Planta

Planta (1984) 161:53-60

9 Springer-Verlag 1984

Evidence for proton]sulfate cotransport and its kinetics in Lemna gibba G1 B. Lass and C.I. Ullrich-Eberius Institut ffir Botanik der Technischen Hochschule, Schnittspahnstrasse 3, D-6100 Darmstadt, Federal Republic of Germany

Abstract. Sulfate uptake into duckweed (Lemna gibba GI) was studied by means of [35S]sulfate influx and measurements of electrical membrane potential. Uptake was strongly regulated by the intracellular content of soluble sulfate. At the onset of sulfate uptake the membrane potential was transiently depolarized. Fusicoccin stimulated uptake up to 165% of the control even at pH 8. It is suggested that sulfate uptake is energized in the whole pH range by a 3H+/sulfate cotransport mechanism. Kinetics of sulfate uptake and sulfate-induced membrane depolarization in the concentration range of 5 l-tM to I mM sulfate at pH 5.7 was best described by two Michaelis-Menten terms without any linear component. The second system had a lower affinity for sulfate and was fully active only at sufficiently high proton concentrations. Key words: Lemna (sulfate uptake) - Membrane potential - Proton/sulfate cotransport.

Introduction

Sulfate, as an essential macronutrient for autotrophic plants, has to be accumulated from very variable concentrations in the soil or in ponds. There are already many studies concerning the elucidation of the mechanism of sulfate uptake in roots (see Nissen 1971, 1973; Nissen and Nissen 1983), in detached leaves of Elodea (Jeschke and Simonis 1965), in cultured tobacco cells (Smith 1976) and in intact Lemna plants (Thoiron et al. Abbreviations and symbols." co = extracellular sulfate concentration; c~=intracellular sulfate concentration; Era=electrical membrane potential difference; AE.,=sulfate-induced, maximal membrane depolarization; AI2H + = electrochemical proton gradient; FW = fresh weight

1969, 1981). Uptake of a divalent anion requires much energy because of the negative charge inside the plant cell plasmalemma. This high energy requirement became apparent from the high sensitivity of divalent-anion uptake towards inhibitors (Legett and Epstein 1956; Shargool and Ngo 1975; Smith 1976) and temperature changes (Jeschke and Simonis 1965; Thoiron et al. 1969). Until now, however, there have been no suggestions about the mechanism of the coupling of the energy supply to the uptake ~echanism in higher plants. Since nitrate and phosphate uptake into duckweed could be shown to proceed by a proton cotransport mechanism along the electrochemical proton gradient (Novacky et al. 1978; Ullrich and Novacky 1981 ; Ullrich-Eberius et al. 1981), it was quite logical to assume a proton cotransport mechanism also for the uptake of the divalent sulfate ion. As the result of observations on extracellular alkalinization, sulfate uptake into Saccharomyces cerevisiae was recently suggested to be a 3H +/SO]- cotransport mechanism, coupled to the extrusion of one K+, and thus resulting in an electroneutral transport (Roomans et al. 1979), In the present study, changes in sulfate-induced membrane potential were compared with uptake kinetics. As an additional test for electrogenic transport the effect of fusicoccin on sulfate uptake was studied. Fusicoccin is known to specifically stimulate the H +-extruding ATPase at the plasmalemma of higher plants and hence to stimulate the electrochemical proton gradient (A~H+)-driven transport. Much effort has already been invested to elucidate the sulfate carrier system, whether the uptake proceeds at one or several sites, with interdependent or independent mechanisms (Nissen 1971, 1973; Nissen and Nissen 1983; Borstlap 1981; Rybova et al. 1982; Cram 1983a, b). At the onset

54

B.Lass and C.I. Ullrich-Eberius: Proton/sulfate cotransport and kinetics

of sulfate uptake in duckweed, only the energydependent component of the electrical membrane potential difference (Era) is changed. This reflects energy-dependent processes only at the plasmalemma, not at the tonoplast. Therefore, we compared the kinetics of sulfate-induced E m changes with those of [3SS]sulfate influx in order to analyze if thermodynamic considerations can contribute to the explanation of influx isotherms. Uptake experiments were restricted to 5-rain periods in order to avoid interference with metabolic processes, as occurred in long-time experiments with Lemna minor (Thoiron et al. 1981).

