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Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance
Biochemical and Biophysical Research Communications xxx (2017) 1e7
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Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance Sheng Xu a, 1, Bin Sun a, 1, Rong Wang a, Jia He a, Bing Xia a, Yong Xue b, *, Ren Wang a, ** a b
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China Eco-Environmental Protection Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2017 Accepted 14 June 2017 Available online xxx
The phytoremediation by using of green plants in the removal of environmental pollutant is an environment friendly, green technology that is cost effective and energetically inexpensive. By using Agrobacterium-mediated gene transfer, we generated transgenic Arabidopsis plants ectopically expressing mercuric transport protein gene (merT) from Pseudomonas alcaligenes. Compared with wild-type (WT) plants, overexpressing PamerT in Arabidopsis enhanced the tolerance to HgCl2. Further results showed that the enhanced total activities or corresponding transcripts of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD) were observed in transgenic Arabidopsis under HgCl2 stress. These results were confirmed by the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) contents and reactive oxygen species (ROS) accumulation. In addition, localization analysis of PaMerT in Arabidopsis protoplast showed that it is likely to be associated with vacuole. In all, PamerT increased mercury (Hg) tolerance in transgenic Arabidopsis, and decreased production of Hg-induced ROS, thereby protecting plants from oxidative damage. The present study has provided further evidence that bacterial MerT plays an important role in the plant tolerance to HgCl2 and in reducing the production of ROS induced by HgCl2. © 2017 Published by Elsevier Inc.
Keywords: Arabidopsis Pseudomonas alcaligenes Mercuric transport protein Mercury tolerance Reactive oxygen species
1. Introduction Mercury (Hg) is one of the most hazardous elemental pollutants, endangering the environment and causing a variety of diseases in humans and animals [1]. It occurs naturally in the environment and can be found in elemental (metallic), inorganic, and organic forms. The most common natural forms of Hg are elemental mercury (Hg0), mercuric sulfide (HgS), mercuric chloride (HgCl2), and methylmercury (MeHg). In response to toxic Hg compounds, microbes have developed alleviation systems to overcome Hg poisoning. They provide a means of bioremediation by taking up these compounds and reducing them to volatile, less-toxicHg0 which diffuses out of the cell [2]. Of the known bacterial heavy metal resistance systems, the mer (mercuric ion resistance) operon is an intensively studied
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Xue), (R. Wang). 1 These authors contributed equally to this work.
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mercurial resistance system consisting of a cluster of linked genes involved in detection and regulation (merR), recognition and mobilization (merP, merT, merC), and enzymatic detoxification (merA) [3e5]. Besides, another important enzyme organomercurial lyase (MerB) catalyzes the protonolysis of the carbonemercury bond. The products of this reaction are a less toxic inorganic species and a reduced carbon compound [6]. Currently, there are five known types of mercury transporters in bacteria: MerC, MerE, MerF, MerH, and MerT [7e11]. While mercury transporters are predicted to have different structures, they all function as mercuric ion transporters. Plants also take up metals [12], and this ability is exploited to clean up metal-polluted environments, using technologies known as phytoremediation, which are potentially regarded as effective and environment friendly alternatives compared to physical remediation methods such as excavation and reburial [13e15]. For example, plant species transformed with merA and merB have been shown to be tolerant to different levels of HgCl2 [6,16e18]. Besides, plants transformed with mercury transporters such as merC and merT genes also have greater capacity to accumulate mercury into their tissues than wild-type (WT) plants, or/and show enhanced
http://dx.doi.org/10.1016/j.bbrc.2017.06.073 0006-291X/© 2017 Published by Elsevier Inc.
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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S. Xu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
tolerance to Hg [19e22]. Moreover, physiological and biochemical processes in plants may be negatively affected by Hg [23]. The toxicity induced by Hg is associated with two major biological events. One is the binding of its ionic forms (Hg2þ) to sulphydryl groups of proteins to modify molecule structure, the other is formation of reactive oxygen species (ROS), which causes oxidative damage to cellular structure and metabolic processes [24e26]. To maintain the homeostasis of ROS, plants have evolved a complex antioxidant network when they are subjected to hazardous environmental conditions [27]. When the redox cellular status is compromised, metabolites such as ascorbate and glutathione (GSH), and antioxidant enzymes such as superoxide dismutases (SODs), ascorbate peroxidases (APXs) and catalases (CATs), help to scavenge excess ROS [28]. The antioxidant systems might play vital roles in protecting plants against Hg-induced ROS stress. In this study, the transgenic Arabidopsis lines overexpressing Pseudomonas alcaligenes merT (PamerT) were generated. To investigate its function, HgCl2 tolerance and accumulation, as well as Hginduced ROS such as H2O2 and O 2 production in the seedlings were determined. Transgenic Arabidopsis plants carrying PamerT were more tolerant to mercury than WT Arabidopsis. Additionally, less TBARS content and lower abundance of ROS, which might be correlated to the enhanced expression of antioxidant enzymes were also found in the transgenic Arabidopsis. These results demonstrated that expression of the bacterial heavy metal transporter MerT may be a useful method for improving plants for the phytoremediation of mercury pollution. 2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis thaliana seeds were surface-sterilized and washed three times with sterile water for 20 min. Afterwards, seeds were cultured on MS agar medium in the presence of different concentrations of HgCl2 (0, 5, 10, 20, 30 and 40 mmol L1). After 2 days of cold (4 C) acclimation, plates were then transferred into a growth chamber with a 16/8 h (22/18 C) day/night regimes at 120 mmol m2 s1 irradiation. For the quantification of roots length, 7-d-old seedlings were transferred to vertically oriented agar plates containing 10 mmol L1 HgCl2. The number of lateral roots, lateral root density, primary root length, and total root length were determined after an additional 7 d of growth using a dissecting microscope and ImageJ software. 2.2. Transformation of Arabidopsis A 0.35-kb fragment containing PamerT gene (GenBank accession no. NC_005909) was cloned into the plant binary vector pYG8468 under the control of the double CaMV 35S (D35S) promoter (Supplementary Fig. S1A). The recombinant plasmid was introduced into Agrobacterium tumefaciens GV3101 and used to transform Arabidopsis via the floral dip method [29]. Independent primary transformants were randomly chosen for a test of hygromycin-resistant segregation in the progeny (T1 generation), and the homozygous T3 lines were selected for further analysis.
