Chemosphere 76 (2009) 623–630
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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Enhancement of Cd tolerance in transgenic tobacco plants overexpressing a
Cd-induced catalase cDNA
ZiQiu Guan a, TuanYao Chai a,*, YuXiu Zhang b, Jin Xu a, Wei Wei a
a
College of Life Science, Graduate University of Chinese Academy of Sciences, Yuquan Rd. 19A, Beijing 100049, China
Department of Biological Engineering, School of Chemical and Environmental Engineering, China University of Mining and Technology (Beijing),
Xueyuan Rd. 11, Beijing 100083, China
b
a r t i c l e
i n f o
Article history:
Received 19 October 2008
Received in revised form 21 April 2009
Accepted 21 April 2009
Available online 26 May 2009
Keywords:
Cadmium
Catalase
Oxidative stress
Transgenic
Tolerance
Cell death
a b s t r a c t
Catalase (CAT), an important enzyme of antioxidant system, was investigated the role in preventing the
plant from Cd-induced oxidative stress caused by reactive oxygen species. A CAT gene from Brassica juncea was cloned and up-regulated in response to Cd/Zn. The CAT cDNA (BjCAT3) under the control of
CaMV35S promoter was introduced into tobacco via Agrobacterium-mediated transformation. Northern
blot analysis verified the BjCAT3 was expressed at high level in different transgenic lines. In morphological observation, we found that seedlings from transgenic tobacco plants grew better and showed longer
root length in the presence of Cd versus wild-type (WT) seedlings. Under 100 lM Cd stress, WT plants
became chlorotic and almost dead while transgenic tobacco plants still remained green and phenotypically normal. The CAT activity of transgenic T1 generations was approximately two-fold higher than that
of WT plants. In WT, endogenous CAT activity is rapidly reduced as a result of 200 lM CdCl2 exposure.
Compared with WT plants, lower level of Cd-induced H2O2 accumulation and cell death were detected
in roots of transgenic plants with high level of CAT activity. All our findings strongly support that overexpressing BjCAT3 in tobacco could enhance the tolerance under Cd stress.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Heavy metal contaminants and metalloids such as Cd, Pb, Hg, As
and Se are increasing environmental problems worldwide (Zhu
et al., 1999). Using hyperaccumulators-plant species that accumulate extremely high concentrations of heavy metals in their shoots
to actively absorb the metal pollutants from environment is known
as ‘‘phytoremediation” (Krämer, 2005). Phytoremediation as an
emerging technology has received great attention for a long time.
Moreover, transgenic plants exhibiting new or improved phenotypes are engineered by the overexpression and/or introduction
of genes from other species, and has shown promising potential
in environment recovery, such as Cd pollutant removal (Van Aken,
2008). Therefore, development of transgenic plants with higher level of metal accumulation and tolerance tailored for remediation
will further enhance feasibility of phytoremediation (Eapen et al.,
2007).
Cd, being a highly toxic metal pollutant for human, animals and
plants, enters into the environment mainly from industrial processes and phosphate fertilizers, and can be accumulated by crops
and eventually transferred into the food chain (Wagner, 1993; Maier et al., 2003). People believed that the toxicological mechanism
* Corresponding author. Tel./fax: +86 10 88256343.
E-mail address: [email protected] (T. Chai).
0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2009.04.047
of Cd closely correlates to the generation of reactive oxygen species
(ROS) in vivo, including H2O2, O
2 and OH . The severe damages of
ROS to the plants include the disturbance of the antioxidative control of the cell, growth inhibition, stimulation of secondary metabolism, cell wall rigidification and lignification, decreasing cellular
viability and even cell death (Halliwell and Gutteridge, 2000; del
Rio et al., 2002; Schützendübel and Polle, 2002; Gratão et al.,
2005). It is currently hypothesized that lipid peroxidation and
accumulation of H2O2 are early symptoms of Cd injury, and that
H2O2 is involved in triggering secondary defenses. To fight against
over-production and accumulation of ROS, plants have evolved a
complex antioxidant enzymatic system. Major ROS-scavenging enzymes of plants are mainly composed of superoxide superoxide
dismutase (SOD), catalase (CAT), peroxydase (POD), and several
NADPH-oxidases (Rodriguez-Serrano et al., 2006; Ortega-Villasante et al., 2007). The cooperative effects between them, together
with sequestering metal ions, are thought to be important to prevent the formation of the highly toxic hydroxyl radical via the metal-dependent Haber–Weiss or the Fenton reactions. However,
once ROS accumulation exceeds the decomposing capability of
these antioxidant enzymes, the imbalance will eventually lead to
the unspecific oxidation of proteins, membrane lipids and DNA
damage (Cho and Park, 2000; Ortega-Villasante et al., 2007). Many
reports indicate that CAT, which decomposes H2O2 into molecular
oxygen and water without the production of free radicals, may play
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Z. Guan et al. / Chemosphere 76 (2009) 623–630
a critical role in plant oxidative defense mechanisms (Mittler,
2002; Talarczyk et al., 2002; Luna et al., 2005).