Material and methods Plant material. Lemna gibba L. strain GI (obtained from the Lemna collection of Professor Kandeler, Vienna, Austria) was grown axenically under short-day conditions (8 h/28~ C light and 16 h/23 ~ C dark) on sucrose-containing (29 mM) nutrient solution, with the macronutrients: 3.96 m M KNO3, 5.47 m M CaClz, 1.47 m M KH2PO4, 1.22 mM MgSO4; pH 4.8 (Pirson and Seidel 1950). Prior to the experiments, the plants were transferred to sucrose-free, axenic nutrient solution, where MgSO 4 was substituted by MgC12. Plants were grown without sulfate for I0 d under long-day conditions (16 h/28 ~ C light and 8 h/23 ~ C dark).

Electrophysiologieal measurements. Intact plants were mounted in a 4-ml Plexiglas chamber, which was perfused with standard experimental perfusion solution (after Higinbotham et al. 1964), containing I mM KC1, 1 mM Ca(NO3)2, 0.25raM MgCI2, and I m M NaHzPO4, • pH 5.7. The perfusion rate was 10 ml rain-1. Glass micropipettes (tip diameter 0.5 tam, tip potential - 5 to - 1 5 mV, and tip resistance 5 to 20 Mr2) filled with 3 M KC1 and the reference salt bridge were connected to the electrometer amplifier (Keithley 604, Munich, FRG) by Ag-AgC1 wires and to a chart recorder (Servogor 320, BBC Goerz, Vienna, Austria). Microelectrodes were inserted into the plants with a Leitz micromanipulator using a horizontally mounted microscope. Membrane-potential measurements were done in complete darkness. Further details are reported in Novacky et al. (1978, 1980). [35S]Sulfate uptake. For uptake experiments, 100 mg fresh weight (FW) of plants per sample were kept in darkness for 18 h in 10 ml sulfate-free standard experimental perfusion solution. Uptake experiments were performed in 5 ml of standard solution without sulfate in 25-ml Erlenmeyer flasks, mounted on a shaking Plexiglas rack at 25 ~ C, with a preincubation time of 1 h in dark or light. Light was supplied by Krypton lamps, at an irradiance of 100 W m -2. Uptake was started by injecting the [35S]sulfate solution. After an incubation time of 5 rain (if not stated otherwise), the reaction was stopped by rapidly sucking off the solution and rinsing five times with 10 ml icecold, unlabeled, sulfate-containing (1 raM) experimental solution for 5 min. Radioactivity in the dried plants was counted on planchets.

Sulfate determination. Endogenous soluble sulfate was determined according to a modified method of Vick (1975). One gram of duckweed was homogenized and extracted with boiling water for I h. After centrifugation the supernatant (8 ml) was

6.0 5.0

%, o3

3.0 "6 ~2.0

\

,0

0 0

2 ~ 6 8 days without SO~-

10

Fig. 1. Decrease of intracellular soluble sulfate in Lemna gibba G1 during sulfate starvation for 10 d under long-day conditions. Mean values of 3-13 experiments

mixed with 4 ml 0.05 M Na-H-phthalate buffer, and filled up with 96% (v/v) ethanol to a 20 ml total volume. The pH was adjusted to 4 with HC1. The reaction was started by addition of 25 mg chloranilic-acid barium salt. The samples were shaken for 18 h at 25 ~ C. After centrifugation of BaSO 4 and the residual barium chloranilate the pink supernatant chloranilic acid was measured at 530 nm.

