Planta (1988)176:68-74

P l a n t a 9 Springer-Verlag 1988

Interaction of sulfate and glutathione transport in cultured tobacco cells Heinz Rennenberg*l, Andrea Polle *, Norbert Martini * *, and Barbara Thoene * Botanisches Institut der Universitfit zu K61n, Gyrhofstrasse 15, D-5000 KSln 41, Federal Republic of Germany

Abstract. Photoheterotrophic and heterotrophic suspension cultures of tobacco ( N i c o t i a n a t a b a c u m L.) were grown with I m M glutathione (reduced; GSH) as sole source of sulfur. Addition of sulfate to both cultures did not alter the rate of exponential growth, but affected the removal of GSH and sulfate in different ways. In photoheterotrophic suspensions, addition of sulfate caused a decline in the net uptake of GSH, whereas sulfate was taken up by the green cells immediately. In heterotrophic suspensions, however, addition of sulfate did not affect the net uptake of GSH and sulfate was only taken up by the cells after the GSH supply in the medium had been exhausted. Apparently, GSH uptake in photoheterotrophic cells is inhibited by sulfate, whereas sulfate uptake is inhibited by GSH in heterotrophic cells. The differences in the effect of GSH on sulfate uptake in photoheterotrophic and heterotrophic tobacco suspensions cannot be attributed to differences in the kinetic properties of sulfate carriers. In short-time transport experiments, both cultures took up sulfate almost entirely by an active-transport system as shown by experiments with metabolic inhibitors; sulfate transport of both cultures obeyed monophasic Michaelis-Menten kinetics with similar app. K m (photoheterotrophic cells: 16.0+2.0 gM; heterotrophic cells: 11.8 + 1.8 gM) and Vmax (photoheterotrophic cells: 323 + 50 nmol. m i n - 1. g - 1 dry weight; heterotrophic cells" 233 + 3 n m o l . m i n - 1.

g-1 dry weight). Temperature- and pH-dependence of sulfate transport showed almost identical patterns. However, the cultures exhibited remarkable differences in the inhibition of sulfur influx by GSH in short-time transport experiments. Whereas 1 m M GSH inhibited sulfate transport into heterotrophic tobacco cells completely, sulfate transport into photoheterotrophic cells proceeded at more than two-thirds of its maximum velocity at this GSH concentration. The mode of action of GSH on sulfate transport in chloroplast-free tobacco cell does not appear to be direct" a 14-h exposure to 1 m M GSH was found to be necessary to completely block sulfate transport; a 4-h time of exposure did not affect this process. Consequently, glutathione does not seem to be a product of sulfur metabolism acting on sulfate-carrier entities by negative feedback control. When transferred to the whole plant, the observed differences in sulfate and glutathione influx into green and chloroplast-free cells may be interpreted as a regulatory device to prevent the uptake of excess sulfate by plants.

Fraunhofer Institut fiir Atmosph/irische Umweltforschung, Kreuzeckbahnstr. 19, D-8100 GarmischPartenkirchen, Federal Republic of Germany 9* Max-Planck-Institut flit Ziichtungsforschung, D-5000 KSln 40, Federal Republic of Germany

Sulfur is available to plants in the soil predominantly in the form of sulfate (see Moss 1978). Most of the sulfate taken up by the roots has to be reduced to the oxidation state-II, before it is incorporated into cellular components (see Schmidt 1979; Anderson 1980). In the intact plant, sulfate reduction is predominantly performed in the chloroplasts of the leaf mesophyll cells (see Schmidt 1979;

P r e s e n t addresses." *

1 To whom correspondence should be addressed D C C D = N,N'-dicyclohexylcarbodiimide; D N P = dinitrophenol; D W = dry weight; FW = fresh weight; GSH = reduced glutathione Abbreviations."