We previously cloned and characterized a cDNA fragment in a
hyperaccumulator species, Brassica juncea, under Cd treatment
(Lang et al., 2005). Analysis of gene sequence revealed that the partial cDNA showed high similarity (90%) to the CAT genes from B.
juncea. We hypothesize that strengthen of antioxidant system
should enhance the plant tolerance to Cd-induced oxidative stress.
Based on the metabolic role of CAT as a H2O2 scavenger, an Agrobacterium-mediated transgenic strategy was chosen to prove the
importance of CAT under Cd-stress. The Cd tolerances were compared between WT (wild type) and transgenic tobacco plants. We
found that overexpression of BjCAT3 in tobacco could increase
resistance to Cd toxicity since high level of CAT activity could
promptly scavenge Cd-induced excessive ROS in time.
2. Materials and methods
2.1. Gene cloning
To isolate the CAT cDNA, we designed the primes for rapid
5amplification
of
cDNA
ends
(RACE)
(30 -gsp:
ATCCAGACAATGGATCCTGCTGAT-3) based on the partial cDNA
fragments which had been obtained by mRNA differential display
technique. The 30 RACE-polymerase chain reaction (PCR) were performed according to the protocol of the SMARTTM RACE cDNA
Amplification Kit (Clontech, USA). The cDNA fragments were
amplified using these primers from poly (A)+ RNA isolated from
leaves of B. juncea treated with Cd. The nested PCR products were
purified and cloned into pGEM-T vector (Promega, USA) followed
by sequencing.
2.2. Gene expression levels under Zn and Cd via Northern blotting and
RT-PCR analyses
B. juncea seedlings were treated with 500 lM ZnCl2 and 200 lM
CdCl2, respectively, and for Cd treatment, the seedlings were set to
different periods of time (0, 2, 6, 12, and 24 h) at greenhouse. After
the treatment of heavy metals, total mRNA was extracted from the
leaves of treated and control B. juncea plants using the Rneasy
Plant Mini Kit according to manufacturer’s instructions (QIAGEN
Company, Germany), and was treated with RNase-free DNase I (final concentration: 0.08 U lL1) to remove DNA contamination.
Northern blot analysis was performed using 10 lg of total RNA
per track. Total RNA was separated on 1.2% denaturing agarose gel
and then was transferred onto a nylon membrane (Roche Company, Switzerland). The membrane was hybridized with digoxigenin (DIG)-labeled CAT cDNA fragments and detected according
manufacturer’s instructions of DIG High Prime DNA Labeling and
Detection Starter Kit II (Roche Company, Switzerland).
For RT-PCR (reverse transcription–PCR) analysis, the concentration of mRNA was accurately quantified by spectrophotometric
measurements and cDNA were synthesized from DNase-treated total RNA with RT System Kit (Promega, USA). Control reactions with
the actin primers were performed to ensure that equal amounts of
RNA were used in each set of reactions.
2.3. Plasmid constructs and plant transformation
The full-length CAT cDNA (BjCAT3) was amplified using the
primers (BjCAT3-up 50 -GGCTCTAGAATGGACCCTTACAAGT-30 and
BjCAT3-dn 50 -ATAGGATCCGATGCTTGGTCTCAC-30 ) with an XbaI site
at the 50 end and a BamHI site at the 30 end (underlined above). The
XbaI/BamHI fragment from BjCAT3 was cloned into the binary vector pBI121. The recombinant plasmid driven by the enhanced
CaMV 35S promoter contains GUS as a reporter gene and the nptII
gene as a selectable maker, which confers the kanamycin resistance. The plasmid was transformed into Agrobacterium tumefaciens strain EHA105 for plant transformation.
Leaf disks from NC89 plants were transformed and the kanamycin-selected plants were regenerated on MS (Murashige and Skoog
Stock) medium by standard methods. The transgenic plantlets
were regenerated on MS medium supplemented with 1 mg L1 6a-naphthaleneacetic acid,
benzylaminopurine,
0.2 mg L1
100 mg L1 kanamycin, and 250 mg L1 carbenicillin, and then
were transferred to a rooting medium (MS medium containing
50 mg L1 kanamycin).
