Effect of Glutathione on Phytochelatin Synthesis in - NCBI

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inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine ...
Received for publication October 16, 1989 and in revised form January 17, 1990

Plant Physiol. (1990) 93, 484-488

0032-0889/90/93/0484/05/$01 .00/0

Effect of Glutathione on Phytochelatin Synthesis in Tomato Cells1 Mary Lou Mendum2, Subhash C. Gupta3, and Peter B. Goldsbrough* Department of Horticulture, Purdue University, West Lafayette, Indiana 47907 (16). Mutants of S. pombe that lack either -y-Glu-Cys synthetase or GSH synthetase do not synthesize PCs in response to cadmium and show reduced growth in the presence of cadmium (13). Finally, it has been demonstrated in cells of Datura innoxia that [35S]GSH is rapidly incorporated into PCs after exposure to cadmium (1). These results confirm that GSH is utilized in production of PCs. The experiments described here further examine the role of GSH in PC synthesis in tomato.

ABSTRACT Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably [-y-Glu-CysJ2-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little [35SJcysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione.

MATERIALS AND METHODS

Cell Cultures Cell suspension cultures of tomato Lycopersicon esculentum Mill. cv VFNT-Cherry were maintained as described (16). Cultures were initiated with an inoculum of 20 mg cells (fresh weight) per mL of medium and grown with shaking at 24 to 260 C. Cells were subcultured weekly and experiments performed 3 or 4 d after inoculation. GSH, BSO, and CdC12 (Sigma Chemical Co.) were added to the medium as filter sterilized solutions.

Plant cells that are exposed to cadmium rapidly accumulate peptides that have the general structure (y-Glu-Cys)n-Gly, where n = 2 to 10 (5, 11, 17). These peptides, variously termed PCs,4 poly(y-glutamyl-cysteinyl)glycines, y-glutamyl metal binding peptides, and cadystins, form intracellular complexes with cadmium. PCs have been identified in a large number of plant species (3) and also in some fungi, including Schizosaccharomyces pombe (12). The y-glutamyl linkages present in these peptides indicate that PCs are not direct translation products of mRNAs. However, the similarity of PCs to GSH (containing a single y-Glu-Cys moiety) suggests that GSH may be involved in the synthesis of PCs, and a number of observations support this hypothesis. Plant species that contain homo-GSH, with a carboxy terminal j3-alanine rather than glycine, synthesize homo-PCs that also contain a carboxy terminal ,3-alanine (6). When PC synthesis is induced by cadmium there is a rapid decline in cellular GSH levels (7, 16). Inhibition of 7y-Glu-Cys synthetase, the penultimate enzyme of the GSH synthesis pathway, by BSO prevents the accumulation of PCs (7, 15-17) although this inhibition can be at least partially overcome by supplying exogenous GSH

Assays for GSH and Phytochelatins Cells to be assayed for GSH and PCs were collected by vacuum filtration on Whatman No. 4 paper and frozen at -70° C. Extracts were prepared by adding an equal volume (1 mL per g fresh weight of cells) of 10% (w/v) 5-sulfosalicylic acid and keeping the mixture on ice for 10 min, during which the extracts were vortexed three times. The lysate was centrifuged at 13,000 g at 4° C for 4 min, and the acid soluble supernatant was either assayed immediately or stored at -70° C for future analysis. GSH and PCs were separated by HPLC on a Beckman Ultrasphere C18 5 ,um 4.6 x 250 mm column using a gradient of acetonitrile in 0.1% trifluoracetic acid at a flow rate of 1 mL/min. The gradient program was 0% acetonitrile for 2 min, 0 to 10% acetonitrile in 2 min, 10 to 20% acetonitrile in 20 min. The column eluant was derivatized with 75 Mm 5,5'-dithiobis(2-nitrobenzoic acid) in 50 mm potassium phosphate (pH 7.6) at a flow rate of 2 mL/ min and monitored at 412 nm (7). Samples of 100 ,L were injected. Using this procedure, cysteine, GSH and PC2 to PC4 were resolved (Fig. 1). Retention times and peak areas were determined with a Hewlett Packard 3380S integrator. PC concentrations are reported as GSH equivalents, based on peak areas of GSH standards. The reproducibility of this method for measuring GSH and PCs is shown in Table I. We have previously shown that recovery of GSH by this extraction method is approximately 90% (16).

