Appl Biochem Biotechnol DOI 10.1007/s12010-015-1549-7
Engineering Tobacco to Remove Mercury from Polluted Soil S. Chang & F. Wei & Y. Yang & A. Wang & Z. Jin & J. Li & Y. He & H. Shu
Received: 17 November 2014 / Accepted: 9 February 2015 # Springer Science+Business Media New York 2015
Abstract Tobacco is an ideal plant for modification to remove mercury from soil. Although several transgenic tobacco strains have been developed, they either release elemental mercury directly into the air or are only capable of accumulating small quantities of mercury. In this study, we constructed two transgenic tobacco lines: Ntk-7 (a tobacco plant transformed with merT-merP-merB1-merB2-ppk) and Ntp-36 (tobacco transformed with merT-merP-merB1merB2-pcs1). The genes merT, merP, merB1, and merB2 were obtained from the well-known mercury-resistant bacterium Pseudomonas K-62. Ppk is a gene that encodes polyphosphate kinase, a key enzyme for synthesizing polyphosphate in Enterobacter aerogenes. Pcs1 is a tobacco gene that encodes phytochelatin synthase, which is the key enzyme for phytochelatin synthesis. The genes were linked with LP4/2A, a sequence that encodes a well-known linker peptide. The results demonstrate that all foreign genes can be abundantly expressed. The mercury resistance of Ntk-7 and Ntp-36 was much higher than that of the wild type whether tested with organic mercury or with mercuric ions. The transformed plants can accumulate significantly more mercury than the wild type, and Ntp-36 can accumulate more mercury from soil than Ntk-7. In mercury-polluted soil, the mercury content in Ntp-36’s root can reach up to 251 μg/g. This is the first report to indicate that engineered tobacco can not only accumulate mercury from soil but also retain this mercury within the plant. Ntp-36 has good prospects for application in bioremediation for mercury pollution. Keywords Phytoremediation . Mercury . Vacuole . Cytoplasm . Chelatin
Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1549-7) contains supplementary material, which is available to authorized users.
S. Chang : A. Wang : Z. Jin : J. Li : Y. He : H. Shu (*) Haikou Experimental Station, Chinese Academy of Tropical Agricultural Sciences, Haikou, China e-mail: [email protected] S. Chang : A. Wang : Z. Jin : J. Li : Y. He : H. Shu The Key Lab of Hainan Banana Genetics and Breeding, Haikou, China F. Wei : Y. Yang : H. Shu Department of Biology, Zhengzhou University, Zhengzhou, China
Appl Biochem Biotechnol
Introduction Mercury pollution is a global environmental problem. With the development of industry, a large amount of mercury has been released into agricultural soil through wastewater [1]. This mercury enters the human body through food and groundwater [2]. Although several physical and chemical methods have been used to remove mercury from soil, these methods are difficult to popularize because of their inherent defects, such as their expense and high risk of introducing secondary pollution [3]. Comparatively, biological remediation, especially plant remediation, costs much less than physical and chemical methods [4]. Furthermore, secondary pollution is not produced by biological remediation [5]. Tobacco is an ideal plant for removing mercury from soil [6]. It has a large biomass and an extensive root system. Furthermore, it can be easily scaled up and engineered. Pseudomonas K-62 is a bacterium that is well known for its high mercury resistance [7]. This phenotype is 1000 times more resistant to phenylmercury than Escherichia coli or Pseudomonas aeruginosa [7]. The mechanism that provides this bacterial strain with its high mercury resistance originates from its two mer operons [8]. In these mer operons, organic mercury and mercury ions are transferred into the bacteria via the transport proteins merT and merP [9]. Organic mercury can be converted into mercury ions by the organomercurial lyases merB1 and merB2 [9]. Mercury ions can then be transformed into elemental mercury by the mercuric reductase merA [10], and this elemental mercury can be volatilized into the air. With the release of elemental mercury into the air, the toxicity to Pseudomonas K-62 is reduced [10]. If these mer genes can be transferred into plants, the resistance to mercury and mercury accumulation in plants might be enhanced significantly. Engineered plants with these mer genes might be able to remove mercury from the soil. When tobacco plants were grown in soil containing 400 μmol/L phenylmercuric acetate (PMA), the dry weights of plants with merA and merB were significantly higher than those of the wild type under the same treatment [11]. Transgenic tobacco showed an approximately 100-fold more efficient mercury accumulation in shoots relative to untransformed plants [6]. Similar results were also found by Shafiul et al [12]. These engineered plants were able to accumulate large quantities of mercury because the mercury ions were reduced to elemental mercury, which was volatilized into the air [6, 12]. However, the mercury that was volatilized into the air might be recycled into the environment, which would still cause public anxiety. To resolve this problem, a ppk gene encoding polyphosphate kinase, which is a key enzyme for synthesizing polyphosphate that can chelate Hg2+, was transferred into tobacco [13]. Tobacco that expressed ppk exhibited a greater resistance to Hg2+ than wild-type plants [13]. The mercury accumulation of the transgenic plants was approximately 150 nmol/g when they were cultured in a medium containing 5 μmol/L of Hg2+ [13]. However, according to the criteria of the Department of Environment in China, 1.0 mg/kg (or 5 μmol/g) is the threshold level for protecting human health. Even when the transgenic tobacco was cultured in medium containing 10 μmol/L of Hg2+, the mercury accumulation in the engineered plants was still only approximately 170 nmol/g [13]. Thus, transgenic plants with this level of mercury accumulation cannot be used in agricultural soil containing more mercury than the threshold value. As a result, an engineered tobacco with stronger mercury accumulation ability is required. In this paper, two plasmids, p(merT-merP-merB1-merB2-ppk) and p(merT-merP-merB1merB2-pcs1), were constructed and transferred into tobacco. The tobacco lines containing p(merT-merP-merB1-merB2-ppk) were named Ntk, and the tobacco lines containing p(merTmerP-merB1-merB2-pcs1) were named Ntp. MerT, merP, merB1, and merB2 were synthesized according to the corresponding sequence in Pseudomonas K-62. PCS1 is a tobacco gene (GenBank accession number AY235426) that encodes phytochelatin synthase, a key enzyme
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for synthesizing phytochelatin that can chelate heavy metals and detoxify heavy metals in plants [14]. Ntk-7 and Ntp-36, two transgenic lines in which seeds can set normally and grow to plant heights that are similar to the wild type, were selected for the experiments. The results showed that the phenotypes of the two transgenic lines and the wild type were similar when cultivated in soil containing no mercury. However, when grown in soil containing 100 μmol/L of Hg2+, the wild type’s height was much lower than the heights of Ntk-7 and Ntp-36. The plant heights of the two transgenic lines grown in soil containing 100 μmol/L mercury were similar to those grown in soil containing no mercury. The mercury accumulation in Ntp-36 was significantly higher than that of Ntk-7 and the wild type. The mechanisms underlying these phenotypes are also discussed.