2.4. Confirmation of the presence of BjCAT3 in transgenic tobacco
plants
For PCR analysis, total genomic DNA was isolated from leaves of
WT and transgenic tobacco plants using the cetyltrimethylammonium bromide method. The primary transgenic plants lines (T0)
were identified by PCR analysis followed by sequencing and then
self pollinated to produce T1 seeds. T-DNA inheritance was scored
by kanamycin segregation analysis in the T1 generation. The surface-sterilized seeds were germinated in MS agar medium containing 100 mg L1 kanamycin. Segregation of the germinated progeny
was scored 2 wk after formed. An <chi> calculation according to
the hypothesis ratio (3:1) was performed.
For Northern blot analysis, total RNA were extracted from WT
and three transgenic lines (CAT2, CAT3 and CAT4) and the hybridization process was performed according above method.
2.5. Morphology of WT and transgenic tobacco plants under Cd
exposure
After germination, seedlings of WT and transgenic tobacco
plants were transferred into MS agar medium containing 50 lM
CdCl2 and 1 wk later, the root inhibition under Cd exposure were
compared. The seedlings of WT and transgenic tobacco plants
without treatment grow for 1 wk were put in MS agar medium
containing different concentrations of CdCl2 (0, 50, and 100 lM)
and the growth situation of seedlings were compared after 2 wk.
2.6. H2O2, malondialdehyde and antioxidant enzyme activities
For determination of H2O2, fresh sample of leave (0.5 g) was
homogenized in 5.0 mL of ice-cold acetone and calculated according to the method described in literature. The amount of H2O2 was
extrapolated using a calibration curve utilizing the 0.1–100 nM
range of H2O2 (30%) standard. Lipid peroxidation was measured
using thiobarbituric acid assay, in which MDA (malondialdehyde)
was quantified as an end product.
Leave tissues of plants were homogenized in a chilled mortar
and pestle with 4 mL of ice-cold extraction buffer and centrifuged
at 15,000 rpm for 30 min at 4 °C. Protein estimation was carried
out using bovine serum albumin at standard. CAT (EC 1.11.1.6)
activity was measured by monitoring the decrease in absorbance
at 240 nm as a consequence of H2O2 consumption and expressed
as amount of H2O2 decomposed per min per mg of protein as reported. POD (EC 1.11.1.7) activity was determined according to
the method using guaiacol as substrate. And the SOD (EC
1.15.1.1) activity was assayed using the photochemical q-nitro
blue tetrazolium chloride method.
2.7. Cd-induced H2O2 localization in situ of tobacco roots
Roots of WT and transgenic tobacco plants treated with 100 lM
Cd for 4 h were excised and immersed in a 1% solution of DAB (3,30 -
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Z. Guan et al. / Chemosphere 76 (2009) 623–630
diaminobenzidine) in water (pH 3.8), and then incubated at room
temperature for 8 h in the absence of light. Roots were washed
by distilled water for at least three times and then bleached by
immersing in the mixture solution with 20% acetic acid, 20% glycerol and 60% ethanol, boiling for 10 min to visualize the brown
spots. Then the samples were fixed using 60% glycerol and examined under the microscope (Olympus IX71, Japan).
(a)
2.8. Detection of cell death in WT and transgenic tobacco roots
(b)
Cell viability was approximated by trypan blue dye exclusion
assay. After treatment with 100 lM Cd for 0, 4, 8, and 48 h, roots
of WT and transgenic plants were immersed into 0.3% trypan blue
solution. 20 min later, the samples were repeatedly washed with
distilled water at least for three times and then immersed into distilled water for 1 h to eliminate the excess dye before microscopy
observation (Olympus IX71, Japan).
Apoptosis and necrosis cell death were determined by fluorescent staining of the nuclei of roots cells of WT and transgenic
plants using combined staining with the chromatin dye, Hoechst
33342, and propidium iodide (PI) (apoptosis and necrosis assay
kit). Hoechst freely passes cells membranes and stains nuclear
DNA blue. The condensed chromatin of apoptotic cells stains more
brightly than the chromatin of normal cells. For necrotic cells, PI,
which is cell membrane impermeable, can give positive staining
with red color. Roots samples treated with 100 lM Cd for 12 h
were examined according the kit manufacturer’s instructions.
