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Plant Physiol. (1999) 119: 73-80
Overexpression of Glutathione Synthetase in Indian Mustard
Enhances Cadmium Accumulation and Tolerance1
Yong Liang Zhu2,
Elizabeth A.H. Pilon-Smits2, 3,
Lise Jouanin, and
Norman Terry*
Department of Plant and Microbial Biology, University of
California, 111 Koshland Hall, Berkeley, California 94720 (Y.L.Z.,
E.A.H.P.-S., N.T.); and Institut National de la Recherche Agronomique,
Laboratoire de Biologie Cellulaire, Route de Saint-Cyr, F78026
Versailles cedex, France (L.J.)
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ABSTRACT |
An
important pathway by which plants detoxify heavy metals is through
sequestration with heavy-metal-binding peptides called phytochelatins
or their precursor, glutathione. To identify limiting factors for
heavy-metal accumulation and tolerance, and to develop transgenic
plants with an increased capacity to accumulate and/or tolerate heavy
metals, the Escherichia coli gshII gene encoding glutathione synthetase (GS) was overexpressed in the cytosol of Indian
mustard (Brassica juncea). The transgenic GS plants
accumulated significantly more Cd than the wild type: shoot Cd
concentrations were up to 25% higher and total Cd accumulation per
shoot was up to 3-fold higher. Moreover, the GS plants showed enhanced
tolerance to Cd at both the seedling and mature-plant stages. Cd
accumulation and tolerance were correlated with the
gshII expression level. Cd-treated GS plants had higher
concentrations of glutathione, phytochelatin, thiol, S, and Ca than
wild-type plants. We conclude that in the presence of Cd, the GS enzyme
is rate limiting for the biosynthesis of glutathione and
phytochelatins, and that overexpression of GS offers a promising
strategy for the production of plants with superior heavy-metal
phytoremediation capacity.
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INTRODUCTION |
Heavy-metal pollution of soils and waters, mainly caused by mining
and the burning of fossil fuels, is a major environmental problem.
Heavy metals, unlike organic pollutants, cannot be chemically degraded
or biodegraded by microorganisms. An alternative biological approach to
deal with this problem is phytoremediation, i.e. the use of plants to
clean up polluted waters and soils (Black, 1995 ; Salt et al., 1995a).
Heavy metals or metalloids can be removed from polluted sites by
phytoextraction, which is the accumulation of the pollutants in the
plant biomass (Kumar et al., 1995). Compared with other remediation
technologies, phytoremediation is less expensive (1000-fold less
expensive than excavation and reburial of soil [Cunningham and Ow,
1996]) and is particularly suitable for treatment of large volumes of
substrate with low concentrations of heavy metals. However, the
presence of heavy metals inhibits plant growth, limiting the
application of phytoremediation. Therefore, one trait that is of great
significance to phytoremediation is the ability of plants to tolerate
the toxic metals that are being extracted from the soil (Goldsbrough,
1998).
GSH plays several important roles in the defense of plants against
environmental threats. Glutathione is not only a substrate for
glutathione S-transferases, enabling neutralization of
potentially toxic xenobiotics (Marrs, 1996), but is also a reductant of
dehydroascorbate (Foyer and Haliwell, 1976). Moreover, GSH is the
precursor for PCs, heavy-metal-binding peptides involved in heavy-metal
tolerance and sequestration (Steffens, 1990). PCs constitute a family
of peptides with the general structure
( -Glu-Cys)n-Gly, where n = 2 to 11. PCs contain a high percentage of Cys sulfhydryl residues, which
bind and sequester heavy-metal ions in stable complexes. PCs were
induced by heavy metals such as Cd in all of the plants tested (Zenk,
1996). The roles of glutathione in heavy-metal tolerance and PC
synthesis have been well illustrated in Cd-sensitive mutants of
Arabidopsis. For example, the Cd-sensitive cad2 mutant was defective in glutathione biosynthesis (Howden et al., 1995).
