inhibitors and inhibited by kinase inhibitors (Schmidt et al., 1995). Moreover, an ABA-insensitive Arabidopsis mutant (abi-1; which displays a number of ABA-related phenotypes including aberrant control of stomatal aperture) has an impaired response of the S-type anion channel to ABA which can be partially restored by the application of kinase inhibitor K-252a (Pei et al., 1997). The ABI1 gene has been shown to encode a protein phophatase 2C homologue (Leung et al., 1994). Although a number of signalling components have now been identified in ABA signalling in guard cells, it remains to be seen how these different signalling components are integrated in vivo to bring about stomatal closure.

Roots:
It was demonstrated, for the first time (Roberts, 1998), that K⁺ channels in roots are also subject to regulation by ABA. In this study, the patch clamp technique was applied to protoplasts isolated from the cortex and stele of maize roots which were either non-stressed (control), water-stressed or pretreated with ABA. It was found that water stress and ABA had no effect on the K⁺ channel activity in the root cortex, but both treatments significantly reduced the activity of the K_out channel in stelar cells. Also, in contrast to water-stressed roots, ABA pretreatment significantly activated K_in activity in the stele (Fig. 2). It was concluded that both ABA and water stress modify the permeability of the plasma membrane of root stelar cells in favour of reduced K⁺ transport to the xylem vessels and enhanced K⁺ accumulation in the root. This is entirely consistent with the K⁺ flux studies in whole root and suggests that ABA regulation of K⁺ transport in higher plant roots is mediated by modulation of K⁺ channel activity.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Whole cell current densities resulting from Kout and Kin channel activity in protoplasts from the cortex and stele of maize roots that were either well-watered (control and high ), water-stressed (WS) or treated with 20 µM ABA approximately 16 h prior to protoplast isolation. Water stress was recorded as a 1.5% reduction in the relative water content of the shoots compared to that recorded in control plants. Detailed methods for plant growth and protoplast isolation have been given elsewhere (Roberts, 1998). Patch clamp conditions were: Extracellular solution contained 15 mM KCl, 0.1 mM CaCl2, 5 mM MES adjusted to pH 6.0. Pipette solution contained 100 mM K gluconate, 3 mM MgCl2, 4 mM EGTA, 3 mM K2ATP, 10 mM HEPES adjusted to pH 7.2 for all experiments except for high in which 3.94 mM CaCl2 was added to give a final Ca2+ activity of 10 µM. Methods for whole-cell patch clamp recordings have been given elsewhere (Roberts, 1998).

 
The similarities between the regulation of K⁺ channel activity in the root stele by water stress and ABA suggested that ABA regulated K⁺ channels as part of the drought response. It is well known that ABA accumulates in plants during drought (Davies et al., 1986) and that ABA mimics a variety of responses during water stress (e.g. stomatal closure: MacRobbie, 1992; and accumulation of solutes in the root: Sharp and Davies, 1979). However, the effects of water stress on the K⁺ channel activity in the root stele were not faithfully mimicked by ABA treatment. Two likely explanations for this observation have been suggested. First, the ABA content of roots from water-stressed plants may differ from that in ABA-treated roots and that K⁺ channel activity is dependent on ABA concentrations. Second, water stress modulates the levels of phytohormones other than that of ABA (e.g. jasmonates, auxin and cytokines; Xin et al., 1997) and that these phytohormones regulate K⁺ channel activity. Consistent with this, is the observation that kinetin inhibits K⁺ efflux from maize root stele (Collins and Kerrigan, 1974) and that auxin regulates K⁺ channel activity in guard cells (Blatt and Thiel, 1994). Clearly, accurate correlation of the activity of K⁺ channels with the levels of various phytohormones will be necessary to investigate further the regulation of K⁺ channels by water stress.

How does K⁺ accumulation in roots help plants to survive drying soils? An early response of plants to water stress is an inhibition of shoot growth while root growth is maintained (Sharp and Davies, 1979; Saab et al., 1990). K⁺ is an essential cytosolic component and thus it would be essential for the maintenance of root growth, whereas ions transported to the shoot would accumulate in the apoplasts of leaves, increasing the extracellular osmotic potential and increasing the water deficit of leaf cells. Water stress also induces a net accumulation of solutes in the roots which is thought to maintain a water potential favouring the uptake of water and cell turgor pressure necessary for root growth. Although organic compounds (i.e. sugars and amino acids) are the major constituents involved in osmoregulation in plant cells during water stress (Morgan, 1984), inorganic ions may also play a role, for example, as an osmolyte (as is observed in guard cells during stomatal closure; see above) or as charge, balancing negative charges associated with the accumulation of amino acids (Jones et al., 1980).