performed using a Bio-Rad iCycler iQ system (Bio-Rad laboratories, Richmond, CA, USA) with One Step SYBR PrimerScript™ RT-PCR Kit (Perfect Real Time; TaKaRa Bio Inc., Dalian, China). AtActin2 was used as the reference control. The specific primers used here were listed in Supplementary Table S1. 2.4. Chlorophyll content determination Chlorophyll a and b contents were quantified according to the method described previously [30]. 2.5. Thiobarbituric acid reactive substances (TBARS) determination Oxidative damage was estimated by measuring the concentration of TBARS as described by Xu et al. [31]. 2.6. Antioxidant enzyme assays Arabidopsis plants (approximately 100 mg) were homogenized in 1 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 12,000g for 20 min at 4 C and the supernatant was used as the crude enzyme extract. Total SOD, POD and CAT activity was measured according to the method described previously [31]. 2.7. Detection of superoxide anion (O 2 ) and hydrogen peroxide (H2O2) and H2O2 levels were visually detected in the plants, O 2 respectively, with nitroblue tetrazolium (NBT) and 3,3’-diaminobenzidine tetrahydrochloride (DAB) as described previously [32]. 2.8. Subcellular localization analysis The complete open reading frame fragment of PamerT without the termination codon was cloned into the expression vector pAN580 for C-terminal green fluorescent protein (GFP) fusion. Meanwhile, the complete coding sequence of Arabidopsis dTIP gene (AT3G16240) was fused with mCherry protein to construct the vacuole-localized marker in a modified vector P16DS:sXVE:mCherryC. Additionally, the well-established fluorescent protein mCherryeHDEL was used as the endoplasmic reticulum (ER) marker [33]. Transformed protoplasts were incubated at room temperature in the dark for about 16 h before observation, and transient expression of GFP and mCherry was observed under a laser scanning confocal microscope (LSM700; Carl Zeiss, Germany). 2.9. Statistical analysis Where indicated, results were expressed as the means ± SE of three independent experiments. Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by Duncan's multiple range test (P < 0.05) when appropriated using the statistical software SPSS 10.0 for Windows. 3. Results
2.3. Total RNA extraction, semi-quantitative reverse-transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) analysis
3.1. Expression of bacterial merT gene in transgenic Arabidopsis
Total RNA was isolated by using RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. The first-strand cDNA was synthesized using reverse transcriptase M-MLV (TaKaRa Bio Inc., Dalian, China). qRT-PCR was
The PamerT gene was transferred into Arabidopsis ecotype Col0 using the Agrobacterium-mediated gene transfer method (Supplementary Fig. S1A). After selection with hygromycin and regeneration, 16 independent PamerT-overexpressing plants (T1
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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Fig. 1. Effects of HgCl2 on root elongation and fresh weight in both wild-type (WT) and PamerT-overexpressing Arabidopsis lines. (A) Primary root length and fresh weights of WT and PamerT-overexpressing plants under 10 mM HgCl2 stress for 16 days. Phenotypes (B) and primary root length and fresh weights (C) of WT and PamerT-overexpressing line M5 plants under different concentrations of HgCl2 stress for 16 days. Error bars indicate standard error (n ¼ 3). Different letters above the error bars indicate that the means are statistically different according to Duncan's multiple test (P < 0.05). Asterisk indicates a significant difference between WT versus PamerT-overexpressing plants (M5) according to student's t-test. Bar ¼ 1 cm.
