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Oxidation of 35S-labelled elemental sulphur by wheat chloroplasts and analysis of 35s-products
Phytochemistry, Vol. 34, No. 6, pp. 1467-1471,1993 Printed in Great Britain.
003I 9422/93$6.00+ 0.00 0 1993Pergamon Press Ltd
OXIDATION OF 35S-LABELLED ELEMENTAL SULPHUR BY WHEAT CHLOROPLASTS AND ANALYSIS OF 35S-PRODUCTS PASCALE JOLIVET, EDITH BERGERON
and
JEAN-CLAUDE
MEUNIER
Laboratoire de Cbimie Biologique, INRA, Centre de Biotechnologie agroindustrielle, 78850 Thiverval-Grignon, France (Received in revised form 13 April 1993)
Key Word Index- Triticum aestioum; Gramineae; chloroplast; “S; aerobiose/anaerobiose; sulphur; sulphate; sulphite; thiosulphate.
elemental
Abstract-35So was offered to wheat intact chloroplasts and 3sS products analysed. Radioactivity in SOi- was determined after precipitation by BaCl,. Bimane derivatives of S compounds were separated by HPLC and detected by fluorimetry and radioactivity monitoring. It has been observed that fresh chloroplasts were able to oxidize 35So to 35SO:- via the production of 3sS20i- and 3sSO:-. From the characteristics of this oxidation, the operation of chloroplastic enzymatic mechanisms is proposed.
It
has been established recently that elemental sulphur So is metabolized by fresh but not by boiled wheat chloroplasts [l]. Although weak, the process was significant and included oxidation since a part of the metabolized So was transformed to sulphate. It is likely that other S compounds less oxidized than sulphate are synthesized from So as intermediates. Sulphur oxidation has been demonstrated in photosynthetic [2] or non-photosynthetic [3] bacteria. The first step is the primary So oxidation to sulphite or thiosulphate under aerobic or anaerobic conditions and several enzymes are involved. Siroheme sulphite reductase has been purified from Thiobacillus denjttijcans [4], hydrogen sulphide: ferric ion oxidoreductase from T. ferrooxidans [5], glutathionedependent oxygenase from T. thiooxiduns [6], glutathione-independent oxygenase from T. neapolitanus [7], and sulphur oxygenase reductase from Desulfirolobus ambiualens [S]. Further oxidation of sulphite or thiosulphate to sulphate is easy in bacteria but similar enzymatic systems have not been reported for higher plants. However, sulphite metabolization by higher plants has been widely studied. In the cytoplasm of mesophyll cells, sulphite is taken up by the chloroplasts via the phosphate translocator [9] and chloroplasts would be the site of both detoxification and toxic effects of SO,. Two pathways would be possible for the metabolism of sulphite inside chloroplasts [lo]. One possibility is the direct reduction of sulphite into hydrogen sulphide which may be metabolized into cysteine or released in part into the atmosphere. The other pathway is the oxidation of sulphite to sulphate; it can proceed via a radical chain reaction involving the light-dependent photosynthetic electron transport [lo, 11). As we have observed oxidation of Na,SO, into SO:- in fresh wheat
chloroplasts both in the light and dark [ 123, the process cannot be explained only by the operation of the photosynthetic electron transport chain. The work described here presents the investigation of S compounds synthesized from So by intact wheat chloroplasts using radioactive labelling with 35S. RESULTS AND DlSCUSSION
Elemental sulphur as a substrate
From previous results on the metabolism of elemental sulphur by higher plants following foliar application or by industrially important bacteria of the genus Thiobacillus, it appeared that the structure and state of this chemically stable and water insoluble element was very important. In wheat, So was applied on foliage as a colloidal dispersion of micronized S in adjuvant (80: 20, Thiovit, Sandoz) and a small part of the So was found to be absorbed and metabolized [13, 141. The same results were obtained when adjuvant was omitted and replaced with Tween 80 solution (Ferron, F., personal communication). In bacteria, it has been observed that So oxidation was markedly increased by the addition of wetting agents and that Tween 80 had the greatest effect [7, 151. The wetting agents allowed the elemental sulphur to become very finely divided. When no wetting agent was present, sulphur remained in a layer on the surface of the reaction mixtures and was apparently only slightly usable in this form [ 151. The crystalline structure of So appeared to be very important [7]. Moreover, as So can exist as several molecular species (S,, etc.), the term mole cannot be used and the patom or natom expression is to be preferred (1 patom= pg of S) [16]. As Tween 80 was used to disperse So, its effect on chloroplast function and integrity, and the chloroplast
1467
P. JOWET et al.
1468
capacity to oxidize So to SOi- was studied at several concentrations (0, 0.025%0, 0.25% and 0.25% in final concentration in the chloroplastic suspension, Table 1). The chloroplast integrity was measured following ferricyanide-dependent 0, evolution before and after an osmotic shock, CO,-dependent 0, evolution and the rate of 14C fixation. It has to be noted that a final concentration of 0.025% Tween did not affect chloroplast integrity unlike higher concentrations. However, the ferricyanide reduction via the photochemical transfer of electrons and the production of SOi- from So were not troubled by the presence of Tween 80. Metabolization rate of 35So For percentage determination of So metabolism, 35So was added to fresh or boiled chloroplasts. After elimination of chloroplastic lamellae by centrifugation, radioactivity was measured in the supernatant. This supernatant radioactivity was expressed as a percentage of total initial radioactivity offered to chloroplasts. In the case of boiled chloroplasts, radioactivity recovered in the supernatant corresponded to ‘solubilized’ S under the experimental
Table
conditions but not to metabolized So (column 2 from Table 2). In the case of fresh chloroplasts maintained in the light, supernatant radioactivity was higher (column 3 from Table 2). The difference between the radioactivity measured in the supernatant of fresh chloroplasts and of boiled ones, called ‘excess’, indicated the transformation of So into soluble S compounds by a chloroplastic process: 3.5 or 8.2% of initial So according to its initial concentration was consumed for 2 hr by this phenomenon in the chloroplastic suspension (0.1 mg chlorophyll ml ‘) and thus corresponded to a metabolism rate of 0.8 chlorophyll (column 7 from or 2patom Shr-‘mg-’ Table 2). A part of this metabolized S was precipitated by BaCl, and thus consisted of SO:-. The total 35So metabolism by chloroplasts was largely diminished in the dark (columns 4, 6, 8 from Table 2): l-2% of initial So consumed i.e. 0.1-l patom S hr- ’ mg- ’ chlorophyll.