Materials and Methods Materials An EHA105 Agrobacterium was used. The tobacco (Nicotiana tabacum, Zhongyan100) used in this paper was donated by the Center of Tobacco Improvement, China (Qingdao, China). The tobacco seeds were surface-sterilized by shaking in 70 % (v/v) ethanol for 30 s followed by shaking in 10 % (v/v) hydrogen peroxide for 10 min and were subsequently washed five times in sterile double-distilled water. The surface-sterilized seeds were transferred to plates containing half-strength MS medium with 0.3 % (w/v) phytogel (pH 5.7). The plates were incubated in a growth chamber with a controlled temperature (22–24 °C), humidity (75–90 %), and light (light intensity 750 μEm−2; 14 h/day/10 h/night). Constructing Ntp and Ntk According to the sequences of plasmid pMR26 (GenBank accession number D83080.2), plasmid pMR28 (GenBank accession number AB013925.1), ppk (D14445.1), and LP4/2A [15], the gene cluster of merT-merP-merB1-merB2-ppk (Fig. S1) was synthesized and inserted into the pRI101-AN plasmid (Takara, Dalian, China). The resulting plasmid was named p(merT-merP-merB1-merB2-ppk). According to the sequence of the pcs1 gene (GenBank accession number AY235426.1), primers NPCF2 (5′-AAATATGCGGCCGCAATGGCGATG GCGGGTTT-3′) and NPCR2 (5′-AAATATGCGGCCGCGGCAAAGCTAGAAGGGAG-3′) were designed. Polymerase chain reaction (PCR) was performed on the complementary DNA (cDNA) from a tobacco leaf. The PCR fragment was purified from an agarose gel and digested with Not I. The p(merT-merP-merB1-merB2-ppk) plasmid was also digested with Not I. The digested PCR fragment was ligated with p(merT-merP-merB1-merB2-ppk) by using a T4 DNA ligase at 4 °C for 24 h. The ligated plasmids were transformed into E. coli DH10B. The positive clones were cultured, and the plasmids were extracted. The plasmids were digested with Not I. The plasmids that could be digested into two bands where one band had a molecular weight of approximately 0.6 kb were sent to Takara for sequencing. The plasmids in which pcs1 had been inserted in the same transcription direction as that of merT were named p(merT-merP-merB1-merB2-pcs1) (Fig. S2). The p(merT-merP-merB1-merB2-ppk) and p(merT-merP-merB1-merB2-pcs1) plasmids were transferred into Agrobacterium EHA105 via the standard procedure [16]. Two-week-old tobacco leaves were cut into 4–6-mm pieces and cocultured with YEB-grown Agrobacterium following standard method. The explants were placed on sterile filter paper to remove the redundant bacteria and cultured on a differentiation medium (MS+2 mg/L 6-BA+0.5 mg/L IAA) in the dark for 2–4 days. The
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explants were then transferred to a differentiation medium containing 300 mg/L cefotaxime sodium and 50 mg/L kanamycin. The explants were maintained at 25 °C with a 16-h light/8-h dark schedule until seedling clumps appeared. The seedlings were cut from the base and transferred onto root medium (MS+0.5 mg/L IAA+300 mg/L cef+50 mg/L kanamycin). After sufficient roots had appeared, the seedlings were transferred to jars containing potting soil for further growth and development in the greenhouse and cultured continuously. The potting soil was obtained from our experimental base, located in Fushan, Chengmai County, Hainan Province, China. Surface soil (0 to 15 cm) was collected, air-dried, and ground to pass a 2-mm sieve. Nitrogen, phosphorous, and potassium (N/P/K=1:1:1) were added to soils as base fertilizers. Identifying the Transgenic Lines The leaves of the transgenic plants were collected, and genomic DNA was extracted. PCR was performed by using primers specific to merB1 (PF: 5′-ATGGACAAGACTATTTATTCCA AAA-3′ and PR: 5′-TACTGGGCTTTCCTCGCAGTCCTCT-3′). If one 0.6-kb band could be amplified, the plant had been transformed successfully. To determine whether all the foreign genes could be expressed normally in the transgenic plant, total RNA was extracted from the leaves of the transgenic plants with a Takara MiniBEST Plant RNA Extraction Kit (Takara, Dalian, China). The cDNA was synthesized with a RevertAid First Strand cDNA synthesis kit (Fermentas, http://www.thermoscientificbio.com/fermentas). MerB1, merB2, merT, merP, and ppk were amplified from the cDNA of Ntk. MerB1, merB2, merT, merP, and pcs were amplified from the cDNA of Ntp. The primers used in these PCR reactions are shown in Table 1. Mercury Accumulation Measurements in Plants and Soils The mercury contents of different tissues were measured according to a previously published method [17]. Immediately prior to flowering, the tobacco leaves or roots were harvested and washed thoroughly in distilled water. The shoots and roots were separated and immersed directly in liquid nitrogen. The frozen plants were dried using a freeze-dryer, and the dry weight was determined. After being ground to a fine powder under liquid nitrogen, the samples Table 1 Primers used to identify transgenic tobacco
Primer name
sequence
PTF
5′-ATGTCTGAACCACAAAACGGGC-3′
PTR PPF
5′-ATAGAAAAATGGAACGACATAG-3′ 5′-ATGAAGAAACTGTTTGCCTC-3′
PPR
5′-CTTCTTCAGCTCAGATGGGT-3′
PB1F
5′-ACAAGACTATTTATTCCAAAA-3′
PB1R
5′-ACATGACAGCAAAACGAACAA-3′
PB2F
5′-ATGAAGCTCGCCCCATATATT-3′
PB2R
5′-CGATTAAACTCCTGGCCCAAG-3′
PPKF
5′-ATGGGTCAGGAAAAGTTATATATCG-3′
PPKR PPCSF
5′-ATATCGACAAAGAACTCAGTAACCG-3′ 5′-ATTGTGGTTTGGCTAGCCTTTCTAT-3′
PPCSR
5′-CCTTGTTATCCTTGCATTTCTTGAG-3′
Appl Biochem Biotechnol
