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Annals of Botany 85: 681±686, 2000 doi:10.1006/anbo.2000.1131, available online at http://www.idealibrary.com on Salinity Eects on the Activity of Plasma Membrane H and Ca2 Transporters in Bean Leaf Mesophyll: Masking Role of the Cell Wall S E R G E Y SH A B A L A *{ and IA N N E W M A N { {School of Agricultural Science, University of Tasmania, Australia and {School of Mathematics and Physics, University of Tasmania, Australia Received: 15 November 1999 Returned for revision: 6 January 2000 Accepted: 25 January 2000 Net ¯uxes of H and Ca2 were measured in the mesophyll tissue of broad bean (Vicia faba L.) leaves and in protoplasts derived from these cells. NaCl at 90 mM enhanced H extrusion in both protoplasts and tissue, but in dierent ways. Proton extrusion was inhibited by vanadate, suggesting the involvement of the plasma membrane H ATPase in cell responses to salinity. There was virtually no eect of NaCl on the net Ca2 ¯ux in protoplasts, while in the tissue a large transient Ca2 eux followed the salt treatment. Salt-induced Ca2 eux was essentially independent of external Ca2 concentrations in the range 0.1 to 10 mM. Also, Ca2 ¯ux responses were `saturated' above 50 mM NaCl. It is suggested that almost all the measured Ca2 ¯ux originates from Na /Ca2 and H /Ca2 ion exchange in the cell wall. This conclusion was supported by the results of modelling cation exchange in the cell # 2000 Annals of Botany Company wall. Key words: Salinity, membrane transporters, wall ion exchange, proton, calcium, Vicia faba. I N T RO D U C T I O N There is much controversy over the way high levels of external NaCl aect plasma membrane Ca2 and H transporters. Some authors have reported rapid elevation of cytosolic free calcium, [Ca2 ]cyt , in response to NaCl treatment (Bittisnich et al., 1989; Lynch et al., 1989; Okazaki et al., 1996), although others attribute these data to methodological drawbacks of dye loading (Cramer and Jones, 1996). The origin of these [Ca2 ]cyt changes is not clear. Both Ca2 transport through the plasma membrane and Ca2 release from intracellular stores have been suggested as possible sources (Cramer et al., 1985, 1987; Lynch et al., 1987; Rengel, 1992). None of the techniques used so far has included direct measurements of ¯uxes through the plasma membrane, so an unambiguous conclusion has not been possible. The problem is further complicated by the possibility that high external Na may exchange with Ca2 in the cell wall. This exchange may confound observations of salinity eects on the activity of plasma membrane Ca2 transporters (Zidan et al., 1991). There are also reports suggesting that NaCl stimulates plasma membrane H -ATPase activity (Nakamura et al., 1992; Ayala et al., 1996; Vera-Estrella et al., 1999), which therefore may enhance H extrusion through the plasma membrane. Thus, as well as the above Na /Ca2 interaction, the extruded H could exchange with cell wall Ca2 (Arif and Newman, 1993). To our knowledge, however, there have been no reports of direct H ¯ux measurements under NaCl application to date. Does NaCl really activate * For correspondence. Fax 613 62262642, e-mail Sergey.Shabala@ utas.edu.au 0305-7364/00/050681+06 $35.00/00 H extrusion, and to what extent does Ca2 /H wall exchange mask plasma membrane H ¯ux kinetics? To answer these questions, a comparison was made between NaCl eects on the net ¯uxes of H and Ca2 measured from bean leaf mesophyll and from protoplasts derived from these cells using our high-resolution noninvasive microelectrode ion ¯ux measurement (MIFE) technique. M AT E R I A L S A N D M E T H O D S Net ¯uxes of H and Ca2 from the mesophyll tissue of broad bean (Vicia faba L. `Coles Dwarf'; Cresswell's Seeds, New Norfolk, Australia) leaves and from protoplasts derived from mesophyll cells were measured non-invasively using ion-selective vibrating microelectrodes. Plants were grown essentially as described by Shabala and Newman (1999). Seven to 10 d old leaves were harvested and the lower epidermis was peeled o. Leaf segments, 5 8 mm, were cut and ¯oated ( peeled-side down) on the experimental solution (unbuered 0.1 mM CaCl2 1.0 mM KCl) as described previously (Shabala and Newman, 1999). The cut segment was mounted in a Perspex holder that provided gentle bending of the plant tissue (to allow a clear view for electrode positioning; see Shabala and Newman, 1999 for details). Flux measurements started 4±5 h after segments were cut, to