Planta 143, 225-231 (1978)

P l ~ n ~ J 9 by Springer-Verlag 1978

Sulfite: Preferential Sulfur Source and Modifier of C 0 2 Fixation in Chlorella vulgaris Gian Franco Soldatini*, Irmgard Ziegler, and Hubert Ziegler Lehrstuhl ffir Botanik, Technische Universitfit Mtinchen, and Institut ftir Toxikologie und Biochemie der Gesellschaft ftir Strahlenund UmweltforschungmbH, Arcisstral3e 21, D-8000 Mtinchen 2, Federal Republic of Germany

Sulfite was added at the time of inoculation to a standard and to a sulfate deficient medium of C h l o r e l l a v u I g a r i s . It was not only used as a sulfur source, but besides this, at concentrations < 1.0 mmol 1- 1, the growth yield was enhanced up to 30% compared to sulfate saturated conditions. Higher sulfite concentrations increasingly inhibited cell growth. Growth rate determinations indicated that the enhancement, and the inhibition respectively, were confined to the very beginning of culture growth; the time period during which the sulfite was not yet oxidized (5-10 h). In contrast, an increased CO2 fixation rate/unit of protein, occurring up to 5.0 mmol l-1 sulfite and a shift towards the 3-carboxylation pathway, are persisting at least during the growth period of 4 days. The preferential uptake of sulfite, also indicated by a marked increase in methionine content of algal protein, presumably causes an increase in thylakoidal sulfolipids, and is such modifying the CO2 fixation. Abstract.

Key words: C h l o r e l l a - COz fixation - Growth yield Methionine - 02 evolution - Sulfolipids - Sulrite action.

Introduction

Sulfite is now considered to be the actual principle site of SO2 action on cell metabolism. In concentrations exceeding 1 mmol 1- x, it reduces photosynthetic CO2 fixation by inhibiting the Calvin cycle enzymes, *

Present address." Istituto di Chimica Agraria, Universit~i Pisa,

Italy Abbreviations." PGA = 3-phosphoglycericacid; APS = adenosine 5'-

phosphosulfate; PEP = phosphoenolpyruvate

e.g. ribulosebisphosphate carboxylase (Ziegler, 1972). In some respects, sulfite has a unique position among toxic compounds; this is manifested in isolated chloroplasts, exposed to non-inhibitory sulfite concentrations of < 1.0 mmol 1- 1. Firstly, it is preferentially taken up by the phosphate translocator of the inner chloroplast membrane (Hampp and Ziegler, 1977), and during illumination, its uptake and binding to the thylakoids is nearly double that of sulfate (Ziegler and Hampp, 1977). Thereby it bypasses the regulatory step of ATP-sulfurylase, which may result in uncontrolled incorporation and sulfur overload (Ziegler and Hampp, 1977). Secondly, in cooperation with the superoxide radicals, which originate in the course of photosynthetic electron transport, it maintains photophosphorylation under N A D P H saturating conditions and thus enhances CO2 fixation (Ziegler and Libera, 1975). It is known that long term exposure to low concentrations of SO2 may result in higher yields, especially under sulfate deficient conditions (e.g. Faller, 1968; Cowling and Lockyer, 1976). However, when 14CO 2 was fed to isolated spinach chloroplasts at low sulfite concentrations, the percentage distribution of ~4C had shifted towards the ~-carboxylation pathway (Libera etal., 1975); thus, metabolic changes are already caused by sulfite at "non-deleterious" concentrations. In order to evaluate the consequences of stimulatory sulfite concentrations, which are found at the level of isolated chloroplasts, and their subsequent effects on total metabolism and growth of an organism, C h l o r e l l a v u l g a r i s was selected as a model. Quantitative effects on growth yields, CO2 fixation capacity, photosynthetic 02 evolution, percentage distribution of fixation products, and amino acid composition were examined in this study.