Dual-stained cells were examined using microscope (Olympus
BX51, Japan).
2.9. Cd content in aboveground tissues of transgenic tobacco plants
After cultured in 1/2 Hoagland nutrient solution for 4 wk, WT
and transgenic tobacco plants were transferred into fresh nutrient
solution containing different concentration (20, 100 and 200 lM)
of CdCl2 for 3 and 6 d, respectively. Four replicates for each treatment were prepared to give a total of 32 pots. For Cd determination, Inductively Coupled Plasma Mass Spectroscopy (ICP-MS)
technology was used following standard methods.
C
2+
C
BjCAT3
BjCAT3
actin
actin
0
2
6
12
Zn
24h
actin
Fig. 1. Effects of Zn/Cd treatments on BjCAT3 transcript levels. (a) Northern blot
analysis with BjCAT3 probes was performed on RNA samples obtained from B.
juncea leaves under 500 lM ZnCl2 and 200 lM CdCl2 exposure for 12 h. Ten
micrograms of total RNA from non-treated and treated B. juncea leaves after
incubated with Zn/Cd, respectively, were used in Northern blot analysis (C, control
non-treated; Cd2+, Cd-treated; Zn2+ and Zn-treated). RNA loading of each sample
was verified by hybridization with the cDNA for the actin. Expression of BjCAT3 was
up-regulated by 500 lM ZnCl2 and 200 lM CdCl2 for 12 h, respectively. (b) Reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis of Cd-induced effects on
BjCAT3 transcripts level over time (0–24 h). Top panels show the changes in
transcript level of BjCAT3 relative to the actin gene (loading control).
transgenic plants and plasmid (positive control), but not in WT
plants (Fig. 2a). The integrations of the BjCAT3 into the genome
of transgenic tobacco plants were further verified by Southern blot
analysis. Restriction digestion of genomic DNA with HindIII resulted in the appearance of bands at different positions in the different transgenic lines, suggesting that these lines were produced
(a)
CK
WT
CAT1 CAT2 CAT3 CAT4 CAT5
(b)
3.1. Up-regulation of BjCAT3 in response to Zn/Cd in B. juncea
The transcript level of the BjCAT3 in response to Zn/Cd was analyzed (Fig. 1a). Northern blot analysis showed the expression of
BjCAT3 was up-regulated by Zn and Cd. To determine whether
the BjCAT3 transcriptional level would be influenced over time by
Cd exposure, expression of BjCAT3 was measured in leaves of B.
juncea after Cd-exposure from 0 to 24 h. The level of BjCAT3 transcripts was increased within 2 h of Cd treatment. This increase
was sustained from 2 to 12 h, but it decreased if we extended
the exposure time to 24 h (Fig. 1b). The result confirmed that
BjCAT3 was up-regulated in leaves in the presence of Cd, which is
in agreement with the result from other independent research
groups, the CAT expression level showed a time and/or dose
dependent manner of Cd exposure (Banjerdkij et al., 2005; Azpilicueta et al., 2007).
3.2. Molecular characterization of the transgenic tobacco plants
Five independent lines were produced by introducing the
expression cassette 35S/CAT/GUS/30 NOS into tobacco leaf disk
using Agrobacterium-mediated transformation. With BjCAT3 as
primers, the expected bands (1.5 kb for BjCAT3) were found in
2+
BjCAT3
M
3. Results
Cd
9.4kb
6.5kb
4.3kb
CAT1 CAT2 CAT3 CAT4 CAT5
(c)
WT
CAT2
CAT3
M
CAT4
BjCAT3
RNA
Fig. 2. Molecular confirmation of the transgenic tobacco plants. (a) Polymerase
chain reaction (PCR) amplification of BjCAT3 gene (1.5 kb) in the leaves from five
independent putative transgenic plants (C, control plasmid; WT wild-type plant).
(b) Southern blot analysis of digested transgenic tobacco genomic DNA hybridized
with BjCAT3 gene probe. Genomic DNA was digested with HindIII and the
digoxigenin (DIG)-labeled BjCAT3 probe was prepared by PCR of plasmid DNA.
Different sizes and locations of the signals suggested that these lined were
produced from different transformation events. The size of molecular weight
markers are indicated on the right. (c) Northern blot analysis with BjCAT3 probe was
performed on RNA samples of WT and transgenic lines CAT2, CAT3, and CAT4, and all
the three lines showed high expression of BjCAT3.
