Planta (1996)198:39-45
P l m a t ~ 9 Springer-Verlag 1996
The mechanism of zinc uptake in plants C h a r a c t e r i s a t i o n o f the low-affinity s y s t e m 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. 5005, 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 ( C h a r a c o r a l l i n a 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. It 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 ( T r i t i c u m a e s t i v u m L.) roots indicated exchange characteristics similar to that of C h a r a . Fractionation of C h a r a 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 5 6 m m o l . m -3 and 32 mmol- m - 3, respectively, for cells grown in a zinc concentration of 0.1 mmol" m - 3 . Influx of 65Zn in C h a r a was linear over several hours, with rapid transfer to the vacuole, but only slow efflux. Influx 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 0.1 m m o l . m -3 and a low-affinity system which showed a linear dependence on concentration up to at least 50 m m o l . m -3. Only the low-affinity system was examined in detail. Influx through this system showed a strong dependence on external p H with an optimum around 7 and was also stimulated by cytoplasmic acidification. Influx was sensitive to metabolic inhibition, but not to blockers of C a 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 N a +. Influx was independent of m e m b r a n e potential difference in cells voltage-clamped at - 65 to - 300 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)232 3297; Tel.: 61 (8)303 5290, E-mail:
[email protected]
Key words:
Chara
-
Cell wall and zinc - Micronutrient
- T r i t i c u m - Zinc uptake
Introduction Despite the importance of zinc as a micronutrient for plant growth, there have been relatively few studies of the mechanism of zinc uptake. It 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. In 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 C h a r a are not fundamentally different from apoplasmic binding in cereal roots, and that by adopting the same techniques used to measure Ca 2 +
R.J. Reid et al.: Zinc influx in Chara
40 influx in Chara, the general properties of m e m b r a n e fluxes of zinc c a n 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 (40-90 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, 0.1 mol.m -3 K2SO4 and 0.5mol'm -3 CaCI2. Unless otherwise stated, experimental solutions were buffered at pH 6.2 with 1 mol.m-3 2-0N-morpholino)ethanesulfonic acid (Mes). Wheat (Triticum aestivum L. cv. Halberd) was grown on a 10% Hoagland's solution for 10 d.
Flux measurements. Influx 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. In the latter method, individual internodal cells were incubated in 6~Zn-APW in a Petri dish (10-12 cells/200ml), then desorbed for 30min 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. Influx of 45Ca was measured using the same techniques as for zinc. Influx 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. Influx solutions were gently agitated and cells were illuminated with dim light (approx. 35 ktmol"m - 2 - s - 1).
Preparation of cell walls. Isolated 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 50-mm apical root segments for 20 min in deionised water. Washed and dried cell wall material was incubated in 10 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. Initial binding and the time-course of desorption were calculated by summing the 65Zn remaining in the tissue after 60 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 1000 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 m a j o r i m p e d i m e n t to m e a s u r e m e n t of u p t a k e of polyvalent cations lies in their extensive extracellular b i n d i n g which obscures m e m b r a n e fluxes. I n order to d e m o n s t r a t e that b i n d i n g characteristics in the freshwater alga Chara were not f u n d a m e n t a l l y different from those of higher plants, a c o m p a r a t i v e study was m a d e of the b i n d i n g a n d d e s o r p t i o n of zinc in cell walls of Chara a n d wheat roots. I n general terms these p a r a m e t e r s were similar for b o t h species although, o n a dry-weight basis, Chara h a d a higher b i n d i n g capacity of 23.2 ___0.4 ~tmol' g - 1 c o m p a r e d to 10.3 + 0.6 ~tmol. g - 1 in wheat. This latter value m a y overestimate the shortterm b i n d i n g in intact roots since the boiling process would have destroyed the endodermis, thereby o p e n i n g up cell wall b i n d i n g sites which are n o t n o r m a l l y exposed to the external solution. D e s o r p t i o n of zinc from Chara was more rapid. Figure 1 shows the time-course from 5-60 min. A l t h o u g h the initial rinses between 0 a n d 5 m i n r e m o v e d between 80 a n d 90% of the original 65Zn activity, the r e m a i n i n g fraction exchanged m u c h more slowly so that after 60 m i n a significant a m o u n t (1% for Chara a n d 2 % for wheat) of the 65Zn r e m a i n e d b o u n d .
