Planta 9 by Springer-Verlag 1977
Planta 135,25-32(1977)
Subcellular Distribution of 35S-Sulfur in Spinach Leaves after Application of 35SO2-4 , 35SO~-, and 35SO 2 Irmgard Ziegler Institut ffir Biochemieder Gesellschaftffir Strahlen- und UmweltforschungmbH. Mfinchen, and Botanisches Institut der TechnischenUniversiffit Miinchen, Arcisstr.21, D-8000 Mfinchen, Federal Republic of Germany
35802, 35802-, and
35SO2-, respectively, were applied to leaves of Spinacia oleracea L. for 60 min in the light. Thereafter, the specific activity was determined in the organelles separated by means of sucrose density gradient centrifugation. In mitochondria and peroxisomes, the specific activity was equally distributed in their protein moieties. After application of 35SOz or 35SO 2-, the chloroplast lamellae are characterized by elevated specific activity, which is not found after application of 35SO2-. Chloroplast stroma shows a low specific incorporation rate after application of either compound, which may be due to the low turnover rate of Fraction I protein,
Abstract.
Key words." Chloroplasts - Chloroplast lamellae Spinaeia - Sulfate incorporation Sulfite incorporation.
Introduction
Like other anions, transported first in the xylem and then in the cell walls of the leaves, sulfate is supposed to be translocated across the plasmalemma into the inner compartments of the leaf cells (cf. L/iuchli, 1972). SO2, penetrating through the stomata, is probably first dissolved in the water of the apoplast. Microautoradiographic studies revealed that afterwards most of the 35S is deposited in the guard cells~of the stomata; much less is found in the epidermis and in the mesophyll (Weigl and Ziegler, 1962). The final transport of 35S compounds is reported to take place in the phloem, according to several authors (Biddulph, 1956; Weigl and Ziegler, 1962; Boukhris, 1972). Transport and incorporation into organic products seem to occur in the same way, regardless of whether the leaves are provided with 802, SO 2-, or SO 2- via roots or via stalks (cf. Ziegler, 1975).
Since the assimilatory sulfate reduction takes place in the chloroplasts (Schmidt and Trebst, 1969; cf. Schiff and Hodson, 1973), and since a sulfite oxidation system is reported to be present in plant mitochondria (Tager and Rautanen, 1955; Stickland, 1961), both organelles appear to take up sulfate as well as sulfite. In addition, indirect evidence for sulfate uptake by chloroplasts is provided by its competition with phosphate (Baldry et al., 1968), whereas in rat liver mitochondria, direct evidence for sulfate and sulfite transport was demonstrated (Crompton et al., 1974). In mammalian liver, rapid incorporation of 35SOZ4 into the Golgi apparatus was recently shown (Katona, 1976). In plants, those parts of the sulfite pool that end up in the sulfur-containing amino acids might be expected to deliver a pattern of specific activity that is about equally distributed among the different organelles. Those that end up in sulfolipids should be found exclusively in the chloroplasts (cf. Benson, 1963). Even though the knowledge of assimilatory sulfate and sulfite reduction in chloroplasts has increased considerably during the past years, the distribution of 35S within the cell and within the organelles after application of 35SO~- or aSSO]- is still unknown. This problem was studied here by means of sucrose density gradient centrifugation.
Materials and Methods Maintainance of Plants and Application of the Isotopes
Spinach was grown as described previously (Ziegler and Libera, 1975), and the plants were used 4-6 weeks after inoculation. Leaves were cut again under water and preilluminated in a water-jacket bath for 30 min with an Ultraphot lamp (18,000lx). For uptake of z5SO4z- or 3sSO~ , 9 leaves (averagetotal area, 20 cmZ; average fresh weight, 0.9 g each) were inserted individuallywith the petioles into test tubes (2.5 ml, containing 4 iiCi 35SO2- or 3sSO]-, respec-
26 tively), which were covered with Parafilm M. The leaves were further exposed to light. The uptake of both sulfate and sulfite was followed every 10 rain for 80 min and proved to be linear during that time. Since the specific activity of sulfite was 2.1 mCi/ retool, the concentration of sulfite (about 0.7 m M ) was so low that no inhibitory effect was to be expected (cf. Ziegler, 1975). For uptake of 35SO2, the test tubes, filled with water, were inserted into an illuminated glass vessel as described by Weigl and Ziegler (1962). After addition of HC1, continuous circulation was achieved by a Neuberger membrane pump. The water vapor of the circulating air was deposited by a Peltier element, supplied by the Siemens company.