0032-0935/78/0143/0225/$01.40

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Material and Methods Culture Methods Chlorella vulgaris (Sammlung Algenkulturen, G6ttingen) was cultivated in the medium described by Kuhl and Lorenzen (1964) at 28 ~ C and 8000 lx in 800 ml vessels (see Fig. 4 in Kuhl and Lorenzen, 1964). They were aerated by passing either air ("air-grown algae") or air+5% CO2 ("CO2-grown algae") from the bottom of the flasks with saturating flow rates. In the sulfur deficient medium, MgSO4 was replaced by MgC12. Sulfite was added after sterilization of the growth medium, together with the inoculation of the algae.

Protein and Chlorophyll DetermNation 3-5 ml of the algal cultures were centrifuged. Protein was determined according to the naphthalene blue-black procedure. Cells, frozen with liquid nitrogen, were resuspended in 50 gl H20, spotted on Whatman I (squares of 2 x 2 cm), and further handled as described by Bramhall et al. (1969). For chlorophyll determination, the algae also were frozen with liquid nitrogen, 5 ml abs. methanol was added and the concentration was calculated from the absorption data according to Vishniac (1957).

14C02 Fixation, 02 Evolution After centrifugation, the algae were resuspended in 0.1 mmol 1 1 KFI2PO4+I.0 mmol 1-1 KNO 3 (Bassham and Kirk, 1973). Aliquots corresponding to 0.01-0.02 mg chlorophyll/ml were kept either in the dark or were preilluminated for 15 min (Ultraphot lamp, 15,000 Ix); NaH14CO3 was added to give a final concentration of 4.83 gmol HCO~-/1.5 ml (with 10 gCi). After 3 min the assay was stopped with ethanol. After evaporation with compressed air, the extraction procedure of Feige et al. (1969) was used. The percentage distribution of products was determined with the same methods as with chloroplasts (see Libera et al., 1975). Photosynthetic 02 evolution was measured with a Clark type 02 electrode as described by Delieu and Walker (1972).

Sulfite Determination To follow the oxidation rate of sulfite in the medium, aliquots of 50 ml were removed and the concentration was measured with a sulfite specific electrode (Orion Research, Digital ionanalyzer 801 1). In parallel, iodometric titrations (see Roy and Trudinger, 1970) were performed.

Automated Amino acid Analys& The protein was hydrolized in 6 N HC1 and the hydrolysate was purified with Celite and by ion exchange chromatography on Dowex 50, W X 8. Thereafter it was dissolved in a total of 2 ml 0.2 N citrate buffer containing 2.5 ~tmol norleucine as the internal standard. An aliquot was run in a Beckman Multichrome Amino acid Analyzer with program 7.

Chemicals Naphthalene blue-black was from Serva. NaH14CO3 was supplied by Amersham-Buchler. All other chemicals were analytical grade

G.F. Soldatini et al. : Sulfite: Preferential Sulfur Source from Merck. In a set of experiments, it was affirmed that the maximum arsenic content indicated for the sodium sulfite (analytical grade) of 0.0001% had neither a stimulatory, nor an inhibitory action. All other trace metals were already in optimum concentrations in the standard medium.

