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First published online February 25, 2005; 10.1104/pp.104.054056 Plant Physiology 137:819-828 (2005) © 2005 American Society of Plant Biologists The Regulation of Anion Loading to the Maize Root Xylem1,[w]Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom
The regulation of anion loading to the shoot in maize (Zea mays) was investigated via an electrophysiological characterization of ion conductances in protoplasts isolated from the root stele. Two distinct anion conductances were identified. In protoplasts from well-watered plants, Z. mays xylem-parenchyma quickly-activating anion conductance (Zm-X-QUAC) was the most prevalent conductance and is likely to load the majority of NO3– and Cl– ions to the xylem in nonstressed conditions. Z. mays xylem-parenchyma inwardly-rectifying anion conductance was found at a lower frequency in protoplasts from well-watered plants than Zm-X-QUAC, was much smaller in magnitude in all observed conditions, and is unlikely to be such a major pathway for anion loading into the xylem. Activity of Z. mays xylem-parenchyma inwardly-rectifying anion conductance increased following a water stress prior to protoplast isolation, but the activity of the putative major anion-loading pathway, Zm-X-QUAC, decreased. Addition of abscisic acid (ABA) to protoplasts from well-watered plants also inhibited Zm-X-QUAC activity within minutes, as did a high free Ca2+concentration in the pipette. ABA was also seen to activate a Ca2+-permeable conductance (Z. mays xylem-parenchyma hyperpolarization activated cation conductance) in protoplasts from well-watered plants. It is postulated that the inhibition of anion loading into the xylem (an important response to a water stress) due to down-regulation of Zm-X-QUAC activity is mediated by an ABA-mediated rise in free cytosolic Ca2+.
The first manifestation of a plant water deficit is a reduction in cell expansion (Sharp et al., 1997  ), particularly in the shoot (Hsiao and Xu, 2000). In maize (Zea mays), the primary root will maintain substantial elongation even at a water potential of –1.6 MPa, whereas shoot development is completely inhibited at around –0.8 MPa (Sharp et al., 1997). It has been proposed that this difference between roots and shoots is due to both an increased ability of the roots to reduce their solute potential as well as a decreased ability of shoots to maintain wall extensibility (Hsiao and Xu, 2000). These effects of water deficit on cell expansion occur before any effects on stomatal conductance become apparent (Davies and Zhang, 1991; Sadras and Milroy, 1996). In this article, we focus on mechanisms underlying reductions in root solute potential in maize under conditions of water deficit. Maize was chosen because of the ease with which the cortex and stele can be physically separated, facilitating study of the separate processes of uptake from the soil and loading into the xylem.
Water stress (and abscisic acid [ABA]) is known to increase solute accumulation within the root by having little effect on initial ion uptake but by significantly inhibiting release of ions into the xylem (Cram and Pitman, 1972
Although control by ABA of root K+ influx and loading into the xylem has been well studied (for review, see De Boer, 1999 The aim of this study was to identify the pathways for the loading of anions into the xylem in maize roots and to investigate the regulation of these pathways by water stress and ABA.
QUAC
A quickly activating current was detected at voltages both positive and negative of ECl across the plasma membrane of protoplasts derived from the stele (Fig. 1A). The quick activation of the current was followed rapidly by a partial inactivation at large voltage deviations from ECl, with the inactivation being more pronounced at larger voltages. The pseudo-steady-state current, measured after 4 s, reversed at –9 mV (Fig. 1B). This is close to ECl (–4 mV) and clearly positive of ETEA (–
The current-voltage relationship of Zm-X-QUAC is not linear (Fig. 1B). Although outward rectification of the current was observed in all ionic conditions tested, it was clear that substantial amounts of Cl– were able to move in both directions. Therefore, depending upon membrane voltage, Zm-X-QUAC may facilitate either Cl– loading into, or Cl– unloading out from, the xylem. Ion substitution experiments confirmed that, in addition to Cl–, Zm-X-QUAC was highly permeable to NO3– and I– (Table I). From the mean conductance measured in the presence of various anions, a selectivity sequence could be calculated of NO3– > Cl– > I– > malate2– > SO42– > citrate3–. This broadly resembled the positive shifts in the reversal potential observed upon substitution of the external anions (Table I).