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Z. Guan et al. / Chemosphere 76 (2009) 623–630
from different transformation events (Fig. 2b). T-DNA inheritance
was scored by kanamycin segregation analysis in the T1 generation.
The T1 seedlings of five T0 lines segregated as a single, dominant,
mendelian character (3:1), and the data are presented in Table 1.
Among of them, CAT2, CAT3 and CAT4 were selected and further
analyzed by Northern blot analysis. The expected band in each
plant was found in transgenic lines but no signal in WT plants,
and overexpression of BjCAT3 was observed in the transgenic lines
(Fig. 2c). All these results clear indicated that foreign gene BjCAT3
was successfully transferred into tobacco and expressed at a high
transcript level.
3.3. Enhancement of the Cd resistance in transgenic tobacco plants
Cd stress can inhibit the plant growth and induce transcript level of CAT (Metwally et al., 2005). However, whether transgenic
plants with BjCAT3 showed resistant to Cd stress need to be further
proven. First, phenotypic/morphological changes were compared
between WT and transgenic plants under Cd treatment. The
growth inhibition of WT and transgenic tobacco plants that responded to the range of external Cd (concentrations: 0, 50,
100 lM) was shown in Fig. 3. After 50 lM Cd treatment for 1 wk,
root inhibition rates were 24% in the transgenic plants group, but
42% in WT group, though the root length inhibition was observed
in both transgenic and WT tobacco plants. When transgenic and
WT tobacco seedlings were exposed to increasing concentrations
of Cd, a progressive inhibition of growth was observed. All WT
plants turned yellow and eventually died while transgenic tobacco
plants were still green and basically normal in morphology with
little injured symptoms under 100 lM Cd exposure. These data
clearly demonstrated that the overexpression of BjCAT3 can significantly enhance resistance to Cd-induced injury in transgenic tobacco plants.
3.4. H2O2 accumulation and lipid peroxidation
H2O2 accumulation and lipid peroxidation are considered as
two major indices involved in Cd-induced injury of plant. Though
the H2O2 level increased in both transgenic and WT tobacco leaves
as function of time, the WT plant exhibited more H2O2 accumulation under same condition of Cd treatment (Fig. 4a). MDA, a product of lipid peroxidation, showed the similar tendency to H2O2
change in transgenic and WT tobacco leaves after Cd treatment
(Fig. 4b). The results suggested that overexpression of BjCAT3 can
strengthen the antioxidant system in plant by enhancing H2O2
decompose capability. Therefore, cell membranes should become
more stable in transgenic ones under Cd exposure.
3.5. Elevation of Cd-induced oxidative stress tolerance in transgenic
tobacco plants
As time went by, that CAT activity in normal plants was inhibited by Cd. In our study, the capacity of CAT to protect the tobacco
Table 1
Genetic analysis transgenic tobacco. Sterile T1 seeds were plated on MS medium
containing 100 mg L1 kanamycin. Two weeks after sowing, the seedlings were
scored for their resistance or sensitivity to kanamycin. R, resistant and S, sensitive
seedlings. v values were calculated with 1 degree of freedom.
Lines
R
S
Ratio
v
P
CAT2
CAT3
CAT4
CAT5
CAT6
81
65
75
59
79
26
20
22
18
25
3:1
3:1
3:1
3:1
3:1
0.015
0.018
0.012
0.011
0.011
0.9
0.9
0.9
0.9
0.9
plant from oxidative stress caused by Cd treatment was evaluated.
In the absence of Cd, the CAT2–4 lines exhibited over 1.5-fold in total
CAT activity in leave crude extracts as compared with WT plants.
Although the activity of CAT was declined under Cd exposure in
both WT and transgenic tobacco plants, CAT activity remains at
the high level at 24 h in transgenic group, even higher than the
CAT level in WT group at the every beginning of the experiment.
65% of CAT enzyme was consumed after the Cd-stress for 24 h, in
contrast, total CAT activity in the CAT24 lines declined only 38%
in the same period. Meanwhile, SOD and POD, which were also involved in the antioxidative protection, were also analyzed. The
activities of SOD and POD both showed increasing trends under
200 lM Cd in WT and transgenic tobacco plants (Fig. 4d and e).
However, the SOD and POD activities in WT tobacco plants sharply
increased, but the smooth ascending manner was found in transgenic group. If the oxidative stresses exceeded the compensatory
protecting mechanism, the plants would show toxicity symptoms,
as the situation in high Cd-treatment group in WT (Fig. 3b, right panel). The results also revealed that high level of CAT activity played
an important role in defense of Cd-induced oxidative stress, which
was consisted with the result mentioned above.