(three replicates of each treatment) were acid digested by step-wise additions of 70 % (v/v) nitric acid, 30 % (v/v) hydrogen peroxide, and concentrated HCl. The mercury content was then determined via flameless cold-vapor atomic adsorption spectrometry with an atomic mercury analyzer (RA-2A, Nippon Instruments, Tokyo, Japan). To measure the mercury content in the soil, soil samples were digested with 4 mol/L nitric acid. The other steps were the same as that for measuring the mercury content in plant samples. Mercury Resistance Measurements in Plants The tobacco seeds were surface-sterilized by shaking them in 10 % hydrogen peroxide for 10 min. They were then washed five times in sterile water. Subsequently, the tobacco seeds were germinated on an MS agar medium for 3 weeks. Some of the seedlings were then transferred to an MS liquid medium containing 10 μmol/L mercuric chloride or methylmercury chloride and cultured for 1 week. The remaining seedlings were transferred to an MS liquid medium without mercury to act as controls. The fresh wet weights of the seedlings were measured. The other method for measuring plant mercury tolerance was performed as follows. The surface-sterilized tobacco seeds were germinated on MS agar medium for 3 weeks. The seedlings were then transferred to soil (50/50 sand and potting soil) in a greenhouse at 22 °C with 16 h of light. All pots were watered twice per week with half-strength Hoagland’s solution. Immediately prior to flowering, the plant heights and fresh wet weights of the aboveground tissues were measured. All measurements were performed for three replicates, and the average values were used. Mercury-Transfer Capacity Measurement in Plants The soil was obtained from our experimental base, located in Fushan, Chengmai County, Hainan Province, China. Surface soil (0 to 15 cm) was collected, air-dried, and ground to pass a 2-mm sieve. Nitrogen, phosphorous, and potassium (N/P/K=1:1:1) were added to the soils as base fertilizers. Mercury chloride was added as a powder to the soil at final concentration of 21 mg/kg. Approximately 50 kg of such soil was transferred into 50-cm diameter 50-cm high plastic pots. For organic mercury treatment, methylmercury chloride was added to the soil at final concentration of 19 mg/kg. Approximately 50 kg of such soil was transferred into 50-cm diameter 50-cm high plastic pots. The tobacco seeds were germinated in vermiculite at a nursery site in a greenhouse at 22 °C with 16 h of light. After 1 month, each seedling was transferred to an individual 7-cm-diameter 13-cm-high plastic cup containing vermiculite. After 1 month, each seedling was transferred to an individual 50-cm diameter 50-cm high plastic pot containing soil treated with mercury chloride or methylmercury chloride. The pots were placed in greenhouse at 22 °C with 16 h of light. The soil was watered once every 2 weeks. Immediately before flowering, the tobacco plants were removed from the pots. The mercury content in the soil was measured as described above.
Results Tobacco Transgenic Lines Ntk and Ntp Were Developed As described in the BMaterials and Methods,^ the fusion genes merT-merP-merB1-merB2-ppk (Fig. S1) and merT-merP-merB1-merB2-pcs1 (Fig. S2) were constructed. The genes were fused with the LP4/2A linker (Figs. S1 and S2). The fusion genes were then cloned into
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plasmid vector pRI101-AN (Takara) (Figs. 1 and 2). The resulting plasmids were named p(merT-merP-merB1-merB2-ppk) and p(merT-merP-merB1-merB2-pcs1). The tobacco transformed with p(merT-merP-merB1-merB2-ppk) was named Ntk (Fig. 3), and the tobacco transformed with p(merT-merP-merB1-merB2-pcs1) was named Ntp (Fig. 4). For each vector, 50 seedlings were selected from the root medium containing kanamycin (50 mg/L) and cultured in soil. Approximately two thirds of the Ntk plants and approximately half of the Ntp plants were clearly shorter than the wild type. Ten Ntp plants and 15 Ntk plants flowered and set seeds normally. Ntk-7 and Ntp-36, two transgenic lines that can set seeds normally and had plant heights similar to that of the wild type, were selected for the following experiments. To identify whether foreign genes were expressed in these two transgenic lines, PCRs were performed from cDNA that was re-transcribed from the total RNA isolated from the transformed seedlings. The results showed that all the foreign genes can be amplified in the selected plants, and there was no corresponding band when the PCR was performed on the wild-type cDNA (Figs. 5 and 6). Thus, all the foreign genes could be expressed successfully in the selected transgenic plants. The Transgenic Lines Had Mercury Tolerance Levels That Were Significantly Higher than That of the Wild Type The results showed that the phenotypes of Ntk-7, Ntp-36, and the wild type were similar when grown in soil without mercury (data not shown). However, when they were grown in soil containing 100 μmol/L mercuric chloride, the plant heights and the fresh weights of the wild type were significantly lower than those of the transgenic lines (Figs. 7 and 8). The phenotypes of Ntk-7 and Ntp-36 were similar when grown in soil containing 100 μmol/L mercuric chloride (Figs. 7 and 8), demonstrating that they had similar mercury tolerances. To explore the transgenic lines’ mercury tolerance, their seeds were germinated on MS agar medium for
Fig. 1 The gene clusters were purified from a 0.8 % agarose gel. M1 is a λ-Hind III DNA marker (Takara, Dalian, China). M2 is a DL2000 DNA marker (Takara). Lane 1 is merT-merP-merB1-merB2-ppk. Lane 2 is merT-merP-merB1-merB2-pcs1
