Planta (1984)160:151-158

P l ~ H ~ 9 Springer-Verlag 1984

Expression of the plant sulphite reductase in cell suspension cultures from Catharanthus roseus L. Jens D. Schwenn and Annette Kemena Lehrstuhl fiir Bioehemie der Pflanzen, Abteilung Biologic, ND3/132, Ruhr-Universit/it, Universit/itsstrasse 150, D-4630 Bochum 1, Federal Republic of Germany

Abstract. Sulphur-heterotrophic growth exhibited a dual response to the expression of sulphate-assimilating enzymes. The level of ATP-sulphurylase (EC 2.7.7.4) appeared "repressed" while sulphite reductase (EC 1.8.7.1) and O-acetyl-L-serine sulphhydrylase (EC 4.2.99.8) were "derepressed" and coordinated in their occurrence. The capability of the cells to reduce adenylylphosphosulphate or 3'phospho adenylylphosphosulphate to cysteine coincided with the activity of sulphite reductase. The expression of these reducing steps lacked correlation with the regulation of ATP-sulphurylase. Key words: Catharanthus- Enzyme regulation (sulphite reductase) - Sulphate reduction - Sulphite reductase.

Introduction Homogeneous sulphite reductases (EC 1.8.7.1) have recently been obtained from spinach leaves by Tamura et al. (1978), Aketagawa and Tamura (1980) and by Krueger and Siegel (/982). The purified enzyme resembled the haemoprotein subunit of the bacterial sulphite reductase (EC 1.8.1.2) in the size of its monomer and in the content of prosthetic groups, e.g. one sirohaem per subunit. It was found devoid of the flavoprotein moiety which only enables the bacterial enzyme to accept electrons from NADPH. The absence of flavines confirmed the earlier data of Asada (1967) and Hennies (1975), who showed that the plant enzyme required ferredoxin rather than pyridine nucleotide as the ultimate electron donor. Abbreviations: APS = adenylylphosphosulphate; MVH = reduced methylviologen; OAS = O-acetyl-L-serine; PAPS = 3'phospho adenylylphosphosulphate

In the past, the physiological significance of a plant sulphite reductase has been questioned by the finding that intact chloroplasts from spinach did not form free sulphite as an intermediate (review: Schwenn and Trebst 1976) when labelled sulphate was administered under conditions otherwise suitable for assimilatory sulphate reduction. Investigations by Schmidt (1973) and Schiff and collaborators (Abrams and Schiff 1973, Schmidt et al. 1974) of assimilatory sulphate reduction in the green alga, Chlorella, led to the proposal of a thiosulphonate reductase as the physiologically relevant enzyme in photosynthetically active organisms rather than the sulphite reductase (survey: Schiff and Fankhauser 1980). This view was extended by the identification of S :sulpho glutathion (Tsang and Schiff 1978) as the intermediate between adenyl phosphosulphate (APS) and the amino-acid sulphur. The reduction of adenylylsulphate by partially purified extracts in vitro and by intact algae in vivo yielded carrier-bound sulphite (designated as "CarS:SO3H" by Abrams and Schiff (1973)). In Chlorella the carrier has been proposed to be identical with glutathione (Tsang and Schiff 1978) where S-sulphoglutathione then represents the intermediate. The sulphite reductase, if present, was suggested to act as a bypass of the main assimilatory path of sulphur through the thiosulphonate reductase requiring the bound sulphite as substrate (Schmidt et al. 1974). The absence of free sulphite during the higher-plant sulphate assimilation was supported by Urlaub and Jankowski (1982) who described a large particulate aggregate of enzymes from Catharanthus cell suspension cultures which reduced adenylylsulphate to sulphide sulphur (incorporated into cysteine) without formation of the free sulphite. Yet, upon prolonged storage of the isolated complex, a ferredoxin-dependent sulphite reductase was released while con-

