Nov 25, 1980 - ABSTRACT. The distribution of sulfur-containing metabolites in cultured tobacco cells was determined by analyzing efflux kinetics. Transported ...
Plant Physiol. (1981) 68, 937-940 0032-0889/8 1/68/0937/04/$00.50/0
Compartmentation of Sulfur Metabolites in Tobacco Cells' USE OF EFFLUX ANALYSIS Received for publication November 25, 1980 and in revised form April 17, 1981
IVAN K. SMITH Department of Botany, Ohio University, Athens, Ohio 45701 cells have small pools of sulfur-containing metabolites (I 1). Usually the pools of sulfur-containing metabolites were labeled The distribution of sulfur-containing metabolites in cultured tobacco by adding either 4 ml 10 mm or 4 ml 10 MM L-[MS]cells was determined by analyzing efflux kinetics. Transported sulfate cysteine (0.1 Ci/mol) through[35S]Na2SO4 a syringe containing a Millipore rapidly labeled the cytoplasmic pools of sulfate (1 hour) and sulfur amino filter. In one experiment, different transport rates were established acids (6 hours). Excess sulfate and amino acids were transported into the by transferring cells to media with different pHs. Cells (10 ml) vacuole. The size and distribution of the amino acid pool was not affected from 8-day-old sulfur-deficient cultures were inoculated into 70 by increasing the sulfate content of the cells. ml of transport medium consisting of 0.1 strength sulfur-deficient Transported cysteine was rapidly degraded to sulfate. The cytoplasmic B5, 10 mm bis-Tris-propane, 0.5 mm CaCl2, and 1% (w/v) sucrose. pools of sulfate and amino acids were completely labeled within 6 hours, The transport medium contained either 0.5 mm [mS]Na2SO4 or 0.5 whereas the vacuolar pools were labeled more slowly. The intracellular mM [35S]cysteine and had a pH ranging from 5 to 8. cysteine pool was elevated by decreasing the pH of the transport medium. Efflux Kinetics. The method of Delmer (4) was used to measure In all experiDents, between 60 and 90% of the label was present in the efflux kinetics. Cells (1 g fresh weight) were harvested by vacuum vacuole. filtration, suspended in 20 ml 0.8 mm KCl and 0.85 mM CaCl2 in 50-ml Erlenmeyer flasks, and shaken at 80 rpm on a rotary shaker. At the times indicated, the cells were harvested by vacuum filtration, the filtrate collected, and the cells resuspended in 20 ml of the same medium. Metabolites remaining in the cells after 4 h were released by boiling the cells for 5 min in 20 ml efflux medium. Tobacco cells convert sulfate to cysteine using the sulfate assimA 5-ml aliquot of the filtrate was placed in 10 ml of liquid ilation pathway (9, 11) and convert cysteine to sulfate using a scintillation fluid to determine total radioactivity. Usually, the degradative pathway (7, 10). My hypothesis is that futile cycling filtrate was passed through a cation exchange column to permit of sulfur from sulfate to cysteine and back to sulfate is reduced by separate determination of sulfate and sulfur-containing amino compartmentation of the two pathways. Compartmentation of acids (10). In some experiments, the sulfur-amino acid fraction sulfur metabolites in plants has been demonstrated. Giovanelli et was oxidized with performic acid and passed through a cation al. (6) presented evidence that Chlorella has two pools of cysteine exchange column to yield a fraction containing cysteic acid and a with different metabolic fates. Wagner (13) showed that the dis- fraction containing methionine sulfone and glutathione sulfone tribution of cysteine and methionine between the cytoplasm and (10). the vacuole differed in Tulipa petals and leaves. My initial approach to compartmentation of sulfur metabolism in tobacco cells RESULTS AND DISCUSSION was to determine the distribution of sulfur metabolites using Effect of DMSO2 on Efflux of Sulfur Metabolites. Delmer (4) analysis of efflux kinetics. proposed that DMSO may be useful for compartmentation analysis in plants, because it renders the plasma membrane more METHODS permeable to small molecules while having far less effect on the Culture and Labeling Conditions. Tobacco XD-cell line (Nico- permeability of the vacuolar membrane. DMSO increased the rate tiana tabacum L. var Xanthi) were cultured in modified B5 of efflux of radioactivity from cells fed either sulfate (Fig. 1) or medium (11). Standard B5 medium contains 25 mt KNO3, 1 mM cysteine (not shown). DMSO (7.5%, v/v) decreased the size of the (NH4)2SO4, and 1 mM MgSO4 as nitrogen and sulfur sources (5). slow compartment, as indicated by the intercept on the ordinate, Components other than these were not changed in tbe modified by 26% when sulfate was to label the cells (Fig. 1) and 31% medium. Modified B5 contained 25 mm KNO3, 1 mm (NH4)2SO4, when cysteine was used.used Reductions of this magnitude were and 1 mM MgCl2. Sulfur-deficient B5 contained 25 mm KNO3, 2 reported by Delmer (4), who measured the kinetics of rubidium, mM NH4CI, and 1 MM MgC12. Stock cultures were maintained in 80 ml modified B5 medium tryptophan, and sugar efflux from tobacco cells. In the case of in a 250-ml Erlenmeyer flask at 25 C on an 80-rpm rotary shaker. rubidium, 5% DMSO converted the fast and intermediate comA 10-ml aliquot of suspension was transferred into 70 ml of sulfur- partment (tl/2 = 7.0 min) into a single fast compartment (tl/2 = 3.8 deficient B5 medium, and the cells were grown for 8 days before min). The advantage of measuring a single fast compartment is initiation of an experiment. Previous work indicates that these outweighed by the absence of a satisfactory explanation for the effect of DMSO on the slow compartment. Consequently, DMSO was not used in subsequent experiments. 1 Supported by the Science and Education Administration of the United ABSTRACT
States Department of Agriculture under Grant 5901-0410-8-0057-0 from the Competitive Research Grants Office.