generation) were obtained. Among them, three transgenic lines (M2, M5, and M12) displaying a 3:1 segregation (hygromycin resistant: hygromycin sensitive) were chosen for further experiments and transgene expression was verified by semi-quantitative RT-PCR (Supplementary Fig. S1B). 3.2. Effects of PaMerT on Hg tolerance and accumulation in transgenic Arabidopsis When grown on normal MS medium, PamerT-overexpressing
plants displayed longer primary root and higher fresh weight (Fig. 1A), but neither of them is significantly different from that of WT plants. In the presence of 10 mmol L1 HgCl2, the growth of WT Arabidopsis was remarkably inhibited (Fig. 1A). For example, the root growth of seedling from WT was largely reduced by HgCl2, only 51.85% of the control. In contrast, the roots of transformants (M2, M5 and M12) were 1.60-, 1.69- and 1.51-fold longer than WT roots respectively under 10 mmol L1 HgCl2. The similar result was also found in the fresh weight of seedlings (Fig. 1A). Additionally, WT plants and one of the transgenic lines (M5) were also subjected
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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S. Xu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
to different concentrations of HgCl2 (Fig. 1B). The results showed that the root growth of PamerT-overexpressing Arabidopsis appeared as healthy on Hg as on mercury-free control plates, and the transgenic line (M5) is more tolerant than that of WT to Hg stress (Fig. 1C). Moreover, Arabidopais seeds were germinated on MS medium for 7 days. After that, WT and PamerT-overexpressing seedlings (M5 line) were transferred to new MS medium in the presence or absence of 10 mM HgCl2 for 5 days. It was found that PamerToverexpressing line M5 showed more tolerant to Hg compared to the WT Arabidopsis (Fig. 2A). For instance, after 5 days of Hg stress, the WT plants showed a sound decrease of chlorophyll contents (with a 32.75% in chlorophyll a and a 57.06% in chlorophyll b) in contrast to the M5 plants (with a 18.65% in chlorophyll a and a 22.43% in chlorophyll b), in which they remained unaltered when compared to their controls (Fig. 2B). Additionally, although the primary root length of WT and transgenic Arabidopsis seedlings did not differ significantly, overexpressing PamerT in Arabidopsis enhances the lateral root formation (including LR number/density) and total root length under both normal and HgCl2-stressed conditions (Supplementary Fig. S2). 3.3. Effects of HgCl2 treatment on lipid peroxidation, transcripts and activities of antioxidant enzymes To confirm the cytoprotective role of PaMerT in alleviation of Hg-induced oxidative stress, we compared the contents of TBARS in WT and transgenic plants under HgCl2 stress. The results showed that HgCl2 stress caused an increment on TBARS contents in both
WT and transgenic plants, while the increase extent of TBARS in PamerT-overexpressing plants is apparently lower than the WT plants (Fig. 2C). Further, changes in several antioxidant enzymes activities and corresponding transcripts in WT and M5 line seedlings were compared. Using qRT-PCR, six Arabidopsis ROS responsive genes (Supplementary Table S1) were examined at the indicated time of HgCl2 treatment. Transcript levels of each gene monitored are expressed as the fold change compared with the expression level of WT at zero time point (Fig. 3A; Supplementary Fig. S3). Time-dependent analysis showed that CSD1 were induced in both M5 line and WT seedlings upon HgCl2 stress, while stronger induction was observed in M5 line than WT seedlings. Under HgCl2 condition, the expression of FSD1 only increased at the end of the treatment (48 h) in WT seedlings, while in M5 line the expression of FSD1 increased at both the 24-h and 48-h time point. Meanwhile, after 24-h treatment, the differences between WT and M5 line seedlings turn relatively smaller under HgCl2 stress. We further noticed that a significant higher transcript level of PER21 and CAT1 were detected in M5 line plants than in WT plants with the treatment of HgCl2 (Fig. 3A). However, there were no significant differences in the transcript levels of PER21 and CAT1 genes between WT and merT-overexpressing (M5) plants in the absence of HgCl2 except at the 24-h time point (Supplementary Fig. S3). On the other hand, more pronounced changes of PER27 were observed in PamerT-overexpressing (M5) than in WT plants under HgCl2 treatment (Fig. 3A), especially after longer period of treatment (48 h). Additionally, CAT2 mRNA increased first and then decreased, but obvious differences existed between WT and M5 at the end of HgCl2 treatment.
Fig. 2. Mercury tolerance in wild-type (WT) and transgenic Arabidopsis seedlings. 7-d-old WT and PamerT-overexpressing line M5 seedlings were transferred to 10 mM HgCl2 for 5 days. Afterwards, photos of Hg treatment of wild-type and transgenic plants were taken. Statistical analysis of chlorophyll contents (B) and TBARS contents (C) at different treatment time were also determined. Error bars indicate standard error (n ¼ 3). Different letters above the bars indicate that the means are statistically different (P < 0.05). Bar ¼ 1 cm.
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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Fig. 3. Time-dependent changes of the transcripts (A) and activity of antioxidant enzymes (B) in wild-type (WT) and transgenic Arabidopsis seedlings upon HgCl2 stress. 7-d-old WT and PamerT-overexpression line M5 seedlings were transferred to 10 mM HgCl2 for the indicated time. Error bars indicate standard error (n ¼ 3). Different letters above the bars indicate that the means are statistically different according to Duncan's multiple test (P < 0.05).
Activities of antioxidant enzymes in WT and transgenic Arabidopsis substantially changed when they were exposed to HgCl2 (Fig. 3B; Supplementary Fig. S4). The CAT activities increased slightly first and then decreased, but obvious differences existed between WT and M5 at different treatment time. Activity of SOD in WT and M5 showed a similar trend, but the activity of M5 was greater than that of WT under HgCl2-stressed conditions. Activities of POD exhibited a sharp difference compared with activities of CAT and SOD (Fig. 3B). Even though, at the last stage of HgCl2 treatment, POD activities of M5 were much greater than that of WT (Fig. 3B). Additionally, the presence of H2O2 and O 2 in seedlings following HgCl2 treatment for 72 h was also detected in situ (Supplementary Fig. S5). The WT seedling leaves exhibited marked brown and blue coloration after Hg treatment, suggesting more H2O2 and O 2 accumulation compared to PamerT-overexpressing plants.
3.4. Localization of PaMerT expressed in Arabidopsis protoplast To confirm experimentally the subcellular localization of the PaMerT protein, we fused the coding region of PaMerT at the Nterminus of a GFP fragment under the CaMV 35S promoter. After being expressed transiently in Arabidopsis protoplasts, the fluorescence signal of GFP itself was visualized in both cytosol and nucleus (Fig. 4A). By contrast, signal from PaMerT-GFP fusion protein was observed in the cytoplasm (Fig. 4B), and is mostly overlapped with the red fluorescence of AtdTIP-mcherry fusion protein (Fig. 4C). Besides, a part of proportion of PaMerT-GFP co-localized with mRFPeHDEL (Fig. 4D). PaMerT is thus very likely to be associated with vacuole and ER.