3.6. Lower Cd-induced H2O2 accumulation in situ in transgenic tobacco
roots
A histochemical method with DAB, which is based on the formation by H2O2 of local brown spots, was performed to detect
in situ the accumulation of H2O2 in WT and transgenic tobacco
roots under Cd treatment. In WT, root appeared dark brown color,
however, the root of transgenic plant showed less staining versus
WT (Fig. 5a).
3.7. Higher cell viability of transgenic tobacco roots under Cd exposure
Trypan blue dye staining is wildly used method for identifying
cell viability, since it is membrane impermeable and generally excluded from viable cells. Trypan blue dye assay showed that more
cells of in WT roots died from Cd stress, while still less dead cells
were found in transgenic plants (Fig. 5b).
Based on the cell viability test using trypan blue dye assay of
root cells after treatment with Cd for 48 h, we further demonstrated
whether overexpression of BjCAT3 can inhibit Cd-induced apoptosis
or necrosis cell death by the Hoechst 33342/PI staining method.
Viable and necrotic cells were identified by intact nuclei with, blue
(Hoechst 33342) or red (PI) fluorescence, respectively. Apoptotic
cells were detected by their fragmented nuclei, which exhibited
either a blue (Hoechst 33342; early apoptosis) or red (PI; late apoptosis) fluorescence (Hashimoto et al., 2003). As shown in Fig. 5c, under control conditions (without Cd treatment), basically all cells of
both WT and transgenic tobacco roots are intact live cells. After
incubation with 100 lM CdCl2 for 24 h, the population of living cells
was decreased, while the apoptotic and necrotic cells were increased (Fig. 5c). However, amount of living cells of transgenic tobacco roots were much more than that of WT roots. The result
demonstrated that Cd could induce apoptosis or necrosis cell death
and overexpression of BjCAT3 could definitely keep cell membrane
intact and protect cells from Cd-induced cell death, which was
accordant with the result of trypan blue dye assay. All the data verified that Cd induced damage in root cells, including H2O2 accumulation, cell membrane injury and cell death, could be alleviated by
BjCAT3 overexpression in transgenic tobacco plants.
3.8. Cd concentration in aboveground tobacco tissues
By using ICP-MS technique, Cd concentrations in aboveground
tissues of plants were determined. Cadmium concentrations in
Z. Guan et al. / Chemosphere 76 (2009) 623–630
627
Fig. 3. Phenotype of WT and transgenic tobacco plants grown under Cd stress. (a) Cd-induced root inhibition of transgenic line CAT3 and WT tobacco plants exposed to 50 lM
CdCl2 for 1 wk after germination. (b) Cd resistance phenotype of transgenic line CAT3 and WT plants were compared on MS agar medium containing 50 and 100 lM CdCl2
treated for 3 wk. Overexpressing of the BjCAT3 gene results in less root inhibition and better growth situation of transgenic tobacco plants compared to WT plants under Cd
exposure (CAT3, transgenic plants; WT, wild-type plants).
the aboveground tissues of WT and transgenic tobacco plants after
exposure of 100 and 200 lM CdCl2 for 3 and 6 d are presented in
Fig. 6. Transgenic tobacco plants exhibited slightly higher Cd concentration but no statistical difference is found.
4. Discussion
Contamination of soil and water with heavy metals and metalloids is an increasing environmental problem worldwide. During
long period evolution, organisms ranging from bacteria and plants
to mammals have developed sophisticated mechanisms to control
metal homeostasis. The ability of organisms protecting themselves
from the metal stress is often associated with inducible increases
in the levels of peroxide detoxification and protective enzymes
(Mongkolsuk et al., 2000; Miller et al., 2008). The aim of this work
is to determine whether overexpression of CAT can enhance plants’
resistance to heavy metal stress (Cd).