Glutathione is synthesized from its constituent amino acids in two
sequential, ATP-dependent enzymatic reactions catalyzed by -ECS and
GS, respectively (Fig. 1). PC synthase subsequently catalyzes the
elongation of the (-Glu-Cys)n by transferring
a -Glu-Cys group to glutathione or to PCs (Zenk, 1996; Chen et al.,
1997).
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| Figure 1.
Regulation of GSH/PC biosynthesis in plants. PS,
PC synthase. Cd enhances the transcription of ECS and activates the PC
synthase enzyme, leading to the production of PCs and the depletion of
GSH. -ECS is also subject to feedback inhibition by GSH.
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Manipulating the expression of enzymes involved in glutathione and PC
synthesis may be a good approach to enhancing heavy-metal tolerance in
plants. The enzyme PC synthase does not appear to be a likely candidate
to be rate limiting for PC synthesis, because it is constitutively
expressed in plants (Steffens, 1990) and activated by the presence of
heavy metals (Zenk, 1996). In any case, to our knowledge, no gene for
PC synthase has yet been cloned. The genes encoding enzymes involved in
GSH synthesis, on the other hand, may hold more promise. The
rate-limiting step for glutathione synthesis in the absence of heavy
metals is thought to be the reaction catalyzed by -ECS, because the
activity of this enzyme is feedback regulated by glutathione and
dependent on Cys availability (Fig. 1).
This view was supported by the observation that overexpression of the
Escherichia coli gshI gene, encoding -ECS, in poplar
resulted in increased foliar glutathione levels (Arisi et al., 1997).
Moreover, expression of tomato -ECS could restore some degree of
heavy-metal tolerance to the cad2 Arabidopsis
mutant. However, overexpression of this gene did not
increase the Cd tolerance of wild-type Arabidopsis plants (Goldsbrough,
1998).
GS is not rate limiting for glutathione synthesis in the absence of
heavy metals. Overexpression of the E. coli gshII gene, encoding GS, did not increase foliar glutathione levels in poplar (Foyer et al., 1995). However, under heavy-metal stress the regulation of glutathione biosynthesis undergoes a significant change. Heavy metals activate the PC synthase enzyme and thus induce the biosynthesis of PCs, resulting in a depletion of cellular glutathione levels (Fig.
1; Zenk, 1996). Consequently, the feedback inhibition of -ECS by
glutathione is released. Furthermore, -ECS expression may be
enhanced by heavy metals (Fig. 1). It was demonstrated that Cd enhances
the transcription of the -ECS gene (Hatcher et al.,
1995). In contrast, Cd may deactivate GS, because GS activity has
previously been shown to be inhibited by Cd, whereas the same Cd
treatment had no effect on -ECS activity (Schneider and Bergmann, 1995). Exposure of maize roots to Cd, in addition to causing a decline
in GSH, caused an accumulation of -Glu-Cys, possibly because the
activity of GS was reduced in vivo (Rauser et al., 1991). Therefore,
under Cd stress the GS enzyme may become rate limiting for the
biosynthesis of glutathione and PCs, and thus overexpression of
gshII may alleviate the depletion of glutathione and enhance
PC synthesis.
To test this hypothesis and to obtain plants with superior Cd
accumulation and tolerance, we overexpressed the E. coli GS enzyme in the cytosol of Indian mustard (Brassica
juncea), which is particularly suitable for phytoremediation
because of its rapid biomass production and large trace-element
accumulation capacity (Dushenkov et al., 1995). Indian mustard was also
shown to produce a PC-Cd-sulfide complex (Speiser et al., 1992). The
transgenic GS plants were compared with untransformed Indian mustard
plants with respect to their Cd accumulation and tolerance, as well as their levels of heavy-metal-binding peptides.
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MATERIALS AND METHODS |
Materials
Indian mustard (Brassica juncea) seeds (accession no.
173874) were obtained from the North Central Regional Plant
Introduction Station (Ames, IA). The gene construct used was described
earlier by Strohm et al. (1995). It contains the Escherichia coli
gshII gene, driven by the double-enhanced 35S cauliflower mosaic
virus promoter, and the nptII gene, conferring kanamycin
resistance.
