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Plant Physiol, December 1999, Vol. 121, pp. 1169-1177
Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced
by Overexpressing
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
LITERATURE CITED
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To investigate rate-limiting factors
for glutathione and phytochelatin (PC) production and the
importance of these compounds for heavy metal tolerance, Indian mustard
(Brassica juncea) was genetically engineered to
overexpress the Escherichia coli gshI gene encoding
-glutamylcysteine synthetase (-ECS), targeted to the plastids.
The -ECS transgenic seedlings showed increased tolerance to Cd and
had higher concentrations of PCs, -GluCys, glutathione, and total
non-protein thiols compared with wild-type (WT) seedlings. When tested
in a hydroponic system, -ECS mature plants accumulated more Cd than
WT plants: shoot Cd concentrations were 40% to 90% higher. In spite
of their higher tissue Cd concentration, the -ECS plants grew better
in the presence of Cd than WT. We conclude that overexpression of
-ECS increases biosynthesis of glutathione and PCs, which in turn
enhances Cd tolerance and accumulation. Thus, overexpression of
-ECS appears to be a promising strategy for the
production of plants with superior heavy metal phytoremediation capacity.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
LITERATURE CITED
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Heavy metals and metalloids such as Cd, Pb, Hg, As, and Se are an
increasing environmental problem worldwide. Plants can be used to
remove heavy metals by accumulating, stabilizing, or biochemically transforming them. This cost-effective and environment-friendly technology has been called "phytoremediation" (Salt et al., 1995
). Hyperaccumulators
plant species that accumulate extremely high concentrations of heavy metals in shootsoffer one option for the
phytoremediation of metal-contaminated sites. However,
hyperaccumulators tend to grow slower and produce little biomass
(Brooks, 1994). An alternative approach is to genetically engineer
fast-growing species to improve their metal tolerance and
metal-accumulating capacity. A suitable target species for this
strategy is Indian mustard (Brassica juncea), which has a
large biomass production, a relatively high trace element accumulation
capacity (Dushenkov et al., 1995), and can be genetically engineered
(Zhu et al., 1999).
Non-protein thiols (NPTs), which contain a high percentage of Cys
sulfhydryl residues in plants, play a pivotal role in heavy metal
detoxification. The reduced form of glutathione (-Glu-Cys-Gly, GSH)
is one of the most important components of NPT metabolism. GSH may play
several roles in heavy metal tolerance and sequestration. It protects
cells from oxidative stress damage, such as that caused by heavy metals
in plants (Gallego et al., 1996; Weckx and Clijsters 1996, 1997). For
example, it has been shown that Cd treatment causes an increase in
lipid peroxidation and lipooxygenase activity (Gallego et al., 1996).
GSH is the direct precursor of phytochelatins (PCs). PCs are heavy
metal-binding peptides involved in heavy metal tolerance and
sequestration (Steffens, 1990). PCs comprise a family of peptides with
the general structure (-Glu-Cys)n-Gly, where
n = 2 to 11 (Rauser, 1995). They were shown to be
induced by heavy metals such as Cd in all plants tested (Zenk, 1996), including Indian mustard (Speiser et al., 1992). The roles of GSH and
PC synthesis in heavy metal tolerance were well-illustrated in
Cd-sensitive mutants of Arabidopsis. For example, the Cd-sensitive cad2 mutant was defective in GSH and PC biosynthesis (Howden
et al., 1995).
GSH is synthesized from its constituent amino acids in two sequential,
ATP-dependent enzymatic reactions, catalyzed by -glutamylcysteine synthetase (-ECS) and glutathione synthetase (GS), respectively. PC
synthase subsequently catalyzes the elongation of the
(-Glu-Cys)n by transferring a -GluCys group
to GSH or to PCs (Zenk, 1996). Genes encoding PC synthase have just
recently been cloned from plants and yeast (Clemens et al., 1999; Ha et
al., 1999; Vatamaniuk et al., 1999). The rate-limiting step for GSH
synthesis in the absence of heavy metals is believed to be the reaction
catalyzed by -ECS, since the activity of this enzyme is
feedback-regulated by GSH and dependent on Cys availability (Noctor et
al., 1998b). This view was supported by the observation that
overexpression of the Escherichia coli gshI gene (which
encodes -ECS) in poplar resulted in increased foliar GSH levels
(Arisi et al., 1997; Noctor et al., 1998a). In contrast, overexpression
of GS did not lead to an increase in GSH levels in poplar (Foyer et
al., 1995) or in Indian mustard (Zhu et al., 1999) in the absence of
heavy metals. However, the Indian mustard GS-overexpressing plants
showed higher levels of GSH and PC2 relative to untransformed plants in
the presence of heavy metals. These GS plants also showed enhanced heavy metal tolerance and accumulation (Zhu et al., 1999).