The activity of Zm-X-QUAC also appears dependent on the presence of cytosolic MgATP. When MgATP is excluded from the pipette, currents declined to negligible levels within 5 min of forming the whole-cell configuration, even with [Ca2+]cyt highly buffered to 100 nM with 20 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; Alexandre and Lassalles, 1992 ; ISF, n = 5; data not shown; see Gilliham, 2002).
Zm-X-QUAC was reversibly inhibited by the presence of niflumate, a known anion channel blocker, on the external face of the protoplast, with an approximate EC50 of 18 µM with a Hill slope of close to –1 (Fig. 1D). GdCl3, a known cation channel blocker, had no inhibitory effect on the inward or outward currents either pre- or postinactivation when applied externally at up to 10 mM (data not shown; see Gilliham, 2002
In some protoplasts isolated from maize stelar tissue, another conductance was measured, this being detected as distinct single channel-like opening/closing transition events during whole-cell patch-clamp experiments. These observations suggest either a low channel density or a low probability of opening (Fig. 2A).
Single channel current-voltage relations showed that the current was mainly carried by anions, with Erev shifting from approximately +23 mV to +53 mV with a shift in ECl from +35 mV to +55 mV (Fig. 2B). The reversal potential for the accompanying cation was consistently very negative of Erev. The unitary conductance determined for the inward current component was 90 pS with 100 mM Cl– in the pipette and 80 pS with 50 mM pipette Cl–. The gating of these apparent channels was strongly voltage dependent, leading to a distinct inward rectification of current through the channels when MgATP was present in the pipette solution (Fig. 2). These channels were therefore given the moniker Z. mays inwardly-rectifying anion conductance (Zm-X-IRAC). Zm-X-IRAC was not only permeable to Cl– but also to NO3– (data not shown; Gilliham 2002 ).
Under varying ionic conditions, but with the maintenance of approximately 100 nM [Ca2+]cyt, Zm-X-QUAC was found in 29% of protoplasts (n = 24/82). Zm-X-IRAC was found in 17% of protoplasts (n = 14/82), and no current was present in 59% (n = 48/82). Both currents were seen in 5% of protoplasts (n = 4/82).
Current densities were calculated at –120 mV (Table II), this being the approximate resting membrane potential of maize stelar tissue (Roberts and Snowman, 2000
Effects of Water Stress, ABA, and High [Ca2+]cyt Current-voltage relations of stelar protoplasts obtained from water-stressed plants clearly showed that mean current densities through Zm-X-QUAC were lower at all voltages compared with those measured in protoplasts isolated from well-watered plants (Fig. 3A). A 12-h pretreatment with 20 µM ABA prior to protoplast preparation, or an increase of [Ca2+]cyt from 100 nM to 1 µM, also inhibited the mean current density of Zm-X-QUAC in well-watered plants at all voltages (Fig. 3, A and B). These reductions are summarized in Figure 3C, the reduction being attributed to both a smaller proportion of protoplasts expressing the conductance and a decrease in conductance in the protoplasts in which currents appeared.
Rapid Effect of ABA on Zm-X-QUAC Mean current densities at –120 mV were compared in protoplasts that were patch clamped at different times after exposure of the intact protoplasts to ABA (Fig. 4A). Within 40 min of the application of 20 µM ABA, the activity of Zm-X-QUAC decreased to 20% of the activity in control protoplasts, from –107 ± 39 mAm–2 (n = 15) to –23 ± 8.8 mAm–2 (n = 3; Fig. 4A).
The time course for ABA control of Zm-X-QUAC was investigated further by the application of 20 µM ABA directly to patch-clamped protoplasts (Fig. 4B). All experiments were performed with a high external Ca2+ concentration ([Ca2+]ext = 20 mM CaCl2) to aid seal integrity. In these conditions, Zm-X-QUAC activity at –120 mV ran down to 66% ± 8% (n = 2) of the maximal current over 20 min. However, if 20 µM ABA was applied to the protoplast 5 min after establishment of the whole-cell configuration (approximately the time at which a protoplast shows maximal Zm-X-QUAC activity), the current at –120 mV was reduced to 26% ± 7% (n = 2) of its maximal value within 15 min of the application of ABA. Therefore, it was clear that ABA significantly increased the rate of rundown of Zm-X-QUAC. If [Ca2+]cyt was highly buffered (i.e. contained 20 mM BAPTA), no appreciable rundown was observed in either control protoplasts or protoplasts to which ABA was added (Fig. 4B). Protoplasts patch clamped with a pipette solution containing high concentrations of BAPTA possessed 94% ± 3% (n = 3) of their Zm-X-QUAC activity after 20 min whether or not they were treated with ABA (Fig. 4B).