Separation of symplasmic and apoplasmic zinc. A moreElectrophysiology. The electrical potential difference (PD) between the vacuole and the outside solution was varied by an external voltage-clamp technique. Individual 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 KC1filled 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 20 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 0.2 s.
detailed analysis of desorption of 65Zn from cell walls a n d the implications for m e a s u r i n g m e m b r a n e fluxes was m a d e using intact cells of Chara, which were pre-incubated in A P W + 10 m m o l . m -3 65Zn for 2 h . The effectiveness of d e s o r p t i o n was d e p e n d e n t on the availability of a n exchangeable cation; p r o l o n g e d rinsing in deionised water released very little of the b o u n d 65Zn (Fig. 2a). D e s o r p t i o n with n o n - r a d i o a c t i v e A P W + 10 m m o l . m - 3 ZnC12 occurred with a half-time of a p p r o x i m a t e l y 20 m i n while 1 m o l . m - 3 LaC13 in A P W displaced a p p r o x i m a t e l y 99.5% of b o u n d 65Zn in 10 m i n (Fig. 2b). S e p a r a t i o n of cell wall a n d cell contents showed that despite the a p p a r e n t efficiency of the d e s o r p t i o n with
R.J. Reid et al.: Zinc influx in Chara
41 0,6
25 2O
. ~ 0.5
i
10 "~
2.5
~
2.0
0.4 ~ 0.3
~ o.2 0.1
1.5 I
1.0
0
5
10
15
20
25
Time 0a) 0.5 i
i
i
0
i
20
i
i
i
40
60
Fig. 3. Time-course of 65Zn influx in Chara. Influx solution = APW pH 6.2 + 10 mmol" m-3 ZnC12. Rinse t = 30 min. n = 10-12 cells
Rinse time (rain)
Fig. 1. Desorption of isolated cell walls of Chara and wheat following incubation for 2 h in 10 mmol.m -3 6SZn. Desorption solution = 1 mol.m-3 LaC13 in APW (unbuffered)
40
4-"
Table 1. Influx of 65Zn to the cytoplasm and vacuole of internodal cells of Chara corallina using two different influx methods. External concentration = 10 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 0.5h Influx time (h)
3O
65Zn influx (pmol- m- 2. s- a) Cell
Cytoplasm
Vacuole
5.9 + 1.3 6.8 + 1.0
2.8 ___0.4 2.9 _ 0.5
3.1 + 1.0 3.8 + 0.5
10 10
7.8 + 0.8 4.4 +__0.7
2.8 _ 0.4 0.8 _ 0.1
5.0 + 0.7 3.6 ___0.7
15 15
Expt. 1
(a) 20
0.25 3
Expt. 2 1 100
0 1
2
3
0.3
0.2 "d 0.1 Vacuole/cytoplasm 0
i
0
iI
i
,
p
2
3
4
Rinse time (h)
Fig. 2a, b. Desorption of internodal cells of Chara in deionised water, 10 m m o l - m - 3 ZnCIz A P W or I tool.m-3 La 3 + in A P W (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 10 mmol' m- a 65Zn (b). Note the scale expansion in b
La 3+, most of the cell-associated radioactivity was still b o u n d in the cell wall (Fig. 2b). This residual cell wall activity exchanged only slowly, while the intracellular c o m p o n e n t was relatively unaffected by rinsing for longer than 20 min.
Uptake and compartmentation of zinc. At a concentration of 10 m m o l . 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 m a y 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 30-min desorption period and the possibility thus existed for loss of intracellular activity during rinsing, thereby underestimating the p l a s m a l e m m a 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 t h r e e - c o m p a r t m e n t c h a m b e r (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 n o t to be due to differences in zinc concentrations in the cultures, since exposure of isolated plants to 10 m m o l . m - a zinc in A P W 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
R.J. Reid et al.: Zinc influx in Chara
42 Table 2. Distribution of zinc in Chara taken directly from culture tanks Zinc mmol. m- 3 Culture solution Vacuole Cytoplasm" Total intracellular Cell walls
0.10 _+0.02 32.2 + 0.1 56.2 _+4.0 33.4 ___2.4 580 • 40b
150
n 100
gmol. m- 2
7.0 ___0.4 8.7 + 0.6
3 4 4 4 4
50
i
50
0
a Calculated assuming that the cytoplasm occupies 5% of the cell volume u Assuming a cell wall thickness of 15 gm
i
100
i
150
200
Zinc (retool. ra~ 2.5 2.0
15 min ( + 5 min rinse) m o r e 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 G F A A S . The cell wall acc o u n t e d for 70% 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 20:1 (Bostrom and Walker 1975) is taken into account, it can be seen that a r o u n d 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 5800 for the cell wall (assuming a wall thickness of 15 pro).
