Dipartimento di Produzione Vegetale, Università degli Studi di Milano, via Celoria 2, I Milano, Italy

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1 Membrane depolarization induces K efflux from Blackwell Science Ltd subapical maize root segments Fabio F. Nocito, Gian Attilio Sacchi and Maurizio Cocucci Dipartimento di Produzione Vegetale, Università degli Studi di Milano, via Celoria 2, I Milano, Italy Summary Author for correspondence: Maurizio Cocucci Tel: Fax: maurizio.cocucci@unimi.it Received: 30 July 2001 Accepted: 12 November 2001 The role of potassium efflux from maize (Zea mays) root segments in maintaining transmembrane electric potential difference (E m ) was studied in vivo, together with the involvement of outward rectifying K channels (ORCs). Measurements were made of the efflux of potassium (K ) from roots when its uptake was competitively inhibited by rubidium (Rb ), of the E m of the root cells by microelectrodes and of the unidirectional fluxes of monovalent cations. The influx of Rb, caesium (Cs ) or ammonium (NH 4 ) into the segments induced an efflux of K. Lithium (Li ) and sodium (Na ) were not taken up and did not induce K efflux. The permeating cations induced membrane depolarizations, which were closely related to the values of K efflux. Two K -channel blockers, tetraethylammoniumchloride and quinidine, inhibited K efflux. The inhibition was accompanied by a higher membrane depolarization induced by Rb, whose influx was not affected. The results suggest that a depolarizing event caused by cation uptake increased K efflux from the cells, probably through the activation of ORCs involved in restoration and stabilization of E m. Key words: potassium channels, potassium efflux, stabilization of E m, Zea mays. New Phytologist (2002) 154: Introduction The involvement of potassium in fundamental processes of plant biology is strictly related to the evolution of different K transport systems in the cell plasma membrane (Maathuis et al., 1997; Chrispeels et al., 1999). Several studies have revealed that the movement of K across the plasma membrane involves a multitude of transport proteins, including carriers and channels (Chrispeels et al., 1999). In the roots of higher plants the uptake of K against its electrochemical gradient (i.e. at micromolar K external concentration, when Nernst s potential for K, E K, is more negative than the transmembrane electric potential difference, E m ) is a carrier-mediated process. The mechanisms of energization of this transport have not yet been completely elucidated. It has been proposed that the active uptake of K into the root cells could be driven by either the H (Maathuis & Sanders, 1994) or the Na (Rubio et al., 1995) electrochemical gradients, via cotransport systems. Unlike carriers, K channels selectively facilitate the movement of potassium ions down their electrochemical gradient. Two classes of K channels have been identified and characterized in plant cells: the inward rectifying K channels (IRCs) and the outward rectifying K channels (ORCs). These types of transporters are different protein entities, characterized by specific kinetic profiles, pharmacological sensitivity and opposite voltage dependence, being activated by either hyperpolarization (IRCs) or depolarization (ORCs) of the plasma membrane (Maathuis et al., 1997). The activity of IRCs and ORCs significantly contributes to several physiological processes, including: K homeostasis in the cells; regulation of membrane voltage; K nutrition; loading and unloading of the xylem; osmoregulation of the cells; stomatal and leaf movements (for an extensive review see Maathuis et al., 1997). The existence in plant root cells of outward-directed K fluxes, mediated by the activity of specific K -channels, is largely confirmed by patch clamp studies conducted on xylem parenchyma cells of barley root stele (Wegner & Raschke, 1994; Wegner & De Boer, 1999), epidermal and cortical cells of Arabidopsis thaliana roots (Maathuis & Sanders, 1995) and cortical and stelar cells of maize roots (Roberts & Tester, 1995). To date, most of the information on the biophysical characteristics and physiological roles of ORCs has New Phytologist (2002) 154:

2 46 been obtained by means of electrophysiological studies conducted on highly simplified systems such as protoplasts, membrane fragments or heterologous expression systems (Maathuis et al., 1997; Ward, 1997; Miller & Zhou, 2000). It is clear that the results obtained by these attractive approaches could acquire more physiological significance if combined with, or compared with, results obtained with techniques that induce as little physiological perturbation as possible (Tester, 1997). With this view, an elegant study has been performed by Gaymard et al. (1998). By studying the phenotype of the appropriate knockout mutant in Arabidopsis, physiological significance has been given to a cloned and electrophysiologically characterized ORC. A totally noninvasive technique for studying ion transport consists of ion-selective electrodes, whose principles have been reviewed by Newman (2001). Nevertheless, even if this technique allows in vivo measurements in whole organs or plants, it does not resolve net ion fluxes into their unidirectional components. The unidirectional radioactive or nonradioactive tracer technique is commonly used for the in vivo study of the activities of ion channels in intact organs or isolated cells (Kochian & Lucas, 1982; Smith et al., 1987; MacRobbie, 1997). Nevertheless, the in vivo characterization of K efflux from the cells is limited by the activity of the high-affinity K uptake system (Maathuis & Sanders, 1996; Rodriguez- Navarro, 2000), which rapidly and efficiently reabsorbs K released from cells. In a previous work we described the effect of Rb on K efflux from subapical maize root segments (Cocucci & Sacchi, 1993). We also showed that incubation of the root segments in the presence of Rb is a suitable experimental condition to detect and characterize the efflux of K from root cells, since Rb efficiently competes with K for the high-affinity K uptake system (Epstein & Hagen, 1952; Sacchi et al., 1997) and thus minimizes reabsorption of K. In our previous work the involvement of ORCs in mediating K efflux from maize root segments was only suggested (Cocucci & Sacchi, 1993). The aim of this work was to study the release of K from subapical maize root segments, evaluated in the presence of Rb, in order to obtain information on the actual involvement of ORC activities in K efflux and on its physiological role. For this purpose, the effects of monovalent cations and of specific inhibitors of K -channel activity on the efflux of K have been analysed. further 24 h. After this period, the main roots of the seedlings were about mm long. Subapical maize root segments (6 mm long), obtained by cutting the main roots at 2 and 8 mm from the tips, were washed for 3 h in aerated 0.5 mm CaSO 4 solution. Batches of 15 segments (about 75 mg f. wt) were then washed twice for 30 min in 10 ml of the same solution, in a water bath thermoregulated at 26 C and agitated at 80 oscillations min 1 ; after these treatments (preincubation) the segments had completely recovered from wounding (Cocucci & Sacchi, 1993). Influxes of monovalent cations into root segments After preincubation, 15 subapical root segments were incubated for 15 or 30 min in 10 ml of a basal medium (0.5 mm CaSO 4, 0.1 mm MES-Ca, ph 6.0) supplemented with 0.5 mm Rb, Li, [ 22 Na ]Na (6.7 MBq mmol 1 ), Cs, or NH 4 (as SO 4 salts) at 26 C and agitated at 80 oscillations min 1. In the cases of Rb, Li and Cs, after 15 or 30 min the segments were collected, washed twice at 4 C for 15 min in the basal medium supplemented with 0.5 mm K and then mineralized at 100 C in 0.5 ml of HNO 3 H 2 SO 4 HClO 4 (5 : 1 : 1, v : v : v). The amounts of Rb, Li or Cs were then determined by atomic absorption spectrophotometry (SpectraAA-20, Varian, Mulgrave, Victoria, Australia), using for each cation the respective SO 4 salt solution as a standard. The Rb assay was performed in the presence of 100 mm KCl as ionization suppressant. The uptake of NH 4 was detected by measuring the depletion of the cation in the medium: after 15 and 30 min of incubation, the total amount of NH 4 was assayed according to Moore & Stein (1954) on aliquots of the external medium. For [ 22 Na ]Na, at the end of the incubation period the segments were washed twice at 4 C for 15 min with