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INTRODUCTION |
In recent years, contamination of soils and waters by toxic heavy
metals and organic pollutants represents a major environmental and
human health problem. The possibility of the removal of these toxic
heavy metals by phytoremediation technology has increased interest in
the knowledge of the physiological and molecular mechanisms of plant
adaptation to high levels of heavy metals. Several heavy metals such as
cadmium and copper are tolerated to certain low levels through the
synthesis of small peptides named phytochelatins, which form a tight
complex with the metal ion to inactivate and store it in the vacuole
(1).
Phytochelatins are enzymatically synthesized from glutathione as a
response of the plant to toxic levels of the heavy metals that produce
a transient decrease of GSH content (2). Glutathione is synthesized by
a two-step ATP-dependent reaction catalyzed by the
-glutamylcysteine synthetase and glutathione synthetase. These
reactions seem to be highly regulated at the transcriptional and
translational levels, as well as by feedback control of the
-glutamylcysteine synthetase by GSH. In
Arabidopsis, a coordinated response of the glutathione
biosynthesis genes to heavy metals has been reported (3). One of the
factors that also regulates GSH biosynthesis is cysteine availability,
because exogenous addition of cysteine has been shown to increase the
GSH content (4). However, little work has been carried out on the
regulation of cysteine biosynthesis under heavy metal stress or
environmental conditions.
The last step of cysteine biosynthesis is catalyzed by the
O-acetylserine(thiol)lyase enzyme
(OASTL)1 that incorporates
sulfide into the O-acetyl-L-serine molecule. This molecule is produced by an acetylation reaction of
L-serine catalyzed by the serine acetyltransferase
enzyme. Both serine acetyltransferase and OASTL activities have been
demonstrated to be localized in the three cellular compartments
involved in protein synthesis, cytosol, chloroplast, and mitochondrion
(5). However, the contribution of each OASTL and serine
acetyltransferase isoform to a particular metabolic pathway is unknown
(6, 7). Recently, the Atcys-3A gene coding for the cytosolic
OASTL isoform from Arabidopsis has been shown to be
regulated by salt stress and the hormone abscisic acid (8).
In this work, we describe the Cd induction of the cytosolic OASTL from
Arabidopsis thaliana and the involvement of this enzyme in
cadmium tolerance, by overexpression of the corresponding cDNA in
transformed plants.
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EXPERIMENTAL PROCEDURES |
Plant Material, Growth, and Treatments--
Wild-type
A. thaliana (ecotype Columbia) plants were grown on
moist vermiculite supplemented with Hoagland medium at 20 °C in the
light and 18 °C in the dark, under a 16-h white light/8-h dark photoperiod.
Cadmium chloride treatments were performed by addition to the Hoagland
medium of CdCl2 to 50 µM final concentration,
unless otherwise indicated. For the tolerance test, seeds were
surface-sterilized and germinated on solid MS medium with and
without CdCl2 at indicated concentrations, after cold
treatment for 1 day to improve germination. The plants were grown in a
growth chamber under the same conditions described above. For circadian
experiments plants were grown on soil under a 16-h white light/8-h dark
photoperiod for 3 weeks, before harvesting every 4 h for 36 h. After this treatment, plants were shifted to constant light and
harvested every 4 h during 1 day.
In Vivo Growth Measurements--
Seeds were germinated on solid
MS medium for 5 days on vertical Petri dishes and transferred onto
medium containing different concentrations of CdCl2. Plants
were weighed after 6 days of further growth. Total seedling
fresh weight was determined after gently drawing the root out of the
agar medium.
Analytical Determination of Cysteine, Glutathione, and
O-Acetylserine(thiol)lyase Activity--
Cysteine and glutathione were
extracted and subsequently quantified by reverse-phase HPLC after
derivatization with monobromobimane (Molecular Probes) following
the methods described by Rauser et al. (9). Plant tissues
were homogenized in cold extraction buffer (4 ml/g fresh weight)
containing 0.1 N HCl and 1 mM EDTA using a mortar and
pestle with liquid nitrogen. Homogenates were centrifuged at
15,000 × g for 15 min in the cold. Thiols were reduced
at 4 °C for 15 min by mixing 400 µl of extracted samples with 600 µl of 200 mM CHES (pH 9.2) and 100 µl of 250 mM NaBH4. A 330-µl aliquot was derivatized in
the dark for 15 min by adding 20 µl of 15 mM
monobromobimane. The reaction was stopped by adding 250 µl of 0.25%
(v/v) methanesulfonic acid at room temperature. Derivatized thiols were
separated and quantified by reverse-phase HPLC (10).