Key words: Glutathione (transport, regulation) N i c o t i a n a - Solanaceae - Sulfate (transport, regulation) - Sulfur nutrition

Introduction

H. Rennenberg et al. : Interaction of sulfate and GSH transport

69

Anderson 1980); therefore, numerous membraneand long-distance-transport processes have to take place to fulfil the plant's needs for reduced sulfur: sulfate taken up by the roots is translocated in the xylem from the roots to the leaves with the transpiration stream (see Willenbrink 1967). In the leaves, sulfate is taken up by the mesophyll cells and transported either into the vacuole for storage, or into the chloroplasts (Rennenberg 1984), where it is reduced and incorporated into cysteine (see Schmidt 1979; Anderson 1980). Cysteine synthesized in the chloroplasts is not only transported into the cytoplasm and used for protein synthesis, a major part of it is incorporated into glutathione (see Rennenberg 1982, 1984). This tripeptide functions as long-distance-transport form of reduced sulfur and is translocated from the leaves to the roots and the growing parts of the stem (Rennenberg et al. 1979; Bonas et al. 1982). In these organs, apparently unable to reduce enough sulfate for their own needs, glutathione is degraded to make reduced sulfur available for protein synthesis (Rennenberg and Bergmann 1979; Bonas etal. 1982; Steinkamp and Rennenberg 1985). The spacial separation of the uptake of sulfate and its reduction in higher plants makes regulatory processes necessary in order to adjust the uptake of sulfate by the roots to the sulfate reduction in the leaves; in addition, sulfate reduction in the leaves has not only to be adjusted to the leaves', but also to the roots' needs for reduced sulfur. In the present investigation, the interaction of sulfate and glutathione transport is studied as a first attempt to obtain information on this interorgan regulation. In these experiments, heterotrophic tobacco cells were used as a model system for root tissue and photoheterotrophic tobacco cells as a model system for leaf-mesophyll cells.

on a stainless-steel sieve (65 gm pore size), washed with sulfatedeficient medium and inoculated into the same medium with 1.73 m M GSH as sulfur source at a density of 1 g_+ 10% fresh weight (FW) per 300 ml. Sulfate-deficient medium was prepared by replacing the sulfate-containing salts in the Murashige and Skoog medium by the corresponding chlorides. Glutathione was added immediately before inoculation as a filter-sterilized solution of pH 5.6-5.8. Suspensions were harvested in 2- to 3-d intervals by vacuum filtration for the determination of growth (FW), the GSH, and the sulfur content of the media. Glutathione was measured colorimetrically as 5,5'-dithiobis-(2nitrobenzoic acid) (DTNB) derivative (Lay and Casida 1976). In suspensions supplied with sulfate in the presence of GSH, the sulfate content of the media was determined turbidometrically as previously described (Rennenberg and Bergmann 1979).

Material and methods Plant material. The tobacco suspension cultures (Nicotiana tabacum L. cv. Samsun) used in the present experiments originate from a callus culture isolated by Bergmann (1960). Photoheterotrophic suspensions were grown in a modified Murashige and Skoog medium (Logemann and Bergmann 1974) at 25 ~ C and 60-70% air humidity under continuous illumination of 485 6 g m o l . m - Z . s -1 (HPS L 65W/150 ultra white; L Fluora 35W/77R, Osram, Mfinchen, F R G ; TL 40W/25, Philips, Eindhoven, The Netherlands). Heterotrophic tobacco cells were cultured under the same conditions on a rotary shaker (100 rpm) in the dark. Aliquots of 20 ml were inoculated every 10th day of cultivation into 300 ml medium in 1-1 Erlenmeyer flasks under sterile conditions.

Analysis of growing tobacco suspensions. Tobacco cells grown for 6-7 d in media with sulfate as sulfur source (Logemann and Bergmann 1974) were harvested under sterile conditions