'Supported in part by U.S. Department of Agriculture grant No. 85-CRCR- I -1653. Journal paper No. 12,318 ofthe Purdue University Agricultural Experiment Station. Present address: Department of Viticulture and Enology, University of California, Davis, CA 95616. 3 Present address: ARS, Northern Region Research Center, 1815 N. University St., Peoria, IL 61604. 4 Abbreviations: PC, phytochelatin; BSO, buthionine sulfoximine. 2

484

PHOTOCHELATIN SYNTHESIS IN TOMATO CELLS

GSH, and individual PCs were measured in cells subjected to these treatments.

Labeling of Phytochelatins Ten mL aliquots of cells (3 d after inoculation) were transferred to 50 mL flasks. BSO was added to one flask to a concentration of 150 ,uM and incubated for 16 h. GSH was then added to 500 ,uM to the BSO treated cells, as well as 150

,uM CdC12 and 50 MuCi [35S]cysteine (New England Nuclear, 1057 Ci/mmol). A second flask received only 150 liM CdC12 and 50 ,tCi [35S]cysteine. The cultures were incubated with shaking for 8 h, and cells collected and extracted with 5sulfosalicylic acid. Each extract (100 AL) was separated by HPLC with postcolumn derivatization. To increase the separation of cysteine and GSH and allow accurate measurement of [35S]cysteine in these compounds, a gradient of 0 to 10% acetonitrile in 10 min, followed by 10 to 20% in 20 min, was used; 1.5 mL fractions (0.5 min) were collected and radioactivity measured by liquid scintillation counting. Using this method, the concentrations and specific activities of cysteine,

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Figure 1. HPLC separation of PCs. An extract prepared from cells treated with 150 gM CdCI2 and 500 ,M GSH for 6 h was analyzed by HPLC with postcolumn derivatization as described in "Materials and Methods." The identified peaks are: 1, cysteine; 2, GSH; 3, PC2; 4, PC3; 5, PC4.

RESULTS

Effect of Exogenous GSH on Cell Growth Because GSH is required for PC synthesis and these peptides are necessary for cadmium tolerance in plant cells, the availability of GSH may be an important element in the adaptation of plants to cadmium stress. BSO specifically inhibits y-Glu-Cys synthetase (4), the first enzyme of GSH biosynthesis, and has been shown to reduce cellular levels of GSH in tomato cells (16). To determine the effect of BSO on growth of tomato cells in the presence of cadmium, cells were grown in medium containing 50 uM CdCl2 and 30 ,uM BSO (Fig. 2). This concentration of BSO does not inhibit cell growth under standard conditions (data not shown). Control cells exposed to cadmium alone reached a normal fresh weight of approximately 190 mg/mL. However, the combination of BSO and cadmium prevented growth, presumably as a result of failure to synthesize PCs. Similar synergistic effects of BSO and heavy metals on plant cell growth have been observed (7, 15, 17). We have previously shown that PC synthesis could be at least partially restored by addition of GSH to the medium (16). Therefore, we examined the effect of supplying GSH to cells treated with BSO and cadmium. Some cell growth was observed when either 10 or 20 ,uM GSH was added. However, concentrations of 50 MAM GSH and above essentially restored growth of BSO treated cells to that of cells grown in the presence of Cd alone.

Restoration of PC Synthesis by GSH Although PC levels were not measured in the experiment shown in Figure 2, one explanation for these results is that exogenous GSH was taken up by cells and utilized for synthesis of PCs in the

Table I. Reproducibility of GSH and PC Measurements Aliquots of tomato cells growing in the presence of 100 Mm CdC12, 7 d after subculturing, were extracted with 5-sulfosalicylic acid and analyzed by HPLC with postcolumn derivatization as described. The mean concentrations of GSH, PC2, PC3, and PC4 of five extractions (±SE) are reported. Concentrationa 0.250 ± 0.001 0.238 ± 0.004 1.077 ± 0.011 0.287 ± 0.004

GSH PC2 PC3 PC4 a Concentrations are reported as mmol/kg fresh weight for GSH, and mmol GSH equivalents/kg fresh weight for PCs.

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Figure 2. Restoration of cell growth by GSH in cells treated with cadmium and BSO. Tomato cells were inoculated into media containing either 50 Mm CdCI2 (0), or 50 Mm CdCI2 and 30 Mm BSO (C). The medium was supplemented with increasing concentrations of GSH. Fresh weight of cells was measured after 7 d. Error bars indicate SE (n = 3).

