Plant Physiol.

(1994) 106: 233-239

Increased Activity of y-Glutamylcysteine Synthetase in

Tomato Cells Selected for Cadmium Tolerance'
Jianjun Chen2 and Peter

B. Coldsbrough*

Department of Horticulture, Purdue University, 1165 Horticulture Building,

West Lafayette, Indiana 47907-1 165

ribosomes, whereas PCs are synthesized enzymically from

GSH.
The pathway of GSH synthesis in plants is similar to that
in other organisms (Rennenberg, 1982). y-GluCys synthetase
(EC 6.3.2.2) combines Glu and Cys to form y-GluCys. This
enzyme is feedback inhibited by GSH and is thought to be
the major regulatory step in GSH synthesis (Meister and
Anderson, 1983; Hell and Bergmann, 1990). GSH synthetase
(EC 6.3.2.3) adds Gly to y-GluCys to produce the tripeptide
(Law and Halliwell, 1986). PC synthesis in plants uses GSH
as a substrate and is catalyzed by a y-GluCys dipeptidyl
transpeptidase, called PC synthase (Grill et al., 1989). This
enzyme transfers y-GluCys, from GSH or PC, to an acceptor
molecule of GSH or PC. GSH-deficient mutants of S. pombe
are unable to produce PCs and have reduced tolerance of Cd
(Mutoh and Hayashi, 1988). Similarly, treatment of plant
cells with BSO, an inhibitor of y-GluCys synthetase, prevents
PC synthesis and also reduces tolerance to Cd (Steffens et
al., 1986; Scheller et al., 1987). It has been shown that
exposure of plants to Cd results in increased activities of
enzymes involved in GSH biosynthesis (Ruegsegger and
Brunold, 1992) and sulfate assimilation (Nussbaum et al.,
1988). Other evidence, however, indicates that increased
production of PCs is not responsible for elevated Cd tolerance
in some plants (de Knecht et al., 1992) and plant cells
(Delhaize et al., 1989), since both the sensitive and tolerant
populations produce equivalent amounts of PCs when exposed to equal concentrations of Cd.
A number of plant cell lines have been selected for increased tolerance to Cd (Steffens et al., 1986; Huang et al.,
1987; Jackson et al., 1987). These cell lines typically contain
elevated concentrations of PCs and the majority of intracellular Cd is present in the form of PC-Cd complexes (Steffens
et al., 1986; Gupta and Goldsbrough, 1991). When grown in
the absence of Cd, selected cell lines maintain varying degrees
of increased Cd tolerance in comparison with the unselected
cells from which they were originally derived. In this paper,
we have systematically compared a selected Cd-tolerant cell
line of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry)
with its nontolerant parental line. Our results indicate that
Cd tolerance in this cell line is correlated with PC synthesis,
and that an increased capacity to produce GSH under Cd
stress may contribute to the increase in Cd tolerance.

Two cell lines of tomato (Lycopersiconesculentum Mill cv VFNTCherry) were systematically compared for their capacity to tolerate
cadmium. Unselected CdS cells died in the presence of 0.3 mM
CdC12. CdR6-O cells, which were selected from CdS, survived and
grew in medium supplemented with 0.3 mM CdCIZ. Growth of
CdR6-O cells under this condition was accompanied by synthesis of
cadmium-bindingphytochelatins and maintenance of cellular glutathione (CSH) levels. CdR6-O cells also exhibited increased tolerance to buthionine sulfoximine, in both the presence and absence
of 0.1 mM CdCI2. The specific activity of y-glutamylcysteine synthetase (EC 6.3.2.2) was approximately 2-fold higher in CdR6-O
cells than in CdS cells, whereas there was no difference between
cell lines in specific activity of CSH synthetase (EC 6.3.2.3). Increased activity of the first enzyme of CSH biosynthesisin CdR6-O
cells, presumably a result of selection for increased cadmium
tolerance, provides an enhanced capacity to synthesize CSH and
to maintain the production of phytochelatins in response to cadmium. This adaptation may contribute to the enhanced cadmium
tolerance of CdR6-O cells.