It should be noted that a mild water stress or application of 50 µM ABA to well-watered plants 12 h prior to protoplast isolation increased the activity and occurrence of Zm-X-IRAC, as did either increasing [Ca2+]cyt, or applying 20 µM ABA to protoplasts obtained from well-watered plants. Occurrence increased from 13.3% (n = 15) in well-watered plants to 26.6% (n = 10) in water-stressed plants; 25% in ABA pretreated plants (all in ESB:ISC; n = 8); and 50% in plants patch clamped with 1 µM [Ca2+]cyt (ESB:ISE; n = 8; data not shown; see Gilliham, 2002
In addition to decreasing the activity of Zm-X-QUAC, ABA also stimulated a conductance at hyperpolarized potentials with time-dependent activation in 82% of cells (n = 18/22; Fig. 5, A and B). These currents were particularly apparent when solutions were designed to maximize the appearance of divalent cation currents. With 20 µM ABA, maximal activation of these currents occurred within 25 to 30 min (Fig. 5, A and B), with average currents in stelar protoplasts in 2.5 mM Ba-HEPES:5 mM BaCl2 (ESO:ISI) at –160 mV reaching 167 ± 30 pA (n = 18). These currents were insensitive to 5 mM H2O2, even after 40 min (n = 5). It should be noted that an ABA-independent activation of the current was also observed in control protoplasts, but at a lower frequency than in ABA-treated protoplasts (25% of protoplasts within 30 min; n = 16).
Activation of this conductance by ABA was repressed by inclusion in the pipette of either 400 µM MgATP (ISM; n = 9/10) or the nonhydrolysable analog MgATP-  -s (ISN; n = 6/6) with 2.5 mM [Ba2+]ext; if protoplasts were exposed to higher concentrations of divalent cations, seal integrity was lost upon current activation at hyperpolarized potentials. The presence of cytosolic Mg2+ had no inhibitory effect upon the activation of the conductance by ABA (n = 5/6; ISP), in contrast to that observed in Vicia faba guard cells (F. Lemtiri-Chlieh, personal communication). Substitution of pipette Cl– with gluconate did not significantly affect the hyperpolarization-activated current (Fig. 5C). Similar results were seen by substitution of pipette Cl– with HEPES (data not shown). (Backfilling the pipette with a solution containing 10 mM KCl around the AgCl half-cell in the above ionic conditions had no effect on the current characteristics.) As the time-dependent current was still active in protoplasts with only channel impermeant anions on the cytosolic side of the membrane, the inward current was most likely to be due to the influx of the external cation, which in this case was Ba2+. In addition, the currents showed clear dependence on the concentration of external cations (Fig. 5D). Accordingly, the conductance allowing this current is referred to as Z. mays xylem-parenchyma hyperpolarization-activated cation conductance (Zm-X-HACC). The magnitude of Zm-X-HACC was dependent on the identity of the external cation. The permeant cation was exchanged while keeping the normality constant (the concentration of charges, i.e. 5 mM K+ = 5 mM of positive charges, as does 2.5 mM Ba2+). The greater permeability of divalent cations compared with monovalent cations of Zm-X-HACC can be seen clearly in Figure 5E. At –160 mV, the current ratio through Zm-X-HACC for Ba2+:Ca2+:Na+:K+ was 23:18:2.2:1.0.
Two anion-selective conductances in the cells of the stele of maize are described, these being distinct in both their activation kinetics and voltage dependence. These conductances share many similarities with two anion conductances found in barley xylem parenchyma cells (Kohler and Raschke, 2000 ), although a third type of anion current found in barley, X-SLAC, was not found in the maize stele. Unlike the aluminum-activated anion channels characterized in the root tips of maize and wheat (Triticum aestivum), they appear not to allow the permeation of significant amounts of organic acid (Piñeros and Kochian, 2001; Zhang et al., 2001). Zm-X-QUAC and X-QUAC from barley share a profound outwardly-rectifying characteristic with an anion channel characterized in wheat root cortical cells (Skerrett and Tyerman, 1994); however, unlike the wheat cortical channel, they also appear to allow significant efflux of anions out of the cell as well as influx at hyperpolarized and depolarized potentials respectively.