Results

Effect of sulfate starvation on uptake and sulfateinduced, maximal membrane depolarization (AE,,,). The endogenous content of soluble sulfate in the plants of the standard culture was about 6 ~tmol SO 2- g-1 FW. Within 10 d of starvation on sulfate-deficient nutrient solution it decreased to about 0.16 pmol SO4a- g-1 FW (Fig. 1). In plants grown on sulfate the sulfate uptake rate was low in light and dark. But it was stimulated more than ten times by a 10-d sulfate-starvation period, not only in light but also in darkness and without any induction period (Fig. 2). Light clearly increased sulfate influx to about 300% (first 5 min) of the rate in darkness in sulfate-starved plants (Fig. 2). In non-starved plants, light stimulation increased sulfate influx from 130% (5 min) to 200% (10-30 rain) of the rate of uptake in the dark. At the onset of sulfate uptake from 1 mM NazSO 4 solution in the dark, the membrane potential of non-starved plants was only slightly changed by not more than 3 mV (Fig. 3 A). Sulfate starvation did not affect the membrane potential itself, so the resting potential in darkness was still between - 2 0 0 and - 2 6 0 mV (Figs. 3, 9). In starved plants, however, the same concentration of 1 mM Na2SO 4 caused an average membrane depolarization of 25 mV in darkness (Fig. 3 B). Depolariza-

B.Lass and C.I. Ullrich-Eberius : Proton/sulfate cotransport and kinetics i

J

55

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Fig. 3, Effect of sulfate on E m in darkness, pH 5.7. Trace A, non-starved Lernna plants; traces B and C, sulfate-starved plants. Solid arrowheads indicate addition and open arrowheads indicate removal of Na2SO 4 or Na-iminodiacetate (NaIDA). Numbers at the traces denote recorded mV

0

5

10

20 t (min)

30

Fig. 2. Time dependence of [3SS]sulfate uptake into non-starved (broken lines) and sulfate-starved (solid lines) Lemna plants, in the light (L) and in darkness (D). co = 1 m M Na2SO4, pH 5.7. Bars indicate SD of four to six experiments

0.2

% tion was followed by a spontaneous repolarization in the presence of sulfate and, upon removal of sulfate, the E m transiently hyperpolarized (Figs. 3 B, C, 9). In the light, the average sulfateinduced AE,, of 11 mV at 1 mM SO42- (data not shown) was much less than in darkness, though uptake was highly light-stimulated. After a washing period of about 15 min, repeated sulfate additions caused equal Em depolarization. To exclude the possibility that the depolarization of the E m might be caused by the accompanying Na § ions, the plants were pretreated with Na-iminodiacetate of corresponding concentrations (iminodiacetate was used as an impermeant anion, according to Raschke and Schnabl (1978), Fig. 3 B, C). Thus, during sulfate addition, the extracellular Na + concentration was kept constant. Figure 3 B and C show the depolarizing effect of 1 and 5 mM sulfate alone, together with the subsequent separated effects of the removal of sulfate and sodium ions. Hence sulfate clearly induced E,, depolarization. p H Dependence. If sulfate uptake is dependent on the electrochemical proton gradient, it should be

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Fig. 4. pH-dependence of [35S]sulfat e uptake by Lemna in darkness. % = 5 0 gM Na2SO4; 1 m M phosphate buffer from pH 3.4 to 7.35 and 5 mM Tris buffer from pH 7.35 to pH 8.6. Bars indicate SD, six to nine experiments

highest at the largest ApH across the plasmalemma. But Fig. 4 shows that in the dark sulfate uptake proceeded with a similar rate over a wide pH range. It decreased unexpectedly slowly with increasing pH. Rates are plotted against the actual pH values during the uptake in the bulk of the experimental solution. At pH 8, 55% of the maximal rate was still measured. Figure 5 and Table 1 show that the sulfate-induced AE m was maximal in the slightly acidic pH range. At pH 8 only 20% of the maximal depolarization occurred. At pH 8.6, the E m did not re-

56

B.Lass and C.I. Ullrich-Eberius: Proton/sulfate cotransport and kinetics

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Fig. 5. pH-Dependence of sulfate-induced E m depolarization and repolarization in Lemna in darkness, co= 1 mM Na2SO 4. pH of standard experimental perfusion solution was adjusted with HCI-NaOH. Solid arrowheads indicate addition, open arrowheads removal of sulfate. Numbers at the traces denote recorded mV

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0 5 15 t (mini 30 Fig. 6. Stimulation of [35S] sulfate uptake (co= 50 gM Na2SO4) by 30 pM fusicoccin (FC) in 0.1% ethanol in the dark, at pH 5.7 (circles) and pH 8 (triangles). pH 8 was adjusted with 10 mM Tris-HC1. Lemna plants were pretreated with FC for 18 h in darkness. Bars indicate SD, six to nine experiments

