The mechanism of zinc uptake in plants

Transcription

1 Planta (1996)198:39-45 P l m a t ~ 9 Springer-Verlag 1996 The mechanism of zinc uptake in plants Characterisation of the low-affinity system Robert J. Reid 1, Justin D. Brookes 1, Mark A. Tester 2, F. Andrew Smith 1 t Botany Department, University of Adelaide, Adelaide, S.A. 55, Australia 2 Department of Plant Sciences, University of Cambridge, Downing St., Cambridge CB2 3EA, UK Received: 6 March 1995/Accepted: 24 April 1995 Abstract. The mechanism of zinc influx was investigated using giant algal cells (Chara corallina Klein ex Will.esk. R.D. Wood), in which it was possible to discriminate clearly between tracer zinc bound in the cell wall and actual uptake into the cell. t was shown that despite lengthy desorption, retention of zinc in slowly exchanging zinc pools in the cell wall can invalidate tracer influx measurements. A comparative study of zinc desorption from isolated cell walls of wheat (Triticum aestivum L.) roots indicated exchange characteristics similar to that of Chara. Fractionation of Chara internodal cells taken directly from cultures showed that most of the cell-associated zinc was in the cell walls. The cytoplasmic and vacuolar zinc concentrations were 56mmol.m -3 and 32 mmol- m- 3, respectively, for cells grown in a zinc concentration of.1 mmol" m-3. nflux of 65Zn in Chara was linear over several hours, with rapid transfer to the vacuole, but only slow efflux. nflux occurred in a biphasic manner, which was tentatively attributed to the operation of two separate transport systems, a high-affinity system which is saturated at.1 mmol.m -3 and a low-affinity system which showed a linear dependence on concentration up to at least 5 mmol. m -3. Only the low-affinity system was examined in detail. nflux through this system showed a strong dependence on external ph with an optimum around 7 and was also stimulated by cytoplasmic acidification. nflux was sensitive to metabolic inhibition, but not to blockers of Ca 2 + and K channels. Other characteristics included a slight sensitivity to Mn 2 + and Fe 2 + but little sensitivity to high concentrations of K or Na +. nflux was independent of membrane potential difference in cells voltage-clamped at - 65 to - 3 mv. Abbreviations: APW = artificial pond water; CCCP = carbonylcyanide m-chlorophenylhydrazone; GFAAS =graphite furnace atomic absorption spectrometry; PD = potential difference; TEA = tetraethylammonium Correspondence to: R.J. Reid; FAX: 61 (8) ; Tel.: 61 (8)33 529, rreid@botany.adelaide.edu.au Key words: Chara - Cell wall and zinc - Micronutrient - Triticum - Zinc uptake ntroduction Despite the importance of zinc as a micronutrient for plant growth, there have been relatively few studies of the mechanism of zinc uptake. t is clear from a recent review of the literature by Kochian (1993) that there is currently little agreement on how zinc crosses the plasmalemma of plant cells. The questions which have not been satisfactorily answered are whether zinc enters via ion channels or via a divalent-cation carrier, the link between uptake and metabolic energy transduction, the existence of an active efflux mechanism, whether fluxes can be described by Michaelis-Menten kinetics with Vm and K... and the possible involvement of phytosiderophores. Most studies of zinc uptake have used solutioncultured roots and radioactive 65Zn either to measure short-term influxes or to estimate fluxes from compartmental analysis of efflux kinetics of tissues equilibrated in 65Zn (Schmid et al. 1965; Chaudry and Loneragan 1972; Santa Maria and Cogliatti 1988). However, it is difficult to assess the reliability of the tracer flux measurements because in none of these studies has a clear distinction been made between extracellular binding and actual membrane influx. n complex tissues such as roots it is difficult to do so, although implicit faith in the effectiveness of desorption treatments to remove extracellularly bound radiotracer seems to be widespread in studies of divalent- and trivalent-cation uptake. Our own experiences with charophyte cells, in which it is indeed possible to assess the effectiveness of desorption (see Reid and Smith 1992a,b; Reid et al. 1993), have led us to question the validity of the application of such practices to short-term uptake experiments with more complex tissues. We demonstrate here that the characteristics of cation binding in cell walls of Chara are not fundamentally different from apoplasmic binding in cereal roots, and that by adopting the same techniques used to measure Ca 2 +