Sucrose Density Fractionation The grinding method used followed the technique described by R o c h a and Ting (1970). The first sediment was obtained by centrifugation at 200 g for 10 min (sediment I), the second at 1700 g for 10 min (sediment II), a n d the third at 20,000g for 15 rain (sediment III). The pellets were gently resuspended in 40% (wt/vol) sucrose, dissolved in 0.05 M Tris-buffer, p H 7.5+ 1 m M E D T A , and layered on linear sucrose density gradients of 40-80% (wt/vol), dissolved as described above. The gradients were centrifuged in a swinging bucket rotor at 25,000 rpm (rav= 11.8 cm) for 4 h in a Beckman L 5-50 preparative ultracentrifuge. All operations were carried out between 2 and 4 ~ C. For fractionation, a glass needle was inserted through the gradient down to the b o t t o m of the tube, and the gradient was pumped continuously by an LKB-Varioperpex. One-ml fractions were collected, the sucrose concentrations of which were controlled by a Zeiss refractometer.
Separation of Chloroplastic Constituents For preparation of grana lameUa, stroma lamella, a n d chloroplast envelopes by gentle osmotic rupture of isolated chloroplasts, followed by discontinuous sucrose density gradient centrifugation, the method of Douce et al. (1973) was employed. The fractions corresponding to the three types of m e m b r a n e s were obtained by pumping as described above.
I. Ziegler: Subcellular Distribution of 35S-Sulfur ever, the absolute uptake rate of each fraction is dependent on the total uptake of the leaf and thus on m a n y external factors, we preferred to document the relative asS content of the fractions (in percentage of total uptake). For this purpose, the corrected values for cpm were used.
Enzyme Determination Cytochrome c oxidase was assayed spectrophotometrically as described by Tolbert et al. (1968), catalase, according to Aebi (1970), and hydroxypyruvate reductase as described by Beevers et al. (1974). For ribulosediphosphate carboxylase activity, the procedure used in earlier experiments (Ziegler, 1972) was applied; ferredoxin:NADP+-oxidoreductase was assayed according to Douce et al. (1973); the Hill reaction with 2,6-dichlorophenol indophenol was performed as described by Jacobi (1974).
Chlorophyll and Protein Determination Chlorophyll was assayed according to A r n o n (1949). For protein determination, either the Lowry method (cf. Layne, 1957) or a modification of the amido black 10 B method, described by Popov et al. (1975), was used. Relative protein determination of the fractions could be performed by this method even in fractions that had been first introduced into the scintillation mixture.
Electron Microscopy The fractions were prefixed with 6% glutaraldehyde. After 2 h, they were diluted and washed with 0.06 M phosphate buffer, p H 7.2. After fixation with 2% osmiumtetroxide, they were washed again with phosphate buffer, followed by desiccation in acetone and embedding in Spurr medium. The chemicals used were: sodium [35S]sulfite, sodium [3S]sulfate, and U-[l*C]malate from A m e r s h a m Buchler; Triton X, TS-1, PPO, and POPOP from Zinsser (Frankfurt); cytochrome c and N A D P H from Boehringer (Mannheim); amido black 10 B from Serva (Heidelberg) ; ribulosediphosphate Na-salt and hydroxypyruvate from Sigma; all other chemicals were analytical grade from Merck.