Cd is a nonessential element that is toxic to all organisms

if it is present at a high enough concentration. Plants and
plant cells growing in culture respond to Cd by synthesizing
a family of peptides with the general structure (yGluCys),Gly, where n = 2 to 11 (reviewed by Rauser, 1990;
Steffens, 1990). These peptides were first identified in Schizosaccharomyces pombe (Kondo et al., 1984) and are produced
in many, perhaps all, plant species (Gekeler et al., 1989). A
variety of names have been given to these peptides including
cadystins (Kondo et al., 1984), PCs (Grill et al., 1985), poly(yglutamylcysteiny1)glycines(Jackson et al., 1987), and y-glutamyl metal-binding peptides (Reese and Winge, 1988). The
synthesis of PCs in response to Cd results in the formation
of intracellular complexes between PCs and Cd. These complexes are believed to be critical for tolerance of plants to Cd
because they sequester Cd in a form that limits damage to
metabolic processes. In this regard, PCs are functionally
analogous to metallothioneins (Grill et al., 1987). However,
metallothioneins are produced by translation of mRNAs on

'

This research was supported by U S . Department of Agriculture

grant No. 90-37264-5449. This i s Joumal Paper No. 14143 of the
Purdue University Agricultural Experiment Station.
Present address: Department of Plant Pathology, University of
Wisconsin-Madison, 1630 Linden Drive, Madison, WI 53706.
* Corresponding author; fax 1-317-494-0391.

Abbreviations: BSO, buthionine sulfoximine; PC, phytochelatin.

233

Chen and Goldsbrough

234
MATERIALS AND METHODS
Cell Culture

Two cell-suspension cultures of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) were used in the experiments
reported here. The unselected cell line, designated CIS, is the
same tomato cell line described in previous publications
(Huang et al., 1987). A Cd-tolerant cell line capable of
growing in the presence of 6 m CdCl,, CdR6, was obtained
from CdS cells by step-wise selection. After growth in medium
containing 6 m~ CdCl, for 6 months, a portion of this cell
culture was transferred to Cd-free medium. This cell line,
designated CdR6-O, was grown in the absence of Cd for at
least 4 years before the experiments reported here were
performed. CdS and CdR6-O cells were grown as previously
described (Gupta and Goldsbrough, 1991)except that all cells
were subcultured after 10 d at a starting density of 30 mg
fresh weight/mL.
A number of experiments were performed to examine the
Cd tolerance of CdS and CdR6-O cells. These included growing
cells in medium containing 0.1 or 0.3 m CdC12, exposing
cells to 0.1 m CdC1, in the presence of increasing concentrations of BSO, and examining the effects of BSO alone on
growth of CdS and CdR6-O cells. In each of these experiments,
a single flask containing sufficient medium for all treatments
was inoculated with cells at a density of 30 mg fresh weight/
mL. Aliquots of this cell culture were then transferred to
sterile flasks and the appropriate amounts of filter-sterilized
solutions of CdC1, or BSO (Sigma) were added. All treatments
were performed in triplicate and repeated in at least two
independent experiments. Samples of cells were removed at
various times to measure cell growth and for analysis of thiolcontaining compounds.
Analysis of CSH and PCs

Cells were collected by filtration and frozen at -7OOC.

Acidic extracts were prepared as described (Gupta and Goldsbrough, 1991). After centrifugation, 120 pL of extract was
mixed with 60 pL of 2 m N-acetyl-Cys and passed through
a 0.2-pm filter. A 150-pL portion of this mixture was analyzed
by HPLC using post-column derivatization with 5,5'-dithiobis(2-nitrobenzoic acid) to detect GSH and PCs as described (Gupta and Goldsbrough, 1991). N-acetyl-Cys was
included as a standard. The concentrations of individual PCs
were determined as mol of y-GluCys/kg fresh weight of cells.