2 4 R.J. Reid et al.: Zinc influx in Chara influx in Chara, the general properties of membrane fluxes of zinc can be described. Materials and methods Plant material. The giant-celled alga Chara corauina Klein ex Will. esk. R.D. Wood was grown in the laboratory in large plastic tanks in tap water on a substrate of garden soil. The cultures were illuminated on a 16 h/8 h light/dark cycle at a photon flux of approximately 35 ~tmol. m-2. s- 1 at the surface of the solution. Before experiments, individual internodal cells (4-9 mm long and approximately 1 mm in diameter) were isolated from the plant and stored overnight in an artificial pond water (APW) containing 1 mol" m -3 NaC1,.1 mol.m -3 K2SO4 and.5mol'm -3 CaC2. Unless otherwise stated, experimental solutions were buffered at ph 6.2 with 1 mol.m-3 2-N-morpholino)ethanesulfonic acid (Mes). Wheat (Triticum aestivum L. cv. Halberd) was grown on a 1% Hoagland's solution for 1 d. Flux measurements. nflux of zinc was measured with 65Zn using the methods described in Reid and Smith (1992a, b) for 4SCa. The advantage of using charophyte internodal cells lies in their large size and cylindrical shape which enables easy separation of cell wall from intracellular contents after the radioactive influx period. Two different techniques were used, one to obtain short-term influxes and a second to measure longer-term accumulation of zinc. n the latter method, individual internodal cells were incubated in 6~Zn-APW in a Petri dish (1-12 cells/2ml), then desorbed for 3min in 1 tool-m-3 LaC13 in APW, the solution being changed six times during this period. The cells were then blotted, allowed to wilt slightly, the ends were excised and a hypodermic needle inserted into one end. The vacuole was expelled by gently injecting an air bubble through the cell, and the cytoplasm was then flushed out by rapidly injecting 1 ml of deionised water, leaving a clear sleeve of cell wall, whose 6SZn activity could be measured separately from that in the cytoplasmic and vacuolar fractions. For the measurement of short-term (i.e. unidirectional) zinc influx, short rinse times were used to reduce the possibility of efflux of 65Zn during the desorption of the cell wall. The increased risk of contamination of the cell contents during the separation of the cell wall was overcome by mounting the cell in a threecompartment chamber in which only the centre compartment contained 65Zn. The ends of the cells remained unlabelled and there was therefore much less likelihood of contamination during the surgical procedures required for separating the cell walls from the cell contents. nflux of 45Ca was measured using the same techniques as for zinc. nflux of 42K was measured using normal tracer techniques with several 1-min rinses to remove extracellular activity. Large volumes of influx solution were needed to prevent depletion of 65Zn by adsorption to cell walls. nflux solutions were gently agitated and cells were illuminated with dim light (approx. 35 ktmol" m-2-s- 1). Electrophysiology. The electrical potential difference (PD) between the vacuole and the outside solution was varied by an external voltage-clamp technique. ndividual internodal cells were mounted in a chamber in which the centre portion of the cell was isolated externally from the two end portions with silicone grease. A KC1- filled glass micropipette was inserted into the vacuole to measure the PD, which was then controlled by passing hyperpolarising or depolarising current between external Ag/AgC1 electrodes located in the centre compartment and one end compartment. When a stable PD was obtained, 65Zn was added to the solution flowing through the centre compartment. After 2 rain the 65Zn was rinsed away and the influx was determined using the cell-fractionation techniques described above. Action potentials were generated by applying a voltage pulse between the Ag/AgC1 electrodes of approximately 3 V for.2 s. Preparation of cell walls. solated cell wall sleeves were