Although -ECS plays an important role in controlling GSH synthesis,
its significance in controlling PC synthesis and heavy metal tolerance
or accumulation remains unclear. It has been reported that
overexpression 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 (WT) Arabidopsis plants (Goldsbrough, 1998). In the present
study we overexpressed the E. coli -ECS enzyme in the chloroplasts of Indian mustard. The transgenic -ECS plants were compared with WT 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 |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
LITERATURE CITED
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Plant Transformation and Characterization
Indian mustard (Brassica juncea, accession no. 173874)
seeds were obtained from the North Central Regional Plant Introduction Station (Ames, IA). Hypocotyl segments from 3-d-old axenically grown
seedlings were transformed as described by Pilon-Smits et al. (1999).
The -ECS gene construct used was described earlier by Noctor et al.
(1998a). It contains the Escherichia coli gshI gene fused to
a pea chloroplast transit sequence and driven by the cauliflower mosaic
virus 35S promoter with a double-enhancer sequence (P70). The construct
also contains the nptII gene, which confers kanamycin
resistance. This construct was shown earlier to successfully target the
E. coli -ECS protein to poplar plastids (Noctor et al.,
1998a).
PCR was used to identify -ECS 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'. The reverse primer
was directed at the gshI gene and had the sequence 5'-GCA CTC GGT TTT CTC AAA CGG-3'.
Total RNA was isolated from 7-d-old seedling shoots using the RNeasy
Plant Mini Kit (Qiagen, Valencia, CA). Northern-blot hybridization was
carried out as described by Krumlauf (1994) using a gshI DNA
probe that was generated by PCR using the primers described above. The
template DNA was a purified plasmid containing the gshI
gene. The PCR product was purified from the gel and labeled with
32P-[dCTP] by random priming using a DNA
labeling kit (Ready-To-Go, Pharmacia Biotech, Piscataway, NJ).
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
g
1 fresh weight. After measurement of the 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, Hercules, CA) by electroblotting. We used the Bio-Rad
Immuno-lite kit for the immunodetection of separated proteins, and
rabbit serum raised against purified E. coli -ECS as the
first antibody (Arisi et al., 1997).
Plant Growth and Tolerance Experiments
Seedling Experiments
Two similar seedling experiments (A and B) were conducted and described here. In experiment A, T2 seeds from transgenic lines ECS2, ECS4, and WT Indian mustard were sterilized by rinsing in 95% ethanol for 30 s, then in 1% hypochlorite solution for 30 min, and subsequently in sterile deionized water five times for 10 min each time, all on a rocking platform. Fifty sterilized seeds were sown in a grid pattern in Magenta boxes (Sigma, St. Louis) on 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).
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. Total NPT concentration and total
glutathione were also measured from this experiment. Seedling
experiment B was essentially the same as experiment A except for the
following: the treatments were 0 and 0.2 mM Cd, the medium
contained 4 g L1 Phytagar, and Cd-treated
seedlings were harvested after 11 d. This experiment focused on
individual thiol measurements. Root length was also measured but data
are not presented since the results were similar to those from
experiment A.
Mature Plant Experiments
Seeds of ECS2, ECS4, and WT 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 to prevent flowering. The plants were watered twice daily, once with tap water and once with one-half-strength Hoagland solution. After 4 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. The plants were placed in channels and quarter-strength Hoagland's nutrient solution (Hoagland and Arnon, 1938) amended with
0.05, 0.075, and 0.1 mM Cd (as CdSO4) was circulated along the plant roots. This setup was maintained under
the same greenhouse conditions. Plants were harvested after 14 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.