avoid wounding eects. The protoplast isolation procedure was modi®ed from Lin and Ehleringer (1997). About 200 mg of peeled leaf tissue was incubated in the enzyme medium (1% cellulase, 0.1% macerozyme, 0.1 mM CaCl2 , 1.0 mM KCl, 0.4 M mannitol) for 2.5 h. The protoplast suspension was ®ltered, centrifuged at 300 rpm for 6 min, resuspended twice, and # 2000 Annals of Botany Company Shabala and NewmanÐSalinity and Cell Wall Exchange R E S U LT S A N D D I S C U S S I O N H ¯ux kinetics In both mesophyll cells and protoplasts, 90 mM NaCl treatment caused an immediate shift in net H ¯ux towards eux (Fig. 1A). For protoplasts, this newly established H ¯ux value was relatively stable over the period of measurements, while for the mesophyll tissue there was a drift towards larger eux, becoming steady after about 50 min. The magnitude of NaCl-induced H eux ( per unit surface area) at the end of the 60 min interval was four±®ve times larger for mesophyll tissue than for protoplasts (Fig. 1A). One reason for this dierence is the dierent geometry of a single protoplast surface compared with a tissue surface for which several cells contribute to the external ¯ux. However, Net H+ flux (nmol m−2 s−1) 40 0 −40 −80 Protoplast 90 mM NaCI −120 A Tissue −160 −200 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min) 100 Net Ca2+ flux (nmol m−2 s−1) then measured in enzyme-free bathing solution (0.1 mM CaCl2 , 1.0 mM KCl, 0.4 M mannitol). A similar bath composition (except mannitol) was used in experiments with mesophyll tissue. In some experiments, Ca2 composition in the bath varied between 0.1 and 10 mM (added as CaCl2 salt); K concentration in the bath remained constant (1.0 mM). NaCl treatment was given as 1M stock added into the 5 ml chamber and mixed to produce the required ®nal concentration of NaCl. In experiments with protoplasts, the bathing solution was replaced by one additionally containing 90 mM NaCl. The period of time required for solution changes was omitted from the analysis and appears as a gap in our ®gures. Speci®c details of microelectrode ion ¯ux measurements using the MIFE2 system (University of Tasmania, Hobart, Australia) have been given previously (Shabala et al., 1997, 1998; Shabala and Newman, 1999). Brie¯y, pulled and silanized electrode blanks, with external tip diameter of 3± 4 mm, were ®lled with commercially available H Ð(95297) or Ca2 Ð(21048) ionophore cocktails (Fluka Chemie AG, Buchs, Switzerland). Back ®lling solutions were 500 mM CaCl2 for calcium, and 15 mM NaCl plus 40 mM KH2PO4 ( pH adjusted to 6.0 using NaOH) for H . Electrodes were calibrated in a known set of standards (calcium from 50 to 500 mM; pH from 4.5 to 8) twiceÐwithout NaCl (used to calculate net ion ¯uxes before salt treatment), and with 90 mM NaCl present in each standard (used to calculate ¯uxes after salt was applied). In both types of standards, the average electrode slopes were 53±54 mV/pH and 27±28 mV/pCa. If a range of NaCl concentrations was used (Fig. 3), an appropriate correction was made to compensate for the changed Ca2 activity due to the changed ionic strength of solution. Electrodes were moved in a slow (10 s cycle) square-wave by a computer-driven hydraulic micromanipulator between two positions, close to (10 mm) and distant from (60 mm) the surface. Net ion ¯uxes were calculated using the MIFE software as described previously (Shabala et al., 1997) from the measured dierence in electrochemical potential for these ions between the two positions using the cylindrical (mesophyll segments) or spherical ( protoplasts) diusion geometry. Signi®cance of dierence between means was based on the use of the Student's t-test. Protoplast 0 −100 −200 90 mM NaCI −300 Tissue B −400 0 3 6 9 12 15 −500 −600 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min) Net H+ flux 682 90 mM NaCl 500 µM vanadate 0 5 10 15 20 C 25 30 35 30 nmol m−2 s−1 40 45 50 55 60 Time (min) F I G . 