Enhancement of Cd tolerance in transgenic tobacco plants overexpressing a Cd-induced catalase cDNA

Chemosphere 76 (2009) 623–630

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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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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 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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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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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

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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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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

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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

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