Cd, a non-essential but extremely phytotoxic metal pollutant,
produces not only significant reduction in the growth of plants,
but also oxidative stress by generating ROS, and even eventually
leads to cell death resulting from oxidative processes such as membrane lipid peroxidation, protein oxidation, enzyme inhibition and
DNA damage (Sandalio et al., 2001; Romero-Puertas et al., 2002;
Razinger et al., 2007). To fight against Cd-leading oxidative stress,
plants adopt complicated defense system However, the compensatory anti-oxidative mechanism appears to be incapable in the high
concentration Cd-exposure, since many studies has shown that
elevated concentrations of heavy metals result in rapid antioxidant
enzymes consumption (Schützendübel and Polle, 2002). Among
the various anti-oxidative enzymes, CAT is one of the most important antioxidant enzymes present in aerobic organisms, and as a
cellular sink for H2O2, maintaining the redox balance during oxida-
tive stress (Willekens et al., 1997). An increase in the CAT activity
in the presence of Cd was reported in many metal hyperaccumulators (Mobin and Khan, 2007; Sun et al., 2007), though the activities
of CAT in normal plant species were inhibited and declined under
Cd exposure (Chaoui et al., 1997; Leon et al., 2002). It implies that
the metal hyperaccumulation plants’ unique natural characters directly associate with the antixidative system in the plant. In our
experiment, it is reasonable that transgenic plants exhibited significantly higher resistance to Cd-stress.
Phytoremediation is considered as one of the most promising
techniques for the contaminated soils recovery because of its
excellent performance, such as efficient, cost-effective, and environment-friendly (Garbisu and Alkorta, 2001; Weber et al., 2001;
Alkorta et al., 2004). However, some shortcomings of hyperaccmulators such as slow growth and little yield production, limit their
application in reality. Unlike to traditional breeding methods,
transgenic technology is a direct and rapid way to improve plant
characters that are a benefit for its utilization. It is reported that
plants will be less influenced by oxidative damage if genes coding
for antioxidant enzymes are over-expressed. For example, overexpression of CAT in tomato could enhance photo-oxidative stress
tolerance (Mohamed et al., 2003), and expressed both SOD and
CAT could enhance tolerance to sulfur dioxide and salt stress of
Chinese cabbage plants (Tseng et al., 2007). In this paper, the
full-length cDNA of the CAT gene (BjCAT3) was cloned and the
BjCAT3 transcript level was up-regulated by Zn/Cd in B. juncea. In
transcript level, BjCAT3 was up-regulated by Zn and Cd stresses,
suggesting that Zn/Cd induced ROS generation, mainly H2O2, which
was responsible for CAT induction.
Therefore, the exogenous CAT gene was transferred into tobacco
plants and successfully expressed in protein level. Though plant
possesses intrinsic antioxidant enzymes such as CAT, SOD and
POD, they are not capable enough in eliminating the over-accumu-
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Z. Guan et al. / Chemosphere 76 (2009) 623–630
225
WT
CAT3
CAT4
200
(a)
150
125
25
100
75
50
25
0
25
WT
CAT3
CAT4
(b)
SOD (Units mg -1 protein)
H2O2 (µmol g-1 FW)
175
15
-1
10
5
15
10
5
(c)
200
WT
CAT2
CAT3
CAT4
(e)
WT
CAT3
CAT4
12
10
8
6
4
2
0
0
4
8
12
24h
160
-1
CAT (Units mg protein)
0
240
(d)
20
0
14
POD (Units mg protein)
-1
MDA (µmol g FW)
20
WT
CAT3
CAT4
120
80
40
0
0
4
8
12
24h
Fig. 4. Effects of Cd exposure on H2O2 contents, lipid peroxidation and antioxidant enzyme activities in WT and transgenic tobacco plants over time (0, 4, 8, 12, and 24 h). (a)
H2O2 accumulation; (b) MDA (malondialdehyde) content; (c) CAT (catalase) activity; (d) SOD (superoxide dismutase) activity and (e) POD (peroxidase) activity (n = 4 per
experiment and each experiment was repeated at least three times). Significant differences between WT and transgenic tobacco plants in each test at the p < 0.05 () and
p < 0.01 () levels are indicated.
lation of ROS when the WT plants were treated with high concentration of Cd, as shown in Fig. 3. By contrast, the transgenic plants
show stronger resistance to Cd toxicity and less visible symptoms
were found. Hence, it is believed that the genetic modification of
antioxidant enzymes by synthesizing new isozymes or increasing
pre-existing enzyme levels, CAT for instance, will effectively scavenge the Cd-induced ROS in the plant. In WT groups, CAT activity
was initially elevated and rapidly reduced after 4 h Cd exposure,
since the intrinsic CAT was too weak to scavenge the over-production of H2O2. While in transgenic group, the CAT, which remained
at a high level after transgenic manipulation, was gradually consumed as a function of time. The CAT activity in transgenic lines
still retains a high level about 1.5 times to that of WT before Cdtreatment. Considering the synergistic effect of antioxidant enzymes in plant, some enzymes, such as POD and SOD were also
examined. Activities of POD and SOD increased after Cd treatment
in both WT and transgenic tobacco plants, however, the increasing
amount of these enzymes were significantly lower than that of WT.