Glutathione and NPT Analysis
Total NPTs and total glutathione were measured
spectrophotometrically in seedlings obtained from seedling experiment
A. For total NPT analysis, 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
homogenized plant sample. The homogenate was centrifuged for 3 min at
13,000g at 4°C. Supernatant (300 µL) was acidified by
adding 50 µL of 37% HCl. NPT contents were measured spectrophotometrically by adding 20 µL of this solution to 1 mL of of
5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent, Ellman, 1959),
followed by the measurement of the
A412. Total glutathione was
measured according to the method of Hermsen et al. (1997).
Cys, -EC, GSH, and PC levels in the seedlings obtained from
experiment B were analyzed by HPLC according to the method of Weber et
al. (1999). NPTs were extracted from 100 mg fresh weight of both shoot
and root samples using 200 µL of 10% sulfosalicylic acid containing
12.6 mM diethylenetriaminepentaacetic acid. The homogenate
was centrifuged, filtered (0.22-µm pore size), and diluted before
injection. As a comparison, the homogenate was also used for total NPT
measurement spectrophotometrically, as described above. The HPLC
equipment included a pump (LC-10AD, Shimadzu, Kyoto), a
C18 column (250 × 4.6 mm, 5-µm particle
size, Altima, Alltech, Deerfield, IL), a pre-column (All-Guard,
Alltech), and a 50-µL injection loop. The detection system included
an amperometric electrochemical detector with a gold/mercury electrode
(model LC-4B, Bioanalytical Systems, West Lafayette, IN). The mobile phase was 9.4 g of monochloroacetic acid and 40 mL of methanol in
1 L of water, pH adjusted to 3.1 to 3.2 with sodium hydroxide pellets.
The system was run at a flow rate of 1 mL min1
at an applied voltage of 0.154 V. The electrode was polished and plated
with mercury the day before analysis according to the instructions
provided. The mobile phase was continuously de-gassed throughout the
operation to eliminate interference from oxygen. The detection limit
for GSH is 5 pmol.
Measurement of -ECS Activity
Protein was extracted from shoots of unstressed seedlings in
experiment B. After grinding in liquid nitrogen, extractions were made
by adding (2 mL g1 fresh weight) buffer (50 mM potassium phosphate and 1 mM
phenylmethylsulfonyl fluoride, pH 8.0), followed by centrifugation at
13,000g for 5 min at 4°C. Protein content in the
supernatant was determined using the standard Bio-Rad assay kit. For
-ECS activity, -EC formation in a reaction mix was determined
with HPLC as described above. The reaction was started by adding 10 µL of the protein extract to 50 µL of -ECS assay solution
containing 100 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 8.0), 50 mM MgCl2, 20 mM glutamate, 1 mM Cys, 5 mM ATP, 10 mM
phosphocreatine, 10 units/mL phosphocreatine kinase, and 0.5 mM dithiothreitol. After 20 and 80 min of
incubation at 30°C, 10 µL of the reaction mixture was added to 40 µL of the mobile phase, followed by either immediate HPLC analysis or
immediate storage in dry ice for HPLC analysis the same day. -EC
formation in 60 min was calculated by subtracting the 20-min -EC
peak from the 80-min -EC peak.
Elemental Analysis
Elemental analysis was carried out after acid digestion of dried
and ground tissue samples as described by Zarcinas et al. (1987). The
concentrations of trace elements in the acid digest were measured by
inductively coupled plasma atomic emission spectroscopy (Fassel, 1978).
Standards (National Institute of Standard and Technology)
and blanks were run with all samples for quality control. Plants that
had not been supplied with trace elements were also analyzed for trace
element concentrations as a negative control.
Statistical analyses were performed using the JMPIN statistical package (SAS Institute, Cary, NC).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
LITERATURE CITED
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Production and Characterization of Transgenic -ECS Plants
Five kanamycin-resistant Indian mustard lines were obtained after
transformation with the gshI construct, and were designated ECS1, ECS2, ECS3, ECS4, and ECS6. All five plant lines showed a product
when PCR was performed using primers directed against the 35S promoter
and the gshI gene (not shown). Progeny of lines ECS2 and
ECS4 showed a kanamycin resistance ratio of 3:1 (100 mg
L1 in one-half-strength Murashige and Skoog
medium) after self-fertilization of the first generation, indicating a
single insert of the gshI gene. Homozygous T2 lines from
individual T1 plants of lines ECS2 and ECS4 were used for subsequent
experiments. These transgenic T1 and T2 plants did not show any
distinguishable difference in visual appearance under unstressed
conditions compared with the WT plants.