Appl Biochem Biotechnol Fig. 2 The original pRI101-AN vector was digested with Not I. M2 is a λ-Hind III DNA marker (Takara). Lane 1 is the original pRI101-AN vector as digested with Not I
Fig. 3 Ntk seedlings grown on a root medium containing kanamycin (50 mg/L)
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Fig. 4 Ntp seedlings grown on a root medium containing kanamycin (50 mg/L)
3 weeks. Some of the seedlings were transferred to an MS liquid medium containing 10 μmol/ L mercuric chloride or 10 μmol/L methylmercury chloride and cultured for 1 week. The remaining seedlings were transferred to an MS liquid medium without mercury as controls. The fresh wet weights of the seedlings were measured. The results showed that the fresh wet weights of the wild type, Ntp-36, and Ntk-7 cultured in an MS liquid medium without mercury were similar (Fig. 9). However, for seedlings cultured in medium containing 10 μmol/L mercuric chloride, the fresh wet weight of the wild type was significantly lower than the
Fig. 5 Identifying the expressions of foreign genes in the transgenic Ntk-7 line. M is the DNA marker DM10000 (CWBIO, Beijing, China). Lanes 1 and 2 show the PCR results for primers PTF and PTR. Lanes 3 and 4 show the PCR results for primers PPF and PPR. Lanes 5 and 6 show the PCR results for primers PB1F and PB1R. Lanes 7 and 8 show the PCR results for primers PB2F and PB2R. Lanes 9 and 10 show the PCR results for primers PPPKF and PPPKR. Lanes 1, 3, 5, 7, and 9 show the PCR results for the cDNA that was re-transcribed from the total RNA isolated from the transgenic line Ntk-7. Lanes 2, 4, 6, 8, and 10 show the PCR results for the cDNA that was re-transcribed from the total RNA isolated from the wild-type tobacco
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Fig. 6 Identifying the expressions of foreign genes in the Ntp-36 transgenic line. M is DNA marker DM10000 (CWBIO). Lanes 1 and 2 show the PCR results for primers PTF and PTR. Lanes 3 and 4 show the PCR results for primers PPF and PPR. Lanes 5 and 6 show the PCR results for primers PB1F and PB1R. Lanes 7 and 8 show the PCR results for primers PB2F and PB2R. Lanes 9 and 10 show the PCR results for primers PPCSF and PPCS R. Lanes 1, 3, 5, 7, and 9 show the PCR results for the cDNA that was re-transcribed from the total RNA isolated from the Ntp-36 transgenic line. Lanes 2, 4, 6, 8, and 10 show the PCR results for the cDNA that was retranscribed from the total RNA isolated from the wild-type tobacco
weights of Ntk-7 and Ntp-36 (Fig. 9). There was no definite difference between the fresh wet weight of Ntk-7 and that of Ntp-36 (Fig. 9). For the seedlings cultured in the medium containing 10 μmol/L methylmercury chloride, the fresh wet weight of the plants can be ordered from lowest to highest as follows: wild type, Ntk-7, and Ntp-36 (Fig. 10). The fresh wet weights of Ntp-36 cultured in the medium containing 10 μmol/L mercuric chloride or methylmercury chloride were similar to those of Ntp-36 cultured in medium without mercury (Figs. 9 and 10). These findings demonstrate that the transgenic tobacco had a higher mercury tolerance than the wild type. The mercury tolerance of plants can be ordered from lowest to highest as follows: wild type, Ntk-7, and Ntp-36. The Mercury Accumulation in Ntp-36 Was Significantly Greater than the Accumulation in the Wild Type and Ntk-7 Immediately prior to flowering, the mercury contents of the tobacco strains grown in soil containing 100 μmol/L mercuric chloride were measured. The results showed that the mercury accumulation was much greater in the transgenic lines than in the wild type (Fig. 11). For example, the mercury content in the roots of the wild type was approximately 80 μg/g, and the values of Ntk-7 and Ntp-36 were 213 and 251 μg/g, respectively (Fig. 11). Furthermore, the mercury accumulation in Ntp-36 was significantly higher than that in Ntk-7. The mercury content in the Ntk-7 roots was much higher than that in the leaves (Figs. 11 and 12). This trend was also observed in the wild type and Ntp-36 (Figs. 11 and 12). These findings demonstrate that most mercury accumulated in the plant roots. Only a small portion of the mercury was transferred to the aboveground tissues. The mercury-accumulation ability of Ntp-36 was
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Fig. 7 The fresh wet weights of plants grown in soil containing 100 μmol/L mercuric chloride compared to the controls (no mercury). The data were collected after the plants flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
greater than that of Ntk-7. Thus, the transgenic plants can accumulate much more mercury than the wild type. Ntp-36 and Ntk-7 Can Take Up Organic Mercury from the Soil in Addition to Inorganic Mercury To identify whether merB1 and merB2 played roles in the transgenic lines, the mercury content of plants grown in soil containing methylmercury chloride was also measured. The results showed that transgenic plants can accumulate much more mercury from soil containing organic mercury than the wild type (Figs. 13 and 14). For example, the mercury content of the roots from the wild type grown in soil containing 100 μmol/L methylmercury chloride was approximately 75 μg/g, and the values of Ntk-7 and Ntp-36 were 202 and 248 μg/g, respectively (Fig. 13). Although the corresponding data in leaves were lower than the concentrations in the roots, the mercury contents in the leaves of the transgenic tobacco were