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of GSH, and the accumulation of PCs was measured (Fig. 3). In the absence of GSH, BSO treated cells failed to synthesize detectable amounts of PCs when challenged with cadmium. After 24 h these cells were discolored and eventually died, although this concentration of cadmium is not lethal to tomato cells in the absence of BSO (10). The addition of 50 AM GSH resulted in a low level of PC synthesis during the first 6 h. However, 500 AM GSH restored PC synthesis in BSO treated cells to a level that was somewhat higher than observed in control cells exposed to cadmium alone. Therefore sustained synthesis of PCs can take place in the presence of an inhibitor of y-Glu-Cys synthetase, provided that GSH is available. In the experiment shown in Figure 2, 50 uM GSH was able to restore growth, whereas in Figure 3 this concentration of GSH resulted in only a small amount of PC synthesis. However, these experiments differed in the concentrations of both BSO and cadmium that were used, and in one the effects on growth were measured over a cell culture cycle of 7 d as opposed to effects on PC synthesis examined over 24 h. These differences in experimental design may account for the apparently contradictory results obtained with the same concentration of GSH. presence

Because GSH is a substrate for PC synthesis and exogenous GSH is capable of increasing the rate of PC synthesis in BSO treated tomato cells above that observed in cells exposed to cadmium alone, we examined the effect of supplying GSH to cadmium treated cells (Fig. 4). In the absence of exogenous GSH, cellular levels of GSH declined to less than 50% of the initial value within 2 h. Addition of 50 or 100 Mm GSH to the medium delayed this depletion of GSH until 4 and 6 h, respectively. Five hundred yM GSH produced a transient increase in cellular GSH levels; 4 h after addition of both cadmium and GSH to the medium, the cellular concentration of GSH had doubled. However, after 24 h the level of GSH in all cells was essentially identical, regardless of the amount of GSH added to the media, suggesting that cellular GSH C-.

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Figure 3. Reversal of BSO induced inhibition of PC synthesis by GSH. Cells were pretreated with 200 Mm BSO for 16 h, and then exposed to 150 Mm CdCI2 with no GSH (0), 50 Mm GSH (A), or 500 Mm GSH (E). Control cells were not pretreated with BSO and received only 150 Mm CdC12 (-).

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Time (hours) Figure 4. Stimulation of PC synthesis by GSH. Cells were exposed to 150 Mm CdCI2 in the presence of 0 (0), 50 AM (A), 100 AM (E), or 500 Mm (0) GSH. Cellular GSH (upper panel) and total PCs (lower panel) were measured by HPLC.

levels are regulated. When GSH was added to cells in the absence of any cadmium stress, there was a temporary increase in cellular GSH which returned to normal within 24 h, and no effect on the level of PCs (data not shown). The elevation of cellular GSH from addition of GSH to the medium increased production of PCs. This effect was apparent for only the first 2 h in cells grown in the presence of 50 Mm GSH. However, addition of 100 and 500 Mm GSH resulted in approximately two- and three-fold increases, respectively, in PC levels after 6 h exposure to cadmium. After 24 h, cells supplemented with 500 gM GSH contained approximately twice the concentration of PCs as control cells. In comparing the levels of GSH and PCs in these cells, it was apparent that the increased rate of PC accumulation occurred during the period when GSH levels were higher than in control, cadmium induced cells. Availability of GSH is therefore a limiting factor in the synthesis of PCs. Effect of Exogenous GSH on Synthesis of Individual PCs Addition of 500 aM GSH to cadmium treated cells stimulated overall production of PCs. However, this effect was not uniformly distributed among all the peptides. There was a disproportionate increase in synthesis of PC2 and, to a lesser extent, PC3 in GSH treated cells, while the levels of PC4 were not affected by the addition of GSH (Fig. 5). For example, cadmium induced cells supplemented with 500 Mm GSH contained more than 3 times the level of total PCs than cells without GSH after 6 h. However, PC2 was elevated five-fold,

PHOTOCHELATIN SYNTHESIS IN TOMATO CELLS

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quantified using post column derivatization, and measured for radioactivity. The concentrations and specific activities of these compounds are given in Table II. Higher levels of PCs were observed in cells supplemented with GSH, as expected. The addition of GSH also increased the relative abundance of PC2 over PC3 in agreement with the data presented in Figure 4. [35S]Cysteine was incorporated into GSH in both treatments, but the specific activity of GSH was lower in BSO treated cells. The specific activities of PCs were also lower in BSO treated cells, whereas cysteine specific activity was very similar in both treatments. If cysteine and glutamate are added sequentially to increase the chain length of PCs, then there should be less difference between treatments in specific activity of larger PCs which incorporate more cysteine. This is not observed, and the approximately tenfold difference in specific activity of each PC between the two treatments reflects the differential labeling of GSH by [35S]cysteine in the presence or absence of BSO. The synthesis of increased amounts of lower specific activity PCs in BSO treated cells can be ac-