When gshI DNA was used as a probe for a northern blot
containing total RNA isolated from T2 seedlings of ECS2 and ECS4, both transgenic lines showed a band of the E. coli gshI
transcript (Fig. 1A). Antiserum raised
against E. coli -ECS was used to analyze transgenic
expression in the transgenic lines at the protein level. On western
blots, shoot tissues from ECS2 and ECS4 lines were both shown to
contain a protein with the same molecular mass as the E. coli -ECS (64 kD), which reacted with the antiserum (Fig. 1B);
no band was detected in WT extract. The expression levels of the
E. coli -ECS protein were similar in ECS4 and in ECS2.
Shoots of both ECS2 and ECS4 plants showed approximately 5-fold-increased -ECS activity compared with WT shoots (Table I).
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-ECS Plants Show Improved Cd Accumulation and Tolerance
Two experiments were conducted to test Cd tolerance, one using
seedlings and one mature plants. For Cd tolerance, we used root length,
which is considered to be a reliable parameter for heavy metal
tolerance (Murphy and Taiz, 1995). After 7 d on agar medium
containing 0.15, 0.20, or 0.25 mM
CdSO4, the -ECS seedlings had longer roots
than WT seedlings (Fig. 2A). For example,
at 0.20 mM Cd, the roots of ECS4 seedlings were more than
2-fold longer (P < 0.001) than those of WT seedlings.
Under control conditions, there were no significant differences in root
length between the -ECS seedlings and the WT seedlings (Fig. 2A).
The shoots of the transgenic seedlings were also taller than shoots of
WT seedlings after Cd treatment (Fig. 2B).
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The Cd tolerance experiments with mature plants were performed with
plants from transgenic lines ECS2, ECS4, and WT. After 14 d of growth
on nutrient solutions containing 0, 0.05, 0.075, or 0.10 mM
CdSO4, the -ECS plants showed superior Cd
tolerance compared with WT: their growth (whole plant biomass) was less inhibited by Cd than the growth of WT plants (Fig.
3), with the exception of ECS2 at 0.1 mM. At 0.05 mM Cd, the relative growth of ECS2
plants was 44% of that of untreated ECS2 controls, while the relative
growth of WT plants was 30%.
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The Cd concentrations in plant roots and shoots were determined for the plants obtained from the mature plant experiments. Both ECS2 and ECS4 plants showed higher Cd concentrations in their shoots than WT plants. For example, when grown at 0.05 mM external Cd, the shoot Cd concentrations in ECS4 plants were 90% higher than in WT plants (P < 0.01, Fig. 4A). The Cd concentrations in roots of ECS2 and ECS4 plants were also slightly higher than in WT plants, but these differences were not significant (Fig. 4B).
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-ECS Plants Contain Higher Levels of GSH, PCs, and Total NPTs
To investigate the effect of -ECS overexpression on the
production of heavy metal binding compounds, total NPTs and
glutathione levels were measured spectrophotometrically in shoot
samples collected from ECS2, ECS4, and WT seedlings used in seedling
experiment A, which were treated with 0.15, 0.20, or 0.25 mM Cd. The glutathione levels were 1.5- to 2.5-fold
higher in both -ECS lines compared with the WT (Fig.
5A). This difference was true for both
Cd-treated and untreated seedlings. ECS2 and ECS4 plants had slightly
higher levels of total NPTs than WT under control conditions (1.36, 1.80, and 1.16 µM g1 fresh
weight, respectively), but this difference was not significant. In
contrast, the total NPT levels in Cd-treated -ECS seedlings were
significantly higher (approximately 1.5-fold) than in WT seedlings
(Fig. 5B).
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The higher total NPT concentrations in both roots and shoots were also
observed in 0.2 mM Cd-treated -ECS seedlings from experiment B (Fig. 6). In this
experiment, individual NPTs including GSH, Cys, -EC, and PCs were
determined with HPLC. As described above, shoot GSH concentrations were
higher in ECS2 and ECS4 seedlings than in WT seedlings (under both 0 and 0.2 mM Cd treatment, Fig. 6, A and B). However, root
GSH concentrations were similar among all three lines (Fig. 6C) under
Cd treatment.