Fig. 8 The heights of plants grown in soil containing 100 μmol/L mercuric chloride compared to the controls (no mercury). The data were collected after the plants flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
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Fig. 9 The fresh wet weights of seedlings cultured in medium containing 10 μmol/L mercuric chloride compared to the controls (no mercury). The data were collected 10 days after the seedlings were transferred to liquid medium with 10 μmol/L mercuric chloride. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp36, respectively
also significantly higher than that of the wild type (Fig. 14). These findings demonstrated that merB1 and merB2 played roles in the transgenic plants. Organic mercury can be reduced into mercuric ions in the transgenic plants. The mercury accumulation in the root of Ntp-36 plants grown in soil containing 100 μmol/L methylmercury chloride was significantly higher than that found in the roots of Ntk-7 with the same treatment (Fig. 13). Similar results were also obtained from the leaves of Ntp-36 and Ntk-7 (Fig. 14). These findings demonstrated that Ntp36 was superior to Ntk-7 in the removal of organic mercury from soil. Ntp-36′ Mercury-Transfer Efficiency Was Higher than Those of Ntk-7 and Wild Type The results showed that after tobacco plants were grown, the mercury content in the soil in which the wild-type seedlings were grown was slightly lower than that in which no plant was
Fig. 10 The fresh wet weights of seedlings cultured in medium containing 10 μmol/L methylmercury chloride compared to the controls (no mercury). The data were collected 10 days after the seedlings were transferred to a liquid medium with 10 μmol/L methylmercury chloride. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
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Fig. 11 The mercury contents in the roots of plants grown in soil containing 100 μmol/L mercuric chloride. The data were collected immediately before the tobacco flowered. Items 1, 2, and 3 correspond to the wild type, Ntk7, and Ntp-36, respectively
grown (Table 2). The mercury transfer ratio (MTR) of the wild-type tobacco grown in soil appended with mercury chloride was only approximately 2.38 % (Table 2). In the soil appended with methylmercury chloride, the MTR of the wild type was also only approximately 2.22 % (Table 2). The MTR of Ntk-7 for mercury chloride was approximately 14.95 % (Table 2). Although this value was higher than that of the wild type, it is lower than that of Ntp36 (Table 2). For plants grown in soil containing methylmercury chloride, the order of MTRs from the highest to the lowest was Ntp-36, Ntk-7, and wild type (Table 2). This demonstrated that Ntp-36 had the best potential for mercury removal from soil.
Discussion Tobacco is an ideal plant for removing mercury from polluted soil. However, although several engineered tobaccos have been constructed, they cannot be used for practical applications [6, 11–13]. Some transgenic plants release elemental mercury directly into the air [6, 11, 12], which can cause public anxiety. Additionally, the mercury accumulation of some transgenic plants is worse than expected [13]. The ideal engineered tobacco should at a minimum meet the following two criteria: The tobacco should be able to (1) take up organic mercury and
Fig. 12 The mercury contents in the shoots of plants grown in soil containing 100 μmol/L mercuric chloride. The data were collected immediately before the tobacco flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
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Fig. 13 The mercury contents in the roots of tobacco strains grown in soil containing 100 μmol/L methylmercury chloride. The data were collected immediately before the tobacco flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
mercuric ions from the soil and (2) store mercury in the plant without discharging it into the environment. In the cells of the two transgenic lines developed for this paper (Ntk-7 and Ntp36), merT and merP enhance the uptake of mercuric ion from the soil by plants [9]. Organic mercury can be reduced by merB1 and merB2 [9]. Polyphosphate, which was synthesized by ppk, binds with the mercuric ions and form chelatins [13]. The toxicity of Hg2+-chelatins was lower than that of Hg2+. Phytochelatins, which were encoded by pcs1, can also bind with mercuric ions and form chelatins [14]. Hg2+-phytochelatins can be transferred into the vacuole, further reducing the mercury toxicity [18]. The mercury resistance of Ntk-7 and Ntp-36 was much higher than that of the wild type with regard to both organic mercury and mercuric ions. The transformed plants grown in soil containing mercury accumulated significantly more mercury than the wild type grown in soil with the same treatment. The transformed plants can remove more mercury from the mercury-polluted soil. Thus, these engineered plants have good prospects for use in removing mercury from the soil. The engineering of metabolic pathways in plants has often required the expression of more than one gene [19]. The use of linker peptides to construct fusion genes is considered to be a good method for multiple gene transformation [15]. LP4/2A is a hybrid linker that contains the first amino acids of LP4 and 20 amino acids from 2A [20]. After translation, the amino acid residues of the linker will be removed from the mature protein by protease in cytoplasm [15].