Plant Physiol. Vol. 106, 1994

Assays lor y-GluCys Synthetase and CSH

Synthetase Activities

Cells were collected by filtration 5 d after inoculation and

immediately frozen in liquid nitrogen. Ten grams of frozen
cells were ground in a chilled mortar in 10 mL cd extraction
buffer (0.1 M Tris-HC1, pH 7.5,5 m~ EDTA) with :he addition
of 1 g of polyvinylpolypyrrolidone (Sigma) and approximately 1 g of acid-washed sand. The slurry was centrifuged
at 15,OOOg for 15 min and the pellet was discarded. After a
second centrifugation at 15,0008 for 15 min, ammonium
sulfate was added to the supematant to 60% saturation.
Precipitated proteins were recovered by centrifugation, redissolved in 50 m~ Tris-HC1, pH 7.5,l m EDTA, and dialyzed
overnight against the same buffer. Insoluble material was
removed by centrifugation and the supematant was used to
determine the activities of y-GluCys synthetase and GSH
synthetase.
For y-GluCys synthetase, the assay contained 100 m TrisHCl (PM 7.5), 50 II~MMgC12, 1 l ~ DTT,
l ~ 10
ATP, 50 m~
Na-L-glutamate, 2 m L-CYS,and 285 bL of protein extract
in a total volume of 500 pL. The assay mixtures, without Cys,
were incubated at 37OC for 10 min. Cys was added and the
reaction was incubated for 45 min. The reaction was stopped
by adding 50 pL of 50% (w/v) 5-sulfosalicylic acid and
transferring the tubes to ice. Denatured proteins were removed by centrifugation and the supematants were assayed
for the presence of y-GluCys by HPLC separation and postcolumn derivatization as described (Gupta and Goldsbrough,
1991). The quantity of 7-GluCys synthesized was determined
by comparison with peak areas of r-GluCys standards (Nacalai Tesque, Kyoto, Japan). Control assays were performed
without the addition of Cys.
For GSH synthetase, the assay contained 100 nw Tris-HC1
(pH 8.0), 50 m KCl, 10 m MgCl,, 5 m ATP, 5 I ~ creatine
M
phosphate, 2 units of creatine kinase, 10 m~ Gly, 1.5 m yGluCys, and 295 pL of protein extract in a total volume of
500 pL. The assay mixtures, without Gly, were incubated at
3OoC for 10 min. Gly was added and the reaction was
incubated for an additional 30 min. The reactions were
tenninated and processed for HPLC analysis as described for
the 7-GlyCys synthetase assay. The quantity of GSH synthesized was determined by HPLC analysis. Control (assayswere
performed without the addition of Cly.
RESULTS

CdRL-O Cells

Determination of Cell Viability

Characterization of

The viability of cells after exposure to 0.3 m CdCl, was

determined by staining with fluorescein diacetate (Widholm,
1972). Because it was difficult to assess staining of individual
cells in larger clumps, cell samples were first filtered through
a 0.4-mm nylon mesh filter. The filtrate was mixed with an
equal volume of fluorescein diacetate solution (0.01%, w/v)
and left at room temperature for 10 min. A minimum of 200
cells were scored for viability in each sample. The results are
reported as a percentage of the viability of control cells grown
in the absence of Cd. Under these conditions, viability was
greater than 90% for both cell lines, with no significant
difference between cell lines.

When grown in the absence of Cd, CdR6-O cells showed a

number of differences from the CdS cells from which they
were originally derived. CdR6-O cells reached a maximum
fresh weight of approximately 110 mg/mL compared to more
than 200 mg/mL for the CdS cell line (Fig. 1A). The difference
between the two cell lines was less pronounced in dry weight
(Fig. lB),, suggesting that there was greater cell expansion in
CdS cells. Both cell lines reached their maximum frmh weights
between 10 and 12 d after inoculation.
In addition to altered growth characteristics, the concentrations of two compounds that are involved in responses to Cd
were also altered in CdR6-O cells. Cellular concmtration of

235

Phytochelatin Synthesis and Cadmium Tolerance

cell lines in concentrations of GSH and PC2 can be accounted

for by the greater gain in fresh weight of CdS cells.
Response of CdRL-O Cells to Cadmium

Exposure of the two cell lines to 0.3 m~ CdC12 demonstrated the increased tolerance of CdR6-O cells to Cd. The
fresh weight of CdS cells declined rapidly when they were
grown in medium containing 0.3 m~ CdC12 (Fig. 2A), and
this was reflected in a decrease in cell viability (Fig. 2B). Four
days after inoculation into medium containing 0.3 m~ CdC12,
no viable CdS cells were detected. Although there was a small
initial decline in fresh weight of CdR6-O cells under the same
conditions, some growth did occur after 6 d of culture. The
Y

.-o,

st.
n

\-

2
i
B

Days a f t e r Inoculation
Figure 1. Growth characteristics of CdS and CdR6-O cell lines. CdS
(O)and CdR6-O (O)cells were inoculated at 30 mg/mL into medium
without CdCI2. Fresh weight (A), dry weight ( B ) , CSH (C), and PCs
(D)were measured during the cell culture period. Error bars indicate
SE of three replicated treatments.