a corresponding nonradioactive solution. The samples were then frozen, homogenized in 1 ml of 0.1 M HNO 3, and heated at 100 C for 10 min. Radioactivity was measured by counting, in a Beckman LS 6000SC (Beckman, Fullerton, CA, USA) scintillation counter, aliquots of the supernatants dissolved in 10 ml of Ready-Solve scintillation cocktail (Beckman). Similar experiments were conducted by incubating the segments in the same basal medium containing 0.5 mm Rb together with equimolar amounts of, respectively, Li, [ 22 Na ]Na (6.7 MBq mmol 1 ), Cs or NH 4. Materials and Methods Plant material Maize (Zea mays L., cv. Dekalb DK 300) caryopses were germinated for 2 d in the dark at 26 C on filter paper saturated with distilled water. Subsequently seedlings selected for uniform growth were transferred to aerated 0.5 mm CaSO 4 solution and maintained in the dark at 26 C for a Measurement of K release Potassium released from the segments was assayed as previously described (Cocucci & Sacchi, 1993). Briefly, 15 subapical root segments were incubated in the conditions described above for the uptake experiments. At the end of the incubation period (15 or 30 min), aliquots of the medium were assayed, by atomic absorption spectrophotometry, to detect changes in K concentration. The assay of K was New Phytologist (2002) 154: 45 51

3 47 performed in the presence of 100 mm CsCl as ionization suppressant. Measurements of transmembrane electric potential difference Transmembrane electric potential (E m ) was measured using a high-impedance electrometer amplifier (World Precision Instrument, model K5-700, New Haven, CI, USA) and microelectrodes pulled from single-barrelled borosilicate glass tubing (World Precision Instrument) and filled with 3 M KCl (adjusted to ph 2 to reduce tip potential). Electrode resistances varied between 10 MΩ and 15 MΩ. Four subapical root segments were placed in a Plexiglass cuvette (total volume 1.5 ml) and maintained under a continuous flow (200 ml h 1 ) of the thermoregulated (26 C) and aerated basal medium. After 30 min, each different monovalent cation (0.5 mm), either alone or together with an equimolar amount of Rb, was added. The electrodes were inserted, perpendicularly to the main root axis, into the epidermal and second to fifth layer cortical cells of the root. Measurement of Rb uptake, K efflux and E m after pretreatment with K -channel blockers The effects of K -channel blockers on Rb uptake, K efflux and E m were evaluated by preincubating subapical root segments for 1 h at 26 C in the basal medium in the presence of 10 mm tetraethylammonium-chloride (TEA ) or 1 mm quinidine. After this period, the segments were rinsed five times with a large volume of the basal medium without the inhibitors and then used for the evaluation of Rb influx, K efflux and E m. Results and Discussion Subapical maize root segments took up Rb, Cs or NH 4 to a similar extent when each cation was present alone at 0.5 mm in the incubation medium. In the same conditions, Li and Na were essentially excluded by the root cells (Table 1). The influxes of Rb, Cs and NH 4 were accompanied by simultaneous efflux of K from the root segments (Table 1). The values of K efflux were highest in Rb and progressively Table 1 Influxes of monovalent cations and their effects on K efflux in subapical maize root segments Monovalent cation (0.5 mm) Cation influx K efflux Rb 6.35 ± ± 0.07 Li 0.07 ± 0.02 Nd Na 0.08 ± ± 0.01 Cs 6.10 ± ± 0.03 NH ± ± 0.12 Fifteen root segments were incubated in 10 ml of 0.5 mm CaSO 4, 0.1 mm MES-Ca (ph 6.0) plus 0.5 mm of the indicated cations (as SO 4 salts). The values are the means ± SE of five experiments in triplicate. Nd, not detectable. lower in NH 4 ( 50%) and Cs ( 70%). No significant efflux of K from the root segments was induced by Li and Na. The possibility that K release may occur through the cut ends of root segments has been excluded by previous experiments (Cocucci & Sacchi, 1993). The in vivo study of the efflux of K from plant tissues and the evaluation of its amount are influenced by the reabsorption of the cation released in the apoplast. Since Rb efficiently competes with K for the high-affinity potassium uptake system (Sacchi et al., 1997), the incubation