Concentration dependence of influx. Influx of zinc showed a biphasic response to changes in the external concentration (Fig. 4). Influx was independent of concentration in the range 0.1-0.4 m m o l . m - 3, which m a y reflect the saturation of a high-affinity transport system. At concentrations between a b o u t 0.4 and 50 m m o l - m -3 influx was linearly related to concentration. The response at higher (albeit unphysiological) concentrations is complicated by the fact that g r o w t h of Chara was found to be reduced by zinc concentrations higher than 10 m m o l . m - 3 (data not shown), and the non-linearity b e y o n d 5 0 m m o l . m -3 (Fig. 4) is therefore likely to be due to metabolic inhibition, which reduces influx (see below).
i
1.5 1.0 t r II
0.5 0
0
0.2
0.4
0.6
0.8
Zinc (retool-m"3)
Fig. 4a, b. Concentration dependence of 65Zn influx at high (a) and low (b) low zinc concentrations. Influx ~ = 2 h, rinse t = 30 min. n = 11 cells
10
zn~
8 6 4 2 0 6
7
8
9
pH 20
~'~ 15
+ (b)
B
~
~
pH dependence. Zinc exists in solution as the free ion Zn z + as well as in soluble complexes. In the simple solutions used in this study, the d o m i n a n t species at low p H was the divalent Zn 2§ with the formation of the m o n o valent Z n ( O H ) § and the neutral Z n ( O H h and insoluble complexes at higher p H (Fig. 5a). Zinc influx showed an o p t i m u m a r o u n d p H 7 (Fig. 5b) but did not c o n f o r m in any simple way with the p H dependent changes in the speciation pattern. Influx was stimulated by cytoplasmic acidification caused by addition of 1 m o l . m - 3 butyric acid at p H 5.
Voltage dependence. P l a s m a l e m m a calcium channels in wheat roots (Pifieros and Tester 1995) and in Chara
5
0 i
5
i
6
~
7
i
8
i
9
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= 10mmol-m -3. Influx t = 2 h, rinse t = 30 min. n = 1~12 cells. Different symbols indicate separate experiments
R.J. Reid et al.: Zinc influx in Chara
43
(Reid and Smith 1992b) are voltage dependent, opening as the membrane is depolarised beyond - 100 mV. In 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. In six cells which were voltage-clamped at PDs between - 9 5 and - 2 5 0 m V during exposure to 1 0 m m o l - m -3 65Zn at p H 6.2, there was no obvious relationship between influx and membrane voltage (R 2 = 0.147). Similarly, for 15 cells which were voltageclamped at PDs between - 6 5 and - 3 0 0 mV during exposure to 50 m m o l . m - 3 6 5 Z n ' the fluxes appeared to be evenly distributed ( R E = 0.074). 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. Influx showed little sensitivity to an increase in the C a 2 + concentration from 0.5 m o l - m - 3 (normal APW) to 5 mol. m - 3, but was partially inhibited by 20 mol. m - 3 N a + or K +, and by 50 m m o l . m - 3 Mn 2 + o r F e 2+. The treatment with high K +, but not high N a +, should have been accompanied by a large depolarisation Table 3. Effect of action potentials on influx of 45Ca and 65Zn. Individual cells were m o u n t e d in segment loading c h a m b e r s and stimulated 10 times during the 20-min radioactive treatment period. 45Ca ( 0 . 5 m o l ' m -3) and 65Zn ( 0 . 0 1 m o l - m -3) were applied in A P W - M e s at p H 6.4. n = 6 cells
Calcium influx (nmol. m- 2. s- 1)
Zinc influx (pmol 9m- 2. s- 1)
Control Stimulated
0.48 + 0.08 6.96 _+0.64
18.3 ___2.7 21.6 + 1.8
% increase
1350
18
100 -
+
80 -
iiiii!ii iiiiiiil
6od ~influx
40 .......... Ca~
..~
20 -
15ii:i~ 0
-
--
APW
+La
+TEA
+CCCP
Fig.7. Effects of channel blockers (0.1mol-m -3 La 3+ and 5 mol- m- 3 TEA) and metabolic inhibition by 10 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. Influx t = 30 min, rinse t = 30 min for 65Zn, 4 min for 4SCa (segment loading technique) and 4 min for 42K. n = 8-12 cells
of the m e m b r a n e P D (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 b l o c k Ca 2 + and K + channels respectively, with appropriate controls for determining the relative effects on 45Ca a n d 4 2 K influxes. L a n t h a n u m inhibited zinc influx by 25% and C a 2 + by 75%, while TEA reduced K + influx by more than 50% but had no significant effect on zinc influx (Fig. 7). Metabolic inhibition by carbonylcyanide m-chlorophenylhydrazone (CCCP) strongly inhibited zinc influx (Fig. 7). Discussion
Measurement of zinc fluxes. By exploiting the mor100
-
~
80-
.~
60-
'
40-
i!!i!!i!!ii ~ 20U
ii!iiiii
0 -
iiiiiiiill APW
Ca 5
Na 20
I
K 20
Mg 50 I
I
mol.m"~
Mn 50
I
Fe 50 I
I
ramol.m-3
Fig. 6. Effects of cations on influx of 65Zn. Influx t = 2 h (pretreatm e n t in A P W for all treatments), rinse t = 30 min. n = 10 cells
phological 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 0.5 m m o l - 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. In Chara cells taken directly from cultures, most of the zinc was found in the cell walls, at a concentration 5800fold higher than in the culture medium, consistent with a D o n n a n potential of approximately - 100 inV. This seemingly high voltage would result from the high density
44 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. It 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 1990) 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). In 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 50 mmol. m - 3 when the external concentration was 0.1 mmol. m-3. Most of the intracellular zinc was stored in the vacuole, which in Chara occupies approximately 95 %0 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
R.J. Reid et al.: Zinc influx in Chara 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? It is clear that zinc influx in the range of approximately 0.5 to 50 m m o l - 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 0 . 1 m m o l . m -3, and a second which may not saturate in the range of physiological tolerance, if at all. In physiological terms, optimal growth of plants in hydroponic culture appears to occur at 0.05-8 m m o l . 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 200 m m o l . 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 P D 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 ( - 120 mV could support a 104-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 10-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.