Analysis of the S compounds produced The determinations of S compounds and particularly of S,O:- and SO:- were carried out by HPLC employ-
1. Effect of several Tween 80 concentrations
Final Tween concentration
14C fixation
0 0.025% 0.25%~ 0.25%
100 108 8.5 0.7
on chloroplast
functionality
CO,-dependent 0, evolution
Chloroplast intactness*
Ferricyanide-dependent 0, evolutiont
SO, productlon
100 95.8 6.8
100 121 44.5
100 112 130 101
100 100 100 100
For each photosynthetic activity, results are compared to the control experiment of Tween 80 (level 100). *Calculated from the measurement of ferricyanide-dependent 0, evolution osmotic shock. tMeasured after an osmotic shock.
without
the presence
before
and after an
Table 2. 3sSo metabolism by wheat chloroplasts (0.1 mg chlorophyll ml-‘) during 2 hr: radioactivity of supernatants, excess of radioactivity in the case of fresh chloroplasts, corresponding So metabolism and radioactivity in SOi- precipitated by BaCI, Supernatant radioactivity (%)t Initial So
boiled chloroplasts
concentration (patom ml- *)*
So metabolism§ (patom hr-’ mg-’
so:-
fresh chloroplasts
Excessf %
light
dark
light
dark
light
dark
(%)
chl)
2
16.7**
24.9
17.7
8.2
1.0
0.83
0.10
I2
11.2
(3.4) lO.Ol? (0.0)
(6.7) 13.5
11.8
3.5
1.8
1.97
1.01
46
(0.4)
;/
radioactivity
(1.1)
*The term mole cannot be used, since sulphur can exist as S,, etc. 1 patom = 32 pg So. tExpressed as a percentage of total radioactivity offered to chloroplasts. SDitTerence between fresh chloroplast and bolled chloroplast supernatant radioactivity. (jS” consumption by a chloroplastic process (excessxinitial So concentration) corrected with time and chlorophyll (/Per cent of metabolized So (excess) which was precipitated by BaCI,. **Mean of six experiments and standard deviation in brackets. ttMean of three experiments and standard deviation in brackets.
concentration.
35S-Labelledelemental sulphur ing their fluorescent monobromobimane derivatives. Sulphate, which does not react with bimane, is not detected by fluorimetry and is determined by the use of a turbidimetric method. It was verified that chloroplasts were devoid of endogenous S,Oi-, SOi- and SOibefore the experiments when detectable amounts were found in fresh chloroplasts treated for 2 hr with 2 patom So (Table 3); amounts of f&O:- and SOi- were low but significant. The lower levels of SZO:- and SOi- observed under O2 could be explained by a faster turnover and an easier total oxidation of So to sulphate in this condition. It was verified previously that the last oxidation step SOi--+SO:was not limiting [12, 171. Logically, this fact led to a higher pool of sulphate under Or. It was possible to calculate the total amount of S involved in the three analysed S compounds, SO:-, SOi- and S,Oi- taking into consideration that two S atoms are present in SZO:-. This recovered S amount was compared to the total So amount consumed by a chloroplastic process estimated as described above. It was observed that total transformed So was recovered into SZOg-, SO;- and SOi- in the dark but only 21-27% in the light (last line of Table 3). It is obvious that in the latter case So had to be incorporated in other S compounds e.g. S-aminoacids via the reductive pathway of sulphate which is a light-dependent process. Some experiments were carried out with much more So (11.2 patom ml-’ instead of 2 patom ml-‘) to permit detection of ‘?l labelling in S compounds by radioactive assay coupled with HPLC separation. In that case, So metabolism had attained 1.97 patom hr- ’ mg- ’ chlorophyll. The SO:- pool was determined as a part of the
Table 3. S compounds production (nmol hr-’ mg-* chlorophyll) from So (initial amount: 2 patom ml-r) by wheat fresh chloroplasts (0.1 mg chlorophyll ml-‘) maintained under different atmosphere conditions, data represent the mean of seven experiments Chloroplasts without So
so:- * soa,-t W-t
Qst So metabolized~ Yield (%)I[
0 0.3 0.1
Chloroplasts incubated with So Oxygen
Nitrogen
light
dark
light
168.5 2.5 5.0 181 833 21.7
117.5 103.0 82.0 12.5 2.5 0 2.0 55.0 6.0 121.5 225.5 96.5 101 833 101 27.1 95.5 120
dark