obtained by removing the ends of internodal cells and injecting deionised water through the lumen. This process appeared to be quite efficient in removing the cell contents since the chloroplasts, which are known to be embedded in a cytoplasmic gel adjacent to the plasma membrane, were completely displaced. Cell wall material was obtained from wheat roots by boiling 5-mm apical root segments for 2 min in deionised water. Washed and dried cell wall material was incubated in 1 mmol'm -3 65Zn in APW for 2 h then desorbed in a solution containing 1 mol.m -3 LaC13 in APW. The rinse solutions were periodically changed and sampled to determine the 65Zn desorbed in each rinse period. nitial binding and the time-course of desorption were calculated by summing the 65Zn remaining in the tissue after 6 min and the preceding rinses. Measurement of zinc content. The zinc concentration in culture solution, cells and cell walls was measured by graphite furnace atomic absorption spectrometry (GFAAS) after extraction in 1 tool" m- 3 nitric acid. Zinc speciation in solution was calculated using GEOCHEM-PC (Parker et al. 1994). Unless specified otherwise, zinc concentrations given in the text refer to the sum of all zinc species in solution. Results are shown as the mean + SE. Results Apoplasmic binding of 65Zn. The major impediment to measurement of uptake of polyvalent cations lies in their extensive extracellular binding which obscures membrane fluxes. n order to demonstrate that binding characteristics in the freshwater alga Chara were not fundamentally different from those of higher plants, a comparative study was made of the binding and desorption of zinc in cell walls of Chara and wheat roots. n general terms these parameters were similar for both species although, on a dry-weight basis, Chara had a higher binding capacity of ~tmol' g- 1 compared to ~tmol. g- 1 in wheat. This latter value may overestimate the shortterm binding in intact roots since the boiling process would have destroyed the endodermis, thereby opening up cell wall binding sites which are not normally exposed to the external solution. Desorption of zinc from Chara was more rapid. Figure 1 shows the time-course from 5-6 min. Although the initial rinses between and 5 min removed between 8 and 9% of the original 65Zn activity, the remaining fraction exchanged much more slowly so that after 6 min a significant amount (1% for Chara and 2% for wheat) of the 65Zn remained bound. Separation of symplasmic and apoplasmic zinc. A moredetailed analysis of desorption of 65Zn from cell walls and the implications for measuring membrane fluxes was made using intact cells of Chara, which were pre-incubated in APW + 1 mmol.m -3 65Zn for 2h. The effectiveness of desorption was dependent on the availability of an exchangeable cation; prolonged rinsing in deionised water released very little of the bound 65Zn (Fig. 2a). Desorption with non-radioactive APW + 1 mmol. m- 3 ZnC12 occurred with a half-time of approximately 2 min while 1 mol. m- 3 LaC13 in APW displaced approximately 99.5% of bound 65Zn in 1 min (Fig. 2b). Separation of cell wall and cell contents showed that despite the apparent efficiency of the desorption with

3 R.J. Reid et al.: Zinc influx in Chara i i i i i i i i Rinse time (rain) Fig. 1. Desorption of isolated cell walls of Chara and wheat following incubation for 2 h in 1 mmol.m -3 6SZn. Desorption solution = 1 mol.m-3 LaC13 in APW (unbuffered) 4-" "d 25 2O 1 "~ 2.5 ~ O (a) Vacuole/cytoplasm i i i, p Rinse time (h) Fig. 2a, b. Desorption of internodal cells of Chara in deionised water, 1 mmol-m-3 ZnCz APW or tool.m-3 La 3 + in APW (a), and effect of rinse time in 1 mol. m- 3 La 3 + in APW on the relative distribution of 65Zn between cell wall and cell contents, of cells incubated for 2 h in 1 mmol' m- a 65Zn (b). Note the scale expansion in b La 3+, most of the cell-associated radioactivity was still bound in the cell wall (Fig. 2b). This residual cell wall activity exchanged only slowly, while the intracellular component was relatively unaffected by rinsing for longer than 2 min.,6.