Plant Transformation
All in vitro plant tissue cultures were grown at 25°C under
continuous light. For transformation, Indian mustard hypocotyl segments
were isolated from 3-d-old axenically grown seedlings (200-300
seedlings per transformation). The segments were immersed for 1 h
in a suspension of the gshII-containing Agrobacterium tumefaciens strain C58pMP90 (A600 = 0.6) suspended in Murashige and Skoog medium; the bacteria were
previously grown for 3 d at 28°C in liquid Luria-Bertani medium
in the presence of 200 µM acetosyringone (3,5-dimethoxy-4-hydroxy-acetophenone; Fluka). After
immersion in the bacterial suspension, the hypocotyls were blotted dry
and transferred to medium containing Murashige and Skoog
salts and vitamins (M5519, Sigma), 4 g L 1
agarose, 10 g L1 Suc, Glc, and mannitol,
200 µM acetosyringone, 2 mg
L1 6-benzylaminopurine, and 0.1 mg
L1 naphthalene acetic acid. After 2 d of
cultivation the hypocotyls were washed for 45 min in standard liquid
Murashige and Skoog medium, blotted dry, and transferred to medium
containing Murashige and Skoog salts and vitamins, 4 g
L1 agarose, 10 g
L1 Suc, Glc, and mannitol, 200 mg
L1 cefotaxime, 100 mg
L1 vancomycin, 20 mg L1
kanamycin, 2 mg L1 6-benzylaminopurine, 0.1 mg
L1 naphthalene acetic acid, and 30 µM AgNO3. After 11 d
the hypocotyls were transferred to the same medium containing 10%
coconut water. Established shoots were transferred to standard
Murashige and Skoog medium containing 30 g
L1 Suc, 100 mg L1
cefotaxime, and 1 mg L1 indole-3-butyric
acid to induce root formation.
Molecular Characterization of Transgenic Plants
PCR was used to identify GS transgenic lines among the
kanamycin-resistant lines obtained. The PCR primers used were the
following: the forward primer was directed against the 35S promoter
with the sequence 5 CCT TCG CAA GAC CCT TCC TC 3, and the reverse primer was directed at the gshII gene with the sequence 5
GGC TGG CAG GTA ATT TTG CGC 3.
For western blotting, 7-d-old seedlings (shoots and roots separately)
were ground in liquid nitrogen and extracted in 50 mM potassium phosphate buffer, pH 8.0, added at 1 mL
g1 fresh weight. After measurement of total
protein concentration (Bradford, 1976), 10 µg of protein from each
sample was separated by SDS-PAGE and blotted onto a Zeta-probe membrane
(Bio-Rad) by electroblotting. We used the Bio-Rad Immun-lite kit for
the immunodetection of separated proteins, and rabbit serum raised
against purified E. coli glutathione synthetase as the first
antibody (Arisi et al., 1997).
Plant Growth and Tolerance Experiments
Experiment I (Seedlings on Agar Medium)
T2 seeds from transgenic lines GS2, GS7, and
GS10 and wild-type Indian mustard were sterilized by rinsing in 96%
ethanol for 30 s, then in 1% hypochlorite solution for 30 min,
and subsequently in sterile, deionized water for 50 min, all on a
rocking platform. Fifty sterilized seeds were sown in a grid pattern in
Magenta boxes (Sigma) on one-half-strength Murashige and Skoog medium containing 10 g L1 Suc, 5 g
L1 Phytagar (Sigma), and different
concentrations of CdSO4 (0, 0.15, 0.20, or 0.25 mM). These relatively high agar Cd concentrations were used
because Cd toxicity in agar is usually much lower than in hydroponic
conditions, possibly because of Cd absorption by agar. Also, the
environmental conditions in agar experiments (rich nutrients, correct
pH, and high moisture) are almost optimal for plant growth, which may
make seedlings more tolerant to Cd. After 7 d at 25°C under
continuous light, the individual seedlings were harvested, washed, and
weighed, and the length of the longest root was measured.