4. Discussion As native plant tolerance to Hg is generally low, strategic focus for improving the phytoremediation potential of plants is the integration of genes from other organisms by genetic engineering. Of the known bacterial Hg resistance systems, the Mer determinant is unique in terms of the orientation of the mercury transporter it encodes [4]. The successful reconstruction and implementation of the mer genes has been demonstrated very effectively in transgenic plants for cleaning Hg-contaminated environments [6,15,34]. In this study, a number of independent transgenic Arabidopsis plants overexpressing PamerT were obtained (Supplementary Fig. S1). As Hg causes a significant reduction of plant growth and biomass [34], we also observed that the primary root length, plant fresh weight and amount of chlorophyll were declined upon HgCl2 stress in Arabidopsis (Figs. 1 and 2). And the higher concentration of HgCl2, the much more decrease in the primary root length and plant fresh weight (Fig. 1). On the other hand, it is obvious that the PamerToverexpressing Arabidopsis was much more tolerant than that of WT plants (Figs. 1 and 2). This result indicated that PamerT may offer a protection effect in plants against toxicity of Hg. Although there is no significant difference in primary root length between WT and transgenic Arabidopsis (Fig. 1), it is worth noting that transgenic Arabidopsis plants overexpressing PamerT have more lateral root numbers than the wild type under both normal and HgCl2-stressed conditions (Supplementary Fig. 2). TBARS content was used as an indicator for lipid peroxidation in vivo, and increase in the level of TBARS in response to HgCl2 has been observed in several plants [24e26]. Consequently, related experiments illustrated that PamerT-overexpressing Arabidopsis plants were able to counteract the increases in TBARS content
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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Fig. 4. Localization of PaMerT-GFP fusion protein transiently expressed in Arabidopsis protoplasts. GFP alone (A) or PaMerTeGFP fusion protein (B) expressed in Arabidopsis protoplasts. (C) Protoplasts co-expressing PaMerT-GFP and AtdTIP-mcherry protein. (D) Protoplasts co-expressing PaMerT-GFP and AtHDEL-mcherry protein. The photographs were taken in the green channel (GFP fluorescence), red channel (mCherry fluorescence), combination of green and red channel, and bright channel. Bar ¼ 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
induced by HgCl2 stress (Fig. 2C). Hg triggers oxidative stress by inducing production of ROS, e.g. O 2 , H2O2, and OH in plants [24e26], and subsequently increases the activity of antioxidant enzymes as well as the content of GSH [35]. Our results also indicated that the transcripts and activities of SOD, POD and CAT were pronounced enhanced in transgenic Arabidopsis compared with WT when exposed to HgCl2 (Fig. 3). Additionally, the higher expression of antioxidant enzymes might lead to the suppression of ROS in transgenic Arabidopsis plants (Supplementary Fig. S5). Compartmentalization of metals in the vacuole is considered to be a part of the tolerance mechanism of some metal hyperaccumulators [36]. In this report, the signal from PaMerT-GFP fusion protein expressed in Arabidopsis protoplast also proved that PaMerT is very likely to be localized in vacuole (Fig. 4). Thus, it is possible that PaMerT might be capable of allocating Hg to vacuole where mercury is less toxic. On the other hand, many studies showed that phytochelatins play a central role in Hg detoxification and it showed that vacuolar transport of phytochelatin-Hg complexes is a prerequisite for Hg tolerance [37]. Thus, the determination of phytochelatin contents would be further carried out.
Based on our results, the acceleration of Hg uptake by overexpression of PaMerT and vacuolar compartmentalization might affect the Hg-induced ROS generation and homeostasis. Moreover, integration of merT gene into ppk-transgenic tobacco not only showed more resistance to Hg2þ than its wild-type progenitors but also accelerated and enhanced Hg uptake into tobacco thus accumulated more mercury [38]. It has been revealed that plants possess several classes of metal transporters that must be involved in metal uptake and homeostasis in general and could play a key role in metal tolerance [39]. Whether similar results caused by PamerT overexpression should be further investigated. In summary, the physiological and antioxidative responses of the PamerT-overexpressing Arabidopsis were analyzed under HgCl2 stress. Our data showed that the antioxidative defense in PamerToverexpressing plants is stronger and persists for a longer time under HgCl2 stress. This might suggest a reestablishment of redox homeostasis, in which MerT-mediated responses would be involved. Furthermore, the development of plants express PamerT by genetically engineering might be useful for the efficient phytoremediation of HgCl2-polluted environments.