152

J.D. Schwenn and A. Kemena: Expression of plant sulphite reductase

comitantly the particles lost their capability to reduce the adenylylsulphate. In the present paper we investigated the level of sulphite-reductase activity in comparison with the level of "APS-reducing activity" in two different cell lines from C a t h a r a n t h u s roseus L. In addition, ATP-sulphurylase and O-acetyl-L-serine (OAS) sulphhydrylase activity were monitored in order to gain a more complete view of the enzymatic status of S-metabolism in each of the cell strains. The differences in the time course and in the level of enzyme activity have been discussed in relation to the physiological role proposed for each enzyme. Since the enzymatic steps of the assimilatory sulphate reduction have recently been proposed to be correlated with the activity of enzymes involved in the assimilation of inorganic nitrogen (Reuveny et al. 1980), we also investigated the development of the nitrate-, nitrite-reductase activity and of the ammonia-assimilating enzyme, glutamine synthetase. Material and methods Growth o f cell suspension cultures. Sulphur-autotrophic cells from Catharanthus roseus L. (3 Don were grown as described by Zenk et al. (1977). For S-heterotrophic growth the sulphate was replaced by sodium sulphide (1.5 mmol 1-1) 1. The cells were assayed only after adaptation to growth on the reduced sulphur for a minimum of six passages (approximately 60 d). Extraction o f soluble protein from the cells. Washed cells were dried briefly on a Biichner funnel and homogenized (1 g of fresh weight per 1 ml) in phosphate buffer (50 mmol 1-1 KH2PO 4 KzHPO4, pH 7.7), 10 mmol 1- a/%mercaptoethanol, I mmol l-1 Na2S205, 0.5 mmol 1-1 ethylenediaminetetraacetic acid (EDTA) and 0.01% Triton X-t00. The homogenate was allowed to rest for a period of 15 rain at 0 ~ C prior to centrifugation (10 rain at 48,000 g). The supernatant was precipitated with saturated ammonium-sulphate (pH 6.8, containing I mmol 1-1 EDTA) at a final concentration of 75% saturation. The crude protein was collected by centrifugation, resuspended in phosphate buffer (50 mmol 1-1, pH 7.7), clarified by a second centrifugation and desalted by gel filtration (Biogel P4, 1.2x 16 cm, BioRad Laboratories, Miinchen, FRG), The protein-containing fractions were pooled and used immediately for the assay of enzymes without further concentration. The E D T A had to be omitted from the buffers after resuspension because of the low activity of glutamate dehydrogenase, see below. The protein content was determined according to Bradford (1976). Assay o f enzymes. The enzyme ATP-sulphurylase (EC 2.7.7.4) was determined as described previously (Schwenn et al. 1979) in its reverse reaction. Extracts from the cells were desalted in the presence of 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1 buffer (50 mmol 1-1, pH 7.5) instead of phosphate buffer. O-Acetyl-L-serine sulphhydrylase (EC 4.2.99.8) was assayed according to Becker et al. (1969). The reaction mixture