metabolized ssSo precipitated by BaCl, and the presence of “SO:was detected as a radioactive peak at the beginning of the radiochromatograms. The S,Og- and SOi- pools were determined by fluorimetric detection and their radioactive labelling measured (Table 4). From the comparison of the results given in Tables 3 and 4, it can be noticed that the SOi- pool increased in the same proportion as the initial So amounts when the increase of SZO:- pool was very high and that of SO:- not significant. Three hypotheses can be proposed to explain the very low SOi- pool size: (i) a very fast oxidation of SOito SO:-; (ii) a slow transformation of S20i- to SOileading to a E&O:- accumulation; (iii) the production of S20:- and SOi- from So by two different pathways not closely related and with the least active being for SO:synthesis. For SO:-, the detection threshold was not always attained and labelling was not systematically observed. On the contrary, it can be seen that S,O:- was strongly labelled, presumably because it was doubly labelled. It has been verified that the observed labelling could not be explained by an isotopic exchange between 3sSo and pre-existent endogenous S compounds. Again, it appeared that the total oxidation of So to SOi- was easier under 0, than under Na since less SzO:- and more label in SO:- were found in the case of experiments carried out under 0,. The S recovered in f&O:-, SO:- and SOi- has been considered and compared to the So metabolism rate (Table 5). A good yield was obtained under N,. Under 0,, a lower recovery yield could indicate that the S metabolic pathways were more diversified. Radiochromatograms of bimane S-derivatives showed some unknown labelled peaks, particularly in the case of samples under light and OZ. In conclusion, these experiments have shown that fresh chloroplasts from wheat were able to oxidize 35So to 35SO:- via the biosynthesis of 35S20:- and 3sSO:-. As this oxidation was effected in the light as well as in the
Table 4. S compounds production (nmol hr-’ mg-r chlorophy:l) and radioactive labelling (lo3 xcounts) from 3sSo (initial amount: 11.2 patom ml-‘) by wheat fresh chloroplasts (0.1 mg chlorophyll ml-‘) maintained under ditferent light and atmosphere conditions Oxygen
so:so;-
*SO:- determined by turbidimetry. tSO:- and S@determined by HPLC as fluorescent derivatives. $Total S amount recovered in SO:-. SOi- and S20:(natom S hr - r mg- ’ chlorophyll). $S” metabolism estimated by determination of soluble radioactivity (natom S hr-’ mg-’ chlorophyll) as in Table 2. 11 Expressed as QS/S’ metabolized.
1469
f&O:-
amount+ labellingt amount5 tabelling~ amount8 labellingt
Nitrogen
light
dark
light
dark
1034 632 7.1 163 168 1047
595 385 4.3 ndS 137 967
826 282 7.6 47 460 2359
387 ndZ 2.5 nd$ 205 959
*Part of metabolized s5So precipitated by BaCl,. PDetection with a flow-through radioactivity monitor. $Not detectable. gFluorimetric detection.
1470
P.
JOLIVET
Table 5. Total So metabolism by wheat chloroplasts maintamed in the presence of So (initial amount: 11.2 patom ml-‘) under different conditions and S recovery into SIO$-, SO: - and so:So metabolism*
S recovery
(fitatom S hr - ’ mg- ’ chlorophyll) Oxygen Nitrogen
light dark light dark
2.25 1.30 1.80 0.84
1.38 0.87 1.76 0.80
*So metabolism was estimated by determination radioactivity as in Table 2.
Yield (%) 61.3 66.9 97.7 95.2 of soluble
dark, it should not therefore be ascribed only to a nonenzymatic aerobic oxidation initiated by the electron transport chain as reported by Asada and Kiso [l 11 and Dittrich et al. [lo]. In the light, in addition to the production of S,O:-, SO:- and SO:-, the reduction of sulphate led to So incorporation into other S compounds which are probably S-amino acids. Joyard et al. [ 181 have observed that spinach chloroplasts contain enzymes involved in the formation of elemental sulphur from SO:and have suggested that a rapid metabolism (presumably oxidation) of produced So was possible within isolated chloroplasts via an enzymatic process. The entry of sulphur into chloroplasts in viuo remains to be established. However, after foliar treatment with crystalline radioactive sulphur, incorporation of So was followed in
several subcellular compartments of the photosynthetic tissue (vacuoles, plastids, cytoplasm, mitochondria) [19] which means that So was able to pass through membrane barriers. In T. ferrooxidans, So is not oxidized on the surface of the outer membrane but in the periplasmic space after passing through the outer membrane by an unknown mechanism [20]. In that case, several proteins were involved in the oxidation of So, one was easily solubilized without detergents and another was purified from the plasma membrane [S, 203.
EXPERIMENTAL
Wheat (Triticum aestioum L., cv Festival) was grown on vermiculite with nutrient soln for neutrophile plants [21]. Irradiation was 180 ~01 me2 s-l, applied during 16 hr. Chloroplasts were prepared from 20 g leaves sampled from 7 or l+day-old plants and homogenized in 100 ml isolation medium (0.1 M PO:- buffer, 0.4 M sucrose, pH 7.8). After filtration through butter muslin, the suspension was centrifuged (10 min, 15OOg). The pellet was resuspended in the isolation medium and layered on the top of a discontinuous sucrose gradient: 2, 1.75, 1.5 and 1 M [22]. After centrifugation (50 min, 50000 g), the intact chloroplast layer was recovered between 1.75 and 1.5 M, half diluted with isolation medium containing 2 mM MgCl, and centrifuged (10 min, 6000 g). The pellet containing the chloroplasts
bt al.