1. Transient changes in the net H (A) and Ca2 (B) ¯uxes in response to 90 mM NaCl treatment. (s), Mesophyll tissue; (d), protoplasts. Each point represents average ¯ux over a 30 s interval. Error bars are + s.e.m. (n 6). The insert in B shows initial changes (at 5 s intervals) in the net Ca2 ¯ux from one representative plant. C, Net H ¯uxes from a mesophyll cell protoplast after 500 mM vanadate pretreatment. Vanadate was added as Na2VO4 stock solution at 5 min; salt treatment was given at 28 min. it is also likely that the protoplast isolation procedure per se aected quantitative characteristics of the measured H ¯uxes. There is a large body of literature reporting that plasma membrane characteristics of isolated protoplasts are very dierent from those of intact cells (Racusen et al., 1977; Pantoja and Wilmer, 1986; Henriksen et al., 1996). The most striking is a dierence in membrane potential (MP). While typical MP values for higher plant cells are found to be in the range ÿ100 to ÿ140 mV, for isolated protoplasts this value is usually close to zero, or even positive (Racusen et al., 1977). Based on the fact that electrical gradients at the plasma membrane are normally Shabala and NewmanÐSalinity and Cell Wall Exchange maintained by the activity of the electrogenic H pump, H extrusion may be diminished in protoplasts following the isolation procedure. If this is the case, NaCl-induced activation of H -extrusion also may be diminished in protoplasts (Fig. 1A). Another point to remember is a strong dependence of protoplast electrophysiological characteristics on external osmoticum (Pantoja and Willmer, 1986; Shabala et al., 1998). As the choice of osmolality of the bath medium is usually empirically-based, even slight deviation from the `optimum' could cause mechanical tension on the plasma membrane and therefore modify activity of membrane transporters (Ding and Pickard, 1993). As already mentioned in the introduction, there are many reports suggesting that NaCl could stimulate H -ATPase activity at the plasma membrane of plant cells (Nakamura et al., 1992; Ayala et al., 1996; Vera-Estrella et al., 1999). To test if such H -pump activity is responsible for the saltinduced eux of H observed in our experiments, 500 mM vanadate (a known inhibitor of plasma membrane H -ATPase; Lew and Spanswick, 1984) was added to the protoplast bathing medium. As expected, such vanadate treatment resulted in suppression of H pumping and caused a signi®cant shift towards net H in¯ux (Fig. 1C). What is even more important, vanadate treatment also prevented NaCl-induced H extrusion (compare Fig. 1A and C). These results suggest that the ATP-dependent H -pump (inhibited by vanadate) forms a signi®cant part of the H extrusion mechanism involved in response to salt stress. Its level of activity is about ÿ50 nmol m ÿ2 s ÿ1 for the protoplast membrane, and about ÿ170 nmol m ÿ2 s ÿ1 for mesophyll tissue surface. Initially this mesophyll H ¯ux exchanges in the cell wall for Ca2 , as discussed below, stabilizing only after that exchange is completed. Ca2 responses Even more striking was the dierence between tissue and protoplast Ca2 ¯ux responses (Fig. 1B); moreover, the observed dierence was qualitative, not only quantitative (as in case of the H ¯ux). While mesophyll tissue responded to the NaCl treatment by an immediate large net Ca2 eux, with the peak Ca2 eux at 2±4 min after NaCl treatment started (see insert to Fig. 1B), there were virtually no changes in the net Ca2 ¯ux measured from the protoplast surface. These data suggest that the presence of the cell wall was crucial for the measured NaCl-induced Ca2 ¯uxes at the tissue level. This is a strong indication that observed Ca2 eux originated from the cation/ calcium exchange in the cell wall, but not from the activity of plasma membrane Ca2 transporters per se. To verify this hypothesis, mesophyll tissue was treated with 100 mM La3 , a known inhibitor of plasma membrane Ca2 channels. It is known that Ca2 in¯ux into the cell is mediated by Ca2 -permeable ion channels which facilitate the rapid movement of Ca2 down its electrochemical gradient (White, 1998), while Ca2 movement out of the cell requires active transport mechanisms such as a Ca2 pump. Therefore, the net Ca2 ¯ux measured in our experiments represents a balance between these two oppositely directed processes. 100 µM La3+ 90 mM NaCl A Net Ca2+ flux 0 683 10 20 200 nmol m−2 s−1 30 40 50 60 70 80 90 Time (min) B 90 mN KCl Net Ca2+ flux 200 nmol m−2 s−1 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min) F I G . 