Transport experiments. For the analysis of the transport of sulfate under standard conditions, tobacco cells grown for 6-7 d in media with sulfate as sulfur source were harvested on a stainless-steel sieve (65 Ixm pore size), washed with, and inoculated into sulfur-deficient medium (10 g + 10% FW per 180 ml medium). After 48 h of sulfur starvation, cells were separated from the medium, washed twice with 100 ml transport medium and inoculated into the same medium (0.2 g_+5% FW per 10 ml). Transport medium was composed of 5 mM bis-tris-propane (Sigma, Mfinchen, FRG), 0.5 m M CaC12 and 1% (w/v) sucrose at pH 7.0 (Jones and Smith 1981). As commercial sucrose contained substantial amounts of sulfate, it was purified on Dowex 1 x 8 (OH-), 50-100 mesh, by a modification of the method described by Hart and Filner (1969). For adaptation, cells were pre-incubated in transport medium for 4 h. Transport was initiated by addition of H235804 (Amersham, Braunschweig, FRG), 3.7 kBq.gmo1-1, to a final concentration of 0.1 mM. The pH of the solution added was adjusted with K O H to 6.0. After 10 rain of incubation at 2 5 ~ on a rotary shaker at 100 rpm, transport of radioactivity was terminated by addition of 15 ml of 5 mM NazSO4 and immediate separation of the cells from the medium by vacuum filtration. In controls, transport was terminated immediately after addition of radioactivity. Cells were washed 4 times with 25 ml transport medium, containing 0.1 mM sulfate, and transferred into scintillation vials with 2.5 ml Unisolve 100 (Zinsser, Frankfurt, F R G ) scintillation fluid. Radioactivity was determined with an LS 7500 (Beckman, Irvine, FRG) or BF 500 Betaszint (Berthold, Wildbad, F R G ) spectrometer. The values given are corrected for the quench of the cells, determined separately in each experiment by addition of known amounts of radioactivity to cells incubated with unlabeled sulfate. Data analysis. The data shown are means of at least three independent experiments with three replicates each. Statistical analysis was performed according to P/itau (1943).

Results

Uptake of sulfate and glutathione during growth of tobacco suspension cultures. Photoheterotrophic tobacco cells transferred from media with sulfate into media with GSH as the sole sulfur source exlhibited exponential growth subsequent to a lag-period of about 24 h (Fig. 1 A). The doubling time of 46 h observed with cells supplied with GSH was slightly higher than the doubling time of cells supplied with sulfate (38 h; Rennenberg 1981). Addition of sul-

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DAYS OF CULTURE Fig. l A, B. Effect of sulfate on the growth of tobacco cell suspensions supplied with G S H as sulfur source. Photoheterotrophic (A) and heterotrophic (B) tobacco cells grown 6-7 d in media with 1.73 m M sulfate were transferred into media with 1.73 m M G S H at a density of 1 g F W . (300 ml m e d i u m ) - 1 (e - e ) . After 4 (heterotrophic cells) or 5 (photoheterotrophic cells) d of growth, sulfate (MgSO 4- 7 H 2 0 ) was added to a final concentration of 1 m M ( o - - o ) . At the times of cultivation indicated, cells were separated from the media and the F W was determined

fate to suspensions fed GSH for 5 d, initially caused a decrease in growth; however, after 24 h, cells grew exponentially at the same rate observed with controls not exposed to sulfate (Fig. 1 A). Suspensions additionally supplied with sulfate grew exponentially up to the l ~ 1 3 t h day (Fig. IA) whereas, in suspensions supplied with GSH, the rate of growth declined after 7-8 d of culture. Similar results were obtained with heterotrophic tobacco cells (Fig. 1 B). When cells were transferred from sulfate to GSH as sulfur source, an initial period of growth was followed by a lag-period of 24-48 h before exponential growth began. Addition of sulfate after 4 d of culture delayed the onset of exponential growth to about 70 h, but did not alter the rate of exponential growth of the cells (Fig. 1 B). In cultures additionally supplied with sulfate, exponential growth proceeded for a longer period of time compared with suspensions fed GSH alone (Fig. 1 B). Despite these similarities in the effect of GSH and sulfate nutrition on growth, green and nongreen tobacco cells showed remarkable differences in the removal of GSH and sulfate from the media. Photoheterotrophic tobacco cells cultured with GSH as sulfur source took up GSH at a much lower rate (Fig. 2A) than heterotrophic cells (Fig. 2B). Addition of sulfate to the medium caused a decline in the uptake of GSH in photoheterotrophic cells (Fig. 2A), but did not affect GSH uptake of heterotrophic suspensions (Fig. 2 B). The sulfate added to the medium after 4 or 5 d of