Results

Growth Yield and Growth Rates

Under the experimental conditions used (standard medium, a i r + 5 % CO2), an initial cell density of 125-150x 103 cells/ml, after 4 days resulted in a growth yield of about 15 x 106 cells/ml, with about 60 txg protein/ml and 3.0 gg chlorophyll/ml. A reexamination of sulfate concentrations needed for optimum growth showed a linear increase up to 0 . 2 m m o l l - t , where saturation conditions were reached. This value is in agreement with that given by Buetow (1965). Thus, the sulfate concentration in the standard medium (1.0 mmol 1-t) is already at a saturation level. Further increase (up to 20.0 mmol 1- 1) had no effect. Addition of sulfite at concentrations < 1.0 mmol 1- ~ clearly enhanced cell numbers as well as protein and chlorophyll yields (Fig. 1). This enhancement occurs irrespective of whether the algae were grown in air or in a i r + 5 % CO2; even the percentage of increase is higher, as is also the growth yield of the control. In the sulfate deficient growth medium, the stimulation by sulfite (compared to controls with optimum supply of sulfate sulfur) is further accentuated and the optimum enhancement is shifted from 0.25 to 1.0 mmol 1- 1 (Fig. 2). If unbuffered sulfite is added to the standard medium, at a concentration of 1.0 mmol 1- 1, it raises the pH from 5.8 to 6.0 (Fig. 3). Elimination of these pH differences by preceding neutralization of the sulfite, and thus keeping the culture medium at the initial pH of 5.8, does not change the growth stimulatory effect, but drastically enhances the inhibitory one (see Fig. 2). This indicates that HSO3 , which predominates at pH 8 0 % at pH >7.5 (Puckett et al., 1973). Whereas at stimulatory sulfite concentrations tl~ere is no change in the ratio of cell numbers/ protein/chlorophyll, at damaging concentrations the ratio protein / chlorophyll is increased, indicating a preferential destruction of chlorophyll. These changes in growth yield, described above, were measured 4 days after inoculation. The cell numbers, taken at different intervals (Fig. 4) demon-

G.F. Soldatini et al. : Sulfite: Preferential Sulfur Source

227

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s t r a t e t h a t t h e i n i t i a l g r o w t h r a t e o f t h e c u l t u r e (i.e. t h e r a t e b e t w e e n 0 - 4 8 h ) is d r o p p i n g m u c h f a s t e r i n t h e s u l f i t e e n h a n c e d c u l t u r e s d u r i n g 4 8 - 1 2 0 h, b u t is s t r o n g l y i n c r e a s i n g w i t h t i m e a t i n h i b i t i n g s u l f i t e c o n c e n t r a t i o n s ( F i g . 4, i n s e r t ) . T h i s i n d i c a t e s t h a t t h e periods of enhancement and of inhibition are confined to the very beginning of the culture growth. This v i e w is c o n s i s t e n t w i t h t h e f a c t t h a t t h e s u l f i t e a d d e d a t i n c o u l a t i o n t i m e h a s b e e n o x i d i z e d a f t e r 5 h (see Section below); thereafter the growth rate gradually recovers and decreases respectively.

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228

G.F. Soldatini et al. : Sulfite: Preferential Sulfur Source

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C O 2 F i x a t i o n , 0 2 Evolution Rate, and Percentage Distribution of Fixation Products

The C O 2 fixation rate of COz grown algae, with optimum sulfate supply and harvested after 4 days, ranged in different series from 180-200 gmol/mg chlorophyll/ h, and the Oz evolution ranged from 200-240 gmol/ mg chlorophyll/h. It is self-evident that the CO2 fixation rate and the Oz evolution rate / unit of culture medium were running parallel with the growth yield. However, at stimulatory sulfite concentrations both rates have increased, if they are calculated per unit of protein (Fig. 5). This indicates an increased CO2 fixation capacity of the algae. Thereby the increase of Oz evolution is only about half of the CO/fixation, that is to say the assimilatory quotient (O2/COz) is decreased. At inhibitory sulfite concentration

(10.0 mmol 1- 1) however, the C O 2 fixation rate drops below the 02 evolution rate. It should be emphasized that stimulation as well as inhibition of cell yields, dependent on different sulfite concentrations (Fig. 1) are not strictly parallel to the CO2 fixation rate/unit of protein (Fig. 5). For example, at 5 mmol 1-1 sulfite there is an enhancement of CO2 fixation and of 02 evolution / unit of protein, whereas the growth yield of the culture has already dropped. The percentage distribution of CO/ fixation products is summarized in Table 1. As already knwon (Graham and Whittingham, 1968), during the induction period, the algae assimilate 1~CO2 predominantly via the fl-carboxylation mechanism. Thus the ratio sugar phosphates + PGA/aspartate is raised from 0.76 during the induction period, to 2.05 at steady state conditions. Under both conditions, precultivation with sulfite causes an increase in percentage of label in aspartate and a decrease in sugar phosphates+PGA. This is clearly expressed by a drop in the ratio of sugar phosphates+PGA/aspartate. Averaged, this drop amounts to 16% of the control values during the induction period, and 18,6% of the control values during steady state conditions. This shift towards fl-carboxylation results in more than 40%14C02 incorporation into aspartate in the case of sulfite precultivated algae, when measured during the induction period.