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A small, but significantly greater amount of -EC was detected in
untreated ECS2 (1.6 nmol g1 fresh weight) and
ECS4 (2.1 nmol g1) seedlings compared with WT
seedlings (0.8 nmol g1) (Fig. 6A). -EC
concentration was also significantly higher in roots of Cd-treated ECS2
(228 nmol g1) and ECS4 (252 nmol
g1) seedlings and in shoots of Cd-treated ECS4
seedlings (145.7 nmol g1) compared with WT
(root 183 nmol g1 and shoot 89.9 nmol
g1). ECS2 shoots (95.8 nmol
g1) showed an intermediate -EC
concentration. While Cys concentrations were significantly lower in
shoots of ECS2 and ECS4 seedlings than in shoots of WT seedlings under
unstressed conditions, they were similar in shoots of ECS2, ECS4, and
WT seedlings under Cd stress (Fig. 6).
Both Cd-treated -ECS and WT seedlings accumulated PC2 in shoots, and
PC2, PC3, and PC4 in roots. ECS4 shoots (1128 nmol µM g1) showed a significantly (P = 0.014) higher PC2 level than WT shoots (870.8 nmol
µM g1) (Fig. 6B). PC2
concentration in ECS2 (961.5 nmol µM
g1) was intermediate (ECS2/WT,
P = 0.100). ECS4 roots also had significantly higher
concentrations of PC2, PC3, and PC4 (Fig. 6C). ECS2 roots only showed
higher levels of PC2 compared with WT roots.
Effects of -ECS Overexpression on Mineral Nutrient Levels
To investigate the effect of Cd on tissue mineral nutrient levels,
the concentrations of B, Ca, Cu, Fe, K, Mg, Mn, Mo, P, S, and Zn were
determined in shoots and roots of mature -ECS and WT plants treated
with 0 or 0.1 mM Cd. Cd treatment had significant effects
on the nutrient levels of some elements in the plants (Table
II). In WT plants, the levels of Ca, Fe,
Mn, P, and S were significantly lower under 0.1 mM Cd
treatment than under non-Cd conditions (levels of B, Cu, Mg, K, Mo, and
Zn were not affected by Cd). Interestingly, the levels of Ca, Fe, Mn,
P, and S were all significantly higher in Cd-treated ECS4 plants than
in Cd-treated WT plants (Table II). ECS2 also had higher concentrations
of Ca, Fe, Mn, P, and S than WT but only Fe and S were statistically significant (Table II). In the absence of Cd there were no significant differences between -ECS and WT plants with respect to tissue concentrations of any of these elements except for Cu and Zn that were
higher in the -ECS plants (data not shown). The root concentrations of all elements tested did not show any significant differences between
-ECS and WT plants.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
LITERATURE CITED
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Overexpression of the E. coli gshI gene in Indian
mustard increased -ECS activity compared with WT. The activity of
this enzyme in transgenic seedling shoots was 5-fold greater than in WT
shoots (Table I). The E. coli protein was most probably
targeted to the chloroplast, as the gene construct contained the pea
rbcS sequence, coding for the chloroplast transit peptide of the rbcS. Using the same construct, Noctor et al. (1998a) concluded that the
E. coli protein was present in the chloroplast of transgenic poplar. In plants treated with Cd, overexpression of the E. coli gshI gene increased the production of -EC, the direct product of the -ECS enzyme, as well as GSH and PCs farther down the pathway. In unstressed plants (i.e. those not treated with Cd), there was increased production of -EC and GSH but not PCs, because PC synthase requires Cd for activation (Zenk, 1996). Both -EC and GSH
concentrations were 2 to 3 times higher in the transgenic lines, ECS2
and ECS4, than in the WT (Fig. 6); this compares to 10- to 15-fold
increases of -EC levels in poplar (Arisi et al., 1997; Noctor et
al., 1998a).