Experiment II (Mature Plants in Hydroponic Solution)
Seeds of line GS7 and wild-type Indian mustard were sterilized and
sown in Magenta boxes as described above. After 5 d on agar the
seedlings were transferred to 4-inch pots containing coarse sand. The
pots were maintained in a greenhouse with controlled temperature
(24°C) and a short-day (9 h) photoperiod. The plants were watered
twice a day, once with tap water and once with one-half-strength Hoagland solution (Hoagland and Arnon, 1938). After 6 weeks of growth
under these conditions, the plants were gently washed in water to
remove the sand adhering to the roots and transferred to a nutrient
film technique setup (Zayed, 1987). The plants were placed in channels,
and one-fourth-strength Hoagland nutrient solution amended with 0.1 mM Cd (as CdSO4) was circulated along the plant roots. Plant growth and Cd accumulation were measured. The
nutrient film technique setup was maintained under the same greenhouse
conditions described above. Plants were harvested after 10 d and
thoroughly washed under running deionized water to remove any trace
elements adhering to the tissue. Total fresh weights of the plants were
measured before and after the experiment to determine the effect of
different concentrations of Cd on growth. Shoot and root tissues were
separated and dried at 70°C for 3 d. The dried tissues were
weighed and then ground in a Wiley mill (Thomas Scientific).
Experiment III (Mature Plants in Hydroponic Solution)
GS7, GS10, and wild-type Indian mustard plants were grown in the
nutrient film technique setup and treated as described in "Experiment
II," except that the Cd treatment was started when the plants were 5 weeks old, and the duration of the Cd treatment was 14 d.
GSH, PC2, and Total Thiol Analysis
Nonprotein thiol, GSH, and PC were measured from the plants used
in experiment II. Extracts were prepared according to the method
described by Galli et al. (1996) by adding 300 µL of a solution
containing 1 M NaOH and 1 mg L1
NaBH4 to 100 mg of a homogenized plant sample.
The homogenate was centrifuged for 3 min at 13,000g at
4°C. Three-hundred microliters of the supernatant was acidified by
adding 50 µL of 37% HCl. Nonprotein thiol contents were measured
photospectrometrically by adding 20 µL of this solution to 1 mL of
5,5-dithiobis(2-nitrobenzoic acid) (Ellman's reagent [Ellman,
1959]), followed by the measurement of A412.
GSH and PCs were analyzed by HPLC using a system similar to that
described by Grill et al. (1987). Low-Mr thiols
were separated on a C18 reverse-phase
column (Waters/Millipore) using a 0% to 20% acetonitrile gradient in
0.05% (v/v) phosphoric acid for 20 min. On-line postcolumn
derivatization with Ellman's reagent was used to specifically monitor
sulfhydryl-containing compounds. The reagent consisted of 0.1 M potassium-phosphate buffer (pH 7.5) and 75 mM Ellman's reagent; the absorption was measured
at 412 nm. The flow rate for the solvent and reagent was 1.0 mL
min1. The amounts of GSH and PC (PC2) were
calculated from standard curves using GSH and PC2 (provided by Dr. M.H.
Zenk, Ludwing-Maximilans-Universität, Munich, Germany) as the
markers.
Elemental Analysis
Elemental analysis was carried out after acid digestion of dried
and ground tissue samples according to the method of Zarcinas et al.
(1987). The concentrations of trace elements in the acid digest were
measured by inductively coupled plasma emission spectroscopy (Fassel,
1978). Standards (National Institute of Standards and Technology) and
blanks were run with all samples for quality control. As a negative
control, plants that had not been supplied with trace elements were
also analyzed for trace-element concentrations.
Statistical analyses were performed using the JMP IN statistical
package (SAS Institute, Cary, NC).