It is noteworthy that the regulation of K⁺ channels in roots is opposite to that in guard cells suggesting that alternative mechanisms underlie the ABA regulation of K⁺ channels in roots. There appear to be at least two distinct mechanisms involved in the regulation of K⁺ channel activity in roots: a ‘long-term’ mechanism via the regulation of transcription and a more rapid ‘short-term’ post-translational mechanism. Similarities have been noted between the effects of ABA and inhibitors of protein synthesis on K⁺ efflux from the root stele (Schaefer et al., 1975) and it was inferred that ABA may be acting via regulation of transcription of ion channels in the stelar cells. Definite proof for this idea has been provided (Gaymard et al., 1998) which showed that the abundance of SKOR mRNA was dramatically reduced 3 h after the application of ABA to Arabidopsis roots. Furthermore, AKT1 expression in the root cortex was unaffected by ABA (which is also consistent with the findings of Roberts, 1998). The gene(s) encoding the K_in channels in root stelar cells have not yet been identified and thus it remains to be seen whether transcriptional regulation by ABA also modulates K_in channel expression. Post-translational control of root cell ion channels is also evident. Application of ABA directly to maize root stelar protoplasts during patch clamp experiments reduced the magnitude of the outward current (Roberts 1998). The mechanisms responsible for this post-translational control are unknown. However, although the effects of ABA on K⁺ channel activity in root cells are opposite to that in guard cells, several observations indicate that components of the ABA signalling pathway may be common to both cell types. In maize root stelar cells, increasing cytosolic Ca²⁺ to 10 µM (from less than 100 nM) modulated K⁺ channel activity in a similar way as was observed in ABA-treated stelar cells (i.e. increased K_in channel activity and decreased K_out channel activity; Fig. 2). The Ca²⁺ sensitivity of the K_out channel in maize roots can be further demonstrated using the calcium ionophore A23187. In such experiments whole cell K_out currents were completely abolished by the addition of extracellular A23187 (Fig. 3), presumably as a result of an A23187-induced elevation of cytosolic Ca²⁺. Down-regulation of K_out channels by cytosolic Ca²⁺ is also reported in barley root XPCs (Wegner and De Boer, 1997a). However, two crucial experiments are required before a role for cytosolic Ca²⁺ in ABA signalling in root stelar cells can be assigned. First, it remains to be seen if ABA elevates cytosolic Ca²⁺ in stelar cells and second, whether experimental modulation of cytosolic Ca²⁺ inhibits ABA-induced regulation of K⁺ channel activity. The activity of K⁺ channels in the root stele can also be modulated independently of cytosolic Ca²⁺. Figure 4 shows that the open probability of K_out channels in maize root stelar cells is increased with increasing cytosolic pH and there is evidence for G protein modulation of K_in channels in barley XPCs (Wegner and De Boer, 1997b). However, it is not known if cytosolic pH or G proteins are regulated by ABA in root cells.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Down-regulation of whole-cell, time-dependent, outward currents (i.e. Kout channel activity) in a maize root stelar protoplast after the addition of 10 µM extracellular A23187. (A) Currents resulting from voltage pulses from +69 to -71 mV (in 20 mV steps). Holding potential was -71 mV. (B) As (A) except 5 min after the addition of A23187. (C) Steady-state current–voltage relationship of time-dependent currents shown in (A) (•) and (B) (). Solutions are as for Fig. 2 (control). Note that the addition of A23187 induces an instantaneously-activating outward current. The ion fluxes which underlie this current are unknown.