O .-

"

is'-'\u

5 _-

GSH increased more rapidly in CdR6-O cells after subculture

into fresh medium (Fig. lC), although the maximum GSH
concentration was the same in both cell lines. In CdS cells,
GSH declined to less than 0.1 mmol/kg fresh weight as the
cells reached stationary phase. In contrast, the lowest GSH
concentration in CdR6-O cells was only 0.3 mmol/kg fresh
weight. This observation suggests that biosynthesis or metabolism of GSH has been altered in the Cd-tolerant cell line. A
low level of PC2 was detected in CdS cells grown in standard
medium (Fig. 1D). In CdR6-O cells, however, the concentration of PC2 was approximately 2-fold higher and did not
decline toward the end of the culture period. PC2 was the
only PC detected in either cell line when they were grown in
the absence of Cd. At least some of the differences between

O -.
5 --

O@

/O

7
/8

0-0
0-0

g'%,

CdS
CdR6-O

Days a f t e r Inoculation
Responses of CdS and CdR6-O cells to 0.3 mM CdCI2. CdS
(O)and CdR6-O (O)cells were inoculated at 30 mg/mL into medium
containing 0.3 mM CdCI2. Fresh weight (A), cell viability (B), CSH
(C), and PCs (D)were measured during the cell culture period. Error
bars indicate SE of three replicated treatments.
Figure 2.

Chen and Coldsbrough

236

Plant Physiol. Vol. 106, 1994

there was little difference between cell lines in accumulation

of GSH and PCs (Fig. 3, B and C). However, the maximum
concentration of PC in either cell line exposed to 0.1 m~
CdCl, was significantly lower than that observed in CdR6-O
cells challenged with 0.3 m~ CdC12.
ToYeranlce of CdRL-O Cells to BSO

Days after Inoculation

Responses of CdS and CdR6-O cells to O. 1 mM CdClz. CdS
(O)and CdR6-O (O)cells were inoculated at 30 mg/mL into medium
containing 0.1 mM CdC12. Fresh weight (A), CSH (B), and PCs (C)
were measured during the cell culture period. Error bars indicate
Figure 3.

SE

of three replicated treatments.

viability of CdR6-O cells declined over the first 5 d to 70% of

the control, but then remained constant during the remainder
of the culture period.
PCs accumulated in CdR6-O cells exposed to 0.3 m~ CdClz
and reached a maximum concentration of 16 m o l yGluCys/kg fresh weight after 8 d (Fig. 2D). Although there
was a similar initial synthesis of PCs in CdS cells, this was
not sustained after the 2nd d. The concentrations of GSH in
CdS and CdR6-O cells under these conditions in general corresponded to the data on PCs (Fig. 2C). In CdS cells, GSH
declined to an undetectable level after 3 d. Although there
was an initial decline in GSH in CdR6-O cells, this was
followed by a gradual increase in GSH concentration, reaching a maximum of approximately 0.45 mmol/kg fresh weight
after 10 d. This pattem of GSH accumulation was quite
different from that observed in the absence of Cd and can
be accounted for by the use of GSH for PC synthesis.
Similar comparisons were made between CdS and CdR6-O
cells grown in the presence of 0.1 m~ CdClz (Fig. 3). Under
these conditions, both cell lines grew to the same maximum
fresh weights that were observed in the absence of Cd (Fig.
3A). In contrast with the results obtained at 0.3 m~ CdClp,