Fig. 2 A - D . Uptake of GSH and sulfate during growth of photoheterotrophic and heterotrophic tobacco cell suspensions. Photoheterotrophic (A, C) and heterotrophic (B, D) tobacco cells were inoculated and grown as described in Fig. 1. At the times of cultivation indicated the sulfate and the GSH content of the media were determined, e - - I , G S H as sulfur source; o-----o, G S H + sulfate as sulfur source

growth with GSH as sulfur source was taken up by photoheterotrophic cells from the beginning (Fig. 2 C), whereas sulfate uptake in heterotrophic suspensions was not observed until the GSH supply in the medium was exhausted (Fig. 2 D). From these experiments it appears that in green tobacco cells the net uptake of GSH is inhibited by sulfate, whereas in heterotrophic tobacco cells the net uptake of sulfate is inhibited by GSH. The latter observation may be explained by differences in the action of GSH as an inhibitor on the sulfate translocator of green and chloroplast-flee cells. Therefore, membrane transport of sulfate and the action of GSH on this process were studied. Sulfate transport in green and chloroplast-free tobacco cells. Both heterotrophic and photoheterotrophic tobacco cells transferred from media with sulfate into transport media showed low rates of sulfate transport (100 n m o l - m i n - l . g - 1 dry weight, DW). Transport velocities were enhanced two- to fourfold by pre-culture in sulfur-free media, indicating that endogenous sulfate pools inhibit sulfate transport (compare Smith 1975, 1976). Therefore, experiments were performed with tobacco cells sulfur starved for 48 h. Under these conditions, sulfate transport showed saturation kinetics with a linear period of at least 15 rain (data not shown). Transport velocities were 310_+ 15 nmol. min- a. (g DW)- 1 for photoheterotrophic and 230 4- 5 nmol-min- 1. (g DW)- 1 for heterotrophic cells (see Fig. 3). The rates of transport at pH 7.0 were linear with increasing inoculation densities up to 4 mg DW. (10 ml)-1 (photoheterotrophic cells) and to 10 mg DW-(10 ml)-1 (heterotrophic cells). The temperature optimum of sulfate

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transport was 30~ for both green and chloroplast-free suspensions. At physiological pH, sulfate transport did not exhibit a pronounced optimum but showed highest rates at pH 7.0 (for photoheterotrophic cells) and pH 5.9-6.4 (for heterotrophic cells), respectively. Transport velocities rapidly declined at alkaline pH (Fig. 3). Sulfate transport obeyed Michaelis-Menten kinetics with an apparent K m of 1.60_+0.20.10 -5 M in photoheterotrophic and 1.18 _+0.18.10- 5 M in heterotrophic cells (Fig. 4). The maximum transport velocities calculated from the Lineweaver and Burk (1914) plots in Fig. 4 (green cells" 323-t50 nmol. m i n - 1. (g DW)- 1 ; chloroplast-free cells: 233 _+3 nmol. m i n - 1. (g DW)- 1) were similar to the transport velocities measured under standard conditions. Although several investigators reported multiphasic influx of sulfate with other species (Nissen 1971, 1973, 1974; Holmern et al. 1974; Vange et al. 1974; Shargool and Ngo 1975; Menon and Varma 1982), no evidence for a multiphasic transport in the concentration range of 0.01 1 mM sulfate was obtained in the present study. This result is consistent with previous experiments with heterotrophic tobacco cells (Hart and Filner 1969; Smith 1975, 1976). Experiments with metabolic inhibitors revealed