Sample Counth~g Aliquots of the fractions were introduced into the scintillation mixture (Triton X/PPO/POPOP/toluene) containing 5% tissue solubilizer TS-1 and counted in a BF betascint 5000. Control experiments with unlabeled tissue fractions revealed that samples, even when kept in the dark for 60 min, showed chemiluminescence, thus pretending radioactivity. Moreover, the intensity of chemiluminescence followed the pattern of organelle distribution exactly. It proved necessary to keep the samples in the dark and cooled at 15~ for 16 h. After that time, the chemiluminescence had disappeared . . . . " ~; ~ " To confirm that true radioactivity Of theisamples, rather than chemiluminescence, was measured, die fractim?s were dried on ashless filter paper in two experiments. The 3sSO3 that evolved after burning in oxygen flasks was trapped and counted as described by R a u s c h e n b a c h and Simon (1974). Due to the different protein and chlorophyll contents of the fractions, the extent of quenching varied over a wide range. The quench correction was made by internal standardization of each fraction with added [14C]malate. In this way, true cpm are obtained. In most cases, a correction for dpm, provided by a computerized program (Diehl Algotronic), was recorded. Since, how-
Results
The Distribution of Organelles Along the Gradient The differential centrifugation, preceding separation by the density gradient, yields three fractions with characteristics summarized in Table 1. The data agree with the electronmicroscopic picture, in that sediment II comprises most of the peroxisomes, whereas the mitochondria predominate in sediment III. Chlorophyll is found in all sediments, but it is derived from chloroplastic material of considerably different states. In sediment I, where the bulk of the chloroplasts is found together with incompletely ruptured cells, the chloroplasts retain their envelopes and show normal ultrastructure. Those of sediment II in general have lost their outer membranes, but their stroma
I. Ziegler: Subcellular Distribution of 35S-Sulfur
27
Fig. l a-d. Electron micrographs of thin sections from organelle fractions: sediment II a; sediment III b; pooled fractions 23 +24 from sediment II e; pooled fractions 23 +24 from sediment III d; P. peroxisomes; M: mitochondria; Chl: chloroplasts or chloroplast lamellae, respectively
Table 1. Percentage distribution of protein, chlorophyll, marker enzymes, and specific radioactivity among the sediments after differential centrifugation of spinach leaf homogenate % of Total
Sediment I Sediment II Sediment III
protein
chlorophyll
hydroxypyruvate reductase
cytochrome c oxidase
ribulosediphosphate carboxylase
Hill activity
25.9 50.0 24.1
32.5 54.2 13.3
12.7 49.5 37.8
11.7 29.6 58.7
68.0 28.0 4.0
24.2 30.0 45.5
% of Total spec. radioactivity
28.0 34.0 37
28
I. Ziegler: Subcellular Distribution of 35S-Sulfur
o/~ of toto.t
content
Density
15
content
O/o of total
Density
t1.4o
1.3o
1.20
1.2o
i
%
i
i
i
J 5
i
i
i
E
i ~ 10
~
i
i
~ i 15
i
i
i
i 20
i
i
i
i
i 25
i
i
t 30
total content
1.10 /. /~ rllcbon
i
Densit,
"/o of
i
J
i
i 5
~
i
r
L
I I 10
I
I
I
I I 15
I
,
I
I 20
i
I
i
i
i 25
I
I
totoL activity
I
I
I 3O
1.10 fracti
on
Density
9 1.4o
15 84
1.30
1.20
1.30
~0
1.20
2
1.10 I
I
I
I
I
5
I
I
~
I
I
10
I
I
J
[
I
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~
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I
20
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$
25
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I
J
o~
1.10
I
30
fraction
. . . .
;
. . . .
1'0 . . . .
,'5 . . . .
2'0 . . . .
2'5 . . . .