As in barley, it is concluded that X-QUAC is most likely to be responsible for the majority of anion efflux into the xylem as it was more frequently detected than Zm-X-IRAC in well-watered conditions and had a greater current density at physiological resting membrane potentials in all conditions. Although Zm-X-QUAC was the most frequently observed conductance, it was observed in only one-third of protoplasts in well-watered conditions. The limited detection of anion conductances, and the fact that these channels were not observed in excised patches, may suggest a heterogeneous or even polar distribution of the channels within xylem associated cells. This observation is consistent with the xylem transfer hypothesis of Kramer (1981)
Water stress, ABA pretreatment, and high [Ca2+]cyt all appeared to inhibit Zm-X-QUAC activity to a similar degree (Fig. 3). If the internal patch solution were only lightly buffered with BAPTA (5 mM), at a level insufficient to maintain [Ca2+]cyt at physiological resting levels (i.e. 100 nM) in the face of high [Ca2+]ext, the activity of Zm-X-QUAC could be seen to decrease over time. The rate of this rundown was increased by application of ABA (Fig. 4B). However, when [Ca2+]cyt was fixed at physiologically resting levels, using 20 mM BAPTA (Alexandre and Lassalles, 1992
One of the first responses of guard cells' membranes when challenged with ABA is a stimulation of HACC activity through a pathway mediated by reactive oxygen species (e.g. Pei et al., 2000
Zm-X-HACC activation by ABA within the time course for inhibition of Zm-X-QUAC suggests a potential role for Zm-X-HACC in an increase in [Ca2+]cyt that would facilitate the decrease in Zm-X-QUAC activity. ABA has been reported to increase [Ca2+]cyt in maize root tissue (Gehring et al., 1990
The activity of Zm-X-IRAC was increased by ABA, water stress, and high [Ca2+]cyt. It has been hypothesized that the equivalent channel found in barley xylem parenchyma Hv-X-IRAC could help to regulate the membrane potential of cells by providing a further pathway for counterions to short circuit the stelar proton pump (Kohler and Raschke, 2000
Some transport proteins in the stele appear to turn over rapidly, in the order of tens of minutes, as revealed by the rapid inhibition of 36Cl– loading into the xylem by addition to intact roots of the amino acid analog, fluorophenylalanine (Schaefer et al., 1975
The requirement for intracellular MgATP for observation of Zm-X-QUAC activity indicates that the conductance could be activated in phosphorylating conditions, a feature common to anion channels (Barbier-Brygoo et al., 2000
During water stress, roots of maize maintain elongation far beyond the point at which the shoot ceases to grow (Sharp et al., 1997 ). This observation correlates with the accumulation of solutes in the roots upon water stress (Sharp and Davies, 1979) and the reduction of xylem loading of solutes by ABA (Cram and Pitman, 1972). It is proposed that these observations constitute a mechanism by which roots have the resources to sustain growth during water stress and so potentially alleviate water stress, while not contributing to the shoot ion load and exacerbating the effects of water stress in the aerial portion of the plant. We propose that a water deficit increases local ABA concentrations, stimulating entry of Ca2+ into the cytosol of stelar cells that down-regulates Zm-X-QUAC activity through an unknown mechanism, so reducing anion entry into the xylem. These effects compare favorably with those observed for other ion transporters in the stele and are summarized in Figure 6. The findings within this article provide an insight into a mechanism by which maize could regulate ion transport between the root and shoot during water stress, contributing to the associated accumulation of ions in the root.