Our results show that plants overexpressing -ECS exhibited increased
Cd tolerance and accumulation. It is presumed that the increased Cd
tolerance and accumulation were due to the enhanced PC production by
-ECS plants, which should lead to a greater capacity to detoxify and
sequester Cd (Steffens, 1990; Zenk, 1996). However, it is possible that
the increased levels of GSH in -ECS plants may have also contributed
to the increased tolerance in -ECS plants. GSH is a major component
of the active oxygen scavenging system of the cell (Noctor and Foyer,
1998), and the relatively high levels of GSH in the tissues of the
-ECS plants could have conferred increased Cd tolerance by
protecting cells from metal-related oxidative stress damage (Gallego et
al., 1996; Weckx and Clijsters 1996, 1997). On the other hand, higher
GSH levels did not confer better oxidative stress resistance in
transgenic poplar (Noctor et al., 1998b), and higher GSH levels may
even make transgenic plants more sensitive to oxidative stress, as
recently observed in -ECS overexpressing tobacco (Creissen et al.,
1999). Therefore, it is uncertain whether GSH can protect oxidative
damage caused by heavy metals. It is also possible that GSH detoxifies
Cd by directly forming a GSH-Cd complex; a
Cd(GS)2 complex was found in Cd-treated yeast (Li
et al., 1997).
Unlike shoots, roots of -ECS and WT plants had similar Cd
concentrations even though PC levels were higher in roots of -ECS plants than in roots of WT plants. One explanation for this is that
roots have much higher Cd concentrations than shoots (at least 6 to 10 times higher), with most of the Cd being bound to the root cell wall
(Salt et al., 1997). Thus, any increase in Cd sequestration due to
greater PC complexation in the -ECS plants compared with WT may have
been masked by the very high Cd concentrations in roots.
Earlier studies have shown that -ECS is rate limiting for GSH
synthesis in "normal" plants (Noctor et al., 1997, 1998b), i.e.
those not stressed with Cd. Our results support this view because
overexpression of -ECS in unstressed Indian mustard plants led to
increased GSH levels. Under conditions of Cd stress, PC synthase is
activated by Cd (Goldsbrough, 1998). Since overexpression of -ECS
increased -EC, GSH, and PC levels in transgenic seedlings, it would
appear that -ECS is rate limiting for both GSH and PC production in
Cd-stressed plants.
In an earlier paper we concluded that GS was not rate-limiting for GSH
production in unstressed Indian mustard plants, since overexpression of
the GS enzyme did not increase GSH levels (Zhu et al., 1999). This is
consistent with the results presented here, which suggest that -ECS
is rate-limiting for GSH production. Under conditions of Cd stress,
however, the earlier research showed that overexpression of GS led to
increased GSH and PC levels, suggesting that GS was limiting GSH
production under those conditions. We explained this by suggesting that
in Cd-treated plants, Cd activated -ECS so that -EC accumulated
and GS became rate limiting (Zhu et al., 1999). This view was supported
in the present work because Cd treatment increased -EC concentration
50- to 100-fold in shoots of both WT and -ECS seedlings (Fig. 6, A
and B).
Cd-caused accumulation of -EC has also been observed in maize
seedlings (Rauser et al., 1991). The fact that -EC levels were so
high, even in the -ECS seedlings, suggests that GSH production in
the Cd-treated plants was limited by GS. On the other hand, the
observations that GSH and PC levels were increased by overexpression of
-ECS and the Cd-caused increase in Cys levels (both in WT and ECS
plants, Fig. 6, A and B) suggest that -ECS is rate-limiting as well.
Thus, it appears that the two enzymes co-limit GSH production under Cd
stress. The observation that Cys synthesis was stimulated by Cd
treatment and by overexpression of -ECS (see following paragraph)
suggests that Cys synthesis does not rate-limit GSH production under
these conditions.
The elevated levels of total shoot S and total NPTs in the -ECS
plants suggests that overexpression of -ECS results in enhanced S
assimilation. In poplar plants overexpressing the same -ECS construct, up-regulation of Cys synthesis was observed: the
-ECS-overexpressing poplar plants showed 2- to 4-fold increased in
vitro activities of APS reductase and Ser acetyltransferase (Noctor et
al., 1998b). Cys concentrations were also increased 5- to 8-fold by Cd
treatment in both -ECS and WT shoots. In addition to -ECS, Cd may
induce several genes encoding enzymes of the S assimilation pathway, such as ATP sulfurylase and APS reductase (Heiss et al., 1999); the
transcript levels of these two enzymes were also found to be enhanced
by Cd in Indian mustard (Lee and Leustek, 1999). Thus, the S
assimilation pathway may be enhanced by both overexpression of -ECS
and by Cd treatment. Both effects may be mediated by an increase in
-EC synthesis.