~.5.4 ~.3 ~ o Time a) Fig. 3. Time-course of 65Zn influx in Chara. nflux solution = APW ph mmol" m-3 ZnC12. Rinse t = 3 min. n = 1-12 cells Table 1. nflux of 65Zn to the cytoplasm and vacuole of internodal cells of Chara corallina using two different influx methods. External concentration = 1 mmol. m-3 ZnC12 in APW. Experiment 1 was conducted using segment loading with a rinse time of 5 min. Experiment 2 was conducted using whole-cell loading with a rinse time of.5h nflux time (h) Expt. 1 65Zn influx (pmol- m- 2. s- a) 25 Cell Cytoplasm Vacuole _ Expt _ _ Uptake and compartmentation of zinc. At a concentration of 1 mmol. m-a, uptake of zinc into single internodal cells of Chara was linear for at least 4 h with a slowing evident after 22 h (Fig. 3). There may also have been a short lag in influx, caused possibly by the initial equilibration of 65Zn in the cell wall. These measurements were made using a 3-min desorption period and the possibility thus existed for loss of intracellular activity during rinsing, thereby underestimating the plasmalemma influx. Reduction in the rinse time to 5 min without increasing contamination from the high 65Zn in the cell wall was achieved by exposing only part of the cell to the radioactive solution using a three-compartment chamber (for full details of the technique, see Reid and Smith 1992a). This showed that there was little loss of influxed 65Zn during the longer rinses and confirmed that influx was linear over at least several hours (Table 1). Actual influx varied markedly between cultures, but this variability appeared not to be due to differences in zinc concentrations in the cultures, since exposure of isolated plants to 1 mmol. m- a zinc in APW for 5 d did not affect their subsequent 65Zn influx (data not shown). Separation of cytoplasm and vacuole of cells exposed to 65Zn for varying periods revealed a rapid transfer of zinc to the vacuole. Even with a short influx period of

4 42 R.J. Reid et al.: Zinc influx in Chara Table 2. Distribution of zinc in Chara taken directly from culture tanks 15 Zinc mmol. m- 3 gmol. m- 2 n 1 Culture solution.1 _+.2 3 Vacuole Cytoplasm" 56.2 _ Total intracellular Cell walls 58 4 b a Calculated assuming that the cytoplasm occupies 5% of the cell volume u Assuming a cell wall thickness of 15 gm Zinc (retool. ra~ 15 min ( + 5 min rinse) more than half of the 6SZn taken up was found in the vacuole (Table 1). The cell fractionation technique was also used to obtain samples of cytoplasm, vacuole and cell wall for measurement of total zinc by GFAAS. The cell wall accounted for 7% of the total cellular zinc (Table 2). The cytoplasmic concentration was found to be higher than that of the vacuole, but when the ratio of vacuole:cytoplasm of 2:1 (Bostrom and Walker 1975) is taken into account, it can be seen that around 85% of the intracellular zinc is stored in the vacuole (Table 2). The accumulation ratios (i.e. the increase over the concentration in the culture solution) for cells from this culture were 322 and 562 for the vacuole and cytoplasm respectively, and approximately 58 for the cell wall (assuming a wall thickness of 15 pro). Concentration dependence of influx. nflux of zinc showed a biphasic response to changes in the external concentration (Fig. 4). nflux was independent of concentration in the range.1-.4 mmol. m- 3, which may reflect the saturation of a high-affinity transport system. At concentrations between about.4 and 5 mmol-m -3 influx was linearly related to concentration. The response at higher (albeit unphysiological) concentrations is complicated by the fact that growth of Chara was found to be reduced by zinc concentrations higher than 1 mmol. m- 3 (data not shown), and the non-linearity beyond 5mmol.m -3 (Fig. 4) is therefore likely to be due to metabolic inhibition, which reduces influx (see below). ph dependence. Zinc exists in solution as the free ion Zn z + as well as in soluble complexes. n the simple solutions used in this study, the dominant species at low ph was the divalent Zn 2 with the formation of the monovalent Zn(OH) and the neutral Zn(OHh and insoluble complexes at higher ph (Fig. 5a). Zinc influx showed an optimum around ph 7 (Fig. 5b) but did not conform in any simple way with the phdependent changes in the speciation pattern. nflux was stimulated by cytoplasmic acidification caused by addition of 1 mol. m-3 butyric acid at ph 5. Voltage dependence. Plasmalemma calcium channels in wheat roots (Pifieros and Tester 1995) and in Chara i t r Zinc (retool- m "3) Fig. 4a, b. Concentration dependence of 65Zn influx at high (a) and low (b) low zinc concentrations. nflux ~ = 2 h, rinse t = 3 min. n = 11 cells ~'~ 15 5 zn ~ ph + (b) B ~ ~ i i ~ i i ph Fig. 5a, b. Speciation of zinc in APW solutions of varying ph as computed by GEOCHEM-PC (a) and effect of external ph and cytoplasmic acidification (by 1 mol- m- 3 butyric acid; BA) on 65Zn influx in Chara (b). Total zinc concentration= 1mmol-m -3. nflux t = 2 h, rinse t = 3 min. n = 1~12 cells. Different symbols indicate separate experiments