Amino acid Composition With respect to total sulfur metabolism, the amount of sulfur containing amino acids is most interesting.

Table 1, Percentage distribution of 14CO z in Chlorella vulgaris after precultivation at different sulfite concentrations added to the standard medium, COz grown algae were harvested after 4 days. Induction period: algae kept for 15 min in the dark, illumination started together with supply of NaH14CO3 . Steady state: preiUumination for 15 rain. For other conditions see " M e t h o d s " . Individual values are the m e a n of 4 experiments, which virtually showed no variation Induction period Control

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1,0 m M

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0.5 m M

1.0 m M

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G.F. Soldatini et al. : Sulfite: Preferential Sulfur Source

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Cysteine and cystine are destroyed by the analytical routine method applied, but methionine clearly can be followed. Its percentage of the total algal protein increases even at low sulfite concentrations supplied during the growth period (Fig. 6). This is especially true for algae grown with sulfite as the only sulfur source, compared to those grown at saturation sulfate levels. Among other amino acids besides methionine, only proline shows a significant increase (Fig. 7). It is present already at the lowest sulfite concentrations and roughly keeps its elevated level over the whole range. This demonstrates that increased proline synthesis is not necessarily bound to osmotic stress conditions, but may also be the result of some change at a regulatory step on the route of proline formation (Schobert, 1977).

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The rate constants for sulfite oxidation, usually given for oxygen saturated conditions (e.g. Fuller and Christ, 1941) cannot be directly transferred to the situation in the present culture medium. Das and Runeckles (1974) found all sulfite oxidized after 48 h, but no earlier records were given. Therefore, the direct oxidation of sulfite was followed in the present culture medium. It is evident (Fig. 8) that up to an initial concentration of 5.0 mmol 1- 1, all sulfite, which can be traced either by the sulfite sensitive electrode or by iodometric titration, had disappeared after 5 h. This oxidation rate is also reflected by the adjustment in pH towards that of the control, if the sulfite was not neutralized before its addition to the culture medium (see Fig. 2).

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The results demonstrate that sulfite can be used not only as a sulfur source, but also exerts specific changes in metabolism. As to sulfite concentrations below 1.0 mmol 1-1, which are not inhibitory on enzymes of the reductive pentose phosphate cycle, its use as a sulfur source is accompanied with stimulation of growth yield, enhanced CO2 fixation r a t e / u n i t of protein, and increased sulfur uptake into organic matter. Growth rate determinations indicate that the enhancement in yield is caused by stimulation during the first division steps; analogously, this is also true for inhibiting concentrations. This period coincides with the presence of sulfite, which is oxidized after a few hours. Thereafter, the increase in cell mass