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RESULTS |
Production and Characterization of Transgenic GS Plants
Seven kanamycin-resistant Indian mustard lines were obtained after
transformation with the gshII construct, and were designated GS1, GS2, GS4, GS6, GS7, GS10, and GS13. All seven plant lines showed a
PCR product when PCR was conducted using primers directed against the
35S promoter and the gshII gene (not shown). Homozygous T2 lines from individual T1
plants of lines GS7, GS10, and GS2, showing different gshII
expression levels (see below), were used for subsequent experiments.
These transgenic T1 and T2
plants did not shown any phenotypic differences from the untransformed Indian mustard plants.
Antiserum raised against E. coli GS was used to analyze the
GS expression in the transgenic lines at the protein level. On western
blots both shoot and root tissues from all of the GS lines contained a
protein with the same molecular mass as that from E. coli GS
(38 kD), which reacted with the antiserum (Fig.
2); no band was detected in the wild-type
extract. The expression levels of the E. coli GS protein
were similar in roots and shoots; line GS10 showed lower E. coli GS expression levels than lines GS7 and GS2 (Fig. 2). Similar
differences in gshII expression between the plant lines were
observed at the mRNA level (results not shown).
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| Figure 2.
Western blot of seedling extracts from the
wild-type (WT) and GS-overexpressing (GS2, GS7, and GS10) transgenic
Indian mustard seedlings (digitized image). Equal amounts (10 µg) of
total protein were loaded onto each lane, separated on a 10% SDS-PAGE
gel, blotted, and treated with antiserum raised against purified
E. coli GS protein. Samples were pooled from 25 seedlings each and grown on one-half-strength Murashige and Skoog
medium. E. coli extract was included as a positive
control.
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GS Plants Show Improved Cd Accumulation and Tolerance
Two types of experiments using seedlings or mature plants were
conducted to test Cd tolerance. For the seedling experiment, seeds of
lines GS7, GS10, and GS2 and from wild-type Indian mustard were sown on
agar medium containing 0, 0.15, 0.20, or 0.25 mM CdSO4, and root length was measured after 7 d. Root length is considered to be a reliable parameter for
trace-element tolerance (Murphy and Taiz, 1995). At all three Cd
concentrations, the GS seedlings had significantly longer roots than
the wild-type seedlings (Fig. 3A). For
example, at 0.15 mM Cd the roots of line GS7 seedlings were
60% longer (P < 0.001) than those of wild-type seedlings. The
transgenic seedlings were also taller than wild-type seedlings under Cd
treatment (Fig. 3B). Under control conditions the GS seedlings had
slightly, but significantly, shorter roots than the wild type (Fig. 3A;
P < 0.05).
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| Figure 3.
Effect of Cd on the growth of wild-type (WT) and
GS-overexpressing (GS2, GS7, and GS10) Indian mustard seedlings grown
for 7 d on agar medium containing different concentrations of
CdSO4. A, Root length (the averages and SE
values of 50 seedlings). B, Five representative seedlings from each
line, grown on 0.20 mM CdSO4 (digitized image).
Using analysis of variance, each of the transgenic lines was shown to
be significantly different (P < 0.05) from the wild type (WT) at
each Cd concentration, with the exception of line GS10 at 0.25 mM Cd.
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The first Cd-tolerance experiment with mature plants was performed with
plants from transgenic line GS7 and the wild type. After 10 d of
growth on 0.10 mM CdSO4 the GS7
plants had attained a 2-fold higher fresh weight than the wild type
(P < 0.01; Fig. 4A). There were no
significant differences between the plant lines with respect to initial
fresh weight (data not shown) or between the final fresh weight of
untreated plants (113.2 ± 13.1 and 122.3 ± 12.3 g for
the wild type and line GS7, respectively). The Cd concentrations were
approximately 40% higher in shoots of GS7 plants compared with the
wild type (P = 0.13; Fig. 4B); no significant differences were
found with respect to root Cd concentration. As a result of the
increased tolerance to Cd and the increased shoot Cd accumulation, the
total amount of harvestable Cd per plant shoot was about 3-fold higher
for GS7 plants than for the wild type (P < 0.01; Fig.
4C).
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| Figure 4.