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Time (hours) Figure 5. Effect of GSH on synthesis of individual PCs. The levels of PC2 (A), PC3 (Ii), and PC4 (0) were measured in cells that were exposed to 150 Mm CdCI2 in the presence of 500 gM GSH (A), 100 Mm

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PC3 three-fold, and PC4 was unchanged. A similar effect, although to a lesser degree, was observed with addition of 100 ,gM GSH. In two additional experiments, the same effect of GSH stimulating the total production of PCs by increasing the accumulation of PC2 and PC3 has been observed. Incorporation of [35S]Cysteine into Phytochelatins It has been established that y-Glu-Cys synthetase is not required for PC synthesis provided that GSH is available. Therefore, in the presence of BSO, PC production could involve either the addition of y-Glu-Cys from GSH to another GSH molecule (or a preformed PC) or the sequential addition of cysteine and glutamate to GSH. To determine which of these is functional, the incorporation of [35S]cysteine into PCs was measured in cells that were pretreated with BSO and then induced to synthesize PCs by the addition of cadmium and GSH. Cysteine, GSH, and PCs were separated by HPLC,

counted for by unlabeled y-Glu-Cys moieties, derived from exogenous GSH, being utilized for PC synthesis. Therefore, at least in the presence of BSO, sequential addition of cysteine and glutamate to GSH does not appear to be the mechanism of synthesis for PC2. Similar results have been obtained in three separate experiments. DISCUSSION Survival and growth of tomato cells in the presence of cadmium are dependent upon the ability of cells to synthesize cadmium binding PCs. Synthesis of these peptides, in turn, depends on the availability of GSH. However, the ability to produce GSH is not essential for either PC synthesis or cadmium tolerance provided that an adequate supply of GSH is available. Exogenous GSH increases the rate of PC accumulation in response to cadmium. This effect is evident during the time that cellular GSH is increased above the level seen in cells treated with cadmium alone. We have not determined if this increased production of PCs has any effect on tolerance to cadmium. In the absence of an exogenous supply, GSH synthesis is required to sustain PC production in response to cadmium. The ability of GSH to restore cadmium tolerance to cells grown in the presence of BSO confirms the functional importance of PCs in response to this stress. Although PCs are required for expression of normal levels of tolerance to cadmium, there are other adaptive responses, including increased activities of enzymes for assimilatory sulfate reduction (14) and changes in protein synthesis (2), that likely play a role in this process. The results presented here indicate that sequential addition of cysteine and glutamate to GSH to form PC2 is not a likely mechanism of synthesis. Therefore addition of y-Glu-Cys moieties to GSH appears likely. Because PC synthesis can occur in the presence of BSO and GSH, y-Glu-Cys required for this process must be derived from GSH. After the completion of these experiments, Grill et al. (8) described the characterization of an enzyme from Silene cucubalus that synthesizes PCs. This enzyme, termed PC synthase, transfers y-Glu-Cys from GSH to (y-Glu-Cys)n-Gly to produce (y-

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Table Il. [wS]Cysteine Labeling of PCs in the Presence of BSO and GSH Tomato cells were pretreated with 150 AM BSO for 16 h before the addition of 150 AM CdCl2, 50 ,ACi [35S]cysteine, and 500 AM GSH. Control cells received only 150 AM CdCl2 and 50 MiCi [35S]cysteine. After 8 h exposure to cadmium, cells were extracted and analyzed for incorporation of [35S]cysteine into GSH and PCs. Specific Activity

Amount Treatment

Cys GSH PC2a PC3 PC4 mmol/kg fresh weight

Cys

GSH

PC2

PC3

PC4

cpm/pmol

BSO pretreatment + CdCI2, 1.06 1.26 0.54 0.58 0.12 48.98 1.92 4.21 12.02 14.71 [35S]Cys, GSH 0.10 0.36 0.13 0.28 0.09 41.19 10.20 42.48 144.66 174.26 CdCI2 + [35S]Cys a PCs are expressed as mmol GSH equivalents/kg fresh weight.