2. A, Eect of 100 mM La3 on net Ca2 ¯ux from bean mesophyll tissue. La3 was added as LaCl3 stock solution at 5 min; 90 mM NaCl treatment was given at 30 min. B, KCl-induced kinetics of net Ca2 ¯ux from bean mesophyll tissue. One representative example out of four is shown. When Ca2 channels were blocked by La3 in our experiments (Fig. 2A), a signi®cant shift towards net Ca2 eux (up to ÿ30 nmol m ÿ2 s ÿ1 30 min after La3 treatment was given) was observed, in full agreement with the above statement. However, blocking the Ca2 channels with La3 had little eect on the NaCl-induced Ca2 ¯ux transient (Fig. 2A). Neither the qualitative course of Ca2 ¯ux responses, nor the magnitude of the peak Ca2 eux were signi®cantly dierent from the control (at the P 0.05 level). Therefore, we conclude that most of the net Ca2 eux measured from NaCl-treated bean mesophyll tissue originated from the cell wall and not from the activity of plasma membrane Ca2 transporters. Hence, we suggest that NaCl-induced changes in cytosolic free Ca2 reported elsewhere (Okazaki et al., 1996; Cramer and Jones, 1996) are likely to have originated from Ca2 released from internal stores. This conclusion is in good agreement with other reports where indirect methods were employed to evaluate the origin of [Ca2 ]cyt changes (Lynch et al., 1987). Further evidence supporting a cell wall origin of the measured Ca2 eux came from experiments when dierent NaCl concentrations were used to induce ¯ux responses from the mesophyll tissue (Fig. 3). No signi®cant dierence was found in the magnitude of the Ca2 ¯ux responses to a wide range of NaCl concentrations ( from 50 to 120 mM; Fig. 3). A possible explanation for such `saturation' kinetics could be that at concentrations above 50 mM most of the exchangeable cell wall Ca2 has been replaced by Na and H ions, and further increase in 684 Shabala and NewmanÐSalinity and Cell Wall Exchange 100 100 0 0 Net Ca2+ flux (nmol m−2 s−1) −100 −100 20 mM −200 −200 −300 −300 −400 −400 −500 −500 −600 −600 −700 50 mM −700 3 5 7 9 11 13 15 3 100 100 0 0 −100 −100 90 mM −200 5 −300 −400 −400 −500 −500 −600 −600 −700 9 11 13 15 150 mM −200 −300 7 −700 3 5 7 9 11 13 15 3 5 7 9 11 13 15 Time (min) F I G . 3. Transient changes in the net Ca2 ¯uxes in response to dierent NaCl treatment in bean mesophyll segments. Average data from ®ve to seven individual segments are shown for each variant. Each point represents average ¯ux over a 5 s interval. Error bars are + s.e.m. external Na concentration had little eect on this process. If plasma membrane transporters were involved, a rising dose-dependent response would be expected. Also small was the eect of variation in the external Ca2 concentrations on the NaCl-induced Ca2 eux. Figure 4 shows typical records obtained for three dierent Ca2 concentrations in the bath (0.1 mM, 1 mM and 10 mM, respectively). No signi®cant dierence was found at the P 0.05 level, indicating that activity of plasma membrane (PM) Ca2 transporters contributed insigni®cantly to the observed kinetics of the net Ca2 ¯uxes in response to salt treatment. The peak value of the Ca2 eux was roughly ÿ570 nmol m ÿ2 s ÿ1 when 90 mM NaCl was applied (Fig. 1B). The steady level of H eux at the end of the transient (when H exchange with wall Ca2 was completed) was only ÿ170 nmol m ÿ2 s ÿ1. As two protons exchange for one Ca2 in the cell wall, only 15% of the observed peak Ca2 eux can be explained by extruded H exchanging for wall Ca2 . The rest of the Ca2 ¯ux due to NaCl treatment must be attributed to displacement of wall Ca2 by sodium ions. This Ca2 displacement from the cell wall is not speci®c to the Na ion. We have found that 90 mM NaCl 0.1 mM 1.0 mM 10 mM 200 nmol m−2 s−1 0 10 20 30 40 50 60 Time (min) F I G . 4. Transient Ca2 ¯ux responses to 90 mM NaCl treatment for dierent Ca2 concentrations in the bath. One representative example (out of ®ve) is given for each variant. Each point represents average ¯ux over a 30 s interval. (s), 0.1 mM Ca2 in the bath; (d), 1.0 mM Ca2 ; (h), 10 mM Ca2 . 90 mM KCl induces Ca2 eux very similar to that caused by 90 mM NaCl (Fig. 2B). Shabala and NewmanÐSalinity and Cell Wall Exchange 685 T A B L E 1. Comparison between WADM modelled ion concentrations in the cell wall, amount of the wall Ca2 exchanged due to solution change, and the observed net Ca2 eux measured in experiments with dierent concentrations of NaCl applied to the leaf mesophyll (Fig. 3) External Na concentration, mM Parameter 0 Donnan potential of the wall, VW (mV) Wall pH Wall Na concentration (mM) Wall Ca2 concentration (mM) Amount of Ca2 exchanged (mM) Ca2 ¯ux changes* 2 min after salt stress (nmol m ÿ2 s ÿ1) ÿ95 3.87 0 329 N/A N/A 20 50 90 120 ÿ76 4.19 435 148 182 ÿ500 ÿ59 4.48 534 117 212 ÿ550 ÿ47 4.69 563 114 215 ÿ570 ÿ37 4.86 588 114 215 ÿ580 Parameters for the model and external ion concentrations are given in the text. *Measured experimentally as shown in Fig. 3. Modelling of wall ion exchange Further support for the cell wall