Growth (A), shoot Cd concentration (B), and shoot
Cd accumulation (C) by wild-type (WT) and GS-overexpressing (GS7)
Indian mustard plants treated with 0.1 mM
CdSO4. In A, the final fresh weight (FW) was used as a
parameter of growth, because the initial fresh weights of the wild-type
and GS plants were not significantly different. Values shown are the
average and SE of eight replicates for individual plants.
P < 0.01 (A); P = 0.13 (B); and P < 0.01 (C).
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In the second experiment using mature plants, 6-week-old GS7, GS10, and
wild-type plants were exposed to 0.05 or 0.10 mM
CdSO4 for 14 d. Again, the GS7 plants showed
superior Cd tolerance: they suffered less growth inhibition by Cd than
the wild type (P < 0.05; Fig. 5A).
For instance, in nutrient solution amended with 0.05 mM Cd,
the relative growth of GS7 plants was 45% of that of untreated GS7
controls, whereas the relative growth of wild-type plants was only
27%. The growth of GS10 plants was intermediate between GS7 and the
wild type, in accordance with the gshII expression levels,
which were lower in GS10 than in GS7 plants (Fig. 2). The shoot Cd
concentrations in GS7 plants were 19% to 22% higher than in wild-type
plants (P < 0.05; Fig. 5B). Similar to Cd tolerance, line GS10
showed intermediate Cd accumulation between GS7 and the wild type (Fig.
5B), in accordance with its lower gshII expression level. No
significant differences were found with respect to root Cd
concentrations.
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| Figure 5.
Growth inhibition (A) and shoot Cd concentrations
(B) of wild-type (WT) and GS-overexpressing (GS7 and GS10) Indian
mustard plants treated with 0.05 or 0.1 mM
CdSO4. In A, the parameter used for growth is the relative
increase in fresh weight of Cd-treated plants as the percentage of
untreated plants of the same line; this parameter was chosen to
normalize the different initial fresh weights for plants from different
lines. Values shown are the average and SE of 12 replicates
(A), or of 6 pooled samples from 2 plants each (B). P < 0.05 for
GS7 versus the wild type, and not significant for GS10 versus the wild
type (A and B). DW, Dry weight.
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GS Plants Have Higher Levels of GSH, PC2, Thiols, S, and Ca
To investigate the effect of GS overexpression on the production
of heavy-metal-binding compounds, the levels of GSH, PC (PC2), and
total thiol were determined in leaf and root samples collected from GS7
and wild-type plants used in the first mature-plant experiment described above, which were treated with 0.1 mM Cd.
The GSH levels were 5-fold higher in roots of Cd-treated GS7 plants
than in wild-type roots (P < 0.05; Fig.
6B). There were no significant
differences between the plant lines with respect to GSH contents in
shoot tissues of Cd-treated plants (Fig. 6B) or in roots or shoots of
untreated plants (Fig. 6A). It is interesting that the GSH levels in
roots from Cd-treated wild-type plants were significantly lower than
those in untreated wild-type plants, whereas the GS7 plants showed
similar GSH levels under both conditions (Fig. 6, A and B).
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| Figure 6.
Tissue concentrations of glutathione (A and B), PC
(PC2, C), and total thiol (D) in wild-type (WT) and GS-overexpressing
(GS7) Indian mustard plants grown in the absence (A) or presence (B-D)
of 0.1 mM CdSO4. Note that tissue PC2 levels in
the absence of Cd were below the detection limit in all of the samples.
Values shown are the average and SE of three replicate
samples from four plants each. A, No significant differences between
line GS7 and the wild type. B, P < 0.05 for roots, not
significant for shoots. C, P < 0.05 for roots, P < 0.01 for
shoots. D, P < 0.01 for roots, not significant for shoots. FW,
Fresh weight.
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In Cd-treated plants the levels of the smallest PC molecule, PC2, were
1.7-fold higher in roots of GS7 plants compared with the wild type
(P < 0.05), and 2.3-fold higher in shoots of GS7 than in the wild
type (P < 0.01; Fig. 6C). In untreated plants no PC2 was
detectable in either plant line.