was suspended in the reaction medium (0.33 M sorbitol;
50mM Hepes; 1 mM MgCl,; 1 mM MnCl,; 2 mM EDTA; pH 7.6). Chloroplast integrity was controlled by three methods: (i) measurement of ferricyanide-dependent 0, evolution before and after an osmotic shock [23]; (ii) measurement of CO,-dependent 0, evolution [23]; (iii) measurement of the rate of 14C fixation [24]. For So metabolism experiments, chloroplasts were shaken in the presence of 5 mM NaHCO, and 3sSo (crystalline, a-rhombic structure, Amersham, France) as a suspension in Tween 80. The total vol. was 1 or 2 ml. Chlorophyll wncn was estimated according to ref. [25] and was ca 0.1 mg ml-‘. The initial concn of 35So was 2 patom ml- ’ (30.6 kBq mg-’ S) in the experiments carried out only for the determination of the percentage of So metabolism. In the case of HPLC analysis of S compound intermediates, much more radioactivity was necessary and 11.2 ,uatom ml- ’ 35So (5.2 MBq mg-’ S) were offered to the chloroplast suspension. Experiments were carried out under 0, or N,, in the light (800 pmol rn-’ s-‘) or in the dark, during 2 hr at 18” and also with boiled chloroplasts. Experiments were stopped with the elimination of chloroplastic lamellae by centrifugation. Radioactivity was determined in the supernatants by scintillation counting and SOi- radioactivity was counted after precipitation with BaCl,. Before turbidimetric SOidetermination according to ref. [12], protein-pigment complexes which were liberated with Tween 80 and interfered with the measurements, were precipitated with 0.5 M TCA Cl]. The determination of S,Oi- and SO:- was carried out by HPLC according to ref. [26]. Aliquots of supernatants (100 ~1) were allowed to react with 10~1 monobromobimane (50 mM) for 15 min at room temp. in the dark. Acetonitrile (100 ~1) was added and the samples were heated to 60” for 10 min to precipitate proteins. Acetic acid (300 ~1, 50 mM) was added before frozen storage. Samples were diluted (1: 25) before injection. Separation of thiol derivatives was achieved on a Ultrasphere ODS 5 pm analytical column (4.6 mmx25 cm, Beckman) by a gradient elution at a constant flow rate of 1.2 ml (Beckman 110A pumps, Altex gradient programmer) using aq. acetic acid (0.25%, pH 3.5) as buffer A and MeOH as solvent B. Elution conditions were 15% B for 5 min, linear increase from 15% B to 23% in 10 min, from 23% to 100% in 15 min, 100% B for 20 min followed by the change to 15% B in 3 min before re-equilibration. Fluorescence detection was carried out with a Jasco fluorimeter (390 nm excitation, 480 nm emission). A flow-through scintillation counter in-line (radioactivity monitor LB 506C Berthold) was coupled to the fluorescence detector. Two simultaneous chromatograms were produced: one showing all of the fluorescent thiol peaks, and another showing the radioactivity present in the different peaks. Sulphate which does not react with bimane and is not detected by fluorimetry, can be detected as a radioactive peak occurring at the beginning of the radiochromatogram. Each result was corrected for decay to the calibration date and the labelling of S compounds was calculated at the time of assay.
3JS-Labelledelementalsulphur
1. Jolivet, P. and Kien, P. (1992) C, R. Ad.
Sci. Paris
314, 231. 2. Truper, H. 6. (1983) in ?7te P~ofosy~fhef~ Bacferia (Clayton, R. R. and Sistrom, N. R., eds), p. 677. Plenum Press. 3. Brune, D. C. (1989) Biochim. Biophys. Acfa 975, 189. 4. Schedel, M. and Truper, H. G. (1979) ~cbi~. Biophys. Acta 56% 454. 5. Sugio, T., Suzuki, II., Oto, A., Inagaki, K., Tanaka, II. and Tano, T. (1991) Agric. Bid. Chem. 55, 2091. 6. Suzuki, I. (1965) ~~ochirn.~~ophys. Acfu 110,97. 7. Taylor, B. F. (1968) Biochim. ~iophys. Acfu 170, 112. 8. Kletzin, A. (1989) J. B~t~io~” 171, 1638. 9. Hampp, R. and ZiegIer, I. (1977) Planta 137, 309. 10. Dittrich, A. P. M., Pfauz, H. and Heher, IJ. (1992) Plant Physid. 98, 738. 11. Asada, K. and Kiso, K. (1973) Eur. J. &o&em. 33, 253. 12. Jolivet, P. and Kien, P. (1992) 6. R. Acad. Sci. Puris 314, 179. 13. ~~s-Delapo~e, S,, Ferron, F., Landry, J. and Costes, C. (1987) Plant Physiol. 85, 1026. 14. Landry, J., Legris-Delaporte, S. and Ferron, F. (1991) Phyf~h~istry 30,729. 15. Adair, F. W. (1966) J. Bactmiol. 92, 899.
1471
16. Suzuki, I., Ghan, C. W. and Takeuchi, T. L. (1992) Appl. Environ, bficrobiol. 58, 3767. 17. Jolivet, P., Bergeron, E. and Kien, P. (1992) Phyfon 32, 59. 18. Joyard, J., Forest, E., BI&e,E. and Deuce, R. (1988) Pia~f P~ysio~.88,961. 19. Legendre, S. and Marty, F. (1987) in Proc. Inf. Symp. Elemental Sulpb~r in Agriculture, Nice, France (Syndicat Frarqais du Soufre, ed.), p_ 689. 20. Sugio, T., Mizunashi, W., Inagaki, K. and Tano, T. (1987) J. Bacterial. 169,4916. 21. CoIe, Y. and Lesaint, C. (1973) Rev. Nortic. 2316,29. 22. Guillot-S~omon, T., Farineau, N., Cant&, C., Oursel, A. and Tuquet, C. (1987) Pbysiol. Planf. 69, 113. 23. Lilley, R. McC., Fitzgerald, M. P., Rienits, K. G, and Walker, D. A. (1975) New Phytol. 75, 1. 24. Jensen, R. G. and Bassham, J. A. (1966) Proc. Nafn. Acad. Sci. U.S.A. 56, 1095. 25. S&mid, G. H. (1971) in Methods in Enzymology (San Pietro, A., ed.), Vol. XXIII, part A, p. 171. Academic Press, New York. 26. Vetter, R. D., Matrai, P. A., Javor, B. and O’Brien, J, (1989) in ~offenic sulfur in the ~n~ironrne~t (Saltzman, E. S. and Cooper, W. J., eds), Vol. 393, p. 243. American Chemical Society, Washington.