Fig. 14 The mercury contents in the shoots of tobacco strains grown in soil containing 100 μmol/L methylmercury chloride. The data were collected immediately before the tobacco flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
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Table 2 Transgenic plants’ mercury-transfer efficiency
Treatment
Initial Hg in soil (mg/kg)
Final Hg in soil (mg/kg)
Mercury transfer ration (%)
WT (HgC12)
21
20.5
WT (CH3HgC1)
19
18.6
2.38 2.22
Ntk-7 (HgC12)
21
17.9
14.95
Ntk-7 (CH3HgC1)
19
17.4
8.42
Ntp-36 (HgC12)
21
17.3
17.62
Ntp-36 (CH3HgC1)
19
15.1
21.58
We found that all the foreign genes in the transgenic plants are well expressed. The expression level of one gene was not significantly different from those of other genes in the cluster, and all genes in the clusters can be expressed simultaneously. These findings demonstrated that the strategy used in this paper for multiple gene transference was successful. The phenotypes of the transgenic lines grown in soil containing mercury or cultured in media containing mercury also confirmed this conclusion. The ppk gene was originally isolated from Enterobacter aerogenes [21]; thus, it is a foreign gene for tobacco. Although polyphosphate kinase (ppk) can catalyze the synthesis of polyphosphate in the plant’s cytoplasm, there might not be any proteins responsible for transporting polyphosphate across the tonoplast in plant cells. Thus, polyphosphate-Hg2+ chelatins might be locked in the cytoplasm. Although the toxicity of polyphosphate-Hg2+ was lower than that of mercury ions, the mercury in these chelatins can still harm the organelles in the cytoplasm. Phytochelatins are native proteins for plants. Therefore, certain proteins located within the tonoplast must be present to transfer phytochelatin-Hg2+ across the vacuolar membrane. Recently, Atabcc1/atabcc2 was cloned and considered to be the candidate genes responsible for transferring phytochelatin-Hg2+ across the tonoplast [18]. With aid of these genes, phytochelatin-Hg2+ chelatins can be transferred into the vacuole and stored there. Because there are no important organelles in the vacuole and the bioactivity of this organelle is very low, the phytochelatin-Hg2+ inside the vacuole will not significantly harm the plant cell. Cells containing phytochelatin-Hg2+ had greater chances of survival than those containing polyphosphate-Hg2+. Plants transformed with pcs1 were more likely to survive and can accumulate more mercury than those transformed with ppk. Our experiments showed that although the mercury resistance of Ntk-7 grown in soil containing mercuric ions was similar to that of Ntp-36 grown in similar soil, the mercury resistance of Ntp-36 grown in soil containing methylmercury chloride was significantly higher than that of Ntk-7 grown under the same conditions. Additionally, the MTR of Ntp-36 for methylmercury chloride was approximately 21.58 %, while that value of Ntk-7 was only approximately 8.42 %. For removing mercury chloride, the MTR of Ntp-36 was also higher than that of Ntk-7. Thus, the mercury removal efficiency of Ntp-36 was better than that of Ntk-7. In our experiments, we found that although the transgenic tobacco plants accumulated much more mercury than the wild type, most of the mercury accumulated in the roots. Similar results were also found in the wild-type plants. But, it is difficult to manually remove tobacco roots from soil. Furthermore, more than 70 % of the biomass of the tobacco plant is in aboveground tissues. If the mercury in the roots cannot be transferred to the aboveground tissues, the utility of these transgenic plants to farmers will be debatable. The approaches needed to overcome this defect and capitalize on the advantages of transgenic plants require further studies.
Appl Biochem Biotechnol Acknowledgments This work was supported by the National Science Foundation of China (No. 41201309).
References 1. Wu, G. H., & Cao, S. S. (2010). Mercury and cadmium contamination of irrigation water, sediment, soil and shallow groundwater in a waste water-irrigated field in Tianjin. China Bulletin Environmental Contamination and Toxicology, 84, 336–341. 2. Cachada, A., Rodrigues, S. M., Mieiro, C., da Ferreira Silva, E., Pereira, E., & Duarte, A. C. (2009). Controlling factors and environmental implications of mercury contamination in urban and agricultural soils under a longterm influence of a chlor-alkali plant in the North–West Portugal. Environmental Geology, 57, 91–98. 3. Dunlevy, S. R., Singleton, D. R., & Aitken, M. D. (2013). Biostimulation reveals functional redundancy of anthracene-degrading bacteria in polycyclic aromatic hydrocarbon-contaminated soil. Environmental Engineering Science, 30(11), 697–705. 4. Sorkhoh, N. A., Ali, N., Al-Awadhi, H., Dashti, N., Al-Mailem, D. M., Eliyas, M., & Radwan, S. S. (2010). Phytoremediation of mercury in pristine and crude oil contaminated soils: contributions of rhizobacteria and their host plants to mercuryremoval. Ecotoxicology and Environmental Safety, 73(8), 1998–2003. 5. Francesca, P., Gianniantonio, P., Meri, B. E. T., Paolo, A., & Leonardo, P. (2011). Mercury mobilization in a contaminated Industrial soil for phytoremediation. Communications in Soil Science and Plant Analysis, 42(22), 2767–2777. 6. Hussein, H. S., Ruiz, O. N., Terry, N., & Daniell, H. (2007). Phytoremediation of mercury and organomercurials in chloroplast transgenic plants: enhanced root uptake, translocation to shoots, and volatilization. Environmental Science & Technology, 41(24), 8439–8446. 7. Kiyono, M., Uno, Y., Omura, T., & Pan-Hou, H. (2000). Role of merT and merP from Pseudomona K-62 plasmid pmr26 in the transport of phenylmercury. Biological & Pharmaceutical Bulletin, 23, 279–282. 8. Kiyono, M., Omura, T., Fujimori, H., & Pan-Hou, H. (1995). Organomercurial resistance determinants in Pseudomonas K-62 are present on two plasmids. Archives of Microbiology, 163, 242–247. 9. Kiyono, M., & Pan-Hou, H. (2006). Genetic engineering of bacteria for environmental remediation of mercury. Journal of Health Science, 52(3), 199–204. 10. Osborn, A. M., Bruce, K. D., Strike, P., & Ritchie, D. A. (1997). Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon. FEMS Microbiology Reviews, 19, 239–262. 11. Ruiz, O. N., Hussein, H. S., Terry, N., & Daniell, H. (2003). Phytoremediation of organomercurial compounds via chloroplast genetic engineering. Plant Physiology, 132(3), 1344–1352. 12. Shafiul, H., Zeyaullah, M., Nabi, G., Srivastava, P. S., & Ali, A. (2010). Transgenic tobacco plant expressing environmental E.coli merA gene for enhanced volatilization of ionic mercury. Journal of Micriobiology and Biotechnolology, 20(5), 917–924. 13. Nagata, T., Ishikawa, C., Kiyono, M., & Pan-Hou, H. (2006). Accumulation of mercury in transgenic tobacco expressing bacterial polyphosphate. Biological & Pharmaceutical Bulletin, 29(12), 2350–2353. 14. Hirata, K., Tsuji, N., & Miyamoto, K. (2005). Biosynthetic regulation of phytochelatins, heavy metal-binding peptides. Journal of Bioscience and Bioengineering, 100(6), 593–599. 15. Sun, H., Lang, Z., Zhu, L., & Huang, D. (2012). Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation. Plant Cell Reports, 31, 1877–1887. 16. Weigel D and Glazebrook J. (2002). Arabidopsis, a laboratory manual. Cold Spring Harbor Laboratory Press. page 123-124. 17. Nagata, T., Nakamura, A., Akizawa, T., & Pan-Hou, H. (2009). Genetic engineering of transgenic tobacco for enhanced uptake and bioaccumulation of mercury. Biological & Pharmaceutical Bulletin, 32(9), 1491–1495. 18. Park, J., Song, W., Ko, D., Eom, Y., Hansen, T. H., Schiller, M., Lee, T. G., Martinoia, E., & Lee, Y. (2012). The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury. The Plant Journal, 69, 278–288. 19. Bock, R. (2013). Strategies for metabolic pathway engineering with multiple transgenes. Plant Molecular Biology. doi:10.1007/s11103-013-0045-0. 20. Francois, I. E., Hemelrijck, W. V., & Aerts, A. M. (2004). Processing in Arabidopsis thaliana of a heterologous polyprotein resulting in differential targeting of the individual plant defensins. Plant Science, 166(1), 113–121. 21. Kato, J., Yamamoto, T., Yamada, K., & Ohtake, H. (1993). Cloning, sequence and characterization of the polyphosphate kinase-encoding gene (ppk) of Klebsiella aerogenes. Gene, 137(2), 237–242.