The observations that CdR6-O cells had an incTeased concentration of GSH at certain stages of the cell culture period
and that CdS cells were unable to maintain G3H and PC
synthesis after exposure to higher concentrations of Cd indicate that CdR6-O cells may have an increased capacity to
produce GSH as substrate for PC production. To test this
hypothesis, cells were grown in medium containing 0.1 m~
CdCl, and increasing concentrations of BSO, and fresh
weights were measured after 10 d (Fig. 4). BSi3 has been
shown :o inhibit plant y-GluCys synthetases. A'j previously
documented (Scheller et al., 1987), the combination of Cd
and BSO had a synergistic inhibitory effect on cell growth.
However, CdS cells were significantly more sens tive to BSO
than the Cd-tolerant CdR6-O cell line. The concentrations of
BSO required to inhibit growth by 50% were 5 and 25 FM,
respectively, for CdS and CdR6-O cells.
BSO has been shown to inhibit the synthesis of PCs in
cells exposed to Cd. One explanation for the differential
sensitivity of CdS and CdR6-O cells to BSO is that CdR6-O cells
have a mechanism to tolerate the toxic effects of Cd that does
not require production of PCs. Altematively, CdR6-O cells
may be more resistant to the inhibitory effects 01' BSO on yGluCys synthetase, thereby allowing synthesis of PCs and
cell growth to occur at higher concentrationsof Bl3O in CdR6O cells than in CdS cells. To distinguish between these two
possibilities, the concentrations of PCs and GSH were measured during the cell culture period of both cell lines grown
in the presence of 0.1 m~ CdClz and 20 PM B5O (Fig. 5).
Under these conditions, GSH levels declined rapidly in CdS
cells and there was little synthesis of PCs before the cells

\ \

10

1O0

1000

Buthionine Sulfoximine (pM)

Figure 4. Tolerance of CdS and CdR6-O cells to BSO in the presence
or absence of Cd. CdS (open symbols)and CdR6-O (filled symbols)

cells were inoculated at 30 mg/mL into medium containing increasing concentrations of BSO either in the presence of 1 O0 p~ CdCI,
(O, O) or without the addition of Cd (V, V).Fresh weights of cells
were measured after 10 d. Error bars indicate SE of three replicated
treatments.

237

Phytochelatin Synthesis and Cadmium Tolerance

1001

./-.

.A.

/-.

O-*-.-.
\

\O
O
\

0-0

I\

CdS
@-a CdR6-O

0-0

O-@

CdS
CdR6-O

GO3 G L

OBSO

20LMBSO

10

Maximum PC Concentration (mmol y-GluCyslkgFW)

Figure 6. Correlation between PC accumulation and cell growth of
CdS and CdR6-O in the presence of Cd. CdS (open symbols) and
CdR6-O (filled symbols) cells were inoculated at 30 mg/mL into
medium containing 0.1 mM CdCI2and increasing concentrations of
BSO (O, O, O p ~ V,
; V, 5 p ~ O,; W, 10 p ~ O,
;
20 p ~ )Samples
.
were removed every other day to measure cell growth and PC
content. Cell growth (maximum fresh weight as a percent of the
no-Cd control) was plotted against the maximum observed PC
concentration for each treatment. Each point represents the mean
of three replicated treatments. FW, Fresh weight.

+,

Days after Inoculation

Figure 5. Responses of CdS and CdR6-O cells to 0.1 mM CdCI2 in
t h e presence of 20 p~ BSO. CdS (O) and CdR6-O (O) cells were
inoculated at 30 mg/mL into medium containing 0.1 mM CdClz and
20 p~ BSO. Fresh weight (A), CSH (B), and PCs (C) were measured
during the cell culture period. Error bars indicate SE of three repli-

cated treatments.
died after 6 d. PC synthesis in CdR6-O cells was delayed by
addition of BSO, but after 2 d there was a steady accumulation of these peptides. This response was paralleled by an
increase in cellular GSH concentration after an initial decline.
The inhibitory effect of 20 ~ L BSO
M
on GSH and PC synthesis
in response to Cd is demonstrated by comparing the results
presented in Figures 3 and 5. PCs accumulated to almost 10
mmol T-GluCyslkg fresh weight 5 d after inoculating CdR6O cells into medium containing 0.1 M CdCI2.The addition of
20 p~ BSO reduced PC accumulation to only 5.6 mmol yGluCys/kg fresh weight after 10 d.
Similar experiments examined the effects of 5 and 10 p~
BSO on growth and PC production in CdS and CdR6-O cells
grown in the presence of 0.1 m~ CdC1,. At each concentration
of BSO, PC accumulation was lower in CdS cells. When cell
growth data were plotted against the maximum PC concentrations observed in cells exposed to Cd and BSO, there was
a clear correlation between PC synthesis and cell growth in
the presence of Cd (Fig. 6). Although PCs were synthesized
in CdS cells exposed to 5 and 10 p~ BSO, their accumulation
was reduced compared to CdR6-O cells, especially during the
first 2 d after inoculation (data not shown). The conclusion

drawn from these results is that CdR6-O cells are able to

produce more PCs than CdS cells in the presence of BSO.
To further examine the increased tolerance of CdR6-O cells
to BSO, cells were tested for their ability to grow in the
presence of this inhibitor without the addition of Cd (Fig. 4).
Although higher concentrations of BSO were required to
inhibit cell growth than when BSO was supplied with Cd,
CdR6-O cells were significantly more tolerant of BSO than
CdS cells. The concentrations of BSO required to inhibit
growth by 50% in the absence of Cd were 0.5 and 1.0 m~
for CdS and CdR6-O cells, respectively. These results suggest
that there is a constitutive alteration in y-GluCys synthetase
in CdR6-O cells.
Activities of Enzymes of