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Fig. 4. Lineweaver-Burk plot of the effect of sulfate concentration on transport velocity. Photoheterotrophic ( o - - o ) and heterotropbic ( o - - o ) tobacco cells grown as described in Fig. 3 were inoculated into transport media p H 7.0 at a density of 0.2 g + 5% FW-(10 ml)-1. After 4 h of pre-incubation, transport was initiated by addition of 35S-sulfate (3.7 kBq. gmol-1) to the final concentrations indicated. Transport was terminated after 10 min of incubation by addition of 15 ml of 5 m M Na2SO4 and immediate separation of the cells from the medium. Radioactivity of the cells was determined by liquid scintillation counting

that the transport of sulfate in heterotrophic and photoheterotrophic tobacco cells is almost entirely an energy-dependent process. It is inhibited by the uncoupling reagents carbonylcyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), by the ATP-synthase inhibitor N,N'-dicyclohexyl carbodiimide (DCCD), and by the respiration inhibitor azide (Fig. 5). Sulfate transport was completely inhibited by 5-10-5 M DCCD or 5. 10 - 6 M CCCP in heterotrophic as well as in photoheterotrophic suspensions. Azide and DNP were much less effective in inhibiting sulfate transport. The requirement for relatively high concentrations of the latter compounds may be a consequence of their slow entry into the cells at pH 7.0 (Kahn 1974; Smith 1975). From these experiments, sulfate transport into heterotrophic and photoheterotrophic tobacco cells is apparently mediated by active-transport systems. Diffusion of sulfate, that has been observed with tissue cultures of Daucus carota (Cram 1983), apparently can be neglected in the tobacco suspensions. From the present studies, the sulfate translocators of green and of chloroplast-free tobacco cells do not exhibit considerable differences in their kinetic properties. Action o f G S H on the transport of sulfate. Sulfate transport of both green and chloroplast-free tobacco cells was not affected by GSH, when 1.73 nM sulfate and 10-7-2.10-3 M GSH were added si-

72

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multaneously to cells pre-cultured and pre-incubated in sulfur-free media (data not shown). Similar results were obtained with cells pre-cultured in media with 1.73 mM sulfate and pre-incubated in sulfur-free or GSH-containing ( 1 0 - ~ - 2 . 1 0 - 5 M ) transport media (data not shown). Sulfate transport was, however, inhibited by glutathione when the peptide was already present in the media during the 48-h period of the pre-culture (Fig. 6). Concentrations of GSH of 2 . 1 0 - 4 M inhibited the sulfatetransport velocity of heterotrophic tobacco cells by 50%, 10- 3 M concentrations inhibited sulfate transport completely. At the latter GSH concentration, sulfate transport into photoheterotrophic cells still proceeded at more than two-thirds of its

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Fig. 6. The influence of GSH concentration on sulfate transport into tobacco cells. Photoheterotrophic ( A - - A ) and heterotrophic ( o - - o ) tobacco cells grown with sulfate as sulfur source for 6 7 d were caltured in the presence of the GSH concentrations indicated as sole source of sulfur for 48 h. Subsequently, cells were inoculated into transport media pH 7.0 containing the same concentrations of GSH at a cell density of 0.2 g • 5% F W . (10 ml)-1. After 4 h of pre-incubation, transport was initiated by addition of 3SS-sulfate (3.7 kBq-p.mol 2) to a final concentration of 0.1 mM. After 10 min of incubation, transport was terminated by addition of 15 ml of 5 m M Na2SO4 and immediate separation of the cells from the media. Radioactivity of the cells was determined by liquid scintillation co unting

10-7 10-6 10-S'10-z, 10-3 INHIBITOR CONCN.[M] Fig. 5A, B. Influence of metabolic inhibitors on sulfate transport of tobacco cells. Photoheterotrophic (A) and heterotrophic (13) tobacco cells grown as described in Fig. 3 were inoculated into transport media pH 7.0 at a density of 0 . 2 g • FW. (10 ml)-1. One hour before the end of the 4-h pre-incubation period NaN3 ( * - - * ) , D N P ( o - - o ) , CCCP (zx--A), or D C C D (o o) was added to the final concentrations indicated. After 1 h exposure to the inhibitors, transport was initiated by addition of 35S-sulfate (3.7 kBq-~tmol 1) to a final concentration of 0.1 raM. Transport was terminated after 10 min of incubation by addition of 15 ml of 5 m M NazSO4 and immediate separation of the cells from the medium. Radioactivity of the cells was determined by liquid scintillation counting