003I 9422/93$6.00+ 0.00 0 1993Pergamon Press Ltd
OXIDATION OF 35S-LABELLED ELEMENTAL SULPHUR BY WHEAT CHLOROPLASTS AND ANALYSIS OF 35S-PRODUCTS PASCALE JOLIVET, EDITH BERGERON
and
JEAN-CLAUDE
MEUNIER
Laboratoire de Cbimie Biologique, INRA, Centre de Biotechnologie agroindustrielle, 78850 Thiverval-Grignon, France (Received in revised form 13 April 1993)
Key Word Index- Triticum aestioum; Gramineae; chloroplast; “S; aerobiose/anaerobiose; sulphur; sulphate; sulphite; thiosulphate.
elemental
Abstract-35So was offered to wheat intact chloroplasts and 3sS products analysed. Radioactivity in SOi- was determined after precipitation by BaCl,. Bimane derivatives of S compounds were separated by HPLC and detected by fluorimetry and radioactivity monitoring. It has been observed that fresh chloroplasts were able to oxidize 35So to 35SO:- via the production of 3sS20i- and 3sSO:-. From the characteristics of this oxidation, the operation of chloroplastic enzymatic mechanisms is proposed.
It
has been established recently that elemental sulphur So is metabolized by fresh but not by boiled wheat chloroplasts [l]. Although weak, the process was significant and included oxidation since a part of the metabolized So was transformed to sulphate. It is likely that other S compounds less oxidized than sulphate are synthesized from So as intermediates. Sulphur oxidation has been demonstrated in photosynthetic [2] or non-photosynthetic [3] bacteria. The first step is the primary So oxidation to sulphite or thiosulphate under aerobic or anaerobic conditions and several enzymes are involved. Siroheme sulphite reductase has been purified from Thiobacillus denjttijcans [4], hydrogen sulphide: ferric ion oxidoreductase from T. ferrooxidans [5], glutathionedependent oxygenase from T. thiooxiduns [6], glutathione-independent oxygenase from T. neapolitanus [7], and sulphur oxygenase reductase from Desulfirolobus ambiualens [S]. Further oxidation of sulphite or thiosulphate to sulphate is easy in bacteria but similar enzymatic systems have not been reported for higher plants. However, sulphite metabolization by higher plants has been widely studied. In the cytoplasm of mesophyll cells, sulphite is taken up by the chloroplasts via the phosphate translocator [9] and chloroplasts would be the site of both detoxification and toxic effects of SO,. Two pathways would be possible for the metabolism of sulphite inside chloroplasts [lo]. One possibility is the direct reduction of sulphite into hydrogen sulphide which may be metabolized into cysteine or released in part into the atmosphere. The other pathway is the oxidation of sulphite to sulphate; it can proceed via a radical chain reaction involving the light-dependent photosynthetic electron transport [lo, 11). As we have observed oxidation of Na,SO, into SO:- in fresh wheat
chloroplasts both in the light and dark [ 123, the process cannot be explained only by the operation of the photosynthetic electron transport chain. The work described here presents the investigation of S compounds synthesized from So by intact wheat chloroplasts using radioactive labelling with 35S. RESULTS AND DlSCUSSION
Elemental sulphur as a substrate
From previous results on the metabolism of elemental sulphur by higher plants following foliar application or by industrially important bacteria of the genus Thiobacillus, it appeared that the structure and state of this chemically stable and water insoluble element was very important. In wheat, So was applied on foliage as a colloidal dispersion of micronized S in adjuvant (80: 20, Thiovit, Sandoz) and a small part of the So was found to be absorbed and metabolized [13, 141. The same results were obtained when adjuvant was omitted and replaced with Tween 80 solution (Ferron, F., personal communication). In bacteria, it has been observed that So oxidation was markedly increased by the addition of wetting agents and that Tween 80 had the greatest effect [7, 151. The wetting agents allowed the elemental sulphur to become very finely divided. When no wetting agent was present, sulphur remained in a layer on the surface of the reaction mixtures and was apparently only slightly usable in this form [ 151. The crystalline structure of So appeared to be very important [7]. Moreover, as So can exist as several molecular species (S,, etc.), the term mole cannot be used and the patom or natom expression is to be preferred (1 patom= pg of S) [16]. As Tween 80 was used to disperse So, its effect on chloroplast function and integrity, and the chloroplast
1467
P. JOWET et al.
1468
capacity to oxidize So to SOi- was studied at several concentrations (0, 0.025%0, 0.25% and 0.25% in final concentration in the chloroplastic suspension, Table 1). The chloroplast integrity was measured following ferricyanide-dependent 0, evolution before and after an osmotic shock, CO,-dependent 0, evolution and the rate of 14C fixation. It has to be noted that a final concentration of 0.025% Tween did not affect chloroplast integrity unlike higher concentrations. However, the ferricyanide reduction via the photochemical transfer of electrons and the production of SOi- from So were not troubled by the presence of Tween 80. Metabolization rate of 35So For percentage determination of So metabolism, 35So was added to fresh or boiled chloroplasts. After elimination of chloroplastic lamellae by centrifugation, radioactivity was measured in the supernatant. This supernatant radioactivity was expressed as a percentage of total initial radioactivity offered to chloroplasts. In the case of boiled chloroplasts, radioactivity recovered in the supernatant corresponded to ‘solubilized’ S under the experimental
Table
conditions but not to metabolized So (column 2 from Table 2). In the case of fresh chloroplasts maintained in the light, supernatant radioactivity was higher (column 3 from Table 2). The difference between the radioactivity measured in the supernatant of fresh chloroplasts and of boiled ones, called ‘excess’, indicated the transformation of So into soluble S compounds by a chloroplastic process: 3.5 or 8.2% of initial So according to its initial concentration was consumed for 2 hr by this phenomenon in the chloroplastic suspension (0.1 mg chlorophyll ml ‘) and thus corresponded to a metabolism rate of 0.8 chlorophyll (column 7 from or 2patom Shr-‘mg-’ Table 2). A part of this metabolized S was precipitated by BaCl, and thus consisted of SO:-. The total 35So metabolism by chloroplasts was largely diminished in the dark (columns 4, 6, 8 from Table 2): l-2% of initial So consumed i.e. 0.1-l patom S hr- ’ mg- ’ chlorophyll.