30
fmctio~
d
Fig. 2a-d. Percentage distribution of protein, chlorophyll, and marker enzymes after linear density gradient centrifugation: protein in sediment II a; protein in sediment III b; chlorophyll in sediment II c; hydroxypyruvate reductase in sediment II ( ), catalase in sediment II (. . . . . . ), cytochrome c oxidase in sediment III ( . . . . . ) d
and their lamellar structures are still present (Fig. 1 a). In sediment III, besides osmiophilic granules, only the free lamellar system remains (Fig. 1 b). Consequently, the percentage of ribulosediphosphate Carboxylase is markedly decreased (Table 1). Moreover, the ratio ferredoxine: NADP § reductase/ribulosediphosphate carboxylase in sediment I is 0.67; it increases to 0.95 in sediment II, and to 6.9 in sediment III. This indicates an increasing portion of lamellar protein but a decreasing one of stroma (Pineau and Douce, 1975); even those parts of ribulosediphosphate carboxylase that might be attached to the lamellar system (Strotmann et al., 1973) seem to be largely lost. After density gradient distribution of sediments II and III, the typical pattern of protein, chlorophyll, and marker enzymes along the gradients is demonstrated in Figures 2a-d. It is evident that the peroxisomes, characterized by hydroxypyruvate reductase and catalase, emerge between fractions 8 and 12 (maximum at a density of 1.25), the mitochondria with cytochrome c oxidase activity between fractions 13 and 21 (maximum at a density of 1.21), and the chlorophyll-containing material between 20 and 25 (maximum at a density of 1.17). In sediments II and III, the latter provides the bulk of protein. Moreover, it coincides with the pattern of ferredoxine:NADP §
reductase and also with the Hill reaction (Fig. 3a, b). Nevertheless, these fractions of chloroplastic origin are markedly different in both sediments, as indicated previously. Electronmicroscopic controls confirm that, in sediment II, these fractions retain the stromal portion of the chloroplasts (Fig. l c) whereas such fractions of sediment III deliver the free lamellar system (Fig. 1 d). The extensive loss of stroma is also indicated by the fact that in the chloroplastic fractions from sediment III the ribulosediphosphate carboxylase activity has dropped to about 12 gmol HCO~-/mg chlorophyll/h, whereas in those of sediment II, the activity level is 50-60 gmol/mg chlorophyll/h. The protein peak found in sediment III at a density of 1.15 corresponds to a plasma membrane fraction described recently (Sinensky and Strobel, 1976; Leonard and Van der Woude, 1976), which was not characterized in any further detail.
Incorporation of 35S Into the Subcellular Fractions Prior to density gradient fractionation no gross differences in the percentage distribution of specific activity
I. Ziegler: Subcellular Distribution of 35S-Sulfur Chtorophytl
29 rag/mr
Chlorophyll
mg/ml
28
fr~ction
O,lOO
1oo
/\
~0-
,.o
.c
!
s
~-
E 50
-._
f~,-
30-
0.05
i
r"=i o,,
20-
10"
,
,
7
,
,
,
,
,
,
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2'~
25
20
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~176
17
a
20
25
b
Fig. 3a and b. Chlorophyll, Hill activity, and ferredoxine: N A D P § oxidoreductase after linear density gradient centrifugation. Sediment II: chlorophyll o . . . . o, Hill activity o - - . . , ferredoxine:NADP § oxidoreductase x - - x a; sediment III b
Table 2. Percentage distribution of specific radioactivity among the organelles after administration of 35SO2, 3sSO32- , and ~sSO,ZPeroxisomes
Mitochondria
Chloroplastic fraction (stroma + lamellae)
Chloroplastic fraction (only lamellae)
30.0 32.5
33.0 35.8
36.5 32.2
not present in sediment II not present in sediment II
29.0 22.9
33.6 24.9
not present in sediment III not present in sediment III
36.5 52.0
Sediment II After feeding of 3s SO,~ After feeding of 3sSO2 or 35SO2 Sediment III After feeding of 3sSO~After feeding of 3sSO~- or 35SO2
% of
totat specific activity
10
/ ~)}
'
~
'
'
5'
~
i
f "
10
"
15
20
"-o
25
fraction
Fig. 4. Percentage distribution of total specific radioactivity after linear density gradient centrifugation: sediment III after feeding of 35SO 2 or 3sSO32 x - - x ; sediment III after feeding of 35SO~- e . . - o ; sediment II after feeding o f 35SO2, 3~SO32-, or 3sSO2O ....