Table 1. pH-Dependence of sulfate-induced membrane depolarization in Lemna in darkness, eo=l mM Na2SO 4. Mean values • SD, numbers of experiments in brackets pH

E,. (mV)

dh,~ (mV)

4.2 5.7 8.0 8.6

-164 -215 -232 -246

18.3_+2.7 (7) 25.3 • 3.9 (12) 5.0_+1.3 (8) 0.0• (5)

shows that 30 jaM fusicoccin (FC) stimulated uptake at low sulfate concentrations (by 41%) and also at the high concentration of 1 m M sulfate (by 33%). Figure 6 shows that FC increased sulfate uptake not only at pH 5.7 but even more so (by 65%) at pH 8.

Table 2. Effect of 30 gM fusicoccin (FC) in 0.1% ethanol on [3SS]sulfate uptake and on E,, (% stimulation of the active component of Era; E D = - 9 0 m V ) . Pretreatment of Lemna plants with FC for 18 h in darkness. Uptake period 5 rain in darkness; Co=0.05mM and ] mM NazSO4, pH 5.7. Mean values • SD, numbers of experiments in brackets co (mM SO2 )

Sulfate uptake (nmol SO~- g- i FW h- 1) Control

+ FC

Stimulation

0,05 1,00

585+__45 (9) 1,103+_75 (3)

823__.70(9) 1,463_+135(3)

41 33

E,,

- 190 mV _+10 (9)

-250 mV _+15 (9)

60

(%)

spond any more, though sulfate influx was still 20% of the maximal rate.

Effect of fusicoccin. Fusicoccin was applied in order to obtain further evidence as to whether sulfate uptake might be energized by AftH + . Table 2

Concentration dependence. Experiments were performed in the range of 5 jaM to 1 m M SO 2-, so as not to exceed the sulfate concentration of the standard nutrient solution, not to induce additional effects by increased cation concentration, and in order to remain in the range of naturally occurring uptake kinetics in plants. Uptake rates did not yield a single hyperbolic saturating isotherm and could not be described by one Michael• ten function, neither in light nor in darkness (Figs. 7, 8). Transformation into a double-reciprocal plot after Lineweaver and Burk yielded two systems, one with high affinity in light and dark ( K r = 8.5 and 5.2 jaM sulfate, respectively), and a second one with a lower affinity in light and dark (KrH = 120 and 180 jaM sulfate, respectively; Figs. 7, 8, Table 3). In the range between 0.1 and 0.2 mM, sulfate-uptake system I seemed to be saturated and system II contributed gradually. No linear component could be observed in this concentration range. Light did not influence Kr, but Vm,x was doubled in comparison with darkness (Table 3). Simultaneously with uptake, the sulfate-induced dE,. also increased with increasing sulfate

B.Lass and C.I. Ullrich-Eberius: Proton/sulfate cotransport and kinetics

Table 3. Apparent transport parameters of [35S]sulfate uptake by Lemna and sulfate-induced membrane depolarization in light and darkness, pH 5.7 and 8, obtained from Lineweaver-Burk plots (mean values of n experiments). Values in brackets calculated after Neal (/972). Kr values are expressed in gM SO~and V,~,~ values in gmol SO2 2 g-1 FW h - I and mV (AE,~)

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180 (260) 120 (200)

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pH 5.7

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Fig. 8A, B. Concentration dependence of [35S]sulfate uptake by Lemna (5 min) in darkness, pH 5.7. A Linear plot; B Lineweaver-Burk plot. Bars indicate SD within a single experiment

Fig. 9. Concentration dependence of sulfate-induced E,~ depolarization in Lemna in darkness, pH 5.7. Solid arrowheads indicate addition, open arrowheads removal of sulfate. Numbers at the traces denote recorded mV

58

B.Lass and C.I. Ullrich-Eberius: Proton/sulfate cotransport and kinetics 30

:~ 20 E

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70

Fig. 10A, B. Linear plot (A) and Lineweaver-Burk plot (B) of concentration-dependent, sulfate-induced E m depolarization in Lemna; darkness, pH 5.7. Bars indicate SD, 4-15 experiments

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