1 Sulphide is converted to elemental sulphur during sterilization of the nutrient

contained: 80mmol 1-1 Tris-HC1 (pH7.5), 0.8mmol 1-1 EDTA, 25 mmol 1-10-acetyl-L-serine if not specified otherwise and 50-200 ~g protein. Cysteine thus formed was determined spectrophotometrically as described by Gaitonde (1967). For the assay of the sulphite reductase, purified OAS sulphhydrylase was used from C. roseus grown on limiting amounts of sulphate (0.5 mmol 1-1). The cells were harvested after 4 d, washed and homogenized in 50 mmol 1-1 Tris-HC1 buffer (pH 7.5), 10 mmol I- 1 fl-mercaptoethanol, 0.5 mmol 1-1 E D T A and 1 mmol 1-1 Na2S20 5. The homogenate was clarified by centrifugation and the supernatant was precipitated with ammonium sulphate (containing 1 mmol 1-1 EDTA) to make a final concentration of 40% saturation. The precipitate was dissolved in Tris-HC1 buffer (no additives) and chromatographed on Sephacryl S 200 ( ~ 2 . 8 x 5 4 c m , Pharmacia, Freiburg, F R G ) equilibrated with Tris-HC1 as above. The most active fractions were rechromatographed on Sephacel ( ~ 1.6 x 10 cm, Pharmacia). The column was developed with a linear gradient of NaC1 from 0 to 300mmol 1-1 in 10mmol 1-1 Tris-HC1 (pH 7.5). The enzyme fractions were precipitated with ammonium sulphate (80% saturation) resuspended in Tris-HC1 buffer and chromatographed on Sepharose 6B ( ~ 1.6 x 32 cm, Pharmacia). The specific activity usually obtained was in the range of 150 to 100 nkat per mg enzyme. The isolated enzyme remains stable for several weeks at - 2 0 ~ without loss of activity. Sulphite reductase (EC 1.8.7.1) was assayed according to Hennies (1975) with reduced methylviologen (MVH) as electron donor. The dye was reduced anaerobically in a home-made glass apparatus containing two platinum electrodes to which a constant electric voltage was applied. The sulphite-dependent oxidation of the MVH was followed at 604 nm in a stoppered cuvette. Additions to the sample (which was made anaerobic by repetitive evacuation and flushing with deoxygenated nitrogen) were injected through the rubber seals with the aid of a microsyringe. Routinely, the reduction of sulphite was coupled to the biosynthesis of cysteine in the presence of purified OAS-sulphhydrylase and OAS. The standard assay contained: t 0 0 m m o l 1-1 phosphate buffer, 0.75 mmol 1-1 sodium sulphite, 40mmol 1-10-acetyl-L-serine, 100-120 nkat purified OAS-sulphhydrylase from C. roseus (representing a 103-fold excess over the sulphite reductase) and sulphite reductase protein 0.5 to 1.0 mg. Adenylylphosphosulphate- and 3'-phospho adenylylphosphosulphate (PAPS)-reducing activity was measured as the formation of [35S]cysteine from 3SS-labetled APS or PAPS in the presence of the OAS-sulphhydrylase system described above. The activity was isolated as the particulate fraction from the cells as published by Urlaub and Jankowski (1982). Reduced ferredoxin was used as the electron donor for the reducing step. The reaction was conducted in 33 mmol 1 1 Tris-HC1 (pH 7.7), 6 mmol 1-1 MgC12, 10mmol ~1-1 glucose-6-phosphate, 1 mmol 1-1 NADP+, 16.6 nkat glucose-6-phosphate dehydrogenase, 33 mmol 1 - 1 0 A S , 8.8 nkat OAS-sulphydrylase, 80 gmol 1 1 [35S]_Ap S (specific activity: 500Bq nmol -x or 80 lamol 1 a [35S]PAPS (specific activity: 266 Bq nmol -~) and enzyme protein 0.7 to 1.5 mg. The APS-assay contained additional glutathione (red) (1.7 mmol 1-1). Ferredoxin was reduced during a preincubation period of 20 min, thereafter the sample was rendered anaerobic and started by the introduction of 35Slabelled APS or PAPS. The reaction was terminated after 30 min with 2 N HC1 and assayed for [35S]cysteine after addition of 40 gmol of unlabelled carrier cysteine. The amino acid was separated from the assay mixture by absorption to Dowex W50 X8, H + (Serva, Heidelberg, FRG). After elution, the labelled cysteine was determined quantitatively in a liquid scintillation counter with appropriate correction for losses and counting efficiency.

J.D. Schwenn and A. Kemena: Expression of plant sulphite reductase Nitrate-reductase activity was determined according to Maldonado etal. (1978). Because of the low activity, 2.5-3.5 mg of plant extract had to used in the assay. Nitrite-reductase activity was measured under conditions identical to those for the sulphite reductase. Non-enzymic oxidation of M V H was counteracted by the introduction of bovine serum albumin. The reaction mixture contained 100 mmol 1 1 phosphate buffer (pH 7.7), 2 g 1 1 bovine serum albumin, 0.16 mmol 1 1 reduced methylviologen and 50 to J00 lag ml 1 enzyme protein of assay. Gtutamate-dehydrogenase activity was extracted in the absence of E D T A and assayed, in general, as described by Bergmeyer (1970). Since only relative amounts of enzyme under the two different growth conditions have to be known for the comparison of their response to both sulphur supplies no attempts were made to activate the enzyme prior to the measurements. Either N A D P H or N A D H at 0.5 mmol 1- t were used to determine the activity from the desalted ammonium-sulphate precipitate containing 50 to 1000 pg of protein. Glutamine-synthetase activity was assayed as described by Shapiro and Stadtmann (1970) in 50 mmol 1-1 imidazol-Hcl buffer (pH7.2), 100mmol 1-1 glutamate, 100retool 1-1 NH2OH, 10 mmol 1-1 ATP, 20 mmol 1-1 MgC12, 25 mmol 1-1 fl-mercaptoethanol and 0.3 to 2.0 mg of protein. The amount of glutamine:~-ketoglutarate-aminotransferase activity in the cells was determined by the method of Miflin and Lea (1975) using the same concentrations of protein as for the glutamine-synthetase activity.