O-Acetylserine(thiol)lyase activity was measured in
crude extracts from wild-type or transformed Arabidopsis
plants following a previously described method (11).
Cadmium Analysis--
Three-week-old plants were
irrigated for 14 days with Hoagland medium supplemented with 250 µM CdCl2, final concentration. The leaves
were collected and dried at 65 °C during 5 h. The dried material was wet-ashed in HNO3-HClO4
(7:1) (v/v) at 65 °C until the volume of the solution was reduced
from 20 to 2 ml. Finally, the samples were analyzed by inductively
coupled plasma atomic emission spectrometry using a Fisons-ARL 3410 sequential multielement instrument.
RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated from leaves, and Northern blot analysis was performed as
previously described (11). The blots were sequentially hybridized with
a gene-specific Atcys-3A probe and then with maize 17 S DNA
for detection of Arabidopsis 18 S rRNA (12). Hybridization
with CCA1 gene probe was used as a positive control of plant
circadian oscillator (13). For quantifying mRNA levels,
radioactivity distributed on the blots was imaged and determined using
an InstantImager electronic autoradiographer (Packard Instrument
Co.). The level of Atcys-3A mRNA was normalized to the level of the 18 S rRNA obtained for each sample.
In Situ Hybridizations--
Probes for in situ
hybridization were labeled with uridine
5'-[
-thio][35S]triphosphate. The Atcys-3A
cDNA insert (11) subcloned into pBluescriptII KS was
amplified by polymerase chain reaction using standard M13 reverse and
forward primers. About 1 µg of the polymerase chain reaction product
was used as template to synthesize 35S-labeled RNA using T7
RNA polymerase (antisense probe) or T3 RNA polymerase (sense probe).
Mature Arabidopsis plants grown under standard conditions or
treated with cadmium chloride (50 µM) for 18 h were
used for in situ hybridization. Leaves, stems, and roots
were cut into small pieces, fixed in 4% formaldehyde, embedded in wax,
and sections were processed and hybridized under conditions as
described previously (12). For autoradiography, slides were coated with
Amersham Pharmacia Biotech Hypercoat LM-1 nuclear emulsion and
exposed for 11 days at 4 °C. The samples were then developed in
Eastman Kodak Co. D19 developer prechilled at 14 °C and were fixed
in 30% sodium thiosulfate for 5 min. After developing, the tissues were stained with 0.05% toluidine blue in water for 0.5 min, rinsed, dehydrated, and air-dried.
For purposes of comparison, the tissue sections from control and
stressed plants were fixed onto a single glass slide and hybridized
with the same labeled probe. Tissue sections were observed in an
Olympus BX50 microscope attached to a JVC TKC-1381 digital CCD
color video camera for image capture. Images were processed and mounted
by using the Olympus MicroImage analysis program and Adobe Photoshop
software, respectively.
Arabidopsis Plant Transformation--
For plant transformation,
the
-glucuronidase gene in the binary vector pBI121
(CLONTECH), under the control of the cauliflower mosaic virus 35 S promoter, was replaced by the full-length
Atcys-3A cDNA (11). A 260-base pair fragment containing
the polyadenylation signal from the nopaline synthase gene of the
Agrobacterium Ti plasmid was placed downstream of the
Atcys-3A gene. The resulting plasmid, named pBIOAS, was
transformed into Agrobacterium tumefaciens strain CV50.
A. thaliana (ecotype Columbia) was transformed by using the
vacuum infiltration method described by Bechtold et al.
(14). Transformed seeds were tested for kanamycin resistance on solid
MS medium containing 50 mg l
1 kanamycin. Integration into
the nuclear genome of the plant was analyzed by genomic Southern blot
analysis. T5 or subsequent generations of each line were
used for the experiments.
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RESULTS |
Effect of Cadmium Treatment on Atcys-3A Transcript
Abundance--
Northern analysis showed a 7-fold induction of
Atcys-3A transcript level in leaves in response to cadmium
chloride treatment when compared with nontreated plants (Fig.