5 R.J. Reid et al.: Zinc influx in Chara 43 (Reid and Smith 1992b) are voltage dependent, opening as the membrane is depolarised beyond - 1 mv. n Chara this state can be achieved either by (i) voltage clamping, (ii) high K + concentrations, or transiently by (iii) electrical stimulation to generate an action potential. n six cells which were voltage-clamped at PDs between -95 and -25mV during exposure to 1mmol-m -3 65Zn at ph 6.2, there was no obvious relationship between influx and membrane voltage (R 2 =.147). Similarly, for 15 cells which were voltageclamped at PDs between -65 and -3 mv during exposure to 5 mmol. m-3 65Zn ' the fluxes appeared to be evenly distributed (R E =.74). Zinc influx in cells which were electrically stimulated to generate action potentials was not significantly different from the control (unstimulated cells). By comparison, influx of 45Ca was stimulated nearly 14-fold by the same electrical stimulation (Table 3). Competition with other cations. The effects on zinc influx of the addition of various monovalent and divalent cations are shown in Fig. 6. nflux showed little sensitivity to an increase in the Ca 2 + concentration from.5 mol-m-3 (normal APW) to 5 mol. m- 3, but was partially inhibited by 2 mol. m- 3 Na + or K +, and by 5 mmol. m- 3 Mn 2 + or Fe 2+. The treatment with high K +, but not high Na +, should have been accompanied by a large depolarisation Table 3. Effect of action potentials on influx of 45Ca and 65Zn. ndividual cells were mounted in segment loading chambers and stimulated 1 times during the 2-min radioactive treatment period. 45Ca (.5mol'm -3) and 65Zn (.1mol-m -3) were applied in APW-Mes at ph 6.4. n = 6 cells Calcium influx (nmol. m- 2. s- 1) Zinc influx (pmol 9 m- 2. s- 1) Control Stimulated 6.96 _ % increase d..~ o APW... Ca ~ 15ii:i ~ +La iiiii!ii iiiiiiil +TEA ~influx +CCCP Fig.7. Effects of channel blockers (.1mol-m -3 La 3+ and 5 mol- m- 3 TEA) and metabolic inhibition by 1 mmol" m- 3 CCCP (ph 5.5) on influx of 65Zn. For comparison of effects of channels blockers, influxes of 45Ca and 4ZK are shown for the relevant treatments. nflux t = 3 min, rinse t = 3 min for 65Zn, 4 min for 4SCa (segment loading technique) and 4 min for 42K. n = 8-12 cells of the membrane PD (Hope and Walker 1975). The similarity of responses to these two cations argues against any PD-mediated effect. Effects of channel blockers and metabolic inhibitors. The possibility that zinc enters (?leaks) through known cation channels was investigated by application of the channel blockers La 3+ and tetraethylammonium (TEA) which block Ca 2 + and K + channels respectively, with appropriate controls for determining the relative effects on 45Ca and 42K influxes. Lanthanum inhibited zinc influx by 25% and Ca 2 + by 75%, while TEA reduced K + influx by more than 5% but had no significant effect on zinc influx (Fig. 7). Metabolic inhibition by carbonylcyanide m-chlorophenylhydrazone (CCCP) strongly inhibited zinc influx (Fig. 7). Discussion 1 - ~ 8-.~ 6- ' 4- ~ 2- U - i!!i!!i!!ii ii!iiiii iiiiiiiill APW Ca Na K Mg Mn Fe mol.m "~ ramol.m -3 Fig. 6. Effects of cations on influx of 65Zn. nflux t = 2 h (pretreatment in APW for all treatments), rinse t = 3 min. n = 1 cells Measurement of zinc fluxes. By exploiting the morphological advantages of the giant charophyte cells, we have been able to show that zinc uptake across the plasmalemma occurs in a biphasic manner with a transition at approximately.5 mmol-m -3. We have tentatively ascribed the concentration dependence to the operation of two separate transport systems and we have described in detail the characteristics of the low-affinity system, which was experimentally easier to investigate (see below). An important aspect of this work was to outline the complexities imposed by plant cell walls on the measurement of unidirectional fluxes of divalent cations, and hence on the validity of conclusions based on these flux measurements. n Chara cells taken directly from cultures, most of the zinc was found in the cell walls, at a concentration 58- fold higher than in the culture medium, consistent with a Donnan potential of approximately - 1 inv. This seemingly high voltage would result from the high density