POD, which catalyzes reactions of organic hydroperoxides, also
functioned as a scavenger for H2O2. High level of CAT activity can
compensate for the consuming of POD for the removal of excess
H2O2 during stress. The balace between SOD and CAT or POD activities in cells is crucial for maintaining the steady-state level of
superoxide radicals and hydrogen peroxide (Bowler et al., 1991).
The H2O2 accumulation and lipid peroxidation were considered
as a consequence of Cd toxicity. Accumulation of H2O2 has been
observed by histochemistry in Cd-treated pea leaves and roots
(Romero-Puertas et al., 2002, 2004). In transgenic tobacco plants,
high level of CAT activity could eliminate the Cd-induced over-production of H2O2 in time by decomposing it to water and molecular
oxygen (Fig. 5a).
Higher level of H2O2 accumulation (Fig. 5a) and root cell death
(Figs. 5b and 5c) were observed in WT tobacco plants compared to
transgenic ones under the same Cd treatment in the root system
(P < 0.05). This suggests that there may be a possible mechanism
by which the H2O2, over-accumulating under Cd stress, as a cellular
indicator involved in the stress-response signal transduction pathway and result in apoptosis or necrosis cell death (Fig. 5c). Heavy
Z. Guan et al. / Chemosphere 76 (2009) 623–630
629
Fig. 5. Cd-induced in situ H2O2 accumulation and cell death in WT and transgenic tobacco roots. (a) Histochemical detection of H2O2 in WT and transgenic tobacco roots after
treated with 100 lM CdCl2 for 8 h. (b) Trypan blue staining of Cd-induced membrane injury and cell death in root tips of WT and transgenic tobacco plants under 100 lM
CdCl2 over time (0, 4, 8, and 48 h). (c) Hoechst 33342/Propidium Iodide (PI) staining of WT and transgenic tobacco plants’ root exposed to 100 lM CdCl2 for 12 h. Hoechst
freely passes cells membranes and stains nuclear DNA blue. The condensed chromatin of apoptotic cells stains more brightly than the chromatin of normal cells. PI is
impermeable to intact membranes and only enters necrosis or late apoptotic cells that have damaged membranes, staining them an orange fluorescent color (CAT3, transgenic
plants; WT, wild-type plants; C, control without Cd treatment).
-1
Cd accumulation (µgg DW)
450
400
350
200 µM
WT
CAT(2-4)
200 µM
300
100 µM
250
100 µM
200
20 µM
150
100
20 µM
50
0
3d
6d
Fig. 6. Cd concentration in aboveground tissues of WT and transgenic tobacco
plants at 20, 100 and 200 lM CdCl2 for 3 and 6 d. Mean ± standard error of the mean
(SEM) (n = 3).
metal exposure, Cd and Hg for instance, could induce abnormality
of physiological parameters and even eventually lead to cell death
in wild type tobacco. Beside tobacco, similar phenomena were also
reported in other species, such as Medicago sativa and alfalfa (Ortega-Villasante et al., 2005, 2007). Higher level of CAT activity in
transgenic tobacco plants can scavenge Cd-induced H2O2 production in time, and remain a steady-state H2O2 level under the same
Cd stress condition. Obviously, overexpression of BjCAT3 plays an
important role on the cell membrane integrality and contributes
to better cell viability in transgenic tobacco plants (Fig. 5b and c)
under heavy metal stress. All these results further supported our
above findings: the transgenic tobacco plants own a more powerful
antioxidant system and are less affected by Cd-induced oxidative
stress.
In conclusion, our present results demonstrated that overexpression of CAT could reduce the phytotoxicity caused by Cd such
as growth inhibition, H2O2 accumulation, lipid peroxidation and
cell death in tobacco. Transgenic tobacco plants exhibited higher
630
Z. Guan et al. / Chemosphere 76 (2009) 623–630
level of CAT activity and less effect on SOD, POD, MDA and H2O2
levels under Cd exposure versus WT. Our findings are the first to
report that the transgenic modification would protect plants from
the toxic effect of Cd-exposure by strengthen the antioxidant system in plant. Development of transgenic technologies, such as the
one described here, might help to improve plant characteristics in
future phytoremediation applications.
Acknowledgements
This research was supported by the National High Technology
Planning Program of China (Grant Nos. 2007AA021404 and
2006AA10Z407), and China National Natural Sciences Foundation
(Grant No. 30570146).
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