2Abbreviation: DMSO, dimethylsulfoxide. 937
938
c .
a)
E c
_
2.84.,
J
.
~~~~~~A
\
2.761 (
2.72
0
2.681 2.64 - - "-"
%a.-,
\~
0
I
2.80
Oz
0'
Plant Physiol. Vol. 68, 1981
SMITH
75%
DMSO
E
,
AO.
1
Iv
200
240
A
40
120
60
160
2.4
0
-
"0'N
B
o
40
80
120
10%
DMSO
-Io\
160
200 240
296
ct:
cy o
2.88 2.84
2.801 c7Ie
40
120
80
160
2.7
200 240 B
..,
2.5
0' 0
I 21
L-
10
20
30
40
50
(MIN.) FIG. 3. Kinetics of release of sulfur-containing amino acids from cells fed sulfate. A, Total sulfur amino acids (nmol/g fresh weight) remaining in the cells; B, slow exponential phase in A was subtracted from the total to yield this secondary plot.
';~~~~~
2.92
2.01
1.61
(MIN.)
FIG. 1. Effect of DMSO on efflux of sulfur metabolites from cultured tobacco cells. Cells were labeled with [5S]04 for 24 h and harvested, and efflux analysis was performed in the presence and absence of DMSO. The soluble sulfur-containing metabolites present in the cells after various washout periods are indicated. 3.00
0
(/)
\9 "\
11
2.3 CK
0%2.1 0
1.9I 17 10
20
30
40
50
(MIN.) FIG. 2. Kinetics of release of sulfate from cells fed sulfate. A, Total sulfate (nmol/g fresh weight) remaining in the cells; B, slow exponential phase in A was subtracted from the total to yield this secondary plot.
Kinetics of Sulfur Metabolite Efflux. The material released by
['S]04-labeled cells was fractionated by ion-exchange chromatog-
raphy into a cationic and a noncationic fraction. Previous works
(10, 11) indicate that greater than 90% of the cationic fraction is cysteine, glutathione, and methionine and that sulfate is the principle component of the noncationic fraction. The kinetics of sulfate efflux (Fig. 2) show a rapid phase followed by a slower exponential phase. Extrapolation of the slower phase to the ordinate yielded the amount of sulfate in the slow compartment initially. The latter amount and the amount remaining in the cells after 240 min were substituted into a first order rate equation to obtain a tl/2 of 862 min for the slow component. Subtraction of the slower phase from the total yielded a second exponential phase (t1/2 = 22 min) and a very rapid phase (tl/2 = 2 min). Extrapolation otthe second exponential phase to the ordinate yielded the amount of sulfate in the intermediate compartment. The kinetics of amino acid release (Fig. 3) indicated two exponential phases, slow (t1/2 = 899 min) and intermediate (tl/2 = 16 min). In accordance with previous interpretations of a three-component serial model, the slow phase is attributed to loss from the vacuoles, the intermediate phase to loss from the cytoplasm, and the rapid phase to loss from the apparent free space (4, 8). The absence of a rapid phase when amino acids were measured is consistent with this interpretation (Fig. 3). Additionally, when [3SJcysteine was used to label the cells, the amino acid efflux curve was composed of three phases, whereas the sulfate efflux curve was only composed of two: slow and intermediate (data not shown). Distribution of Sulfate and Sulfur-Containing Amino Acids in Tobacco Cells. Cells were fed 0.5 mm ['S]Na2SO4 for various periods, and compartment analysis was performed as described in "Methods." The cytoplasmic pool ofsulfate was almost completely labeled within 1 h, after which time additional sulfate was transported into the vacuole (Fig. 4A). Equilibration of sulfate between
Plant Physiol. Vol. 68, 1981
COMPARTMENTATION OF SULFUR METABOLITES I, , 12001
939 +
A
0
1000 800
0
a
I
600-
4001 LA:
7
12
0
E c
24
36
2001 LL
16
6
2 4 6 8 10
16
2
24
36
0
E
C:
B
300 P
\+
200
1oo0 2 4 6 8 10
12
24
36
HOURS FIG. 4. Distribution of sulfur-containing metabolites in cells fed sulfate. Cells were fed 0.5 mM [35S]O4 for various periods. The distribution of label between cytoplasm (@ -) and vacuole (O-O) was determined by analyzing efflux kinetics. A, Sulfate; B, sulfur amino acids. Table I. Estimated Concentration of Sulfate in Cultured Tobacco Cells The data in Figure 4 were used to estimate sulfate concentration. The intermediate component was attributed to the cytoplasm representing 20% of the volume of the cells, and the slow component was attributed to the
vacuole.