Results

While ATP-sulphurylase and OAS-sulphhydrylase activity in the cell homogenates can be determined routinely by reliable standard procedures, it became necessary to improve the determination of sulphite-reductase activity for two main reasons: (a) the enzyme from C. roseus is apparently not readily soluble but is integrated into particles (Urlaub and Jankowski 1982), (b) the rate in vitro may also be limited by the release of its product as observed for the enzyme from Escherichia coli (Rueger and Siegel 1975; Janick et al. 1983). The sulphite reductase was most effectively extracted from the plant cell material when Triton X-100 had been added to the homogenizing buffer (Fig. 1). Nitrite reductase was also recovered at low (0.1%, v/v) concentrations while the inhibitory effect upon the OAS-sulphhydrylase was still neglectable. Most of the detergent was removed by precipitating the protein with ammoniumsulphate and by gel filtration so that it could not interfere with the assays. O-Acetyl-L-serine was observed to stimulate the activity of partially purified sulphite reductase 2. The routine assay was complemented with purified OAS-sulphhydrylase and O-acetyl-L-serine (Fig. 2). This coupled assay determined the ac2 This stimulation was lost after removal of OAS-sulphhydrylase activity by gel and ion-exchange chromatography; details not shown

153

I 1,5

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2,0

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0 011

015

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Fig. 1. Solubilization by Triton X-100 of sulphite reductase, nitrite reductase and OAS-sulphhydrylase from Catharanthus roseus cell homogenates. Basal sulphite-reductase activity can be stimulated by OAS due to endogeneous OAS-sulphhydrylase present in the crude cell extracts. Triton X-100 at 0.1% has been used for further work as a compromise between enzyme recovery and apparent activity, o - - o NO 2 reductase [102 nmol M V H ox.ml 1 extract, min-1]; n - - n SO 3 reductase [10 nmol M V H ox-m1-1 extract.min-1]; m - - m residual SO 3 reductase [nmol M V H ox-m1-1 extract-min 1]; ~ , - - o OAS-sulphhydrylase [lamol cysteine, m l - 1 extract, r a i n - 1]; right sc. : x - - x protein ling. m l - 1]

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ratio of 0AS su[phhydrytase fo S0~reducfase Fig. 2. Activation of sulphite-reductase activity by the complete sulphide-trapping system consisting of purified OAS-sulphhydrylase and OAS. Inset: Saturation by OAS of different amounts of sulphite reductase in the absence of purified OAS-sulphhydrylase; o - - o 120 gg, n - - n 270 lag and a - - ~ 350 lag of protein after precipitation and gel chromatography

154

J.D. Schwenn and A. Kemena: Expression of plant sulphite reductase

3,0-

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1,570

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-30

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0,5-20

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age of cul.fure [days]

age of culture [days]

Fig. 3. Growth of Catharanthus roseus L. cells in batch suspension cultures on sulphate or sulphide as source of sulphur. e - - o Content of sulphate in the growth medium, closed symbols correspond to values of S-autotrophic and open symbols to values of S-heterotrophic growth conditions as described in Material and methods. D - - D protein [mg-ml-~]; e - - o sulphate [mmol-1 1]; right sc. : o - - ~ dry weight per culture [g-100 ml 1]