The thiol levels (including Cys, -Glu-Cys, GSH, and PC2) in roots of
Cd-treated GS7 plants were approximately 2-fold higher than in the wild
type (P < 0.01; Fig. 6D). There were no differences between the
plant lines with respect to shoot thiol content in Cd-treated or
untreated plants. In both GS7 and wild-type plants the total thiol
levels were 3-fold higher in shoots and 10-fold higher in roots of
Cd-treated plants compared with untreated plants of the same line.
The GS7, GS10, and wild-type plants used in the second mature-plant
experiment (0 or 0.1 mM Cd treatment) were analyzed for shoot and root levels of Ca, Cu, Fe, Mg, Mn, S, and Zn. S and Ca were
present at higher levels in shoots of Cd-treated GS7 plants compared
with wild-type shoots (Fig. 7; P < 0.10); GS10 plants showed intermediate values between the wild type and
line GS7. There were no differences among the plant lines with respect
to root Ca or S concentrations in Cd-treated or untreated plants (data
not shown). The Ca levels in roots and shoots of Cd-treated plants were
significantly lower than those in untreated plants in both GS and the
wild type (P < 0.001); S levels were not significantly affected
by Cd in either plant line. No differences were found between GS and
wild-type plants with respect to the levels of Mg, Zn, Cu, Mn, and Fe
(data not shown).
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| Figure 7.
Shoot tissue concentrations of S and Ca in
wild-type (WT) and GS-overexpressing (GS7 and GS10) Indian mustard
plants treated with 0.1 mM CdSO4. Values shown
are the average and SE of six pooled samples from two
plants each. S, P < 0.10 for GS7 versus the wild type, not
significant for GS10 versus the wild type; Ca, P < 0.10 for GS7
versus the wild type and for GS10 versus the wild type. S and Ca
concentrations in roots were not significantly different between the
wild-type and transgenic plants, either under control conditions or
after Cd treatment. DW, Dry weight.
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DISCUSSION |
Our results show that overexpression of the E. coli
gshII gene in Indian mustard resulted in enhanced production of
GSH and PCs and improved Cd accumulation and tolerance. The
physiological and biochemical effects observed were correlated with the
ghsII expression levels. These results suggest that in the
presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and PCs, and that the levels of glutathione and/or PCs determine the plant's capacity to accumulate and tolerate Cd.
Under unstressed conditions GS does not appear to be rate limiting for
glutathione synthesis, because the glutathione levels did not differ
significantly in unstressed GS7 and wild-type plants (Fig. 6A), and
there was no detectable PC in either plant line. These results agree
with those of Foyer et al. (1995), who found that overexpression of the
same gene did not affect poplar glutathione levels. Under Cd stress,
however, the GS enzyme appears to become rate limiting for the
biosynthesis of glutathione and PCs. In roots of Cd-treated wild-type
plants, the GSH levels were 3-fold lower (caused by depletion
attributable to PC synthesis) than in unstressed wild-type plants. In
the tissues of GS plants, the rate limitation was overcome and GSH
levels were the same in Cd-treated and untreated plants (Fig. 6, A and
B). Furthermore, the PC2 levels in roots and shoots of the GS plants
were about 2-fold higher than in wild-type plants (Fig. 6C).
The roots appear to be the major site of PC synthesis. First, the PC2
levels were higher in roots than in shoots of Cd-treated wild-type and
transgenic plants (Fig. 6C). Second, the depletion in glutathione was
observed only in the roots of wild-type plants, but not in the shoots
(Fig. 6, A and B). Third, the Cd-induced increase in total thiol (a
substantial fraction of which represents PCs of various lengths) was
10-fold in roots and only 3-fold in shoots. The notion that PCs are
produced mainly in the root was also suggested by Salt et al. (1995b).