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[hi Fig. 7. Inhibition of sulfate transport by GSH in heterotrophic tobacco cells. Heterotrophic tobacco cells were grown with sulfate as sulfur source for 6-7 d and pre-cultured with 1 m M GSH as sole source of sulfur for the periods of time indicated. Subsequently, cells were inoculated into transport media pH 7.0, 10- 3 M GSH at a density of 0 2 g • 5% F W . 10 mr- L After 4 h of pre-incubation transport was initiated by addition of 35S-sulfate (3.7 k B q - p m o l -~) to a final concentration of 0.1 raM. After 10 min of incubation, transport was terminated by addition of 15 ml of 5 m M NazSO4 and immediate separation of the cells from the media. Radioactivity of the cells was determined by liquid scintillation counting. The inset shows a semiJogarithmic plot of the initial decline in sulfate transport velocity

maximum velocity. These data show that the sulfate transport of heterotrophic tobacco is inhibited by G S H at physiological concentrations; sulfate transport into green tobacco cells seems to be much less sensitive to GSH. The mode of action

H. Rennenberg et al. : Interaction of sulfate and GSH transport

73

of GSH on sulfate transport does not appear to be direct, as the cells have to be exposed for more than 4 h to the peptide to achieve inhibition of sulfate transport. With increasing time of pre-culture with 1 m M GSH, sulfate-transport velocity of heterotrophic tobacco cells declined exponentially (Fig. 7). After 10 h of pre-culture with 1 m M GSH, maximum inhibition is achieved. Under these conditions the cells had been exposed to GSH for a total of 14 h, i.e. 10 h of pre-culture and 4 h of pre-incubation.

by negative-feedback control (see Rennenberg 1984). Previous investigations have shown that mechanisms regulating synthesis and degradation of carrier proteins control the availability of transport entities for sulfate (Cacco et al. 1980). Further experiments will show whether GSH also plays a role in synthesis and-or degradation of sulfate carriers. However, from the present experiments it cannot be excluded that differences in the internal, cytoplasmic glutathione concentration are responsible for the observed differences in the inhibition of sulfate transport between heterotrophic and photoheterotrophic cells. In previous experiments, photoheterotrophic tobacco cells were successfully used as a model system for leaf mesophyll cells and heterotrophic tobacco cells as a model system for root cells (Rennenberg et al. 1979; Bonas et al. 1982). When the present observations with photoheterotrophic and heterotrophic tobacco cells are transferred to the intact plant, they may provide evidence for a regulatory device for preventing the uptake of excess sulfate by the roots. In the presence of more than optimal amounts of sulfate in the soil, initially excess amounts of sulfate would be taken up by the roots and transferred from the roots to the leaves. In the leaves, excess sulfate can cause surplus synthesis of GSH (Bergmann and Rennenberg 1978; Rennenberg and Bergmann 1979; de Kok et al. 1981). As a consequence, GSH may be translocated to the roots in amounts exceeding the roots' needs for reduced sulfur. In the roots, GSH may accumulate under these conditions and prevent further uptake of sulfate by inhibiting its transport into the root cells. Obviously, such a regulatory system is unable to balance sudden fluctuations in sulfate nutrition and reduction. In the long term it may, however, be able to prevent a plant from taking up sulfate in amounts exceeding its capability for sulfate reduction and storage. Certainly, whole-plant experiments are necessary to test this hypothesis.