In conclusion, this study shows that the -EC enzyme is rate-limiting
for the synthesis of GSH in Cd-stressed and unstressed plants, and that
overexpression of -ECS increased the production of -EC, GSH,
total NPTs, and PCs in Cd-treated plants. We conclude that the greater
Cd tolerance and accumulation in the transgenic plants compared with WT
were due primarily to the increased level of PCs. Thus, overexpression
of -ECS appears to be a promising strategy for enhancing the
efficiency of Cd phytoextraction from polluted soils and wastewater.
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ACKNOWLEDGMENTS |
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We thank Dr. A.C.M. Arisi for providing -ECS antibodies and
Dr. M.H. Zenk for generously providing PC standard references.
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FOOTNOTES |
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Received April 8, 1999; accepted September 4, 1999.
1 This work was supported by the Electric Power Research Institute (grant no. W04163 to N.T.) and a graduate scholarship from the University of California, Berkeley (to Y.Z.).
* Corresponding author; e-mail nterry{at}nature.berkeley.edu; fax 510-642-3510.
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LITERATURE CITED |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
LITERATURE CITED
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-glutamylcysteine synthetase.
Plant Physiol
112: 1071-1078
[Abstract]-GluCys, and glutathione levels in maize seedlings: distribution and translocation in normal and cadmium-exposed plants.
Plant Physiol
97: 128-138
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Y. Li, O. P. Dankher, L. Carreira, A. P. Smith, and R. B. Meagher The Shoot-Specific Expression of {gamma}-Glutamylcysteine Synthetase Directs the Long-Distance Transport of Thiol-Peptides to Roots Conferring Tolerance to Mercury and Arsenic Plant Physiology, May 1, 2006; 141(1): 288 - 298. [Abstract] [Full Text] [PDF]
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S. D. Lindblom, S. Abdel-Ghany, B. R. Hanson, S. Hwang, N. Terry, and E. A. H. Pilon-Smits Constitutive Expression of a High-Affinity Sulfate Transporter in Indian Mustard Affects Metal Tolerance and Accumulation J. Environ. Qual., April 3, 2006; 35(3): 726 - 733. [Abstract] [Full Text] [PDF]
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D.-Y. Kim, L. Bovet, S. Kushnir, E. W. Noh, E. Martinoia, and Y. Lee AtATM3 Is Involved in Heavy Metal Resistance in Arabidopsis Plant Physiology, March 1, 2006; 140(3): 922 - 932. [Abstract] [Full Text] [PDF]
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S. L. Singla-Pareek, S. K. Yadav, A. Pareek, M.K. Reddy, and S.K. Sopory Transgenic Tobacco Overexpressing Glyoxalase Pathway Enzymes Grow and Set Viable Seeds in Zinc-Spiked Soils Plant Physiology, February 1, 2006; 140(2): 613 - 623. [Abstract] [Full Text] [PDF]
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N. Fusco, L. Micheletto, G. Dal Corso, L. Borgato, and A. Furini Identification of cadmium-regulated genes by cDNA-AFLP in the heavy metal accumulator Brassica juncea L. J. Exp. Bot., November 1, 2005; 56(421): 3017 - 3027. [Abstract] [Full Text] [PDF]
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 Y. Li, O. P. Dhankher, L. Carreira, D. Lee, A. Chen, J. I. Schroeder, R. S. Balish, and R. B. Meagher Overexpression of Phytochelatin Synthase in Arabidopsis Leads to Enhanced Arsenic Tolerance and Cadmium Hypersensitivity Plant Cell Physiol., December 15, 2004; 45(12): 1787 - 1797. [Abstract] [Full Text] [PDF]
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-Glutamylcysteine Synthetase
Cd) (A), in shoots of 0.2 mM Cd treated
seedlings (S, +Cd) (B), in roots of 0.2 mM Cd-treated
seedlings (R, +Cd) (C), and total NPTs in shoots or roots of 0.2 mM Cd-treated or unstressed seedlings from experiment B. White bars, Control; hatched bars, ECS2; black bars, ECS4. *,
Significant difference (P < 0.05) from the WT of
the same treatment. FW, Fresh weight.