Fig. 4A, B. Specific activity of sulphur-assimilating enzymes as exhibited under A S-autotrophic and B S-heterotrophic growth in the batch culture. Activities were determined from at least two different growth periods in triplicates. + - - + APS reducing - ; x - - x PAPS - activity [nmol.mg -1" 60 m i n - 1] ; o - - o OAS sulphhydrylase [gmol, m g - 1 m i n - 1] ; D - - D sulphite reductase [10 nmol mgmin]; right sc.: o - - o ATP sulphurylase [nmol . m g - 1. rain- 1]

tivity of sulphite reductase as the formation of cysteine from the sulphide of the reaction product of the enzyme. An excess of 800 to 1,200 arbitrary units of OAS-sulphhydrylase over sulphite reductase was required to render the rate of reduction independent of the sulphide-trapping system. In the presence of high concentrations of OAS (30 mmol 1-1), the formation of cysteine became proportional to the oxidation of methylviologen with a stoichiometry of 5.5 to 6.5 mol MVH oxidized per mol cysteine formed. Up to 1.2 mg protein from the cell extract could be measured in this assay, provided that the concentrations of MVH and of sulphite were saturating (apparent Km for MVH:70" 10 . 6 mol 1-1, apparent Km for SO3:250.10 -6 mol 1-1, details not shown). The assay was suitable for following the rates even when the content of protein in the cells changed drastically during the growth period (Fig. 3). The content of protein of the S-heterotroph culture exceeded the S-autotroph culture considerably with a maximum of extractable protein between the third and fifth day of growth. Yet, the production of dry matter in both cultures appeared little affected by the different sulphur supplies. In accordance with our previous observation, the level of ATP-sulphurylase and of OAS-sulphhydrylase

(Schwenn et al. 1979; Bergmann et al. 1980) passed through a relatively small maximum 3 during the early phase of logarithmic growth (Fig. 4A). It deteriorated in the S-autotrophs within 4 d. Thereafter, the specific activity of both enzymes was maintained at a low level ("repressed") until after the ninth day of growth where it regained its original height. Compared with these enzymes, the sulphite-reductase activity developed inversely: a minimum of activity was observed at the beginning of logarithmic growth and a maximum when the cells reached their stationary phase. Growth on sulphide instead of sulphate caused the content of OAS-sulphhydrylase to increase rather than decrease (Fig.4B). While the level of ATP-sulphurylase exhibited a constantly reduced level under these S-heterotrophic conditions, the OAS-sulphydrylase exceeded the autotrophic enzyme level by 60% after 3 d of growth; thereafter, it dropped rapidly to the original content with a time course comparable to the S-autotrophs. The development of sulphite reductase was not seriously altered under these conditions, yet, the level of this enzyme was increased by up to 96% (after 4 d). The time 3 The time required to attain this maximum was observed to be dependent upon the amount of cells used for inoculation

J.D. Schwenn and A. Kemena: Expression of plant sulphite reductase

course of developing sulphite reductase was mirrored by the particulate adenylylsulphate-reducing activity. However, the latter represented not more than 0.1-0.2% of the sulphite-reductase activity throughout all phases of growth. Enzyme levels in a batch culture are known to change rapidly during growth (Yeoman et al. 1977). Hence, regulatory phenomena which are interpreted as a consequence of the changed growth condition, have to be seen in relation to the enzyme level as it occurred in the original culture. This normalization of enzyme levels from the S-autotroph cultures in comparison with the S-heterotroph culture showed that the ATP-sulphurylase activity was "repressed" until the end of the logarithmic growth with a small increase towards the stationary phase (Fig. 5). The increase coincided with the exhaustion of the sulphate from the medium. The OAS-sulphhydrylase, as the final enzyme in the sequence of enzymatic steps in this pathway, went through a maximum of activity at the early phase of logarithmic growth (third day) and then adjusted to the level of activity of the ATP-sulphurylase. Sulphite-reductase activity showed its maxim u m closer to the middle of the logarithmic growth (fourth day). It began to decline shortly before the onset of the stationary phase of growth. Rather unexpectedly, the level of sulphite-reductase activity appeared "derepressed" during the whole period of growth on sulphide. An apparent derepression was also observed for the N-assimilating enzymes when sulphate was replaced by sulphide, although the supply of nitrogen was not changed. The nitrite reductase was found to be most active during the early phases of growth (first to fourth day), whereby the growth on sulphide increased the level of the enzyme by 56% (mean value for the complete growth period of 10 d, Fig. 6). Its activity was following a time course which can be described as antagonistic to the sulphite reductase. Nitrate-reductase activity developed with a time course different from that of nitrite reductase in that the maximum level of enzyme occurred at the end of the logarithmic growth. Of the ammonia-incorporating enzymes, glutamine-synthetase activity developed with a time course resembling that of the nitrite reductase. Sulphur-heterotrophic growth raised the level of this enzyme by 210% during early logarithmic growth. After passing through a minimum (fifth to sixth day), it adjusted at 105% above that of the S-autotrophs (eighth day). The time course of glutamate-dehydrogenase activity in the cells, however, remained virtually unaffected by the change in the sulphur supply. On average, the level of ac-