Discussion

Numerous investigators have studied sulfate transport in chloroplast-free tissue cultures or roots cells, but little is known about the uptake of sulfate by green tissues or by subcellular compartments of green cells (for a review, see Rennenberg 1984). Nissen (1971) compared the uptake of sulfate by roots and by leaf slices of barley and found similar kinetic properties of the transport systems of both organs. In the present investigation, this observation is confirmed with green and chloroplast-free tobacco cells in suspension culture. The uptake of sulfate by both tissues is reduced by metabolic inhibitors and therefore appears to be an active, carrier-mediated process. Only small differences in temperature and pH dependency were observed between green and chloroplast-free tobacco cells (Fig. 3). In both tissues the uptake of sulfate is mediated by a monophasic transport system (concentration range 10- 5-10- 3 M) with similar apparent K m. From these experiments it appears that green and chloroplast-free tobacco cells do not differ considerably in the kinetic properties of the transport systems of sulfate. Considerable differences were found in the regulation of sulfate transport between green and chloroplast-free tobacco cells. Experiments with growing suspension cultures showed that the net uptake of sulfate is inhibited in the presence of 1 m M GSH in heterotrophic, but not in photoheterotrophic tobacco cells. This result was confirmed in transport experiments, where 1 0 - 3 M GSH completely inhibited sulfate transport in heterotrophic cells. In photoheterotrophic cells, sulfate transport was only reduced by about 25% at this GSH concentration. As a relatively long time of exposure was necessary to obtain inhibition of sulfate transport by GSH, this peptide does not appear to act directly on sulfate-carrier entities. However, this observation is inconsistent with the hypothesis that GSH is one of the reduction products of sulfur metabolism acting on sulfate carriers

Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged. I. Grundel provided excellent technical assistance.

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H. Rennenberg et al. : Interaction of sulfate and GSH transport

of glutathione in suspension cultures of Nieotiana tabacum. Z. Pflanzenphysiol. 88, 175-185 Bonas, U., Schmitz, K., Rennenberg, H., Bergmann, L. (1982) Phloem transport of sulfur in Ricinus. Planta 155, 82-88 Cacco, G., Ferrari, G., Saccomani, M. (1980) Pattern of sulfate uptake during root elongation in maize: its correlation with productivity. Physiol. Plant. 48, 375-378 Cram, W.J. (1983) Characteristics of sulfate transport across plasmalemma and tonoplast of carrot root cells. Plant Physiol. 72, 204-211 de Kok, L., de Kan, P.L.J., Tfinczos, O., Kuiper, P.J.C. (1981) Sulphate induced accumulation of glutathione and frosttolerance of spinach leaf tissue. Physiol. Plant. 53, 435-438 Hart, J.W., Filner, P. (1969) Regulation of sulfate uptake by amino acids in cultured tobacco cells. Plant Physiol. 44, 1253-1259 Holmern, K., Vange, M.S., Nissen, P. (1974) Multiphasic uptake of sulfate by barley roots. II. Effects of washing, divalent cations, inhibitors, and temperature. Physiol. Plant. 31, 302-310 Jones, S.L., Smith, I.K. (1981) Sulfate transport in cultured tobacco cells. Effect of calcium and sulfate concentration. Plant Physiol. 67, 445-448 Kahn, J.S. (1974) Physiological adaption of Euglena graeilis to uncouplers and inhibitors of oxidative phosphorylation. Arch. Biochem. Biophys. 164, 266 274 Lay, M.-M., Casida, J. (1976) Dichloroacetamide antidotes enhance thiocarbamate sulfoxide detoxification by elevating corn root glutathione content and glutathione-S-transferase activity. Pestic. Biochem. Physiol. 6, 442-456 Lineweaver, H., Burk, D. (1914) The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658 666 Logemann, H., Bergmann, L. (1974) EinfluB yon Licht und Medium auf die ,,Plating Efficiency" isolierter Zellen aus Calluskulturen von Nicotiana tabacum var. ,,Samsun". Planta 121, 283-292 Menon, V.K.N., Varma, A.K. (1982) Sulfate uptake in the cyanobacterium Spirulina platensis. FEMS Microbiol. Lett. 13, 141 146 Moss, M.R. (1978) Sources of sulfur in the environment; the global sulfur cycle. In: Sulfur in the environment, part I: The atmospheric cycle, pp. 23-50, Nriagu, J.O., ed. Wiley, New York Nissen, P. (1971) Uptake of sulfate by roots and leaf slices of barley: mediated by single, multiphasic mechanisms. Physiol. Plant. 24, 315 324

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Received 29 January; accepted 11 April 1988