Analysis of the S compounds produced The determinations of S compounds and particularly of S,O:- and SO:- were carried out by HPLC employ-
1. Effect of several Tween 80 concentrations
Final Tween concentration
14C fixation
0 0.025% 0.25%~ 0.25%
100 108 8.5 0.7
on chloroplast
functionality
CO,-dependent 0, evolution
Chloroplast intactness*
Ferricyanide-dependent 0, evolutiont
SO, productlon
100 95.8 6.8
100 121 44.5
100 112 130 101
100 100 100 100
For each photosynthetic activity, results are compared to the control experiment of Tween 80 (level 100). *Calculated from the measurement of ferricyanide-dependent 0, evolution osmotic shock. tMeasured after an osmotic shock.
without
the presence
before
and after an
Table 2. 3sSo metabolism by wheat chloroplasts (0.1 mg chlorophyll ml-‘) during 2 hr: radioactivity of supernatants, excess of radioactivity in the case of fresh chloroplasts, corresponding So metabolism and radioactivity in SOi- precipitated by BaCI, Supernatant radioactivity (%)t Initial So
boiled chloroplasts
concentration (patom ml- *)*
So metabolism§ (patom hr-’ mg-’
so:-
fresh chloroplasts
Excessf %
light
dark
light
dark
light
dark
(%)
chl)
2
16.7**
24.9
17.7
8.2
1.0
0.83
0.10
I2
11.2
(3.4) lO.Ol? (0.0)
(6.7) 13.5
11.8
3.5
1.8
1.97
1.01
46
(0.4)
;/
radioactivity
(1.1)
*The term mole cannot be used, since sulphur can exist as S,, etc. 1 patom = 32 pg So. tExpressed as a percentage of total radioactivity offered to chloroplasts. SDitTerence between fresh chloroplast and bolled chloroplast supernatant radioactivity. (jS” consumption by a chloroplastic process (excessxinitial So concentration) corrected with time and chlorophyll (/Per cent of metabolized So (excess) which was precipitated by BaCI,. **Mean of six experiments and standard deviation in brackets. ttMean of three experiments and standard deviation in brackets.
concentration.
35S-Labelledelemental sulphur ing their fluorescent monobromobimane derivatives. Sulphate, which does not react with bimane, is not detected by fluorimetry and is determined by the use of a turbidimetric method. It was verified that chloroplasts were devoid of endogenous S,Oi-, SOi- and SOibefore the experiments when detectable amounts were found in fresh chloroplasts treated for 2 hr with 2 patom So (Table 3); amounts of f&O:- and SOi- were low but significant. The lower levels of SZO:- and SOi- observed under O2 could be explained by a faster turnover and an easier total oxidation of So to sulphate in this condition. It was verified previously that the last oxidation step SOi--+SO:was not limiting [12, 171. Logically, this fact led to a higher pool of sulphate under Or. It was possible to calculate the total amount of S involved in the three analysed S compounds, SO:-, SOi- and S,Oi- taking into consideration that two S atoms are present in SZO:-. This recovered S amount was compared to the total So amount consumed by a chloroplastic process estimated as described above. It was observed that total transformed So was recovered into SZOg-, SO;- and SOi- in the dark but only 21-27% in the light (last line of Table 3). It is obvious that in the latter case So had to be incorporated in other S compounds e.g. S-aminoacids via the reductive pathway of sulphate which is a light-dependent process. Some experiments were carried out with much more So (11.2 patom ml-’ instead of 2 patom ml-‘) to permit detection of ‘?l labelling in S compounds by radioactive assay coupled with HPLC separation. In that case, So metabolism had attained 1.97 patom hr- ’ mg- ’ chlorophyll. The SO:- pool was determined as a part of the
Table 3. S compounds production (nmol hr-’ mg-* chlorophyll) from So (initial amount: 2 patom ml-r) by wheat fresh chloroplasts (0.1 mg chlorophyll ml-‘) maintained under different atmosphere conditions, data represent the mean of seven experiments Chloroplasts without So
so:- * soa,-t W-t
Qst So metabolized~ Yield (%)I[
0 0.3 0.1
Chloroplasts incubated with So Oxygen
Nitrogen
light
dark
light
168.5 2.5 5.0 181 833 21.7
117.5 103.0 82.0 12.5 2.5 0 2.0 55.0 6.0 121.5 225.5 96.5 101 833 101 27.1 95.5 120
dark