1, A and B).
Although the maximum level of induction was observed 18 h after
treatment, a significant increase in RNA accumulation after 1 h
was observed. Concomitant with the increasing mRNA levels, OASTL
enzyme activity increased by 2.5-fold after 18 h of exposure to
metal (Fig. 1C). The time course of changes in
Atcys-3A transcript and OASTL enzyme activity levels could
reflect a diurnal rhythm of this gene. To check this possibility,
circadian experiments were performed on plants not exposed to Cd, using
as a positive control the CCA1 (circadian
clock-associated 1) gene (13). We
were unable to observe any fluctuation of Atcys-3A gene
expression over a 60-h period, suggesting that the pattern of induction
of Atcys-3A mRNA is specific to the heavy metal
treatment (data not shown).

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Fig. 1.
Regulation of the cytosolic
O-acetylserine(thiol)lyase transcript abundance and
enzyme activity in leaves of cadmium-treated Arabidopsis
plants. CdCl2 (50 µM) was added to
the irrigation medium of 3 week-old Arabidopsis plants at
the indicated times. Atcys-3A mRNA accumulation in leaf
extracts was determined by Northern blot analysis. The experiment was
repeated three times, and a representative hybridized blot is shown
(A). The level of Atcys-3A mRNA and 18 S rRNA
was quantified for the representative hybridized blot shown in
A as indicated under "Experimental Procedures," and the
level of Atcys-3A was normalized to the level of the 18 S
rRNA obtained for each sample (B).
O-acetylserine(thiol)lyase activity. The values are
averages of at least three independent experiments, and the error
bars represent the standard deviations. The symbol * indicates
significant differences (p < 0.05) from the data at
t = 0 (C).
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To gain insight into this response, the effect of cadmium chloride on
Atcys-3A expression was investigated at the tissue level by
in situ hybridization. In roots, the transcript of the
O-acetylserine(thiol)lyase Atcys-3A gene was
localized in the cortex, but the amount of detected signal was higher
in cadmium-treated plants as compared with nontreated plants (Fig.
2, C and D). In
stem sections, the level of expression of Atcys-3A was
almost undetectable above background in control plants (Fig. 2,
E and F). However, it was possible to detect
signal in the cortex and the vascular tissue of cadmium-stressed plants (Fig. 2, G and H). In leaf, the basal level of
expression observed in all cell types was increased throughout the leaf
lamina after cadmium treatment (Fig. 2, L-O). Although the
in situ hybridization technique is not a quantitative
method, for purposes of comparison we hybridized the tissue sections on
the same slide and therefore processed them under the same conditions.
The high level of Atcys-3A mRNA detected in trichomes of
untreated plants (12, 15) was maintained or even increased in
cadmium-treated Arabidopsis (data not shown).

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Fig. 2.
Effect of heavy metal on the
expression of the cytosolic O-acetylserine(thiol)lyase
gene in Arabidopsis tissues. Transverse sections
(10 µm) of A. thaliana leaves were hybridized in
situ with 35S-labeled antisense (A-O) or
sense (P-R) RNA probes. A and B,
bright- and dark-field micrographs, respectively, of untreated roots,
hybridized with antisense Atcys-3A 35S-RNA.
C and D, bright- and dark-field micrographs,
respectively, of roots treated with 50 µM
CdCl2 for 18 h, hybridized with antisense
Atcys-3A 35S-RNA. E and F,
bright- and dark-field micrographs, respectively, of untreated stems,
hybridized with antisense Atcys-3A 35S-RNA.
G and H, bright- and dark-field micrographs,
respectively, of stems treated with 50 µM
CdCl2 for 18 h, hybridized with antisense
Atcys-3A 35S-RNA. I and K, bright-
and dark-field micrographs, respectively, of untreated leaf, hybridized
with antisense Atcys-3A 35S-RNA. L
and M, bright- and dark-field micrographs, respectively, of
leaf treated with 50 µM CdCl2 for 18 h,
hybridized with antisense Atcys-3A 35S-RNA.
N and O, magnification (× 20) of the images in
L and M, respectively. P and
R, bright- and dark-field micrographs, respectively, of
untreated leaf, hybridized with sense Atcys-3A
35S-RNA.