GSH Synthesis

The activities of y-GluCys synthetase and GSH synthetase

were measured in extracts prepared from CdS and CdR6-O
cells grown in Cd-free medium. The specific activity of yGluCys synthetase was approximately 2-fold higher in CdR6O cells than in CdS cells (Table I). However, there was no
differencebetween cell lines in the effect of BSO on inhibition

Activities of enzymes of CSH synthesis in CdS and

CdR6-O cells
y-CluCys synthetase and GSH synthetase were assayed in extracts prepared from CdS and CdR6-O cells 5 d after inoculation.
Results are the means (&SE) of four and two assays for y-CluCys
synthetase and GSH synthetase, respectively. Enzyme activities are
reported as nmol of product formed per min per mg of protein.

Table 1.

Enzyme

CdS

CdR6-O

y-GluCys synthetase

0.54 (0.04)
3.32 (0.08)

0.97 (0.09)
3.32 (0.22)

CSH synthetase

Chen and Goldsbrough

238

of this activity in vitro (data not shown). The same protein

extracts were used to measure GSH synthetase activity. For
this enzyme no difference in specific activity was observed
between the two cell lines. The specific increase in y-GluCys
synthetase activity in CdR6-O cells provides a direct explanation for the increased tolerance of CdR6-O cells to BSO.
DISCUSSION

In this paper we have compared two cell lines that differ

in Cd tolerance. CdR6-O cells were derived from a cell line
selected for growth in medium containing 6 m~ CdCI,. CdR6O cells grow in the presence of 0.3 m~ CdCl,, whereas
progenitor CdS cells do not survive this treatment. The Cd
tolerance of CdR6-O cells has not remained stable, but has
declined over time. When tested previously, growth of CdR6O cells was unaffected by 0.3 m~ CdCI, (Gupta and
Goldsbrough, 1991). After approximately 2 more years of
growth without Cd, in the experiments described here,
growth of CdR6-O cells is reduced by 0.3 m~ CdCl,. Nevertheless, one can clearly distinguish between CdS and CdR6-O
cells under these conditions, in terms of viability and growth.
The cell lines differ in other aspects, including accumulation
of biomass under control growth conditions. Metal-tolerant
plants have also been shown to produce less biomass and
have reduced fitness compared to their nontolerant counterparts when grown in normal soils (Antonovics et al., 1971;
Cook et al., 1972; Wilson, 1988; Wu, 1989). Reduced growth
of both tolerant plants and cells may result from the metabolic
adaptations that give rise to increased tolerance.
The specific activity of 7-GluCys synthetase is higher in
CdR6-O than in CdS cells. This modification provides a direct
explanation for the increased tolerance of CdR6-O cells to
BSO and may account for the increased level of GSH in the
later stages of the CdR6-O cell culture period. 7-GluCys
synthetase is considered to be the rate-limiting step in GSH
synthesis because it is subject to feedback inhibition by GSH
(Hell and Bergmann, 1990). One response of plants to Cd is
an increase in activity of this enzyme (Ruegsegger and Brunold, 1992), reflecting the requirement for substrates for PC
synthesis. Increased activity of y-GluCys synthetase in CdR6O cells may provide a similar increase in the capacity to make
GSH, and this may be a factor contributing to the increased
Cd tolerance of these cells. When the GSH biosynthesis
pathway is either challenged by Cd or inhibited by BSO, the
higher activity of y-GluCys synthetase in CdR6-O cells will
allow synthesis of GSH to continue in the presence of higher
concentrations of Cd or BSO. Other modifications to yGIuCys synthetase in CdR6-O cells, such as reduced feedback
inhibition by GSH, could also increase production of GSH in
response to Cd. A more detailed comparison of the enzyme
from CdS and CdR6-O cells is required. Steffens and Williams
(1989) reported a similar increase in y-GluCys synthetase
activity in an independently selected Cd-tolerant cell line of
tomato. This cell line was also characterized as having an
increased cellular GSH concentration. This adaptation in
GSH synthesis in selected Cd-tolerant cells would allow
production of PCs to continue after exposure to higher concentrations of Cd, whereas in unselected cells the capacity to