R.J. Reid et al.: Zinc influx in Chara A n a l t e r n a t i v e m e c h a n i s m for the low-affinity system, for which there is a g o o d e x p e r i m e n t a l basis, is a p r o t o n l i n k e d c a r r i e r system. A 2 H + / Z n z+ e x c h a n g e w o u l d be consistent with the responses of influx to changes in internal a n d e x t e r n a l p H (i.e. the p r o t o n efflux g r a d i e n t w o u l d b e c o m e m o r e f a v o u r a b l e with increasing external p H o r c y t o p l a s m i c acidification). Z i n c influx does i n d e e d increase u n d e r these c o n d i t i o n s , at least u p to p H 7, while the fall-off at higher p H coincides with the fall in the c o n c e n t r a t i o n of the Z n 2 § species. T h e lack of response of zinc influx to m e m b r a n e P D also suggests an e l e c t r o n e u tral system and, d e p e n d i n g on the free zinc c o n c e n t r a t i o n in the c y t o p l a s m , the exchange m a y require energy input, especially at e x t e r n a l p H b e l o w 7 where the p H g r a d i e n t w o u l d be i n w a r d l y directed. T h u s a 2 H + / Z n 2 + - A T P a s e w o u l d be consistent with all of the c u r r e n t results.
Investigation of the high-affinity zinc uptake system. W h i l e zinc u p t a k e at zinc c o n c e n t r a t i o n s a b o v e a b o u t 0.5 m m o l . m - 3 (nutrient-sufficient conditions) w o u l d be d o m i n a t e d b y the low-affinity system, p l a n t s g r o w i n g und e r n u t r i e n t - l i m i t i n g c o n d i t i o n s (e.g. n a t u r a l soils) w o u l d d e p e n d on the high-affinity system. O u r c u r r e n t investigation e x t e n d e d o n l y as low as 0.1 m m o l . m - 3 zinc; m a i n tenance of a c c u r a t e external s o l u t i o n c o n c e n t r a t i o n s b e l o w this level is m o r e of a p r o b l e m a n d the l i k e l i h o o d of a high p H sensitivity i m p o s e s further limitations. Z i n c chelates m i g h t be a p p r o p r i a t e , b u t the a c c u r a c y of the c o n c e n t r a t i o n involves a n act of faith in the reliability of the t h e r m o d y n a m i c c o n s t a n t s used to c o m p u t e free i o n c o n c e n t r a t i o n s . A m o r e sinister p r o b l e m m a y be the pene t r a t i o n of zinc in the c h e l a t e d form r a t h e r t h a n as the free ion. M o s t chelate buffering systems utilise a huge excess (e.g. 105 in N o r v e ! l a n d W e l c h 1993) of the chelated form which is often less c h a r g e d t h a n the free ion. Hence, even low relative m e m b r a n e p e r m e a b i l i t i e s to the c h e l a t e d forms c o u l d result in g r e a t e r u p t a k e of the chelate t h a n of the free ion. It is suspicious t h a t n u t r i t i o n a l r e q u i r e m e n t s b a s e d on e x p e r i m e n t s using chelate buffering are often several o r d e r s of m a g n i t u d e l o w e r t h a n in e x p e r i m e n t s with unbuffered zinc. A c o m p a r a t i v e s t u d y using buffered a n d unbuffered zinc is o b v i o u s l y the wisest course of a c t i o n u n d e r 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.
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