The most likely explanation for the increased Cd tolerance of the GS
plants is that these plants produced more PCs. The 2-fold increase in
PC levels in the shoots and roots of the GS transgenic plants is
expected to lead to a greater capacity to detoxify and sequester Cd,
because PCs bind heavy metals, followed by sequestration of the Cd-PC
complex in the vacuole (Zenk, 1996). The Cd-PC complex is further
complexed in the vacuole with sulfide. It has been suggested that metal
tolerance by plants may be limited by the availability of reduced S for
Cys and sulfide synthesis (Goldsbrough, 1998). It is interesting that
the levels of total S were higher in the shoots of the GS plants
compared with the wild type (Fig. 7A). Furthermore, because GSH has an
important role in plant-stress tolerance, the higher GSH levels in the
roots of the GS plants may have contributed to their increased Cd
tolerance.
Cd significantly reduced the tissue Ca concentration in both wild-type
and GS plants, but overexpression of GS diminished this decrease in Ca
in shoots (Fig. 7B). The Ca concentration in roots was not
significantly different between the wild-type and transgenic plants
(data not shown). Two recent studies have focused on the interaction of
Cd2+ and Ca2+ (Ibekwe et
al., 1996; Beyersmann and Hechtenberg, 1997).
Cd2+ is a Ca2+ channel
blocker, and Cd2+ interferes with the
Ca2+ messenger system by binding calmodulin, a
Ca2+-binding protein that regulates a variety of
enzymes and cell processes (Cheung, 1984). The increased levels of
Cd-binding peptides in the GS plants may contribute to reducing the
effect of Cd on the Ca-calmodulin interaction.
The GS plants accumulated more Cd (at higher concentrations) than
wild-type plants in their shoots, but not in their roots. The
translocation of Cd from the root to the shoot through the xylem is
thought to be driven by transpiration (Salt et al., 1995b). Because
more Cd was bound by PCs and stored in the vacuole in the GS plants,
vital biochemical and physiological processes were less damaged than in
wild-type plants. This led to increased total leaf surface area in GS
plants and, therefore, to the accumulation of more Cd (as a result of
greater transpiration per plant). Furthermore, GS plants may have
absorbed more Cd because they sustained less damage by Cd to the root
surface. Root water uptake was shown to be one of the primary
mechanisms affected by Cd stress in plants (Marchiol et al., 1996). The
higher PC levels in the roots of the GS plants may have alleviated this
negative effect of Cd on root water uptake.
In conclusion, this study provides insight into the regulation of GSH
and PC biosynthesis and heavy-metal sequestration. In addition, we have
successfully developed transgenic plants that have an increased
capacity for Cd accumulation and tolerance. These GS plants offer great
promise for enhancing the efficiency of Cd phytoextraction from
polluted soils and wastewater. These plants may also show increased
tolerance to, and accumulation of, other heavy metals, because
PCs are thought to play a role in tolerance of a range of heavy metals,
especially nonessential heavy metals such as mercury and lead
(Goldsbrough, 1998).
| |
FOOTNOTES |
1
This work was supported by a grant from the
Electric Power Research Institute (no. W04163 to N.T.) and by a
TALENT stipend from the Dutch Organization for Scientific
Research to E.A.H.P.-S.
2
These authors contributed equally to this
work.
3
Present address: Department of Biology, Colorado
State University, A/Z Building, Fort Collins, CO 80523.
*
Corresponding author; e-mail nterry{at}nature.berkeley.edu; fax
1-510-642-3510.
Received June 29, 1998;
accepted October 14, 1998.
| |
ABBREVIATIONS |
Abbreviations:
-EC, -glutamyl-Cys.
-ECS, -glutamyl-Cys synthetase.
GS, glutathione synthetase.
PC, phytochelatin.
PC2, (-Glu-Cys)2-Gly.
| |
ACKNOWLEDGMENTS |
We thank Dr. M.H. Zenk for generously providing PC2
standard references, Dr. A.C.M. Arisi for providing GS antibodies, and X.L. Du and Dr. G. Noctor for help with biochemical analyses. We also
thank Dr. M. de Souza for reviewing the manuscript.
| |
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