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant No. 81603240), and the Jiangsu Provincial Public Institutions Program for Research Conditions and Building Capacity (BM2015019). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2017.06.073. References [1] J.C. Hansen, A.P. Gilman, Exposure of Arctic populations to methylmercury from consumption of marine food: an updated risk-benefit assessment, Int. J. Circumpol. Health 64 (2005) 121e136. €bler, Microbial transformations of mercury: potentials, [2] T. Barkay, I. Wagner-Do challenges, and achievements in controlling mercury toxicity in the environment, Adv. Appl. Microbiol. 57 (2005) 1e52. [3] A.M. Osborn, K.D. Bruce, P. Strike, et al., Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon, FEMS Microbiol. Rev. 19 (1997) 239e262. €€ [4] V.B. Mathema, B.C. Thakuri, M. Sillanpa a, Bacterial mer operon-mediated detoxification of mercurial compounds: a short review, Arch. Microbiol. 193 (2011) 837e844. [5] N.V. Hamlett, E.C. Landale, B.H. Davis, et al., Roles of the Tn2l merT, merP, and merC gene products in mercury resistance and mercury binding, J. Bacteriol. 174 (1992) 6377e6385. [6] S.P. Bizily, C.L. Pugh, A.O. Summers, et al., Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 6808e6813. , L.G. Dover, G.S. Besra, et al., Sequence and analysis of a plasmid[7] M. Schue encoded mercury resistance operon from Mycobacterium marinum identifies MerH, a new mercuric ion transporter, J. Bacteriol. 191 (2009) 439e444. [8] M. Kiyono, Y. Sone, R. Nakamura, et al., The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli, FEBS Lett. 583 (2009) 1127e1131. [9] C.A. Liebert, A.L. Watson, A.O. Summers, The quality of merC, a module of the mer mosaic, J. Mol. Evol. 51 (2000) 607e622. [10] A.P. Morby, J.L. Hobman, N.L. Brown, The role of cysteine residues in the transport of mercuric ions by the Tn501 MerT and MerP mercury-resistance proteins, Mol. Microbiol. 17 (1995) 25e35. [11] J.R. Wilson, C. Leang, A.P. Morby, et al., MerF is a mercury transport protein: different structures but a common mechanism for mercuric ion transporters, FEBS Lett. 472 (2000) 78e82. ~o, A. Polle, P.J. Lea, et al., Making the life of heavy metal-stressed [12] P.L. Grata plants a little easier, Funct. Plant Biol. 32 (2005) 481e494. [13] S.P. Bizily, C.L. Rugh, R.B. Meagher, Phytodetoxification of hazardous organomercurials by genetically engineered plants, Nat. Biotechnol. 18 (2000) 213e217. [14] R.B. Meagher, Phytoremediation of toxic elemental and organic pollutants, Curr. Opin. Plant Biol. 3 (2000) 153e162. [15] O.N. Ruiz, H. Daniell, Genetic engineering to enhance mercury phytoremediation, Curr. Opin. Biotechnol. 20 (2009) 213e219. [16] D. Che, R.B. Meagher, A.C.P. Heaton, et al., Expression of mercuric ion reductase in Eastern cottonwood (Populus deltoides) confers mercuric ion reduction
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Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance Sheng Xu a, 1, Bin Sun a, 1, Rong Wang a, Jia He a, Bing Xia a, Yong Xue b, *, Ren Wang a, ** a b
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China Eco-Environmental Protection Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2017 Accepted 14 June 2017 Available online xxx
The phytoremediation by using of green plants in the removal of environmental pollutant is an environment friendly, green technology that is cost effective and energetically inexpensive. By using Agrobacterium-mediated gene transfer, we generated transgenic Arabidopsis plants ectopically expressing mercuric transport protein gene (merT) from Pseudomonas alcaligenes. Compared with wild-type (WT) plants, overexpressing PamerT in Arabidopsis enhanced the tolerance to HgCl2. Further results showed that the enhanced total activities or corresponding transcripts of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD) were observed in transgenic Arabidopsis under HgCl2 stress. These results were confirmed by the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) contents and reactive oxygen species (ROS) accumulation. In addition, localization analysis of PaMerT in Arabidopsis protoplast showed that it is likely to be associated with vacuole. In all, PamerT increased mercury (Hg) tolerance in transgenic Arabidopsis, and decreased production of Hg-induced ROS, thereby protecting plants from oxidative damage. The present study has provided further evidence that bacterial MerT plays an important role in the plant tolerance to HgCl2 and in reducing the production of ROS induced by HgCl2. © 2017 Published by Elsevier Inc.
Keywords: Arabidopsis Pseudomonas alcaligenes Mercuric transport protein Mercury tolerance Reactive oxygen species
1. Introduction Mercury (Hg) is one of the most hazardous elemental pollutants, endangering the environment and causing a variety of diseases in humans and animals [1]. It occurs naturally in the environment and can be found in elemental (metallic), inorganic, and organic forms. The most common natural forms of Hg are elemental mercury (Hg0), mercuric sulfide (HgS), mercuric chloride (HgCl2), and methylmercury (MeHg). In response to toxic Hg compounds, microbes have developed alleviation systems to overcome Hg poisoning. They provide a means of bioremediation by taking up these compounds and reducing them to volatile, less-toxicHg0 which diffuses out of the cell [2]. Of the known bacterial heavy metal resistance systems, the mer (mercuric ion resistance) operon is an intensively studied
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Xue), (R. Wang). 1 These authors contributed equally to this work.
[email protected]
mercurial resistance system consisting of a cluster of linked genes involved in detection and regulation (merR), recognition and mobilization (merP, merT, merC), and enzymatic detoxification (merA) [3e5]. Besides, another important enzyme organomercurial lyase (MerB) catalyzes the protonolysis of the carbonemercury bond. The products of this reaction are a less toxic inorganic species and a reduced carbon compound [6]. Currently, there are five known types of mercury transporters in bacteria: MerC, MerE, MerF, MerH, and MerT [7e11]. While mercury transporters are predicted to have different structures, they all function as mercuric ion transporters. Plants also take up metals [12], and this ability is exploited to clean up metal-polluted environments, using technologies known as phytoremediation, which are potentially regarded as effective and environment friendly alternatives compared to physical remediation methods such as excavation and reburial [13e15]. For example, plant species transformed with merA and merB have been shown to be tolerant to different levels of HgCl2 [6,16e18]. Besides, plants transformed with mercury transporters such as merC and merT genes also have greater capacity to accumulate mercury into their tissues than wild-type (WT) plants, or/and show enhanced
http://dx.doi.org/10.1016/j.bbrc.2017.06.073 0006-291X/© 2017 Published by Elsevier Inc.