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Effect of Cadmium Treatment on Cysteine and Glutathione Content in
Leaf Tissues--
Other workers have demonstrated that cadmium induces
the biosynthesis of phytochelatin peptides from GSH. To investigate
whether cadmium treatment induces the biosynthesis of the precursor
molecules, cysteine and GSH, we determined by HPLC analysis the content
of both compounds in leaf tissues from cadmium-treated
Arabidopsis. It was clearly observed that the GSH content
increased about 2-fold after 18 h of treatment with 50 µM cadmium and dropped to the basal level within 24 h of treatment. However, the level of cysteine was not significantly
increased. Similarly to the mRNA accumulation, a rapid
increase that peaks after 1 h was also observed (Table I). However, after exposure of higher
concentrations of cadmium for 24 h, a depletion of the cysteine
and GSH levels was observed. At 100 µM Cd, the level of
cysteine declined 35% with respect to untreated plants, and a decrease
of 30% was observed in the GSH level. Larger decreases in either
cysteine (43% depletion) or GSH (34% depletion) levels were detected
at the highest concentration of 500 µM Cd used (data not
shown). These results are in concordance with the behavior of the GSH
content upon Cd treatment of Arabidopsis also observed by
other authors (3). Circadian fluctuation of cysteine or GSH levels
(without Cd treatment) was not observed (data not shown).
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Table I
Cysteine and glutathione content upon cadmium treatment
Arabidopsis plants were incubated with 50 µM
CdCl2 for the indicated times, and the leaves were collected
for cysteine and GSH determination as described under "Experimental
Procedures." Values represent nmol/g fresh weight of leaf, and means
and standard deviations of at least four replicate experiments are
shown. The symbol * indicates significant differences
(p < 0.05) from the data at t = 0.
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Cadmium Tolerance in Transformed Arabidopsis Plants Overexpressing
the Atcys-3A Gene--
We have produced
Arabidopsis-transformed plants overexpressing the
Atcys-3A gene via Agrobacterium-mediated
transformation. Full-length Atcys-3A cDNA was fused in
sense orientation to the cauliflower mosaic virus 35 S promoter to
obtain constitutive expression of the gene. Six independent transformed
lines, tested by Southern blot, were used for further analysis.
Northern blot analysis of the transformed plants showed up to a 9-fold
increase in Atcys-3A mRNA accumulation in the pBIOAS-10
line compared with control-transformed plants expressing the pBI121
plasmid (lines pBI121-C1, -C3, -C5, and -C7) (Fig.
3A). The transformed plants also showed a concomitant increase in OASTL activity in leaf extracts (Fig. 3B).

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Fig. 3.
O-Acetylserine(thiol)lyase
transcript abundance, enzyme activity, and cadmium tolerance of
Arabidopsis-transformed plants expressing the
Atcys-3A gene. A, Northern blot
analysis of control pBI121 (lines C1, C3,
C5, and C7) and transformed pBIOAS (lines
1, 2, 3, 4, 5, and
10) plants. Total RNA (30 µg) isolated from leaf was
hybridized with a gene-specific probe for Atcys-3A. As
control of the RNA loading, ethidium bromide (EtBr) staining
of the gel is also shown. B,
O-acetylserine(thiol)lyase activity in leaves of 3 week-old
control pBI121 and transformed Atcys-3A Arabidopsis plants.
The values are averages of at least three independent experiments, and
the error bars represent standard deviations. The symbol *
indicates significant differences (p < 0.05) from the
C1 control plants. C, cadmium tolerance tests of seedling
from wild-type (w-t; a), control pBI121 line C1
(b), and transformed pBIOAS lines 1 (c), 2 (d), 4 (e), and 10 (f) grown on solid
MS media containing 250 µM CdCl2 for 15 days.
Seedlings were visualized with an Olympus SZ4045TR stereo
microscope.
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We have tested the effect of the cadmium ion on the transformed plants
by growing seeds on solid MS medium containing 250 µM
CdCl2. The transformed lines showing the higher levels of
overexpression of the Atcys-3A gene were able to germinate
and grow on this medium (Fig. 3C). Wild-type or transformed
pBI121-C1 control plants germinate poorly in the presence of the metal,
and those plants that did develop cotyledons did not produce leaves and
died after 5-7 days (Fig. 3C).