155 2,0

1,0

0

,

0

2

3

~

6 age of culfure

8 9 [days]

Fig. 5. Changes in enzyme activity relative to the activity during S-autotrophic growth when sulphate is replaced by sulphide (data from Fig. 4A, B). o: ATP-sulphurylase, D: sulphite reductase, : OAS-sulphhydrylase

~3,0-

=tO2 , 0 c-

1 ,01 0 0 age ofcutture

[days]

Fig. 6. Changes in the activity of nitrogen-assimilating enzymes as caused by the different sulphur supplies. The data are obtained from two enzyme patterns (as described in the legends to Figs. 4 and 5) and treated by using the activity under Sautotrophic growth for normalization, o: Nitrate reductase, + : nitrite reductase, o: glutamine synthetase, D: glutamine:~ketoglutarate-aminotransferase activity

tivity was reduced by 25% in the S-heterotrophs. No changes in the ratio of NADH- and NADPHdependent activity have been observed. Discussion

The physiological importance of an assimilatory sulphite reductase in algae or higher plants has been doubted in principal for three reasons: (a) Chlorella wild type and mutants were reported to reduce a carrier-bound sulphite (designated "CarS:SO3H") using a thiolsulphonate reductase in preference to free sulphite using sulphite reductase (Schiff and Fankhauser 1980); (b) isolated intact chloroplasts, as the major site of assimilatory

156

J.D. Schwennand A. Kemena: Expressionof plant sulphitereductase

sulphate reduction by higher plants, did not form free sulphite from sulphate which sulphite reductase would require as substrate (Schwenn and Trebst 1976) and (c); sulphite reductase did not exist per se in cell extracts from Catharanthus cell cultures but appeared to be released from a particulate enzyme complex (designated "APS-reducing activity"; Urlaub and Jankowski 1982) when sonicated or exposed to low ionic strength. In the present investigation of C. roseus cell cultures, we compared the level of sulphite-reductase activity with the particulate adenylylsulphatereducing activity as they were expressed during growth of the culture under S-autotrophic or Sheterotrophic conditions. As judged from the activities observed in vitro, the level of APS-reducing activity was considerably lower than the level of sulphite reductase. The amount of sulphite reductase exceeded the amount of this complex by a factor of 102 when assayed under optimized reaction conditions. This relative excess of sulphite reductase has previously been observed, although to a much smaller extent, with the purified APS-reducing particles. When frozen or sonicated they released twice as much sulphite-reductase activity than they contained APS-reducing activity before the treatment (Urlaub and Jankowski 1982). The authors suggested that the low recovery of APSreducing activity from the Catharanthus cells could have been the consequence of a slow decomposition of the complex during isolation or storage. Since sulphite reductase was liberated from this complex while concomitantly the capability to reduce APS to cysteine was lost, sulphite reductase may indeed have been necessary for the reduction. The large excess of sulphite reductase as observed in this investigation may be attributed to the use of detergent. In this respect, the possibility should not be overlooked that extraction of sulphite reductase with this detergent may have increased the apparent activity by activation and by a more efficient isolation. An appreciable excess of sulphite reductase, however, would make it difficult to render the rate of APS reduction dependent on this particular activity as a rate-limiting step. Moreover, a high amount of sulphite reductase as an obligate component of an APS-reducing enzyme complex would scarcely allow free sulphite to accumulate as its intermediate if not liberated "accidentally" as bound sulphite by exogeneous thiols. Tsang and Schiff (1976) have already shown that during the transfer of the activated sulphate group from APS onto a not-yet-identified protein (APS-sulphotransferase activity?) S-sulpho compounds can be released.