metabolized ssSo precipitated by BaCl, and the presence of “SO:was detected as a radioactive peak at the beginning of the radiochromatograms. The S,Og- and SOi- pools were determined by fluorimetric detection and their radioactive labelling measured (Table 4). From the comparison of the results given in Tables 3 and 4, it can be noticed that the SOi- pool increased in the same proportion as the initial So amounts when the increase of SZO:- pool was very high and that of SO:- not significant. Three hypotheses can be proposed to explain the very low SOi- pool size: (i) a very fast oxidation of SOito SO:-; (ii) a slow transformation of S20i- to SOileading to a E&O:- accumulation; (iii) the production of S20:- and SOi- from So by two different pathways not closely related and with the least active being for SO:synthesis. For SO:-, the detection threshold was not always attained and labelling was not systematically observed. On the contrary, it can be seen that S,O:- was strongly labelled, presumably because it was doubly labelled. It has been verified that the observed labelling could not be explained by an isotopic exchange between 3sSo and pre-existent endogenous S compounds. Again, it appeared that the total oxidation of So to SOi- was easier under 0, than under Na since less SzO:- and more label in SO:- were found in the case of experiments carried out under 0,. The S recovered in f&O:-, SO:- and SOi- has been considered and compared to the So metabolism rate (Table 5). A good yield was obtained under N,. Under 0,, a lower recovery yield could indicate that the S metabolic pathways were more diversified. Radiochromatograms of bimane S-derivatives showed some unknown labelled peaks, particularly in the case of samples under light and OZ. In conclusion, these experiments have shown that fresh chloroplasts from wheat were able to oxidize 35So to 35SO:- via the biosynthesis of 35S20:- and 3sSO:-. As this oxidation was effected in the light as well as in the
Table 4. S compounds production (nmol hr-’ mg-r chlorophy:l) and radioactive labelling (lo3 xcounts) from 3sSo (initial amount: 11.2 patom ml-‘) by wheat fresh chloroplasts (0.1 mg chlorophyll ml-‘) maintained under ditferent light and atmosphere conditions Oxygen
so:so;-
*SO:- determined by turbidimetry. tSO:- and S@determined by HPLC as fluorescent derivatives. $Total S amount recovered in SO:-. SOi- and S20:(natom S hr - r mg- ’ chlorophyll). $S” metabolism estimated by determination of soluble radioactivity (natom S hr-’ mg-’ chlorophyll) as in Table 2. 11 Expressed as QS/S’ metabolized.
1469
f&O:-
amount+ labellingt amount5 tabelling~ amount8 labellingt
Nitrogen
light
dark
light
dark
1034 632 7.1 163 168 1047
595 385 4.3 ndS 137 967
826 282 7.6 47 460 2359
387 ndZ 2.5 nd$ 205 959
*Part of metabolized s5So precipitated by BaCl,. PDetection with a flow-through radioactivity monitor. $Not detectable. gFluorimetric detection.
1470
P.
JOLIVET
Table 5. Total So metabolism by wheat chloroplasts maintamed in the presence of So (initial amount: 11.2 patom ml-‘) under different conditions and S recovery into SIO$-, SO: - and so:So metabolism*
S recovery
(fitatom S hr - ’ mg- ’ chlorophyll) Oxygen Nitrogen
light dark light dark
2.25 1.30 1.80 0.84
1.38 0.87 1.76 0.80
*So metabolism was estimated by determination radioactivity as in Table 2.
Yield (%) 61.3 66.9 97.7 95.2 of soluble
dark, it should not therefore be ascribed only to a nonenzymatic aerobic oxidation initiated by the electron transport chain as reported by Asada and Kiso [l 11 and Dittrich et al. [lo]. In the light, in addition to the production of S,O:-, SO:- and SO:-, the reduction of sulphate led to So incorporation into other S compounds which are probably S-amino acids. Joyard et al. [ 181 have observed that spinach chloroplasts contain enzymes involved in the formation of elemental sulphur from SO:and have suggested that a rapid metabolism (presumably oxidation) of produced So was possible within isolated chloroplasts via an enzymatic process. The entry of sulphur into chloroplasts in viuo remains to be established. However, after foliar treatment with crystalline radioactive sulphur, incorporation of So was followed in
several subcellular compartments of the photosynthetic tissue (vacuoles, plastids, cytoplasm, mitochondria) [19] which means that So was able to pass through membrane barriers. In T. ferrooxidans, So is not oxidized on the surface of the outer membrane but in the periplasmic space after passing through the outer membrane by an unknown mechanism [20]. In that case, several proteins were involved in the oxidation of So, one was easily solubilized without detergents and another was purified from the plasma membrane [S, 203.
EXPERIMENTAL
Wheat (Triticum aestioum L., cv Festival) was grown on vermiculite with nutrient soln for neutrophile plants [21]. Irradiation was 180 ~01 me2 s-l, applied during 16 hr. Chloroplasts were prepared from 20 g leaves sampled from 7 or l+day-old plants and homogenized in 100 ml isolation medium (0.1 M PO:- buffer, 0.4 M sucrose, pH 7.8). After filtration through butter muslin, the suspension was centrifuged (10 min, 15OOg). The pellet was resuspended in the isolation medium and layered on the top of a discontinuous sucrose gradient: 2, 1.75, 1.5 and 1 M [22]. After centrifugation (50 min, 50000 g), the intact chloroplast layer was recovered between 1.75 and 1.5 M, half diluted with isolation medium containing 2 mM MgCl, and centrifuged (10 min, 6000 g). The pellet containing the chloroplasts
bt al.