O
I. Ziegler: Subcellular Distribution of 3sS-Sulfur
30
"1, of totaL content
1.5 M
1.2 M
sucrose
sucrose
0.93 M sucrose
I
15
I
I
0.6 M sucrose
M sucrose
0.05
[
Table3. Percentage distribution of specific radioactivity in osmotically ruptured chloroplasts after discontinuous sucrose density gradient centrifugation
7_
10
Chloro- Chloro- Supernatant plast plast (stroma-bound3sS lamellae envelopes +free 35SO~+ 3sSO~-) Feeding of 35SO~Feeding of 3sSO~-
57.2 21.0
6.4 11.5
33.1 61.8
5
I
I
I
I
I
5
I
I
I
I
I
10
I
I
I
I
~
15
I
I
I
I
I
2O
I
I
I
I
25 froctio n
Fig. 5. Percentage distribution of protein after discontinuous density gradient centrifugation of osmotically ruptured chloroplasts
among sediments I, II, and III can be seen (Table 1). After density gradient fractionation of sediments II and III, the sum of specific activities found in the peroxisomal, in the mitochondrial, and in the chloroplastic fractions, respectively, was ascertained and is given in Table 2 as a percentage of total activity found. It is evident that mitochondria and peroxisomes are identical in their specific activities; their percentage of the total activity correlates with their percentage of total protein. Thereby, the specific activity of the residual peroxisomes in sediment III does not differ from that of the majority found in sediment II, and the same holds true for the mitochondria already sedimented in II, compared with their bulk in sediment III. The chloroplastic fractions show quite different behavior (Table 2). Those derived from sediment II, and representing relatively intact chloroplasts, show a low specific activity; it is markedly increased in the lamellar fractions obtained from sediment III. This, however, only holds true if 3sSO2 or 35SO~was applied (Fig. 4). After application of 35SO]-, the specific activity in the lamellar fractions, delivered by sediment III, is as low as in the stroma-containing chloroplasts of sediment II. This suggests that 3sS, delivered only by sulfite or by SO2, but not by sulfate, is preferentially incorporated into the lamellar system. For further examination, intact chloroplasts were subjected to gentle osmotic swelling, followed by centrifugation in a discontinuous sucrose density gradient. The procedure yielded a separation pattern of grana and stroma lamellae and of the chloroplast envelopes as described by Douce et al. (1973). In Figure 5 the profile of protein distribution along the gradient demonstrates the location of the organelles. In the 0.05 M sucrose layer at the top of the gradient,
all activity derived from the stroma protein and from free sulfate or sulfite, should be expected. The data in Table 3 confirm that 3sS derived from sulfite is incorporated to a much greater extent into both lamellar fractions than is 35S derived from sulfate. Besides, the envelope fraction comprises a small portion of the total specific activity.
Discussion
The incorporation rate of 35S into the peroxisomes and the mitochondria, regardless of whether SO2, sulfite, or sulfate is applied, indicates that the sulfur is distributed equally among their protein moieties; no sulfur-containing compound specific for one of the two organelles seems to be accumulated. The abundance of [35S]sulfate occurring after application of 35SO2 (Weigl and Ziegler, 1962), is surely not associated preferentially with one organelle in comparison to the others, though in plants, sulfite oxidation seems to be linked to the mitochondria (Tager and Rautanen, 1955; Stickland, 1961). The experiments do not provide information as to whether the proteins of peroxisomes and of mitochondria being equally labeled, are derived exclusively from assimilatory sulfate reduction in the chloroplasts, or whether, like in Euglena (Brunold and Schiff, 1976) sulfate reduction is also associated partly with microbodies and partly with mitochondria. After application of 3sS, regardless of whether it is supplied as SO2, sulfite, or sulfate, low specific activity is characteristic for chloroplasts that retain their stroma, that is, if stromal protein is included in the calculation of the specific activity. Willenbrink (1967) reported that chloroplast protein contains 10-15% less sulfur than other leaf protein. However, J the low specific activity after short-term application of 35S more likely may be attributed to the remarkable lack of turnover in Fraction I protein (Huffaker and Peterson, 1974), which compromises up to 50% of leaf