origin of the measured Ca2 ¯ux induced by NaCl treatment comes from modelling the Na -induced exchange of ions in the cell wall using the Weak Acid Donnan Manning (WADM) model described by Ryan et al. (1992). This model incorporates three main components. (1) The pH-dependent dissociation of weak acids (of assumed pK) ®xed in the wall, described by the Henderson-Hasselbalch equation: Adiss 10 pHW ÿpK ; Asites 1 10 pHW ÿpK where Asites HA Adiss is the total concentration of ionizable sites in the wall at pH pHW . We assumed Asites 800 mM, and pK 3 for this study (Arif and Newman, 1993). (2) The classical Donnan equilibrium between the wall (with concentration of available ®xed charges A) and the external solution, described by the Donnan potential VW of the wall, which relates the wall and external concentrations of each ion. If the concentration (strictly activity) ratio, wall/external, of an ion of valency z is x, then VW RT=zF1n x (3) The special character of divalent Ca2 in binding to available weak acid anions is called `condensation' in the WADM model. The Ca2 which binds thereby reduces the anions available for the Donnan equilibrium from Adiss to A, according to the equation A Adiss (0.5/x), where x is a linear charge density parameter (see Richter and Dainty, 1990, who found a value of 0.7 for x). Under our conditions the overall eect of this binding is 520% of the calculated results and does not aect their qualitative validity. The above equations, with overall charge balance and use of activity coecients with ion concentrations, were solved iteratively to determine the equilibrium concentrations of all ions in the cell wall. This was done for the pre-treatment solutions (0.1 mM CaCl2 ; 1 mM KCl; pH 5.5) and for each solution having increased Na concentration ( from 20 to 120 mM). Some results of this modelling are shown in Table 1. Application of non zero external Na concentrations caused a signi®cant increase in the cell wall pH (up to 1.0 pH for 120 mM NaCl; Table 1) and a dramatic threefold decrease in the wall Ca2 concentrations. However, the amount of exchanged Ca2 plateaued for external Na concentrations larger than 50 mM. The model predicts, therefore, that the amount of Ca2 exchanged in the cell wall for two dierent NaCl concentrations, 50 and 120 mM, will not be signi®cantly dierent. Being displaced from the cell wall, this exchangeable Ca2 will be measured as net Ca2 ¯ux by the microelectrode technique. Therefore, if no PM transporters contribute to the Ca2 ¯ux, no dierence between net Ca2 ¯uxes for 50 mM and 120 mM NaCl treatments is expected. This is exactly what was found in our experiments (Fig. 3; Table 1). Physiological implications An important conclusion from these observations is that NaCl-induced changes in cytosolic free Ca2 reported elsewhere (Cramer and Jones, 1996; Okazaki et al., 1996) are likely to have originated from Ca2 released from internal stores, but not from the activity of the PM Ca2 transporters. Further study should be aimed at elucidating the signalling pathways involved in this process. Also, displacement of Ca2 from the cell wall not only confused assessment of the activity of plasma membrane Ca2 transporters in the mesophyll cells, but also masked the actual magnitude and timing of Na -stimulated H ¯ux from the tissue. Only when Ca2 transients are complete (40±50 min after the start of NaCl treatment), does net H eux represent the actual H ¯ux through the plasma membrane. The cell wall ionic exchange, which can be quantitatively modelled, must be kept in mind when mechanisms of salt stress perception are studied. 686 Shabala and NewmanÐSalinity and Cell Wall Exchange AC K N OW L E D G E M E N T S This work was supported by the Australian Research Council grants to Dr Sergey Shabala and Dr Ian Newman. L I T E R AT U R E C I T E D Arif I, Newman IA. 1993. Proton eux from oat coleoptile cells and exchange with wall calcium after IAA or fusicoccin treatment. Planta 189: 377±383. Ayala F, O'Leary JW, Schumaker KS. 1996. Increased vacuolar and plasma membrane H -ATPase activities in Salicornia bigelovii Torr. in response to NaCl. Journal of Experimental Botany 47: 25±32. Bittisnich D, Robinson D, Whitecross M. 1989. Membrane-associated and intracellular free calcium levels in root cells under NaCl stress. In: Dainty J et al., ed. Plant membrane transport: the current position. Amsterdam: Elsevier, 681±682. Cramer GR, Jones RL. 1996. 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