/f\-L-1t '\ 2 4 6 8 10
16
24
36
HOURS FIG. 5. Distribution of sulfur-containing metabolites in cells fed Lcysteine. Cells were fed 0.5 mM L-[1Slcysteine for various periods. Seventyfive percent of the cysteine had been removed from the medium by 24 h. The arrow indicates the addition of labeled cysteine to reestablish a concentration of 0.5 mm. The label in the cytoplasm (@-) and vacuole (O-O) is illustrated. A, Sulfate; B, sulfur amino acids.
graded to sulfate, as previously reported (7, 10, 11). The sulfate completely labeled the cytoplasmic pool within 6 h, and a major Labeling Period fraction of the sulfate entered the vacuole (Fig. 5A). Maximum Medium Cytoplasm Vacuole labeling of the cytoplasmic amino acid pool occurred in 6 h in both cysteine- and sulfate-fed cells (Figs. 4B and 5B). I would mM h expect that the cytoplasmic pool of cysteine would be labeled 0 0.50 0 0 within 1 to 2 h. The slower labeling of the total amino acid pool 0.49 0.70 0.07 1 is probably due to the slow synthesis of glutathione and methio0.86 3 0.48 0.21 nine (10, 11). Experiments are in progress to confirm these sup0.67 0.46 0.34 6 positions. 0.56 9 0.45 0.48 The Effect of Sulfate and Cysteine Transport Rate on the 0.42 0.48 0.47 12 Distribution of Sulfur Metabolites. The results reported above 0.41 0.36 0.34 24 0.35 were obtained by adding labeled compounds to cells in culture 0.29 0.28 36 media where the transport rates are relatively low. Higher transthe cytoplasm and the vacuole occurred within 9 h (Table 1). The port rates were established by transferring cells to a simpler cellular sulfate concentration declined as the medium was depleted transport medium. The objective was to determine the distribution of sulfate (Table I). In this experiment (performed at pH 6.0), the of metabolites in cells overloaded with either sulfate or cysteine. The sulfate transport rate increases when the pH of the medium accumulation of sulfate was minimal. In contrast, accumulation was observed in later experiments conducted at pH 8.0. There is is increased from 5 to 8 (Fig. 6). The rate of sulfate transport into no evidence for either exclusion of sulfate from the vacuole or the cells had no effect on the intracellular distribution of sulfate, accumulation of sulfate in the vacuole above the concentration in i.e. from 80 to 85% was located in the vacuole (Fig. 6A). The the cytoplasm. The cytoplasmic pool of sulfur amino acids was higher rate of sulfate transport at pH 8 did result in a significant more slowly labeled (Fig. 4B). A significant fraction of the newly elevation of the cytoplasmic pool. The 3-fold difference in sulfate synthesized amino acids entered the vacuole (Fig. 4B). Similar content, however, had no effect on the total amino acid pool or results were obtained by Wagner (13), who showed that, in Tulipa the distribution of this pool (Fig. 6B). The insensitivity of the leaves, 82% of the cysteine and 75% of the methionine were sulfur amino acid pool to fluctuating intracellular sulfate has been reported in tobacco cells (10) and in Lemna (3). This suggests that localized in the vacuole. The distribution of label in [3S]cysteine-fed cejs (Fig. 5) was in vivo regulation of the sulfate assimilation pathway maintains similar to that obtained with sulfate. Cysteine was rapidly de- stable pool sizes. Several in vitro studies have examined potential Concentration
SMITH
940 1000,
Plant Physiol. Vol. 68, 1981 2400 i
A
I
0
0
2000
800
A
0
0
1600 F
600
0\
400
1200F
200
0
8001-
1EL:9 5
6
400
8
7
:
B
200
5
E
6
7
8
7
8
I 0-
c
E
150 F
c
2000
B/
1600 100
1200 50
F
800
5
6
7
400
8
pH FIG. 6. Effect of the pH of the medium on the size and distribution of metabolite pools in cells fed sulfate. Cells were harvested and placed in transport medium containing 0.5 mM ['S]04 for 24 h. The label in the cytoplasm ( - *) and vacuole (0-O) is illustrated. A, Sulfate; B, sulfur amino acids. Table II. Estimated Concentration of Sulfur Metabolites in Cells Fed
/MSJCysteine
5
pH FIG. 7. Effect of the pH of the medium on the size and distribution of metabolite pools in cells fed L-cysteine. Cells were harvested and placed in transport medium containing 0.5 mM L-[ISlcysteine for 24 h. The label in the cytoplasm (@-) and vacuole (0 -0) is illustrated. A, Sulfate; B, sulfur amino acids.