A proposal of the functional and presumably structural relationship between sulphite reductase and the APS-reducing activity is strongly indicated by the coincidence in the time course of their expression under S-heterotrophic growth conditions. The reduction of APS and of PAPS or sulphite to sulphide was synchronously increased towards the end of the logarithmic growth. In this respect, the reducing steps resembled the OAS-sulphhydrylase which appeared derepressed at a maximum approx. 24 h before the sulphite reductase. The ATP-sulphurylase remained repressed throughout the logarithmic growth with a minor increase towards the end of the stationary phase. According to previous observations (Reuveny and Filner 1977; Schwenn et al. 1979), the ATP-sulphurylase remains repressed under S-autotrophic conditions, whereas sulphate deficiency seems to initiate a derepression (Bergmann et al. 1980). Symptoms of a repression have been obtained in Catharanthus when the inorganic sulphate was replaced by high concentrations of cysteine or methionine while lower concentrations also led to a derepression of the ATP-sulphurylase and of the enzyme(s) involved in the formation of the intermediary sulphite (Schwenn et al. 1979). The apparent derepression of sulphite reductase and of OAS-sulphhydrylase, as observed in the present investigation, could have imaged this type of derepression when sulphide is used as source of sulphur. Yet conditions of sulphur starvation cannot completely be excluded because of the tendency of sulphide to evaporate at neutral pH or to oxidize to elemental sulphur. Despite this experimental limitation, a coordinate expression of sulphite reductase and OAS-sulphhydrylase may exist in plants. It is noteworthy, that Catharanthus cells lack correlation between the "reducing" and "activating" enzyme(s). This finding may have further importance for the plant cell because the path of sulphate is branched into the formation of sulphate esters and in the biosynthesis of cysteine. However, the absence of a correlated expression has also been observed to occur in Salmonella typhimurium (Kredich 1971). Reuveny et al. (1980) proposed that the regulation of cysteine biosynthesis is correlated with the regulation of nitrogen assimilation in tobacco cell cultures. We have examined the expression of enzymes involved in the assimilatory reduction of inorganic nitrogen in S-autotroph and S-heterotroph C. roseus cell cultures. The nitrate-reductase activity appeared most drastically affected by the change in the sulphur supply with a nearly threefold increase in specific activity during the early phase of growth. The activities of nitrate reductase, hi-

J.D. Schwenn and A. Kemena: Expression of plant sulphite reductase

trite reductase and glutamine synthetase coincided in their time course of activity, indicating a coordinate type of expression. All three enzymes showed a rapid decay with a minimum of activity towards the end of the logarithmic growth. It is noteworthy, that sulphite- and nitrite-reductase activity were expressed as antagonists. This different expression of activity may be seen in support of the recent proposal by Krueger and Siegel (1982) t h a t - based on molecular and immunological diversity - both enzymes represent two distinct molecular entities in the plant. In view of the reciprocal connection between the sulphur and nitrogen assimilating pathways, however, our data disagree with the proposal of a positive control of the initial steps, e.g. ATP-sulphurylase and nitrate reductase. The latter appeared derepressed to a maximum when the ATP-sulphurylase was completely repressed. Since cysteine or methionine in S-heterotrophically grown cell cultures were below detection (Schwenn et al. 1983) they may be excluded as regulating end products in that they repress the ATP-sulphurylase and derepress the nitrate reductase. On the other hand, basic amino acids, in particular arginine, accumulated in the S-heterotrophs three to nine times above the level of S-autotrophs after 5 d of growth without effect upon the expression of the ATPsulphurylase. In conclusion, we will have to regard regulatory models of sulphate metabolism in plant cell cultures like Catharanthus as inadequate as long as they are based exclusively on the determination of ATP-sulphurylase activity, in particular because of the lack of correlation between the activating and the reducing steps. Further work is needed which will have to show the biosynthesis of m R N A corresponding to each individual activity in order to elucidate the regulatory pattern of sulphate-assimilating enzymes in plants. We gratefully acknowledge the financial support by the Deutsche Forschungsgemeinschaft. We also have to thank G. Jankowski for her help in the preparation of the particulate APS-reducing activity.

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Received 8 June; accepted 22 August 1983