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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tolerance to Hg [19e22]. Moreover, physiological and biochemical processes in plants may be negatively affected by Hg [23]. The toxicity induced by Hg is associated with two major biological events. One is the binding of its ionic forms (Hg2þ) to sulphydryl groups of proteins to modify molecule structure, the other is formation of reactive oxygen species (ROS), which causes oxidative damage to cellular structure and metabolic processes [24e26]. To maintain the homeostasis of ROS, plants have evolved a complex antioxidant network when they are subjected to hazardous environmental conditions [27]. When the redox cellular status is compromised, metabolites such as ascorbate and glutathione (GSH), and antioxidant enzymes such as superoxide dismutases (SODs), ascorbate peroxidases (APXs) and catalases (CATs), help to scavenge excess ROS [28]. The antioxidant systems might play vital roles in protecting plants against Hg-induced ROS stress. In this study, the transgenic Arabidopsis lines overexpressing Pseudomonas alcaligenes merT (PamerT) were generated. To investigate its function, HgCl2 tolerance and accumulation, as well as Hginduced ROS such as H2O2 and O 2 production in the seedlings were determined. Transgenic Arabidopsis plants carrying PamerT were more tolerant to mercury than WT Arabidopsis. Additionally, less TBARS content and lower abundance of ROS, which might be correlated to the enhanced expression of antioxidant enzymes were also found in the transgenic Arabidopsis. These results demonstrated that expression of the bacterial heavy metal transporter MerT may be a useful method for improving plants for the phytoremediation of mercury pollution. 2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis thaliana seeds were surface-sterilized and washed three times with sterile water for 20 min. Afterwards, seeds were cultured on MS agar medium in the presence of different concentrations of HgCl2 (0, 5, 10, 20, 30 and 40 mmol L1). After 2 days of cold (4 C) acclimation, plates were then transferred into a growth chamber with a 16/8 h (22/18 C) day/night regimes at 120 mmol m2 s1 irradiation. For the quantification of roots length, 7-d-old seedlings were transferred to vertically oriented agar plates containing 10 mmol L1 HgCl2. The number of lateral roots, lateral root density, primary root length, and total root length were determined after an additional 7 d of growth using a dissecting microscope and ImageJ software. 2.2. Transformation of Arabidopsis A 0.35-kb fragment containing PamerT gene (GenBank accession no. NC_005909) was cloned into the plant binary vector pYG8468 under the control of the double CaMV 35S (D35S) promoter (Supplementary Fig. S1A). The recombinant plasmid was introduced into Agrobacterium tumefaciens GV3101 and used to transform Arabidopsis via the floral dip method [29]. Independent primary transformants were randomly chosen for a test of hygromycin-resistant segregation in the progeny (T1 generation), and the homozygous T3 lines were selected for further analysis.
performed using a Bio-Rad iCycler iQ system (Bio-Rad laboratories, Richmond, CA, USA) with One Step SYBR PrimerScript™ RT-PCR Kit (Perfect Real Time; TaKaRa Bio Inc., Dalian, China). AtActin2 was used as the reference control. The specific primers used here were listed in Supplementary Table S1. 2.4. Chlorophyll content determination Chlorophyll a and b contents were quantified according to the method described previously [30]. 2.5. Thiobarbituric acid reactive substances (TBARS) determination Oxidative damage was estimated by measuring the concentration of TBARS as described by Xu et al. [31]. 2.6. Antioxidant enzyme assays Arabidopsis plants (approximately 100 mg) were homogenized in 1 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 12,000g for 20 min at 4 C and the supernatant was used as the crude enzyme extract. Total SOD, POD and CAT activity was measured according to the method described previously [31]. 2.7. Detection of superoxide anion (O 2 ) and hydrogen peroxide (H2O2) and H2O2 levels were visually detected in the plants, O 2 respectively, with nitroblue tetrazolium (NBT) and 3,3’-diaminobenzidine tetrahydrochloride (DAB) as described previously [32]. 2.8. Subcellular localization analysis The complete open reading frame fragment of PamerT without the termination codon was cloned into the expression vector pAN580 for C-terminal green fluorescent protein (GFP) fusion. Meanwhile, the complete coding sequence of Arabidopsis dTIP gene (AT3G16240) was fused with mCherry protein to construct the vacuole-localized marker in a modified vector P16DS:sXVE:mCherryC. Additionally, the well-established fluorescent protein mCherryeHDEL was used as the endoplasmic reticulum (ER) marker [33]. Transformed protoplasts were incubated at room temperature in the dark for about 16 h before observation, and transient expression of GFP and mCherry was observed under a laser scanning confocal microscope (LSM700; Carl Zeiss, Germany). 2.9. Statistical analysis Where indicated, results were expressed as the means ± SE of three independent experiments. Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by Duncan's multiple range test (P < 0.05) when appropriated using the statistical software SPSS 10.0 for Windows. 3. Results
2.3. Total RNA extraction, semi-quantitative reverse-transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) analysis
3.1. Expression of bacterial merT gene in transgenic Arabidopsis
Total RNA was isolated by using RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. The first-strand cDNA was synthesized using reverse transcriptase M-MLV (TaKaRa Bio Inc., Dalian, China). qRT-PCR was
The PamerT gene was transferred into Arabidopsis ecotype Col0 using the Agrobacterium-mediated gene transfer method (Supplementary Fig. S1A). After selection with hygromycin and regeneration, 16 independent PamerT-overexpressing plants (T1
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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Fig. 1. Effects of HgCl2 on root elongation and fresh weight in both wild-type (WT) and PamerT-overexpressing Arabidopsis lines. (A) Primary root length and fresh weights of WT and PamerT-overexpressing plants under 10 mM HgCl2 stress for 16 days. Phenotypes (B) and primary root length and fresh weights (C) of WT and PamerT-overexpressing line M5 plants under different concentrations of HgCl2 stress for 16 days. Error bars indicate standard error (n ¼ 3). Different letters above the error bars indicate that the means are statistically different according to Duncan's multiple test (P < 0.05). Asterisk indicates a significant difference between WT versus PamerT-overexpressing plants (M5) according to student's t-test. Bar ¼ 1 cm.