 

View larger version (15K): [in this window] [in a new window]   Fig. 4. Increasing cytosolic pH activates Kout channels from maize root stelar protoplasts. (A) Recordings of Kout channel activity from an inside-out patch. pH of the cytosol was either 8.0, 7.2 or 6.7. The holding potential was +19 mV (cytosol with respect to outside) and upward deflections represent channel openings. Pipette solution was the same as the extracellular solution in Fig. 2. The bath solution was the same as the pipette solution in Fig. 2 except pH was adjusted to 8.0, 7.2 or 6.7 using TRIS base. (B) Voltage-dependence of steady state-single channel open probability recorded at various cytosolic pH. Data are from the patch shown in (A) (see Roberts and Tester, 1995, for calculation of open probability). Data are fitted to a Boltzman equation of the form P=/[1+ exp(Vm-V0.5)/S) where Po is the open probability, is the maximal Po, Vm is the test voltage, V0.5 is the voltage at which Po is half maximal and S is a slope factor equivalent to RT/F where is the minimal gating charge and R, T and F have their usual meanings. Data were obtained with the cytosolic pH clamped at 8.0 (•), 7.2 () and 6.7 (). For the data presented, at pH 8.0 V0.5=0 mV; =2 and =0.9; at pH 7.2 V0.5=+44 mV; =1.8 and =0.9; at pH 6.7 no fit was obtained.

 
It is noteworthy that stelar cells from ABA-pretreated maize roots had increased K_in channel activity (Fig. 2), whereas the addition of ABA directly to stelar protoplasts during patch clamp experiments had no effect on the inward current (see Fig. 4, Roberts, 1998). Also, using a pipette (cytosolic) solution containing 10 µM Ca²⁺ to record whole cell currents increased K_in channel activity (Fig. 2), but elevation of cytosolic Ca²⁺ (using A23187) after achieving the whole cell configuration of the patch clamp technique did not increase K_in activity (data not shown). The most likely explanation for these observations is that during patch clamp experiments, the pipette solution replaces the cytosol and washes out cytosolic factors which may play an integral role in the ABA signalling pathway. By analogy to guard cells, the cytosolic factors may be phosphatases—indeed, it would be interesting to determine whether abi-1 mutants have aberrant ABA regulation of K⁺ channel regulation in root cells.

Membrane potential regulation
Regulation of channel-mediated K⁺ transport across roots is dependent on the resting membrane potential of root cells; both as a regulatory factor of channel activity and as part of the driving force for passive K⁺ transport. Previously, technical difficulties have prevented accurate measurement of stelar cell membrane potentials. The authors' best estimates came from studies using microelectrodes forced through the cortex and into the stele of maize roots (Dunlop, 1982) and from measurements of the potential difference between the cortical and stelar apoplasts of Plantago roots (DeBoer and Prins, 1985). These studies showed that the membrane potential of stelar cells is more depolarized than that recorded for cortical cells.

In the present study, impalement microelectrodes were used to record membrane potentials from intact cortex and stele tissue derived from control and ABA-treated maize roots. To gain free access to the stelar cells, the cortex and endodermis were removed by hand (Gronwald and Leonard, 1982; Roberts and Tester, 1995). Furthermore, isolated steles were allowed to recover for three hours prior to electrode impalement (see Fig. 5 legend), from which stable membrane potentials could be recorded. This circumvented problems associated with forcing the microelectrode through several layers of cortical cells (i.e. soiling and possibly blocking the electrode tip with cell debris) and greatly increased the likelihood of recording accurate membrane potentials. It was confirmed that the membrane potentials of cortical cells were more hyperpolarized than those observed for stelar cells in control roots (Table 1). The addition of ABA to maize roots had no effect on the membrane potentials of cortical cells, at least in 1 mM extracellular K⁺. In contrast, the membrane potential of stelar cells from ABA-treated roots was significantly more negative than those from control roots. Figure 5 shows typical recordings from the root stele in varying extracellular K⁺ concentrations. In 10 mM K⁺ the average membrane potential in control roots (i.e. not treated with ABA) was -71±4 mV (n=5), reducing extracellular K⁺ to 10 µM hyperpolarized the cell to -110±3 mV (n=5). In ABA-treated roots, the stelar cells possessed membrane potentials ranging from 104±6 (n=5) and -145±2 (n=4) mV in 10 mM and 10 µM K⁺, respectively. These results are summarized in Table 1. The dependence of the membrane potential on extracellular K⁺ indicated that the major conductance across the plasma membrane results from K⁺ transport.