I. Ziegler: Subcellular Distribution of 3sS-Sulfur
protein (cf. Kawashima and Wildner, 1971) and thus, appropriately more of the chloroplast protein. In contrast to chloroplast stroma, the lamellae undergo rapid sulfur incorporation after application of 35302 or 35SO2-, but not after that of 35SO2-. At present, two possible explanations are at hand: In Chlorella pyrenoidosa, the sulfolipids were highly specifically labeled after 40 min in the light (see Benson, 1963). In higher plants, these are located in the lamellar fragments of the chloroplasts. Thus, the more rapid incorporation after application of [35S]sulfite than after [3SS]sulfate is consistent with the view that the sulfonic group of the sulfoquinovosyl moiety is derived directly from sulfite (e.g., enolpyruvate ---, /?-sulfonylpyruvate; Davies et al., 1966), but not from sulfate, which is first reduced to S2-. Another reason for elevated specific activity after application of 35SO2- may be represented by the "sulfite binding sites" that are present in the thylakoids (Schwenn et al., 1976). Infiltration of exogenous 3sSO~- ions m a y saturate these sulfite binding groups by mere addition to Car. 5 or by cleavage of the disulfide form of the carrier (cf. Schiff and Hodson, 1973) much more rapidly and to a greater extent than does the less concentrated sulfate via APS and PAPS. The identification of the status of 35S in the chloroplastic components should elucidate the preferential uptake of 35SO~- into the lamellae. In this context, it is evident that SO2 exerts a more manifold action than do pollutants not involved in plant metabolism. This is especially true for low concentrations, which may even prove to be beneficial with respect to CO2 fixation (Libera et al., 1973 ; Zieger and Libera, 1975). The skillful technical assistance by Frau E. Schoepe is gratefully acknowledged. The electronmicroscopic procedures were made by Frau K. Blase. I am indebted to Dr. P. Rauschenbach (Inst. ffir Biochemie, Technical University, Mfinchen) for most valuable discussions and advice with respect to scintillation techniques.
References Aebi, H.: Katalase. In: Methoden der enzymatischen Analyse. Bergmeyer, H.U., ed., pp. 636 647. Weinheim: Verlag Chemic 1970 Arnon, D.I. : Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (I949) Baldry, C.W., Cockburn, W., Walker, D.A. : Inhibition, by sulfate, of the oxygen evolution associated with photosynthetic assimilation. Biochim. Biophys. Acta 153, 476 483 (1968) Beevers, H., Theimer, R.R., Feierabend, I. : Microbodies (Glyoxysomen, Peroxysomen). In: Biochemische Cytologie der Pflanzenzelle. Jacobi, G., ed. Stuttgart: G. Thieme 1974 Benson, A.A.: The plant sulfolipid. Adv. Lipid Res. 1, 387 394 (1963)
31 Biddulph, S.F.: Visual indications of S 35 and p32 translocation in the phloem. Amer. J. Bot. 43, 143-148 (1956) Boukhris, M.: Localisation histo-autoradiographique du 3sSO2dans les tissus de l'Erodium glaucophy!!um. , C.R: &cad. Sci, 275, 2651 2654 (1972) Brunold, Ch., Schiff, J.A.: Studies of sulfate utilization by algae. 15. Enzymes of assimilatory sulfate reduction in Euglena and their cellular organization. Plant Physiol. 57, 430-436 (1976) Crompton, M., Palmieri, F., Capano, M., Quagliariello, E.: The transport of sulphate and sulphite in rat liver mitochondria. Biochem. J. 142, 127 137 (1974) Davies, W.H., Mercer, E.I., Goodwin, T.W.: Some observations on the biosynthesis of the plant sulfolipid by Euglena graeilis. Biochem. J. 98, 369-373 (1966) Douce, R., Holtz, R.B., Benson, A.A.: Isolation and properties of the envelope of spinach chloroplasts. J. Biol. Chem. 248, 7215 7222 (1973) Huffaker, R.C., Peterson, L.W.: Protein turnover in plants and possible means of its regulation. Ann. Rev. Plant Physiol. 