generation) were obtained. Among them, three transgenic lines (M2, M5, and M12) displaying a 3:1 segregation (hygromycin resistant: hygromycin sensitive) were chosen for further experiments and transgene expression was verified by semi-quantitative RT-PCR (Supplementary Fig. S1B). 3.2. Effects of PaMerT on Hg tolerance and accumulation in transgenic Arabidopsis When grown on normal MS medium, PamerT-overexpressing
plants displayed longer primary root and higher fresh weight (Fig. 1A), but neither of them is significantly different from that of WT plants. In the presence of 10 mmol L1 HgCl2, the growth of WT Arabidopsis was remarkably inhibited (Fig. 1A). For example, the root growth of seedling from WT was largely reduced by HgCl2, only 51.85% of the control. In contrast, the roots of transformants (M2, M5 and M12) were 1.60-, 1.69- and 1.51-fold longer than WT roots respectively under 10 mmol L1 HgCl2. The similar result was also found in the fresh weight of seedlings (Fig. 1A). Additionally, WT plants and one of the transgenic lines (M5) were also subjected
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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to different concentrations of HgCl2 (Fig. 1B). The results showed that the root growth of PamerT-overexpressing Arabidopsis appeared as healthy on Hg as on mercury-free control plates, and the transgenic line (M5) is more tolerant than that of WT to Hg stress (Fig. 1C). Moreover, Arabidopais seeds were germinated on MS medium for 7 days. After that, WT and PamerT-overexpressing seedlings (M5 line) were transferred to new MS medium in the presence or absence of 10 mM HgCl2 for 5 days. It was found that PamerToverexpressing line M5 showed more tolerant to Hg compared to the WT Arabidopsis (Fig. 2A). For instance, after 5 days of Hg stress, the WT plants showed a sound decrease of chlorophyll contents (with a 32.75% in chlorophyll a and a 57.06% in chlorophyll b) in contrast to the M5 plants (with a 18.65% in chlorophyll a and a 22.43% in chlorophyll b), in which they remained unaltered when compared to their controls (Fig. 2B). Additionally, although the primary root length of WT and transgenic Arabidopsis seedlings did not differ significantly, overexpressing PamerT in Arabidopsis enhances the lateral root formation (including LR number/density) and total root length under both normal and HgCl2-stressed conditions (Supplementary Fig. S2). 3.3. Effects of HgCl2 treatment on lipid peroxidation, transcripts and activities of antioxidant enzymes To confirm the cytoprotective role of PaMerT in alleviation of Hg-induced oxidative stress, we compared the contents of TBARS in WT and transgenic plants under HgCl2 stress. The results showed that HgCl2 stress caused an increment on TBARS contents in both
WT and transgenic plants, while the increase extent of TBARS in PamerT-overexpressing plants is apparently lower than the WT plants (Fig. 2C). Further, changes in several antioxidant enzymes activities and corresponding transcripts in WT and M5 line seedlings were compared. Using qRT-PCR, six Arabidopsis ROS responsive genes (Supplementary Table S1) were examined at the indicated time of HgCl2 treatment. Transcript levels of each gene monitored are expressed as the fold change compared with the expression level of WT at zero time point (Fig. 3A; Supplementary Fig. S3). Time-dependent analysis showed that CSD1 were induced in both M5 line and WT seedlings upon HgCl2 stress, while stronger induction was observed in M5 line than WT seedlings. Under HgCl2 condition, the expression of FSD1 only increased at the end of the treatment (48 h) in WT seedlings, while in M5 line the expression of FSD1 increased at both the 24-h and 48-h time point. Meanwhile, after 24-h treatment, the differences between WT and M5 line seedlings turn relatively smaller under HgCl2 stress. We further noticed that a significant higher transcript level of PER21 and CAT1 were detected in M5 line plants than in WT plants with the treatment of HgCl2 (Fig. 3A). However, there were no significant differences in the transcript levels of PER21 and CAT1 genes between WT and merT-overexpressing (M5) plants in the absence of HgCl2 except at the 24-h time point (Supplementary Fig. S3). On the other hand, more pronounced changes of PER27 were observed in PamerT-overexpressing (M5) than in WT plants under HgCl2 treatment (Fig. 3A), especially after longer period of treatment (48 h). Additionally, CAT2 mRNA increased first and then decreased, but obvious differences existed between WT and M5 at the end of HgCl2 treatment.
Fig. 2. Mercury tolerance in wild-type (WT) and transgenic Arabidopsis seedlings. 7-d-old WT and PamerT-overexpressing line M5 seedlings were transferred to 10 mM HgCl2 for 5 days. Afterwards, photos of Hg treatment of wild-type and transgenic plants were taken. Statistical analysis of chlorophyll contents (B) and TBARS contents (C) at different treatment time were also determined. Error bars indicate standard error (n ¼ 3). Different letters above the bars indicate that the means are statistically different (P < 0.05). Bar ¼ 1 cm.
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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Fig. 3. Time-dependent changes of the transcripts (A) and activity of antioxidant enzymes (B) in wild-type (WT) and transgenic Arabidopsis seedlings upon HgCl2 stress. 7-d-old WT and PamerT-overexpression line M5 seedlings were transferred to 10 mM HgCl2 for the indicated time. Error bars indicate standard error (n ¼ 3). Different letters above the bars indicate that the means are statistically different according to Duncan's multiple test (P < 0.05).
Activities of antioxidant enzymes in WT and transgenic Arabidopsis substantially changed when they were exposed to HgCl2 (Fig. 3B; Supplementary Fig. S4). The CAT activities increased slightly first and then decreased, but obvious differences existed between WT and M5 at different treatment time. Activity of SOD in WT and M5 showed a similar trend, but the activity of M5 was greater than that of WT under HgCl2-stressed conditions. Activities of POD exhibited a sharp difference compared with activities of CAT and SOD (Fig. 3B). Even though, at the last stage of HgCl2 treatment, POD activities of M5 were much greater than that of WT (Fig. 3B). Additionally, the presence of H2O2 and O 2 in seedlings following HgCl2 treatment for 72 h was also detected in situ (Supplementary Fig. S5). The WT seedling leaves exhibited marked brown and blue coloration after Hg treatment, suggesting more H2O2 and O 2 accumulation compared to PamerT-overexpressing plants.