of subapical maize root segments with Rb greatly reduces the possibility of a significant K re-influx. Therefore, we evaluated the efflux of K by incubating the root segments in solutions containing each different cation, together with an equimolar amount of Rb. Table 2 shows that, when the segments were incubated in the presence of 0.5 mm Li plus an equimolar concentration of Rb, the values of K efflux were similar to those recorded in the presence of Rb alone (Table 1). The Li was not absorbed by root segments and did not affect Rb influx (Table 2). Similar results were obtained for Na in the presence of Rb (Table 2). It has been suggested that, at a Na external concentration less than 1 mm, K is actively absorbed by a Na K symporter (Rubio et al., 1995). In the presence of Na alone in the incubation medium, the activity of this system could reduce the amount of K released from the cells. Nevertheless, the very low influx of Na into root segments observed under our experimental conditions (Table 2) allows Table 2 The K efflux and cation influxes in subapical maize root segments incubated with Rb, Li, Na, Cs or NH 4 plus an equimolar amount of Rb Rb cation (0.5 mm 0.5 mm) K efflux Rb influx Cation influx Rb Rb 2.69 ± ± 0.17 Rb Li 2.37 ± ± 0.37 Nd Rb Na 2.41 ± ± ± 0.01 Rb Cs 1.80 ± ± ± 0.09 Rb NH ± ± ± 0.13 Fifteen root segments were incubated in 10 ml of 0.5 mm CaSO 4, 0.1 mm MES-Ca (ph 6.0) together with Rb plus the different cations (as SO 4 salts) at the concentrations indicated. The values are the means ± SE of four experiments in triplicate. Nd, not detectable. New Phytologist (2002) 154:

4 48 us to exclude a possible underestimate of K efflux. We can thus conclude that, for Li and Na, the lack of a detectable K efflux was not attributable to active reabsorption of K released. At Cs concentrations up to 0.5 mm, the uptake of this cation involves the high-affinity K uptake system that discriminates only poorly between K, Rb and Cs (Sheahan et al., 1993; Sacchi et al., 1997). As a consequence, the efflux of K measured in the presence of Cs, as in the presence of Rb, should not be affected by a significant K reabsorption. Comparison of Tables 1 and 2 shows that the presence of Rb inhibited the influx of Cs into the root segments, but the overall influx of Rb plus Cs (6.26 µmol h 1 g 1 f. wt) observed when the two cations were present together in the incubation medium was similar to that observed when each cation was administered alone. By contrast, K efflux in the presence of Rb plus Cs was affected, being lower ( 45%) than the sum of the effluxes induced by Rb and Cs alone. In the presence of NH 4 plus Rb, the efflux of K was higher than that measured in NH 4 alone (Tables 1 and 2). In the former condition, the sum of the influxes of Rb and NH 4 (7.43 µmol h 1 g 1 f. wt) was slightly higher than the single values of the influxes measured in Rb or NH 4 alone (17% or 24%, respectively), whereas the K efflux was enhanced by 57% or 222%, respectively. These data suggest that, in the presence of NH 4 alone, the efflux of K was underestimated because of a K reabsorption component. These results suggest that the influx of monovalent cations into subapical maize root segments is accompanied by an efflux of K from the cells. Moreover, with the exception of Cs, there seems to be a relationship between the values of cation influxes and those of K effluxes. The involvement of ORCs in mediating K efflux is strongly suggested when: (1) the electrochemical gradient of K is outward directed (i.e. E K > E m ); (2) the efflux of K increases as E m increases; (3) the efflux of K is inhibited by specific K -channel blockers. In the experimental conditions used in this work, the external K concentration ranged from zero (at the beginning of incubation) to a maximum value of about 19 µm (after a 30- min period in 0.5 mm NH 4 plus 0.5 mm Rb ). In plant cells the cytosolic concentration of K typically ranges from 80 to 120 mm (Vorobiev, 1967; Maathuis & Sanders, 1993; Walker et al., 1995). It can thus be calculated that, under our conditions, E K was always more negative than 214 mv (i.e. in the case of NH 4 Rb, 59 mv log mm : 80 mm). Figure 1 shows the values of E m measured in cortical cells of maize