25, 363-392 (1974) Jacobi, G.: Chloroplasten. In: Biochemische Cytologic der Zelle, pp. 72-108, Jacobi, G., ed. Stuttgart: G. Thieme I974 Katona, E.: Incorporation of inorganic sulfate in rat-liver Golgi. Europ. J. Biochem. 63, 583-590 (1976) Kawashima, N., Wildner, S.G.: Fraction I protein. Ann. Rev. Plant Physiol. 21, 325-358 (1971) Lfiuchli, A. : Translocation of inorganic solutes. Ann. Rev. Plant Physiol. 23, 197-218 (1972) Layne, E. : Spectrophotometric and turbidimetric methods for measuring proteins. In: Methods in E~zymology, Vol. 3, pp. 447-454, Colowick, S.P., Kaplan, N.Y., eds. New York: Academic Press 1957 Leonard, R.T., Van der Woude, W.H. : Isolation of plasma membranes from corn roots by sucrose densitiy gradient centrifugation. An anomalous effect of Ficoll. Plant Physiol. 57, 105-114 (1976) Libera, W., Ziegler, H., Ziegler, I.: F6rderung der Hill-Reaktion und der COz-Fixierung in isolierten Spinatchloroplasten dutch niedere Sulfitkonzentrationen. Planta 109, 269 279 (1974) Pineau, B., Douce, R.: Analysis of the protein composition of spinach chloroplast envelopes. In: Proc. lII Internat. Congr. Photosynth. pp. 1667-1673, Avron, M., ed. Amsterdam: Elsevier 1975 Popov, N., Schmitt, M., Schulzech, S., Matthies, H. : Eine st6rungsfreie Mikromethode zur Bestimmung des Proteingehaltes in Gewebehomogenaten. Acta Biol. Med. Germ. 34, 1441 1446 (1975) Rauschenbach. P., Simon, H. : Sample preparation with an automated oxygen flask combustion apparatus for liquid scintillation counting of 3H-, 14C-, and/or 3sS-labelled material. In: Liquid Scintillation Counting, Vol. 3, pp. 158-163. Crook, M.A. Johnson, P., eds. London-New York: Hyden 1974 Rocha, V., Ting, I.P. : Preparation of cellular plant organelles from spinach leaves. Arch. Biochem. Biophys. 140, 398-407 (1970) Schiff, J., Hodson, R.C.: The metabolism of sulfate. Ann. Rev. Plant Physiol. 24, 381-414 (1973) Schmidt, A., Trebst, A. : The mechanism of photosynthetic sulfate reduction by isolated chloroplasts. Biochim. Biophys. Acta 180, 529-535 (1969) Schwenn, J.D., Depka, B., Hennies, H.H.: Assimilatory sulfate reduction in chloroplasts: Evidence for the participation of both stromal and membrane-bound enzymes. Plant Cell Physiol. (Tokyo) 17, 165-171 (1976) Sinensky, M., Strobel, G.: Chemical composition of a cellular fraction enriched in plasma membranes from sugarcane. Plant Sci. Letters 6, 209-214 (1976) Stickland, R.G.: Oxidation of reduced pyridine nucleotides and /
32 of sulphite by pea root mitochondria. Nature (Lond.) 190, 648-649 (1961) Strotmann, H., Hesse, H., Edelmann, K. : Quantitative determination of coupling factor CF 1 of chloroplasts. Biochim. Biophys. Acta 314, 202-210 (1973) Tager, J.M., Rautanen, N.: Sulphite oxidation by a plant mitochondrial system. Biochim. Biophys. Acta 18, 100-121 (1955) Tolbert, N.E., Oeser, A., Kisaki, T., Hageman, R.H., Yamazaki, R.K.: Peroxisomes from spinach leaves containing enzymes related to glycolate metabolism. J. Biol. Chem. 243, 5179-5184 (1968) Weigl, J., Ziegler, H.: Die rfiumliche Verteilung von 35S und die Art der markierten Verbindungen in Spinatblfittern nach Begasung mit 3sSO 2. Planta (Berl.) 58, 435447 (1962)
I. Ziegler: Subcellular Distribution of 35S-Sulfur Willenbrinck, J. : Uber Beziehungen zwischen Proteinumsatz und Schwefelversorgung der Chloroplasten. Z. Pflanzenphysiol. 56, 427-438 (1967) Ziegler, I.: The effect of SO~ - on the activity of ribulose-l,5diphosphate carboxylase in isolated spinach chloroplasts. Planta (Berl.) 103, 155-163 (1972) Ziegler, I.: The effect of SO z pollution on plant metabolism. Res. Rev. 56, 79-105 (1975) Ziegler, I., Libera, W.: The enhancement of CO2-fixation in isolated chloroplasts by low sulfite concentration and by ascorbate. Z. Naturforsch. 30c, 634-637 (1975)
Received 6 December 1976; accepted 5 January 1977