Plant growth, protoplast isolation, and patch-clamp electrophysiology were carried out as described by Roberts and Tester (1995) and Roberts (1998), with the following modifications (for more detailed methods, see Gilliham, 2002). Protoplasts were adhered to a coverslip treated with 75,000 molecular weight poly-D-Lys (Sigma, Gillingham, UK) in an irrigation chamber (of 200-µL volume) with a fast volume exchange (1 mL min–1). The generation of voltage test pulses, recording of whole-cell currents, and storage of data were undertaken using an Axon 200A amplifier under the control of Clampex 8.21 (Axon Instruments, Union City, CA) and a Pentium II computer. Whole-cell currents, digitized at 5 kHz by a Digidata 1200A (Axon Instruments) interface, were filtered with a corner frequency of 2 kHz (8-pole Bessel filter) and stored on the computer. Capacitance and series resistance were compensated using the amplifier. Seal resistance was monitored regularly and data was only analyzed if protoplasts had a seal resistance of over 1 G . Current-voltage relations were obtained from current traces, resulting from voltage step protocols, using the analysis package Clampfit 8 (Axon Instruments). Data analyses were conducted using Excel 2000 (Microsoft, Redmond, WA) and all graphs constructed and data fitted using Origin 5 (Microcal, Northampton, MA) or Prism 3.0 (GraphPad, San Diego). Variations in data are always presented as the SE of the mean.
Where appropriate, current densities were obtained per protoplast by dividing the current of a protoplast, either pre- or postinactivation, by its membrane area obtained from a measurement of the protoplast membrane capacitance. The plant membrane was assumed to have a specific capacitance of 7.6 mFm–2 (Homann and Tester, 1997
Recordings from outside-out patches were analyzed as in Kohler and Raschke (2000)
It is worth noting that the water stress imposed in this study was small and slow, with the identical treatment imposed by Roberts (1998)
A variety of solutions were used to investigate both the anion and cation currents in an attempt to reduce the artifacts that may be associated with the use of particular impermeant ion species and channel blockers and hence increase the robustness of the data. The main species can be found in appropriate figure legend or text. TEA+, Cs+, Ca2+, and Gd3+ have been used to reduce K+ or cation currents, external Ca2+ was used to maintain a giga- Variation in the data is always presented as SEM, calculated as the standard deviation divided by the square root of the number of replicates.
We thank Drs. Alex Webb and Fouad Lemtiri-Chlieh and members of the Department of Plant Sciences ion transport supergroup for helpful discussions, Mr. John Banfield and Mr. Paul Freeman for technical support, and the useful guidance of anonymous referees. Received September 25, 2004; returned for revision December 9, 2004; accepted December 22, 2004.
1 This work was supported by the Biotechnology and Biological Sciences Research Council (studentship to M.G. and research development fellowship to M.T.). 
2 Present address: Australian Centre for Plant Functional Genomics, PMB 1, Glen Osmond, South Australia 5064, Australia.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.054056. * Corresponding author; e-mail mark.tester{at}acpfg.com.au; fax 61–8–8303–7102.
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ABSTRACT
RESULTS
) and negative of ECa (+136 mV). When Erev was plotted against ECl with varying internal and external ionic conditions, it is clear that Erev closely follows ECl (
, Instantaneous current; 
, noninactivating component. Reversal potential for the noninactivating component = –9 mV. C, Reversal potential of Zm-X-QUAC shifts with changes in ECl. Data are shown as a mean of Erev values with SEM (n = 4 for each ECl). ECl was varied by changing both external and internal solutions. In ascending order, solutions contained (in mM): 60 Cl– in the bath and 50 Cl– in the pipette (ESD:ISC), ECl = –4 mV; [Cl–]ext:[Cl–]cyt 50:50 (ESC:ISA), ECl = 0 mV; 60:100 (ESD:ISB), ECl = +10 mV; and 10:50 (ESE:ISA), ECl = +35 mV. Line drawn is Erev = ECl. D, Niflumate inhibits noninactivating component of Zm-X-QUAC (n = 7). Sigmoidal (Hill equation) fit used; EC50 = 17.5 µM, Hill slope = –0.93.
) 50:50 (ESC:ISE). 
, protoplasts prepared from whole plants watered with 20 µM ABA 12 h prior to harvest (ABA, n = 8); and 
), which contained weakly Ca2+ buffered internal solution (5 mM BAPTA, ISA), protoplasts treated the same way but with ABA added (
soil). This initiates: (1) increased root ABA concentration, possibly through the detection of cell volume changes within root cells (Jia et al., 2001