Glu-Cys)n+ 1-Gly. PCs are also able to act as the y-Glu-Cys donor in this reaction. A similar dipeptidyl transpeptidase activity for synthesis of PCs in tomato cells would be consistent with the results presented here. We had previously shown that de novo protein synthesis in response to cadmium was not required for production of PCs (16). PC synthase in S. cucubalus is present in the absence of cadmium, and the enzyme is activated by the addition of cadmium (8). We have obtained similar results for this enzyme in tomato cell suspension cultures (J Zhou, ML Mendum, PB Goldsbrough, unpublished observations). The observation that additional GSH results in a disproportionate increase in synthesis of PC2, as opposed to a uniform increase in levels of all PCs, is in agreement with a single enzyme being involved in the production of PCs of increasing size. If GSH and PCs are competitors for addition of y-Glu-Cys, the relative concentrations of these substrates will determine which PCs are synthesized. Increasing the cellular GSH concentration therefore stimulates production of PC2. While it is clear that synthesis of PCs is essential for cadmium tolerance, it is perhaps surprising that PC synthase is present in the absence of cadmium. This may be a result of growth in culture, and the presence and distribution of the enzyme in plants needs to be determined. Constitutive production of this enzyme may indicate that PCs have an essential role in plant metabolism, unrelated to cadmium tolerance (9, 17). ACKNOWLEDGMENTS We thank D. Altman and J. Gaska-Straub for help in preparing the manuscript, Dr. D. Kuhn for valuable discussions, and E. Hatch and B. Leinberger for excellent technical assistance. LITERATURE CITED 1. Berger JM, Jackson PJ, Robinson NJ, Lujan LD, Delhaize E (1989) Precursor-product relationships of poly(-y-glutamylcysteinyl)glycine biosynthesis in Datura innoxia. Plant Cell Rept 7: 632-635 2. Delhaize E, Robinson NJ, Jackson PJ (1989) Effects ofcadmium on gene expression in cadmium-tolerant and cadmium-sensitive Datura innoxia cells. Plant Mol Biol 12: 487-497 3. Gekeler W, Grill E, Winnacker E-L, Zenk MH (1989) Survey

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of the plant kingdom for the ability to bind heavy metals through phytochelatins. Z Naturforsch 44c: 361-369 Griffith OW, Meister A (1979) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butylhomocysteine sulfoximine). J Biol Chem 254: 7558-7560 Grill E, Gekeler W, Winnacker E-L, Zenk MH (1985) Phytochelatins: the principal heavy-metal complexing peptides of higher plants. Science 230: 674-676 Grill E, Gekeler W, Winnacker E-L, Zenk MH (1986) Homophytochelatins are heavy metal-binding peptides of homoglutathione containing Fabales. FEBS Lett 205: 47-50 Grill E, Winnacker E-L, Zenk MH (1987) Phytochelatins, a class of heavy-metal-binding peptides from plants, are functionally analogous to metallothioneins. Proc Natl Acad Sci USA 84: 439-443 Grill E, Loffler S, Winnacker E-L, Zenk MH (1989) Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific -y-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc NatI Acad Sci USA 86: 6838-6842 GrillE, Thumann J, Winnacker E-L, Zenk MH (1988) Induction of heavy-metal binding phytochelatins by inoculation of cell cultures in standard media. Plant Cell Rept 7: 375-378 Huang B, Hatch E, Goldsbrough PB (1987) Selection and characterization of cadmium tolerant cells in tomato. Plant Sci 52:

211-221 11. Jackson PJ, Unkefer CJ, Doolen JA, Watt K, Robinson NJ

(1987) Poly(y-glutamylcysteinyl)glycine: its role in cadmium resistance in plant cells. Proc Natl Acad Sci USA 84: 66196623 12. Kondo N, Imai K, Isobe M, Goto T, Murasugi A, Wada-Nakagawa C, Hayashi Y (1984) Cadystin A and B, major unit peptides comprising cadmium binding peptides induced in a fission yeast-separation, revision of structures and synthesis. Tetrahedron Lett 25: 3869-3872 13. Mutoh N, Hayashi Y (1988) Isolation of mutants of Schizosaccharomyces pombe unable to synthesize cadystin, small cadmium-binding peptides. Biochem Biophys Res Commun 151: 32-39 14. Nussbaum S, Schmutz D, Brunold C (1988) Regulation of assimilatory sulfate reduction by cadmium in Zea mays L. Plant Physiol 88: 1407-1410 15. Reese RN, Wagner GJ (1987) Effects of buthionine sulfoximine on Cd-binding peptide levels in suspension-cultured tobacco cells treated with Cd, Zn, or Cu. Plant Physiol 84: 574-577 16. Scheller HV, Huang B, Hatch E, Goldsbrough PB (1987) Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells. Plant Physiol 85: 1031-1035 17. Steffens JC, Hunt DF, Williams BG (1986) Accumulation of

non-protein metal-binding polypeptides (y-glutamyl-cysteinyl),-glycine in selected cadmium-resistant tomato cells. J Biol

Chem 261: 13879-13882