230

approaches control levels; thus the increase in yield, found after 4 days represents an initial, continously vanishing pool, from the very first stimulation period. In contrast, the stimulation in COz fixation capacity/unit of protein, the change of CO2 assimilation towards the /3-carboxylation mechanism, as well as the increased methionine content of algal protein are maintained at least up to 4 days. Thus, these criteria outlast the period of sulfite present in the culture medium and the period of increased growth rate. The dissociation of growth rate stimulation from the other metabolic changes, furthermore is indicated by the fact that stimulation of CO2 fixation capacity tolerates higher initial sulfite concentrations (up to 5.0 mmol 1-1) than does growth rate. Thus, a metabolic step subsequent to CO2 fixation must be especially sensitive. The sulfite dependent enhancement of growth rate partially may be due to its direct stimulatory effect by maintaining photophosphorylation under N A D P H saturating conditions (Ziegler and Libera, 1975). In addition, facilitated uptake and binding of sulfite to the thylakoids during illumination (Ziegler, 1977; Ziegler and Hampp, 1977) may improve sulfur supply compared to the sulfate. Principally, economization of ATP, which is needed for the formation of APS during the initial step of photosynthetic sulfate reduction, but is not needed for incorporation of sulfite (see Schiff and Hodson, 1973) may improve ATP supply for COz fixation. However, since sulfate reduction amounts to about 1/50 of the CO2 fixation rate (Trebst and Schmidt, 1969), the energetic economization may be minimal. The sulfite induced increase in CO2 fixation capacity is paralleled by a marked decrease in the assimilatory quotient (02 / CO2) and a decrease in the ratio of sugar phosphates+PGA/aspartate. Both criteria may be connected with each other (acid formation needs O2) and indicate a shift towards the ~-carboxylation pathway of CO2 fixation. This shift, found in isolated chloroplasts (Libera et al., 1975) during the actual presence of sulfite, was explained by the fact that ribulosebisphosphate carboxylase is more sensitive to competitive inhibition with respect to CO2 towards sulfite (Ziegler, 1972) than is PEP-carboxylase (Ziegler, 1973). This explanation fails in the case of sulfite grown algae examined after 4 days in a sulfite free incubation medium. Since the causative agent had disappeard about 90 h before, induced enzyme formation also cannot be responsible for the changes in CO2 metabolism found. According to all experience, induced enzyme formation in algae does not persist longer than 5-15 h after withdrawal of the inducing agent, e.g. increased CO2 concentration

G.F. Soldatini et al. : Sulfite: Preferential Sulfur Source

(Hogetsu and Miyachi, 1977; Nelson and Tolbert, 1969). Rather, preferential formation of a long lasting sulfur compound seems to be a candidate for preserving the ability of enhanced CO2 fixation capacity and the shift towards /%carboxylation pathway. Among these, sulfolipids, which are essential constituents of the thylakoids (see Schiff and Hodson, 1973) are the most likely compounds. In particular, since their biosynthesis via addition of sulfite to enolpyruvate or to a 5-6-unsaturated glucose seems to be the more likely route than via reduced sulfur compounds such as cysteine (see Schiff and Hodson, 1973). Experiments to prove this hypothesis are under way. The preferential uptake and further metabolism of sulfite sulfur is also clearly demonstrated by the increase in methionine content, occurring even at low sulfite concentrations and in the presence of saturating sulfate concentrations. As pointed out in the case of isolated chloroplasts (Ziegler and Hampp, 1977), bypassing the regulatory step of ATP-sulfurylase, results in an imbalance in the C/S ratio, which seems to be a most characteristic feature of sulfite action already occurring at non-deleterious and even growth enhancing concentrations. One author (G.F.S.) is indebted to Alexander von Humbold Stiftung for a grant. The work was supported by the Deutsche Forschungsgemeinschaft.

References Bassham, J.A., Kirk, M. : Sequence of formation of phosphoglycolate and glycolate in photosynthesising Chlorella pyrenoidosa. Plant Physiol. 52, 407~11 (1977) Bramhall, S., Noack, N., Wu, N., Loewenberg, J.R.: A simple colorimetric method for determination of protein. Anal. Biochem. 31, 146-148 (1969) Buetow, D.E. : Growth, survival and biochemical alteration of Euglena gracilis in medium limited in sulfur. J. Cell Comp. Physiol. 66, 235-242 (1969) Cowling, D.W., Lockyer, D.R. : Growth of perennial ryegrass (Lolium perenne L.) exposed to low concentration of sulfur dioxide. J. Exp. Bot. 27, 411~17 (1976) Das, G , Runeckles, V.C. : Effects of bisulfite on metabolic development in synchronous Chlorella pyrenoidosa. Environmental Res. 7, 353-362 (1974) Delieu, T., Walker, D.D.: In improved cathode for the measurement of photosynthetic oxygen evolution by isolated chloroplasts. New Phytol. 71, 201-225 (1972) Faller, N.N., Herwig, K., Kiihn, H. : Die Aufnahme von Schwefeldioxyd (35SO2) aus der Luft. I. EinfluB auf den pflanzlichen Ertrag. Plant Soil 33, 177-191 (1970) Feige, B., Gimmler, H., Jeschke, W.D., Simonis, W. : Eine Methode zur dfinnschichtchromatographischen Auftrennung von ~4Cund 32P-markierten Stoffwechselprodukten. J. Chromatogr. 41, 80-90 (1969) Fuller, E.C., Christ, R.H.: The rate of oxidation of sulfite ions by oxygen. J. Amer. Chem. Soc. 63, 1644-1650 (1941)