was suspended in the reaction medium (0.33 M sorbitol;
50mM Hepes; 1 mM MgCl,; 1 mM MnCl,; 2 mM EDTA; pH 7.6). Chloroplast integrity was controlled by three methods: (i) measurement of ferricyanide-dependent 0, evolution before and after an osmotic shock [23]; (ii) measurement of CO,-dependent 0, evolution [23]; (iii) measurement of the rate of 14C fixation [24]. For So metabolism experiments, chloroplasts were shaken in the presence of 5 mM NaHCO, and 3sSo (crystalline, a-rhombic structure, Amersham, France) as a suspension in Tween 80. The total vol. was 1 or 2 ml. Chlorophyll wncn was estimated according to ref. [25] and was ca 0.1 mg ml-‘. The initial concn of 35So was 2 patom ml- ’ (30.6 kBq mg-’ S) in the experiments carried out only for the determination of the percentage of So metabolism. In the case of HPLC analysis of S compound intermediates, much more radioactivity was necessary and 11.2 ,uatom ml- ’ 35So (5.2 MBq mg-’ S) were offered to the chloroplast suspension. Experiments were carried out under 0, or N,, in the light (800 pmol rn-’ s-‘) or in the dark, during 2 hr at 18” and also with boiled chloroplasts. Experiments were stopped with the elimination of chloroplastic lamellae by centrifugation. Radioactivity was determined in the supernatants by scintillation counting and SOi- radioactivity was counted after precipitation with BaCl,. Before turbidimetric SOidetermination according to ref. [12], protein-pigment complexes which were liberated with Tween 80 and interfered with the measurements, were precipitated with 0.5 M TCA Cl]. The determination of S,Oi- and SO:- was carried out by HPLC according to ref. [26]. Aliquots of supernatants (100 ~1) were allowed to react with 10~1 monobromobimane (50 mM) for 15 min at room temp. in the dark. Acetonitrile (100 ~1) was added and the samples were heated to 60” for 10 min to precipitate proteins. Acetic acid (300 ~1, 50 mM) was added before frozen storage. Samples were diluted (1: 25) before injection. Separation of thiol derivatives was achieved on a Ultrasphere ODS 5 pm analytical column (4.6 mmx25 cm, Beckman) by a gradient elution at a constant flow rate of 1.2 ml (Beckman 110A pumps, Altex gradient programmer) using aq. acetic acid (0.25%, pH 3.5) as buffer A and MeOH as solvent B. Elution conditions were 15% B for 5 min, linear increase from 15% B to 23% in 10 min, from 23% to 100% in 15 min, 100% B for 20 min followed by the change to 15% B in 3 min before re-equilibration. Fluorescence detection was carried out with a Jasco fluorimeter (390 nm excitation, 480 nm emission). A flow-through scintillation counter in-line (radioactivity monitor LB 506C Berthold) was coupled to the fluorescence detector. Two simultaneous chromatograms were produced: one showing all of the fluorescent thiol peaks, and another showing the radioactivity present in the different peaks. Sulphate which does not react with bimane and is not detected by fluorimetry, can be detected as a radioactive peak occurring at the beginning of the radiochromatogram. Each result was corrected for decay to the calibration date and the labelling of S compounds was calculated at the time of assay.
3JS-Labelledelementalsulphur
1. Jolivet, P. and Kien, P. (1992) C, R. Ad.
Sci. Paris
314, 231. 2. Truper, H. 6. (1983) in ?7te P~ofosy~fhef~ Bacferia (Clayton, R. R. and Sistrom, N. R., eds), p. 677. Plenum Press. 3. Brune, D. C. (1989) Biochim. Biophys. Acfa 975, 189. 4. Schedel, M. and Truper, H. G. (1979) ~cbi~. Biophys. Acta 56% 454. 5. Sugio, T., Suzuki, II., Oto, A., Inagaki, K., Tanaka, II. and Tano, T. (1991) Agric. Bid. Chem. 55, 2091. 6. Suzuki, I. (1965) ~~ochirn.~~ophys. Acfu 110,97. 7. Taylor, B. F. (1968) Biochim. ~iophys. Acfu 170, 112. 8. Kletzin, A. (1989) J. B~t~io~” 171, 1638. 9. Hampp, R. and ZiegIer, I. (1977) Planta 137, 309. 10. Dittrich, A. P. M., Pfauz, H. and Heher, IJ. (1992) Plant Physid. 98, 738. 11. Asada, K. and Kiso, K. (1973) Eur. J. &o&em. 33, 253. 12. Jolivet, P. and Kien, P. (1992) 6. R. Acad. Sci. Puris 314, 179. 13. ~~s-Delapo~e, S,, Ferron, F., Landry, J. and Costes, C. (1987) Plant Physiol. 85, 1026. 14. Landry, J., Legris-Delaporte, S. and Ferron, F. (1991) Phyf~h~istry 30,729. 15. Adair, F. W. (1966) J. Bactmiol. 92, 899.
1471
16. Suzuki, I., Ghan, C. W. and Takeuchi, T. L. (1992) Appl. Environ, bficrobiol. 58, 3767. 17. Jolivet, P., Bergeron, E. and Kien, P. (1992) Phyfon 32, 59. 18. Joyard, J., Forest, E., BI&e,E. and Deuce, R. (1988) Pia~f P~ysio~.88,961. 19. Legendre, S. and Marty, F. (1987) in Proc. Inf. Symp. Elemental Sulpb~r in Agriculture, Nice, France (Syndicat Frarqais du Soufre, ed.), p_ 689. 20. Sugio, T., Mizunashi, W., Inagaki, K. and Tano, T. (1987) J. Bacterial. 169,4916. 21. CoIe, Y. and Lesaint, C. (1973) Rev. Nortic. 2316,29. 22. Guillot-S~omon, T., Farineau, N., Cant&, C., Oursel, A. and Tuquet, C. (1987) Pbysiol. Planf. 69, 113. 23. Lilley, R. McC., Fitzgerald, M. P., Rienits, K. G, and Walker, D. A. (1975) New Phytol. 75, 1. 24. Jensen, R. G. and Bassham, J. A. (1966) Proc. Nafn. Acad. Sci. U.S.A. 56, 1095. 25. S&mid, G. H. (1971) in Methods in Enzymology (San Pietro, A., ed.), Vol. XXIII, part A, p. 171. Academic Press, New York. 26. Vetter, R. D., Matrai, P. A., Javor, B. and O’Brien, J, (1989) in ~offenic sulfur in the ~n~ironrne~t (Saltzman, E. S. and Cooper, W. J., eds), Vol. 393, p. 243. American Chemical Society, Washington.