root segments incubated with the different cations in the absence or in the presence of an equimolar concentration of Rb. In all the experimental conditions analysed, E m was less negative than 214 mv and thus we can conclude that the electrochemical gradient of K was always outward directed. The presence of 0.5 mm of either Li or Na in the incubation medium slightly depolarized the membrane of the root cortical cells (4 or 3 mv, respectively), according to their nil or very low uptake, whereas permeating cations induced significant depolarizations of the plasma membrane, with the following sequence: Rb (32 mv) > NH 4 (28 mv) > Cs (22 mv) (Fig. 1). For all the cations tested, the simultaneous presence of Rb induced further depolarization with respect to that induced by each cation alone. In the cases of Li and Na, the extent of depolarization was the same as that induced by 0.5 mm Rb alone (Fig. 1), suggesting that the observed effect on E m was Fig. 1 Effects of different cations on the transmembrane electric potential differences (E m ) of maize root subapical cells. Arrows indicate the addition to the basal medium of 0.5 mm of each cation, alone or plus an equimolar amount of Rb. The values are means of five experiments in quadruplicate; SE did not exceed ±1%. New Phytologist (2002) 154: 45 51

5 49 K efflux (µmol h 1 g 1 FW) Table 3 Effects of selective K channel blockers on K efflux and Rb influx in subapical maize root segments Inhibitor K efflux Rb influx Nil 2.46 ± ± 0.15 Tetraethylammoniumchloride 1.55 ± ± 0.10 Quinidine 1.15 ± ± 0.15 Fifteen root segments pretreated for 1 h with 10 mm tetraethylammonium-chloride or 1 mm quinidine were incubated in 10 ml of 0.5 mm CaSO 4, 0.1 mm MES-Ca (ph 6.0) plus 0.5 mm Rb. The values are the means ± SE of four experiments in triplicate Fig. 2 Relationship between membrane depolarization ( E m ) and K efflux in maize root subapical segments. mainly due to the Rb absorption component, which was not affected by the presence of Li or Na (Tables 1 and 2). The E m analysis indicated that permeating monovalent cations induced different extents of depolarization of the plasma membrane, which showed a strong relationship with the values of K effluxes from the root segments (Fig. 2). This relationship appeared to be closer than that existing between cation influxes and K effluxes and extended also to Cs. The low efflux of K measured in the presence of Cs, whose influx was essentially the same as that of Rb (Table 1), was probably due to the lower depolarising effect of Cs (Fig. 1). The electrochemical gradient for Rb and Cs is always inward directed and then potentially competent in driving the uptake of these nonphysiological ions through passive transport systems such as channels. However, the uptake of Cs, at 0.5 mm, into maize root segments involves the activity of the high-affinity K uptake system only and thus induces E m perturbations which are different than those induced by Rb, whose uptake is mediated by both high- and low-affinity systems (Sacchi et al., 1997). It has been proposed that the high-affinity K uptake is mediated by a H K symport mechanism. The in vivo activity of this system is less depolarizing than that of the low-affinity mechanism (Maathuis & Sanders, 1994), despite the fact that two positive charges are transported across the membrane per potassium ion taken up. This because the influx of H through the symporter is balanced by the activity of the dominant electrogenic H - ATPase (Rodriguez-Navarro et al., 1986). The addition of K, at micromolar concentrations, to K -starved roots largely elicits net H release into the medium (Kochian et al., 1989). We can exclude the possibility that the lower K efflux measured in the presence of Cs might be due to a direct inhibitory effect of this cation on transport systems involved in K efflux. The results reported in this work were obtained in short-time experiments (incubation time 30 min) at 0.5 mm external concentration. Considering the value of Cs influx, it is unlikely to be the case that the internal concentration of Cs might affect the activity of transport systems involved in K efflux. We can also rule out the hypothesis that the external Cs concentration might affect the