3.4. Localization of PaMerT expressed in Arabidopsis protoplast To confirm experimentally the subcellular localization of the PaMerT protein, we fused the coding region of PaMerT at the Nterminus of a GFP fragment under the CaMV 35S promoter. After being expressed transiently in Arabidopsis protoplasts, the fluorescence signal of GFP itself was visualized in both cytosol and nucleus (Fig. 4A). By contrast, signal from PaMerT-GFP fusion protein was observed in the cytoplasm (Fig. 4B), and is mostly overlapped with the red fluorescence of AtdTIP-mcherry fusion protein (Fig. 4C). Besides, a part of proportion of PaMerT-GFP co-localized with mRFPeHDEL (Fig. 4D). PaMerT is thus very likely to be associated with vacuole and ER.
4. Discussion As native plant tolerance to Hg is generally low, strategic focus for improving the phytoremediation potential of plants is the integration of genes from other organisms by genetic engineering. Of the known bacterial Hg resistance systems, the Mer determinant is unique in terms of the orientation of the mercury transporter it encodes [4]. The successful reconstruction and implementation of the mer genes has been demonstrated very effectively in transgenic plants for cleaning Hg-contaminated environments [6,15,34]. In this study, a number of independent transgenic Arabidopsis plants overexpressing PamerT were obtained (Supplementary Fig. S1). As Hg causes a significant reduction of plant growth and biomass [34], we also observed that the primary root length, plant fresh weight and amount of chlorophyll were declined upon HgCl2 stress in Arabidopsis (Figs. 1 and 2). And the higher concentration of HgCl2, the much more decrease in the primary root length and plant fresh weight (Fig. 1). On the other hand, it is obvious that the PamerToverexpressing Arabidopsis was much more tolerant than that of WT plants (Figs. 1 and 2). This result indicated that PamerT may offer a protection effect in plants against toxicity of Hg. Although there is no significant difference in primary root length between WT and transgenic Arabidopsis (Fig. 1), it is worth noting that transgenic Arabidopsis plants overexpressing PamerT have more lateral root numbers than the wild type under both normal and HgCl2-stressed conditions (Supplementary Fig. 2). TBARS content was used as an indicator for lipid peroxidation in vivo, and increase in the level of TBARS in response to HgCl2 has been observed in several plants [24e26]. Consequently, related experiments illustrated that PamerT-overexpressing Arabidopsis plants were able to counteract the increases in TBARS content
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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Fig. 4. Localization of PaMerT-GFP fusion protein transiently expressed in Arabidopsis protoplasts. GFP alone (A) or PaMerTeGFP fusion protein (B) expressed in Arabidopsis protoplasts. (C) Protoplasts co-expressing PaMerT-GFP and AtdTIP-mcherry protein. (D) Protoplasts co-expressing PaMerT-GFP and AtHDEL-mcherry protein. The photographs were taken in the green channel (GFP fluorescence), red channel (mCherry fluorescence), combination of green and red channel, and bright channel. Bar ¼ 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
induced by HgCl2 stress (Fig. 2C). Hg triggers oxidative stress by inducing production of ROS, e.g. O 2 , H2O2, and OH in plants [24e26], and subsequently increases the activity of antioxidant enzymes as well as the content of GSH [35]. Our results also indicated that the transcripts and activities of SOD, POD and CAT were pronounced enhanced in transgenic Arabidopsis compared with WT when exposed to HgCl2 (Fig. 3). Additionally, the higher expression of antioxidant enzymes might lead to the suppression of ROS in transgenic Arabidopsis plants (Supplementary Fig. S5). Compartmentalization of metals in the vacuole is considered to be a part of the tolerance mechanism of some metal hyperaccumulators [36]. In this report, the signal from PaMerT-GFP fusion protein expressed in Arabidopsis protoplast also proved that PaMerT is very likely to be localized in vacuole (Fig. 4). Thus, it is possible that PaMerT might be capable of allocating Hg to vacuole where mercury is less toxic. On the other hand, many studies showed that phytochelatins play a central role in Hg detoxification and it showed that vacuolar transport of phytochelatin-Hg complexes is a prerequisite for Hg tolerance [37]. Thus, the determination of phytochelatin contents would be further carried out.
Based on our results, the acceleration of Hg uptake by overexpression of PaMerT and vacuolar compartmentalization might affect the Hg-induced ROS generation and homeostasis. Moreover, integration of merT gene into ppk-transgenic tobacco not only showed more resistance to Hg2þ than its wild-type progenitors but also accelerated and enhanced Hg uptake into tobacco thus accumulated more mercury [38]. It has been revealed that plants possess several classes of metal transporters that must be involved in metal uptake and homeostasis in general and could play a key role in metal tolerance [39]. Whether similar results caused by PamerT overexpression should be further investigated. In summary, the physiological and antioxidative responses of the PamerT-overexpressing Arabidopsis were analyzed under HgCl2 stress. Our data showed that the antioxidative defense in PamerToverexpressing plants is stronger and persists for a longer time under HgCl2 stress. This might suggest a reestablishment of redox homeostasis, in which MerT-mediated responses would be involved. Furthermore, the development of plants express PamerT by genetically engineering might be useful for the efficient phytoremediation of HgCl2-polluted environments.
Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073
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Please cite this article in press as: S. Xu, et al., Overexpression of a bacterial mercury transporter MerT in Arabidopsis enhances mercury tolerance, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.06.073