G.F. Soldatini et al. : Sulfite: Preferential Sulfur Source Graham, D., Whittingham, C.P. : The path of carbon during photosynthesis in Chlorella pyrenoidosa at high and low carbon dioxide concentrations. Z. Pflanzenphysiol. 58, 418427 (1968) Hampp, R., Ziegler, I. : Sulfate and sulfite translocation via the phosphate translocator of the inner envelope membrane of chloroplasts. Planta 137, 309-312 (1977) Hogetsu, D., Miyachi, S.: Effects of CO2 concentration during growth on subsequent CO2 fixation in Chlorella. Plant Cell Physiol. 18, 347 352 (1977) Kuhl, A., Lorenzen, H. : Handling and culturing of Chlorella. In: Methods in Cell Physiol. Vol. I, p. 159 187. Ed. D.M. Prescott. New York: Academic Press 1964 Libera, W., Ziegler, I., Ziegler, Ziegler, H. : The action of sulfite on the HCO3--fixation and the fixation pattern of isolated chloroplasts and leaf tissue slices. Z. Pflanzenphysiol. 74, 420433 (1975) Nelson, E.B., Tolert, N.E. : The regulation of glycolate metabolism in Chlamydomonas reinhardii. Biochim. Biophys. Acta 184, 263-270 (1969) Puckett, V.J., Nieboer, E., Flora, P., Richardson, D.M.S. : Sulphur dioxide: its effect on photosynthetic 14C fixation in lichens and suggested mechanisms of phytotoxicity. New Phytol. 72, 141-154 (1973) Roy, A.B., Trudinger, P.A. : The biochemistry of inorganic compounds of sulphur. Cambridge: The University Press 1970 Schiff, J.A., Hodson, R.C. : The metabolism of sulfate. Ann. Rev. Plant Physiol. 24, 381414 (1973)

231 Schobert, B.: The influence of water stress on the metabolism of Diatoms. III. The effect of different nitrogen sources on proline accumulation. Z. Pflanzenphysiol. 85, 463-470 (1977) Trebst, A., Schmidt, A. : Photosynthetic sulfate and sulfite reduction by chloroplasts. Progr. Photosynth. Res. Ill, 1510-1516 (1969) Vishniac, W. : Methods for study of the Hill reaction. In: Methods in Enzymology Vol. IV, pp. 342-355. Colowick, S.P., Kaplan, N.O. eds. New York: Academic Press 1957 Ziegler, I.: The effect of SO3- on the activity of ribulosediphosphate carboxylase in isolated spinach chloroplasts. Planta 103, 155-163 (1972) Ziegler, I. : Effect of sulphite on phosphoenolpyruvate carboxylase and malate formation in extracts of Zea mays. Phytochem. 12, 1027-1030 (1973) Ziegler, I. : Subcellular distribution of 3SS-sulfur in spinach leaves after application of 35SO4-, 35SO3-, and s5SO2. Planta 135, 25-32 (1977) Zicgler, I., Hampp, R. : Control of a5SO42- and 35SO32- incorporation into spinach chloroplasts during photosynthetic CO2 fixation. Planta 137, 303-307 (1977) Zicgler, I., Libera, W. : The enhancement of CO2 fixation in isolated chloroplasts by low sulfite concentration and by ascorbate. Zeitschr. f. Naturforsch. 30e, 634-637 (1977)

Received 18 July; accepted 7 August 1978