Plant Physiol. Vol. 106, 1994

synthesize both GSH and PCs would be inhibited more

severely by the same concentration of Cd.
The importance of PCs for Cd tolerance has been clearly
demonstrated by analysis of Cd-sensitive mutants of S. pombe
(Mutoh and Hayashi, 1988). Mutants that are unable to
synthesize PCs, yet have normal levels of GSH, are Cd
sensitive. Similarly, mutants that contain PCs bu are &able
to form the more stable, sulfide-containing complexes have
the samle phenotype. Several observations indicate that synthesis of PCs is a mechanism utilized by plants and plant
cells to adapt to Cd stress. These include the induction of PC
synthesis in response to Cd (Grill et al., 1987; Sc heller et al.,
1987), the binding of intracellular Cd by these peptides
(Bemhaird and Kagi, 1987; Jackson et al., 1987), the presence
of elevated concentrations of PC-Cd complexes in selected
Cd-tolerant cell lines (Gupta and Goldsbrough, 1991), and
the reduced tolerance of plant cells that are unable to synthesize I T S as a result of treatment with BSO (Steffens et al.,
1987; Mendum et al., 1990). In light of these observations, it
would be surprising if PCs were not involved in the normal
adaptive response to Cd.
Other observations indicate that altered synthesis or accumulation of PCs is not responsible for increased Cd tolerance
in selected plants or cell lines. Cd-tolerant Sillme vulgaris
plants synthesized less PCs than sensitive plans when exposed to the same concentration of Cd (de Krlecht et al.,
1992). 117Datura innoxia, the initial rate of PC synthesis was
the same in unselected and tolerant cell lines, but PC-Cd
complexes formed more rapidly in the tolerant cells (Delhaize
et al., 1989). In several experiments reported here there is an
increased accumulation of PCs in CdR6-O cells. When exposed
to 0.1 m M CdCI2,however, there was little difference between
CdR6-O and CdS cells in accumulation of PCs. Two observations indicate that this concentration of Cd is inwfficient to
reveal the advantage that is provided by increased y-GluCys
synthetase activity. First, 0.1 m~ CdC12 has no effect on
growth of either cell line. Second, both cell lines maintain
very similar cellular concentrations of GSH under these conditions. Therefore, the ability of CdS cells to synthesize PCs
was not compromised by exposure to 0.1 m~ CdlCl,. When a
greater demand is placed on the capacity to synthesize GSH
or PCs, such as that imposed by BSO or higher coi~entrations
of CdCl?,the advantage afforded to CdR6-O cells by increased
y-GluCys synthetase activity is apparent.
Metal-tolerant plants have evolved various mechanisms to
respond to Cd or other heavy-metal ions (Woolhouse, 1983;
Bradshaw, 1984; Baker, 1987). Our results suggest that Cd
tolerance of tomato cells depends on the potentid of cells to
synthesize PCs, which then form complexes with Cd. This
potential is dependent, at least in part, on the capacity to
synthesize GSH under stressful conditions. At least two approaches are possible to obtain definitive proof that increased
GSH and/or PC synthesis is sufficient to increase Cd tolerance. The first is to identify and characterize mutants that
have altered Cd tolerance and determine if the mutations
affect production of these sulfhydryl compounds in plants.
Although the variants identified on metal-contaminated mining sites, for example, represent one source of such material,
the use of a model system such as Arabidopsis may be
advantageous (Howden and Cobbett, 1992). Akematively,

Phytochelatin Synthesis and Cadmium Tolerance

transgenic plants with altered capacities to synthesize either

GSH or PCs will also allow this hypothesis to be directly
tested.
ACKNOWLEDGMENTS

We thank Dr. Edward Ashworth for providing facilities to test cell

viability, Colleen Martin for preparing the manuscript, and Bernie
Leinberger for her excellent technical assistance.
Received January 28, 1994; accepted May 10, 1994.
Copyright Clearance Center: 0032-0889/94/106/0233/07.
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