6 44 R.J. Reid et al.: Zinc influx in Chara of fixed negative charges in the cell wall and is in agreement with measurements and predictions based on cationexchange capacity in a variety of plant cell types (Walker and Pitman 1976). The consequence of this high zinc concentration in the cell wall is that following incubation in solutions containing divalent cation tracers there will be a very high apoplasmic content which needs to be quantitatively removed in order to estimate accurately how much tracer has crossed the plasmalemma. There are two components of this system which contrive to make such estimates difficult to obtain. Firstly, for micronutrient concentrations the membrane fluxes are very low in comparison with cell wall exchange so that even with lengthy incubations the intracellular component is small and may represent less than 1% of the cell wall content before rinsing. Secondly, there appear to be a variety of cation exchange pools in the cell wall, some of which exchange only very slowly; removal of the final 1-2% of extracellular tracer requires long rinse times with the associated risk of underestimating unidirectional influx due to efflux during rinsing. These considerations are of minor importance when using a system in which the cell walls can be separated from the contents without significant exchange between the fractions. Thus, giant algal cells have many advantages over complex cell systems such as roots for the study of divalent- and trivalentcation flux mechanisms. t might be argued that a micronutrient-uptake mechanism in a freshwater alga is unlikely to be relevant to higher plants. However, the function of zinc uptake is presumably to provide zinc for essential cytoplasmic processes and there is no evidence that these cytoplasmic processes are radically different between charophyte algae and higher plants. Moreover, given the apparent close evolutionary relationship between charophytes and higher plants (Manhart and Palmer 199) it seems reasonable to propose similar transport mechanisms for nutrients fulfilling a similar purpose in both plant types. The literature on mechanisms of zinc uptake by higher plants is by no means large, but there exist order-of-magnitude differences in reported rates and Kms (see review by Kochian 1993). n the absence of any simple method for demonstrating the effectiveness of desorption in roots, and from our own data on wheat root cell walls, it is difficult to decide whether the previously published kinetics apply to uptake to intracellular or to slowly exchanging extracellular zinc PoOls. Compartmentation of zinc in Chara. Measurements with GFAAS showed that the zinc concentration in the cytoplasm was approximately 5 mmol. m- 3 when the external concentration was.1 mmol. m-3. Most of the intracellular zinc was stored in the vacuole, which in Chara occupies approximately 95 % of the total cell volume. The cytoplasmic zinc concentration was slightly higher than in the vacuole but in the absence of data on the free zinc concentration in these compartments it is not possible to speculate further on possible intracellular transport mechanisms. What is obvious though, is that zinc transport to the vacuole is much faster than efflux across the plasma membrane. The fact that 65Zn influx was constant over several hours indicates a low tracer efflux across the plasma membrane, whereas transfer of 65Zn to the vacuole was rapid. This implies either a large unidirectional uptake of zinc to the vacuole or rapid exchange between cytoplasmic and vacuolar pools. Two uptake systems? t is clear that zinc influx in the range of approximately.5 to 5 mmol-m-3 is linearly dependent on concentration. The kinetics at higher concentrations are obscured by the rapid onset of toxicity