View larger version (9K): [in this window] [in a new window]   Fig. 5. Typical membrane potential recordings in stelar cells from control (A) and ABA-treated (B) maize roots. Stele tissue was isolated as described previously (Roberts and Tester, 1995). Horizontal bar below each recording denotes perfusion with varying extracellular K+ (in mM). Bath solution contained X mM KCl, 0.1 mM CaCl2, 5 mM MES adjusted to pH 6.0 with TRIS base. Methods for membrane potential recordings are as described previously (Maathuis and Sanders, 1993). Plants were grown as described previously (Roberts, 1998) except for hydroponic culture solution which consisted of 1 mM KCl, 0.5 mM CaSO4, 5 mM MES adjusted to pH 6.0 with TRIS base. Plants were harvested at 8 or 9 d. ABA-treated plants were grown as control plants with 20 µM ABA added to the hydroponic solution 14–16 h prior to harvesting. Root steles were isolated from the cortex and incubated in hydroponic solution for approximately 3 h prior to membrane potential recordings.

 

View this table: [in this window] [in a new window]   Table 1. Membrane potentials (in mV) of intact cortical and stelar cells from control and ABA-treated maize roots Plant growth, stele isolation and recording solutions were as described for Fig. 5. Mean values (±SEM) are shown and the number of replicates is indicated in the brackets.

 
The physiological significance of the ABA regulation of the membrane potential is to favour the activation of K_in channels and inhibit K_out channel activity in root stelar cells. Moreover, the more negative membrane potential in ABA-treated cells increases the driving force for net K⁺ uptake. Using data from Table 1 and assuming cytosolic K⁺ to be 100 mM, the Nernst equation predicts that the membrane potential of ABA treated stelar cells is sufficiently negative to permit channel-mediated K⁺ uptake from solutions containing between 0.1 and 1 mM, whereas in control roots passive K⁺ uptake by stelar cells is limited to solutions containing more than 1 mM K⁺. Moreover, the predicted enhanced capacity for K⁺ uptake of isolated steles from ABA-treated maize roots can be demonstrated using radiotracers (Fig. 6). In these experiments, the stele from ABA-pretreated maize roots had significantly greater rates of K⁺ uptake compared to that observed in control roots. Predictably, ABA had no effect on K⁺ uptake by the epidermis or cortex of maize roots (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 6. K+ uptake rates for control (open bars) and ABA-treated (solid bars) intact isolated maize root steles. 86Rb+ was used as a tracer for K+ and the protocol for measuring 86Rb+ uptake was as described previously (Kochian and Lucas, 1982). Values are the mean (±SEM) of at least five separate experiments. Plant growth and root stele isolation was as for Fig. 5. Extracellular recording solution was as described in Fig. 5 and contained 1, 5 or 10 mM K+.

 
The mechanism by which ABA hyperpolarizes the maize root stelar cells is unknown. In guard cells, ABA induces a rapid membrane depolarization which is mediated primarily by anion channel activation and subsequent anion efflux. It is unlikely that anion channel activation will induce plasma membrane hyperpolarization because the electrochemical potential for most anions favours anion efflux. An attractive alternative mechanism for membrane hyperpolarization in roots is via the stimulation of the H⁺-ATPase activity. To date, there is little evidence to support this except that ABA has been reported to stimulate a transient increase in proton efflux into the xylem apoplast of onion roots (Clarkson and Hanson, 1986).

In summary, the complementary regulation of K⁺ fluxes, K⁺ channels and membrane potentials in root cells by ABA supports the hypothesis that K⁺ channels form an important pathway for the translocation of K⁺ from the soil solution to the shoots. Further investigation of their regulation will provide more detailed insights into the role of these ion channels in both nutrient acquisition and in adaptation responses to abiotic stresses. It is clear that fundamental differences exist in the regulation of K⁺ channels (and membrane potential) in guard cells and root cells. The different regulation appears physiologically appropriate for the specialized function of each cell type and highlights the limitations of making generalizations and extrapolations from a single (model) cell type.

	Acknowledgments

 
I thank Dale Sanders and Frans Maathuis for useful discussions and comments on the manuscript. Part of the research was supported by an AFRC grant awarded to Mark Tester.

	Notes

 
¹ To whom correspondence should be addressed. Fax: +44 1904 434317. E-mail: skr4{at}york.ac.uk

² Present address: Wolfson Laboratory for Plant Molecular Biology, School of Biological Sciences, University of Birmingham, Birmingham B15 2TT, UK.

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