The high cadmium resistance shown by the transformed lines
overexpressing Atcys-3A could suggest that cysteine
availability is a main factor for tolerance. In this sense, we
investigated whether exogenous addition of cysteine to pBI121 control
seedlings is sufficient to mimic the cadmium resistance observed in the Atcys-3A-overexpressing transformed seedlings. To avoid
reaction in the medium between the heavy metal and the thiol group of
cysteine, we used exogenous addition of cystine, which lacks the free
thiol group. Addition of 200 µM cystine to control plants
was sufficient to support their growth in the presence of 250 µM cadmium, although not to the extent observed in the
transformed lines (Fig. 4).

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Fig. 4.
Effect of cystine feeding on cadmium
tolerance. Seeds from control pBI121 lines C1 (A and
E), C3 (B and F), C5 (C and
G), and C7 (D and H) were plated on
solid MS media containing 250 µM CdCl2 in the
presence (A-D) or absence (E-H) of 200 µM cystine. Seedlings were visualized after 15 days of
growth with an Olympus SZ4045TR stereo microscope.
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Cadmium analysis by atomic emission spectrometry of mature transformed
Atcys-3A plants growing for 14 days on cadmium-containing media showed an increase of cadmium accumulation in leaves
compared with control plants (Table II).
Cadmium accumulation in different lines correlates with mRNA and
OASTL activity levels, with line pBIOAS-10 having the highest level of
cadmium accumulation, a 72% higher cadmium concentration than control
plants.
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Table II
Cadmium concentration in leaves of transformed Arabidopsis lines
overexpressing the Atcys 3A gene
Mature Arabidopsis plants were incubated with 250 µM CdCl2 for 14 days, and leaves were then
collected from different pots of the same plant line for Cd
determination, as described under "Experimental Procedures."
Values ± S.E. are shown from five independent measurements. The
symbol * indicates significant differences (p < 0.05)
from the control plants. DW, dried weight.
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Characterization of the Cadmium Tolerance of Transformed pBIOAS
Line 10--
Two different Cd resistance tests were performed. In the
first test, transformed seeds were germinated in the presence of a
range of Cd concentrations and compared with wild-type. Transformed seeds were able to germinate in the presence of CdCl2 up to
400 µM, producing green leaves. By contrast, wild-type
seedlings either failed to germinate or germinated but were unable to
produce leaves, at 250 µM CdCl2 and bleached
white and died at a cadmium concentration of 400 µM (Fig.
5A). In the other test, we
studied the inhibition of growth of wild-type and line pBIOAS-10
seedlings upon Cd treatment. pBIOAS-10 was able to survive over 300 µM concentration, whereas wild-type died. Dead seedlings
showed a fresh weight of around 2 mg (Fig. 5B).

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Fig. 5.
Tolerance of transformed pBIOAS line 10 to
cadmium. A, sensitivity of wild-type and transformed
pBIOAS line 10 seedlings to Cd. Seeds were germinated on solid MS media
containing different concentrations of CdCl2 as indicated
and were grown for 15 days. Seedlings were then visualized with an
Olympus SZ4045TR stereo microscope. B, growth of wild-type
and transformed pBIOAS line 10 seedlings upon Cd treatment.
Five-day-old seedlings germinated on Cd-free MS medium were
transferred to medium containing CdCl2 at the indicated
concentrations. Total seedling fresh weights were determined after an
additional 6 days of growth. Each data point represents the mean of
five independent measurements, in which ten seedlings were weighed.
Error bars represent the S.D. Curve fitting showing the
tendency of the data (dashed lines, wild-type in
red and line 10 in blue) was calculated by
nonlinear regression using the program Microsoft Excel 98.
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The glutathione level was also determined in the transformed pBIOAS
line 10 and compared with the level in wild-type plants. In the absence
of cadmium, the concentrations of GSH in the transformed line and
wild-type were similar. However, treatment with 250 µM CdCl2 for 18 h produced opposite effects in wild-type
and the transformed line, a 30% decrease of the GSH content in
wild-type, and an increase of 54% in the line 10 (Fig.
6).

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Fig. 6.
Glutathione content in wild-type and
transformed pBIOAS line 10 leaves upon cadmium treatment. Mature
plants were incubated in the absence ( Cd) or the presence
of 250 µM CdCl2 (+Cd) for 18 h, and leaves were collected for GSH determination. Error
bars represent the standard deviations. The symbol * indicates
significant differences (p < 0.05) from data of the
same plant in absence of Cd.