symptoms, and at lower concentrations by technical limitations associated with maintaining accurate solution concentrations. On the basis of the data obtained so far, it seems likely that two uptake systems are involved, one of which saturates below.1mmol.m -3, and a second which may not saturate in the range of physiological tolerance, if at all. n physiological terms, optimal growth of plants in hydroponic culture appears to occur at.5-8 mmol.m -3 (Carroll and Loneragan 1968; Asher 1987), at which concentration the high-affinity system would be saturated. Many plants, however, grow normally at much higher concentrations of zinc, and some will tolerate concentrations up to 2 mmol.m-3 (Sela et al. 1989). The function of the low-affinity system is difficult to define since it appears to admit zinc freely at toxic concentrations, although toxicity would itself limit influx as a consequence of metabolic inhibition (cf. effect of CCCP). Zinc influx by this system showed a ph optimum around 7, was sensitive to changes in cytoplasmic ph and was dependent on metabolism. On the other hand, influx was independent of membrane PD and showed little sensitivity to high Ca 2 +, Na + or K + and only moderate sensitivity to Mn 2 + and Fe 2 + when these were added at a 5-fold higher concentration than zinc. Mechanism of zinc transport across the plasmalemma. At first sight it seems unnecessary to invoke an active transport system for zinc since under almost all cell conditions zinc influx will be thermodynamically favoured. Like Ca 2 +, uptake of the Zn 2 species could be driven by the negative membrane voltage (- 12 mv could support a 14-fold accumulation). The dependence on metabolism is therefore difficult to explain, although it is known that passive movement of ions through some plasmalemma K channels is controlled by ATP (Katsuhara and Tazawa 1992; Wu and Assmann 1995). The possibility that zinc uptake occurs via leakage through the same channels that pass Ca 2 + seems to be discounted by the current results, which showed that zinc influx was insensitive to blockage of Ca 2 channels by La 3 was not voltage sensitive or stimulated by action potentials and was not inhibited by a 1-fold increase in the concentration of Ca 2 +. Similarly, the low sensitivity to TEA and to increased concentrations of Na and K argues against zinc penetration throug h K channels, at least for uptake by the low-affinity system. Several authors have speculated on the existence of ion channels for micronutrient cations in plants (e.g. Kochian et al. 1991; Welch et al. 1993) and the current work does not dismiss the possibility that they do exist; the results simply show that the low-affinity system does not use the known K and Ca z channels.

7 R.J. Reid et al.: Zinc influx in Chara 45 An alternative mechanism for the low-affinity system, for which there is a good experimental basis, is a protonlinked carrier system. A 2H+/Zn z+ exchange would be consistent with the responses of influx to changes in internal and external ph (i.e. the proton efflux gradient would become more favourable with increasing external ph or cytoplasmic acidification). Zinc influx does indeed increase under these conditions, at least up to ph 7, while the fall-off at higher ph coincides with the fall in the concentration of the Zn 2 species. The lack of response of zinc influx to membrane PD also suggests an electroneutral system and, depending on the free zinc concentration in the cytoplasm, the exchange may require energy input, especially at external ph below 7 where the ph gradient would be inwardly directed. Thus a 2H+/Zn2+-ATPase would be consistent with all of the current results. nvestigation of the high-affinity zinc uptake system. While zinc uptake at zinc concentrations above about.5 mmol. m -3 (nutrient-sufficient conditions) would be dominated by the low-affinity system, plants growing under nutrient-limiting conditions (e.g. natural soils) would depend on the high-affinity system. Our current investigation extended only as low as.1 mmol. m-3 zinc; maintenance of accurate external solution concentrations below this level is more of a problem and the likelihood of a high ph sensitivity imposes further limitations. Zinc chelates might be appropriate, but the accuracy of the concentration