Cells were exposed to [35SJcysteine for 24 h at pHs 5, 6, 7, or 8. An efflux analysis was performed and the samples fractionated. The intermediate component was attributed to the cytoplasm representing 20%Yo of the volume of the cells, and the slow component was attributed to the vacuole. Cysteine Medium
pH
Cyto-
Vacuole
plasm
Melfthioneate
Cyto
Vacuole
plasm
Cyto-
11.9 6.6
3.1 1.7
3.3 2.4 2.3 0.9
0.6 0.5 0.6 0.4
1. BERTAGNOLLI BL, RT WEDDING 1977 Purification and initial kinetic character-
2.
4.
plasm
5.
0.3 0.4 0.4 0.3
3.4 2.8 3.2 2.6
3.0 3.5 3.8 3.8
sites of regulation (1, 2, 9-12). The cysteine transport rate decreases when the pH of the medium is increased from 6 to 8 (Fig. 7). The high rates of cysteine transport at low pH caused a large increase in both the cytoplasmic and the vacuolar pools of amino acids. This increase was due totally to an increase in cysteine (Table II). The present experiments were not designed to determine flux, and, therefore, it is not known what effect cysteine concentration has on the flux of sulfur through methionine into protein. It is clear, however, that the amount of label in soluble methionine and glutathione is unaffected by the intracellular cysteine concentration (Table II). A significant proportion of the cysteine entering cells is degraded to sulfate, which confirms our earlier work (7, 10).
of different forms of O-acetylserine sulfhydrylase from seedlings of two species of Phaseolus. Plant Physiol 60: 115-121 BRUNOLD C, A SCHMIDT 1978 Regulation of sulfate assimilation in plants. 7. Cysteine inactivation of adenosine 5-phosphosulfate sulfotransferase in Lemna minor L. Plant Physiol 61: 342-347 DATKO AH, SH MUDD, J GIOVANELLI, PK MACNICOL 1978 Sulfur-containing compounds in Lemna perpusilla 6746 grown at a range ofsulfate concentrations. Plant Physiol 62: 629-635 DELMER DP 1979 Dimethylsulfoxide as a potential tool for analysis of compartmentation in living plant cells. Plant Physiol 64: 623-629 GAMBORG OL 1970 The effects of amino acids and ammonium on the growth of plant cells in suspension culture. Plant Physiol 45: 372-375 GIOVANELLI J, SH MUDD, AH DATKO 1978 Homocysteine biosynthesis in green plants. Physiological importance of the transsulfuration pathway in Chlorelta sorokiniana growing under steady state conditions with limiting sulfate. J Biol ization
Vacuole
mM
5 6 7 8
LITERATURE CITED
3.
Glutathione
6
6.
Chem 253: 5665-5677 7. HAINGTON HM, IK SMITH 1980 Cysteine metabolism in cultured tobacco cells.
Plant Physiol 65: 151-155 8. MACROBBIE EAC 1971 Fluxes and compartmentation in plant cells. Annu Rev Plant Physiol 22: 75-96 9. REuvENY Z, P FILNER 1977 Regulation of adenosine triphosphate sulfurylase in cultured tobacco cells; effects of sulfur and nitrogen sources on the formation and decay of the enzyme. J Biol Chem 252: 1858-1864 10. SMITH IK 1975 Sulfate transport in cultured tobacco cells. Plant Physiol 55: 303307 11. Sm1TH IK 1980 Regulation of sulfate assimilation in tobacco cells. Effect of nitrogen and sulfur nutrition on sulfate permease and O-acetylserine sulfhydrylase. Plant Physiol 66: 877-883 12. SMrrH IK, JF THOMPSON 1971 Purification and characterization of L-serine transacetylase and O-acetyl-L-serine sulfhydrylase from kidney bean seedlings (Phaseolus vulgaris). Biochim Biophys Acta 227: 288-295 13. WAGNER GJ 1979 Content and vacuole/extravacuole distribution of neutral sugar, free amino acids, and anthocyanin in protoplasts. Plant Physiol 64: 8893