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DISCUSSION |
Heavy metals such as cadmium, mercury, lead, and arsenic are
highly reactive with sulfydryl groups and can affect a vast array of
biochemical processes. It is now well established that the thiolate
peptides, the phytochelatins, play an essential role in the
detoxification of cadmium and some other heavy metals. Thus, the
Arabidopsis cad1 mutant deficient in PC synthase is highly
sensitive to cadmium ion (16). Arabidopsis plants treated with heavy metals such as cadmium and copper respond by increasing transcription of genes involved in GSH synthesis and reduction (gsh1, gsh2, and gr1) (3).
Furthermore, increased levels of
-glutamylcysteine synthetase and
glutathione synthetase have also been observed in cadmium-treated
species such as maize (17), tobacco, and tomato cells (18, 19) and
Brassica juncea (20). Because phytochelatin synthesis
produces a detectable depletion in GSH content (3), it is expected that
the GSH precursor thiol molecule, cysteine, has to be produced at
higher rates to support GSH biosynthesis under cadmium treatment.
The data reported here clearly show a transient increase of the
O-acetylserine(thiol)lyase upon cadmium treatment, and this effect is observed at the transcriptional and enzymatic level. Although
the increase in OASTL activity is only 2.5-fold, we have to point out
that these measurements represent the sum of the three OASTL isoforms
identified in the cell. In situ hybridization data also show
that the transcript is significantly induced in the whole leaf lamina
and in the cortex and the vascular tissue of root and stem. In B. juncea, coordinate changes of expression for several sulfur
assimilation enzymes, including OASTL, have been observed in response
to cadmium treatment (20, 21). All these data support the idea that
plants respond to cadmium toxicity by inducing the genes required for
phytochelatin synthesis, the genes involved in cysteine, and in
glutathione biosynthesis (3).
In addition, because cadmium and other heavy metals are very reactive
with thiol molecules, the reduced availability of free cysteine for GSH
biosynthesis can significantly reduce and limit the amount of
phytochelatin synthesis. Addition of exogenous cystine supports plant
growth in cadmium-containing media and suggests an important role for
the cysteine molecule. In fact, in transformed plants overexpressing
the Atcys-3A gene, the tolerance to cadmium ion also
increases, up to 400 µM CdCl2, in the case of
the line pBIOAS-10. By increasing the OASTL mRNA and enzyme level,
the cysteine synthesis machinery within the transformed plants seems to
be able to supply the required precursor for GSH synthesis and
therefore for PC synthesis. Although the level of
O-acetylserine, the precursor of cysteine, has been
considered to be a limiting step for cysteine biosynthesis, we have not
observed differences in metal tolerance by adding
O-acetylserine to the MS media (data not shown). The effect
of induction of cysteine biosynthesis in Cd resistance can be clearly
observed in the transformed line pBIOAS-10 where high levels of OASTL
mRNA and catalytic activity correlate with a higher capacity for Cd
accumulation. Characterization of this transformed line has shown an
enhanced cadmium tolerance when compared with wild-type, probably
because of its capacity to respond to the Cd treatment with a
dramatic increase of GSH biosynthesis.
The manipulation of O-acetylserine(thiol)lyase gene
expression and, therefore, cysteine biosynthesis in transformed plants enhances the capacity of plants for toxic metal sequestration, which
may be useful for phytoremediation of heavy metal-contaminated environments. Overexpression of PC synthase in yeast leads to increased
tolerance to heavy metals, but no data have been reported about
tolerance in plants (22, 23). It will be interesting to determine
whether manipulation of the PC synthase in Arabidopsis is
sufficient to support metal tolerance or whether its precursor, cysteine, is the limiting step in the process. However, the limiting step could be different between species; thus in B. juncea
GSH biosynthesis and not cysteine availability seems to be the limiting step, although an increase in OASTL expression is also observed on
cadmium treatment (20). Overexpression of
-glutamylcysteine synthetase in this species enhances cadmium tolerance to the same level
(24) as that reported here. To conclude, in Arabidopsis, cysteine availability seems to be one of the limiting steps for GSH
and, therefore, for PC biosynthesis.