involves an act of faith in the reliability of the thermodynamic constants used to compute free ion concentrations. A more sinister problem may be the penetration of zinc in the chelated form rather than as the free ion. Most chelate buffering systems utilise a huge excess (e.g. 15 in Norve!l and Welch 1993) of the chelated form which is often less charged than the free ion. Hence, even low relative membrane permeabilities to the chelated forms could result in greater uptake of the chelate than of the free ion. t is suspicious that nutritional requirements based on experiments using chelate buffering are often several orders of magnitude lower than in experiments with unbuffered zinc. A comparative study using buffered and unbuffered zinc is obviously the wisest course of action under these circumstances. The authors are grateful to Danny Brock for his initial study of exchange of zinc in cell walls, to Dawn Verlin for technical assistance, and to Zdenko Rengel for comments on the manuscript. This work was supported by the Australian Research Council. References Asher C (1987) Effects of nutrient concentration in the rhizosphere on plant growth. n: X th Congress nt Soc Soil Sci Symposium, vol 5. SSS, Hamburg, pp Bostrom TE, Walker NA (1975) ntercellular transport in plants.. The rate of transport of chloride and the electric resistance. J Exp Bot 26: Carroll MD, Loneragan JF (1972) Responses of plant species to concentrations of zinc in solution.. Growth and zinc content of plants. Aust J Agric Res 19: Chaudhry FM, Loneragan JF (1972) Zinc absorption by wheat seedlings and the nature of its inhibition by alkaline earth cations. J Exp Bot 23: Katsuhara M, Tazawa M (1992) Calcium-regulated channels and their bearing on physiological activities in Characean cells. Phil Trans R Soc London Ser B 338:19-29 Kochian LV (1993) Zinc absorption from hydroponic solutions by plant roots. n: Robson AD (ed) Zinc in soils and plants. Kluwer, Dordrecht Boston London, pp Kochian LV, Norvell WA, Shaft JE, Chaney RL (1991) Characterizing root iron uptake using a ferrous chelate to buffer free Fe 2+ ion activity in solution. (Abstr) Plant Physiol 96: Suppl 142 Hope AB, Walker NA (1975) The physiology of giant algal cells. Cambridge University Press, London Manhart JR, Palmer JD (199) The gain of two chloroplast trna introns marks the green algal ancestors of land plants. Nature 345: Norvell WA, Welch RM (1993) Growth and nutrient uptake by barley (Hordeum vulgare L. cv. Herta): Studies using an N-(2- hydroxyethyl)ethylenedinitrilotriacetic acid-buffered nutrient solution technique. Plant Physiol 11: Parker DR, Norvell WA, Chaney RL (1994) GEOCHEM-PC: A chemical speciation program for BM and compatible computers. n: Loeppert RH, Goldberg S, Schwab AP (eds) Chemical equilibrium reaction models. American Society of Agronomy, Madison, Wisconsin Pifieros M, Tester M (1995) Characterisation of a voltage-dependent Ca 2 +-selective channel from wheat roots. Planta, in press Reid R J, Smith FA (1992a) Measurement of calcium fluxes in plants using 45Ca. Planta 186: Reid RJ, Smith FA (1992b) Regulation of calcium influx in Chara. Effects of K +, ph, metabolic inhibition and calcium channel blockers. Plant Physiol 1: Reid RJ, Tester M, Smith FA (1993) Effects of salinity and turgor on calcium influx in Chara. Plant Cell Environ 16: Santa Maria FE, Cogliatti DH (1988) Bidirectional Zn-fluxes and compartmentation in wheat seedling roots. J Plant Physiol 132: Schmid WE, Haag HP, Epstein E (1965) Absorption of zinc by excised barley roots. Physiol Plant 18: Sela M, Garty J, Tel-Or E (1989) The accumulation and the effect of heavy metals on the water fern Azolla filiculoides. New Phytol 112:7-12 Walker NA, Pitman MG (1976) Measurement of fluxes across membranes. n: Luttge U, Pitman MG (eds) Encylopedia of plant physiology, NS, vol. 2. Springer-Verlag, Berlin Heidelberg New York, pp Welch RM, Norvell WA, Schaefer SC, Shaft JE, Kochian LV (1993) nduction of iron(ll) and copper() reduction in pea (Pisum sativum L.) by Fe and Cu statusl Does the root-cell plasmalemma Fe()-chelate reductase perform a general role in regulating cation uptake. Planta 19: Wu W-H, Assmann SM (1995) s ATP required for K channel activation in Vicia guard cells? Plant Physiol 17: 11-19