same systems, since the addition of an equimolar amount of Rb induces further K release from the segments (Tables 1 and 2). This group of results allows us to suggest that, apart from a relationship between cation influx and membrane depolarization, a closer link may be established between the extent of the depolarization per se and the amount of K efflux (Fig. 2). The involvement of ORCs in mediating K efflux from the root segments was verified by using two specific K -channel blockers effective both in vivo and in vitro: TEA and quinidine (Maathuis & Sanders, 1995; Roberts & Tester, 1995; Murphy et al., 1999). As reported in Table 3, preincubation of root segments with the two chemicals inhibited the efflux of K without (or in the case of TEA, only slightly) affecting the influx of Rb. In particular, quinidine was more efficient than TEA in inhibiting K efflux ( 53% and 37%, respectively). The suggestion that ORCs are involved in mediating K efflux from subapical maize root segments seems to be supported by our results. This allows us to suggest that incubation in the presence of Rb is a suitable method to study the activity of ORCs in vivo and to estimate the values of K efflux from plant materials. When expressed on the basis of surface unit (diameter of root segments about 1 mm), the effluxes of K, evaluated in our experimental conditions ranged from 60 nmol m 2 s 1 (Cs alone) to 220 nmol m 2 s 1 (Rb plus NH 4 ). These values are consistent with those determined in vivo in different plant materials by a noninvasive ion-selective microelectrode technique (Shabala, 2000). New Phytologist (2002) 154:

6 50 E m (mv) Nil TEA Quinidine Fig. 3 Effects of 1-h pretreatment with 10 mm tetraethylammonium-chloride (TEA ) or 1 mm quinidine on Rb -induced shifts in membrane potential (E m ) in maize root subapical cells. The values are the means ± SE of three experiments in quadruplicate. Some histological and functional considerations should be taken into account concerning the physiological role of the ORC activities detected under our experimental conditions. The root portion used in our experiments corresponds to the elongation zone of the primary root of maize seedlings, which is constituted by cells that are programmed to yield the mature root architecture. As a consequence, K efflux is probably the result of the combined activity of ORCs localized in the plasma membrane of undifferentiated cells that will differentiate into root tissues such as epidermis and cortical or xylem parenchyma, where these channels play different physiological roles (Wegner & Raschke, 1994; Maathuis & Sanders, 1995). However, the elongation zone is a root portion where the uptake of mineral nutrients takes place to a large extent. Absorption of nutrient cations such as NH 4 and K is an electrogenic, and thus potentially depolarizing, process. In the same way, nutrient anions, such as NO 3, SO 4 and H 2 PO 4, are absorbed by depolarizing mechanisms involving electrogenic H anion symporters (Chrispeels et al., 1999). Net efflux of K has also been described in the root hairs of A. thaliana, as a response to membrane depolarization induced by high H influx into the cells (Babourina et al., 2001). The K efflux from the cells of the root elongation zone might have a role in the restoration and stabilization of E m during the uptake of nutrient ions. This hypothesis is consistent with the data reported in Fig. 3. The presence of Rb in the incubation medium induced a higher membrane depolarization in maize root segments pretreated with TEA or quinidine (about 10 mv) than in the controls. Treatment with either TEA or quinidine did not affect the H -extrusion activity of root segments (data not shown) thereby excluding an effect of these compounds on the active component of E m. In conclusion, our results suggest that, in subapical cells of maize root, membrane depolarization induces an ORCmediated K efflux that can be considered, as in animal cells (Hille, 1992), an energy buffer able to restore and stabilize E m after the depolarizing events linked to cation uptake. The ORC-mediated K efflux may be detected in vivo by incubating the roots in the presence of Rb. 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