Engineering Tobacco to Remove Mercury from Polluted Soil S. Chang & F. Wei & Y. Yang & A. Wang & Z. Jin & J. Li & Y. He & H. Shu
Received: 17 November 2014 / Accepted: 9 February 2015 # Springer Science+Business Media New York 2015
Abstract Tobacco is an ideal plant for modification to remove mercury from soil. Although several transgenic tobacco strains have been developed, they either release elemental mercury directly into the air or are only capable of accumulating small quantities of mercury. In this study, we constructed two transgenic tobacco lines: Ntk-7 (a tobacco plant transformed with merT-merP-merB1-merB2-ppk) and Ntp-36 (tobacco transformed with merT-merP-merB1merB2-pcs1). The genes merT, merP, merB1, and merB2 were obtained from the well-known mercury-resistant bacterium Pseudomonas K-62. Ppk is a gene that encodes polyphosphate kinase, a key enzyme for synthesizing polyphosphate in Enterobacter aerogenes. Pcs1 is a tobacco gene that encodes phytochelatin synthase, which is the key enzyme for phytochelatin synthesis. The genes were linked with LP4/2A, a sequence that encodes a well-known linker peptide. The results demonstrate that all foreign genes can be abundantly expressed. The mercury resistance of Ntk-7 and Ntp-36 was much higher than that of the wild type whether tested with organic mercury or with mercuric ions. The transformed plants can accumulate significantly more mercury than the wild type, and Ntp-36 can accumulate more mercury from soil than Ntk-7. In mercury-polluted soil, the mercury content in Ntp-36’s root can reach up to 251 μg/g. This is the first report to indicate that engineered tobacco can not only accumulate mercury from soil but also retain this mercury within the plant. Ntp-36 has good prospects for application in bioremediation for mercury pollution. Keywords Phytoremediation . Mercury . Vacuole . Cytoplasm . Chelatin
Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1549-7) contains supplementary material, which is available to authorized users.
S. Chang : A. Wang : Z. Jin : J. Li : Y. He : H. Shu (*) Haikou Experimental Station, Chinese Academy of Tropical Agricultural Sciences, Haikou, China e-mail: [email protected] S. Chang : A. Wang : Z. Jin : J. Li : Y. He : H. Shu The Key Lab of Hainan Banana Genetics and Breeding, Haikou, China F. Wei : Y. Yang : H. Shu Department of Biology, Zhengzhou University, Zhengzhou, China
Appl Biochem Biotechnol
Introduction Mercury pollution is a global environmental problem. With the development of industry, a large amount of mercury has been released into agricultural soil through wastewater [1]. This mercury enters the human body through food and groundwater [2]. Although several physical and chemical methods have been used to remove mercury from soil, these methods are difficult to popularize because of their inherent defects, such as their expense and high risk of introducing secondary pollution [3]. Comparatively, biological remediation, especially plant remediation, costs much less than physical and chemical methods [4]. Furthermore, secondary pollution is not produced by biological remediation [5]. Tobacco is an ideal plant for removing mercury from soil [6]. It has a large biomass and an extensive root system. Furthermore, it can be easily scaled up and engineered. Pseudomonas K-62 is a bacterium that is well known for its high mercury resistance [7]. This phenotype is 1000 times more resistant to phenylmercury than Escherichia coli or Pseudomonas aeruginosa [7]. The mechanism that provides this bacterial strain with its high mercury resistance originates from its two mer operons [8]. In these mer operons, organic mercury and mercury ions are transferred into the bacteria via the transport proteins merT and merP [9]. Organic mercury can be converted into mercury ions by the organomercurial lyases merB1 and merB2 [9]. Mercury ions can then be transformed into elemental mercury by the mercuric reductase merA [10], and this elemental mercury can be volatilized into the air. With the release of elemental mercury into the air, the toxicity to Pseudomonas K-62 is reduced [10]. If these mer genes can be transferred into plants, the resistance to mercury and mercury accumulation in plants might be enhanced significantly. Engineered plants with these mer genes might be able to remove mercury from the soil. When tobacco plants were grown in soil containing 400 μmol/L phenylmercuric acetate (PMA), the dry weights of plants with merA and merB were significantly higher than those of the wild type under the same treatment [11]. Transgenic tobacco showed an approximately 100-fold more efficient mercury accumulation in shoots relative to untransformed plants [6]. Similar results were also found by Shafiul et al [12]. These engineered plants were able to accumulate large quantities of mercury because the mercury ions were reduced to elemental mercury, which was volatilized into the air [6, 12]. However, the mercury that was volatilized into the air might be recycled into the environment, which would still cause public anxiety. To resolve this problem, a ppk gene encoding polyphosphate kinase, which is a key enzyme for synthesizing polyphosphate that can chelate Hg2+, was transferred into tobacco [13]. Tobacco that expressed ppk exhibited a greater resistance to Hg2+ than wild-type plants [13]. The mercury accumulation of the transgenic plants was approximately 150 nmol/g when they were cultured in a medium containing 5 μmol/L of Hg2+ [13]. However, according to the criteria of the Department of Environment in China, 1.0 mg/kg (or 5 μmol/g) is the threshold level for protecting human health. Even when the transgenic tobacco was cultured in medium containing 10 μmol/L of Hg2+, the mercury accumulation in the engineered plants was still only approximately 170 nmol/g [13]. Thus, transgenic plants with this level of mercury accumulation cannot be used in agricultural soil containing more mercury than the threshold value. As a result, an engineered tobacco with stronger mercury accumulation ability is required. In this paper, two plasmids, p(merT-merP-merB1-merB2-ppk) and p(merT-merP-merB1merB2-pcs1), were constructed and transferred into tobacco. The tobacco lines containing p(merT-merP-merB1-merB2-ppk) were named Ntk, and the tobacco lines containing p(merTmerP-merB1-merB2-pcs1) were named Ntp. MerT, merP, merB1, and merB2 were synthesized according to the corresponding sequence in Pseudomonas K-62. PCS1 is a tobacco gene (GenBank accession number AY235426) that encodes phytochelatin synthase, a key enzyme
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for synthesizing phytochelatin that can chelate heavy metals and detoxify heavy metals in plants [14]. Ntk-7 and Ntp-36, two transgenic lines in which seeds can set normally and grow to plant heights that are similar to the wild type, were selected for the experiments. The results showed that the phenotypes of the two transgenic lines and the wild type were similar when cultivated in soil containing no mercury. However, when grown in soil containing 100 μmol/L of Hg2+, the wild type’s height was much lower than the heights of Ntk-7 and Ntp-36. The plant heights of the two transgenic lines grown in soil containing 100 μmol/L mercury were similar to those grown in soil containing no mercury. The mercury accumulation in Ntp-36 was significantly higher than that of Ntk-7 and the wild type. The mechanisms underlying these phenotypes are also discussed.
Materials and Methods Materials An EHA105 Agrobacterium was used. The tobacco (Nicotiana tabacum, Zhongyan100) used in this paper was donated by the Center of Tobacco Improvement, China (Qingdao, China). The tobacco seeds were surface-sterilized by shaking in 70 % (v/v) ethanol for 30 s followed by shaking in 10 % (v/v) hydrogen peroxide for 10 min and were subsequently washed five times in sterile double-distilled water. The surface-sterilized seeds were transferred to plates containing half-strength MS medium with 0.3 % (w/v) phytogel (pH 5.7). The plates were incubated in a growth chamber with a controlled temperature (22–24 °C), humidity (75–90 %), and light (light intensity 750 μEm−2; 14 h/day/10 h/night). Constructing Ntp and Ntk According to the sequences of plasmid pMR26 (GenBank accession number D83080.2), plasmid pMR28 (GenBank accession number AB013925.1), ppk (D14445.1), and LP4/2A [15], the gene cluster of merT-merP-merB1-merB2-ppk (Fig. S1) was synthesized and inserted into the pRI101-AN plasmid (Takara, Dalian, China). The resulting plasmid was named p(merT-merP-merB1-merB2-ppk). According to the sequence of the pcs1 gene (GenBank accession number AY235426.1), primers NPCF2 (5′-AAATATGCGGCCGCAATGGCGATG GCGGGTTT-3′) and NPCR2 (5′-AAATATGCGGCCGCGGCAAAGCTAGAAGGGAG-3′) were designed. Polymerase chain reaction (PCR) was performed on the complementary DNA (cDNA) from a tobacco leaf. The PCR fragment was purified from an agarose gel and digested with Not I. The p(merT-merP-merB1-merB2-ppk) plasmid was also digested with Not I. The digested PCR fragment was ligated with p(merT-merP-merB1-merB2-ppk) by using a T4 DNA ligase at 4 °C for 24 h. The ligated plasmids were transformed into E. coli DH10B. The positive clones were cultured, and the plasmids were extracted. The plasmids were digested with Not I. The plasmids that could be digested into two bands where one band had a molecular weight of approximately 0.6 kb were sent to Takara for sequencing. The plasmids in which pcs1 had been inserted in the same transcription direction as that of merT were named p(merT-merP-merB1-merB2-pcs1) (Fig. S2). The p(merT-merP-merB1-merB2-ppk) and p(merT-merP-merB1-merB2-pcs1) plasmids were transferred into Agrobacterium EHA105 via the standard procedure [16]. Two-week-old tobacco leaves were cut into 4–6-mm pieces and cocultured with YEB-grown Agrobacterium following standard method. The explants were placed on sterile filter paper to remove the redundant bacteria and cultured on a differentiation medium (MS+2 mg/L 6-BA+0.5 mg/L IAA) in the dark for 2–4 days. The
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explants were then transferred to a differentiation medium containing 300 mg/L cefotaxime sodium and 50 mg/L kanamycin. The explants were maintained at 25 °C with a 16-h light/8-h dark schedule until seedling clumps appeared. The seedlings were cut from the base and transferred onto root medium (MS+0.5 mg/L IAA+300 mg/L cef+50 mg/L kanamycin). After sufficient roots had appeared, the seedlings were transferred to jars containing potting soil for further growth and development in the greenhouse and cultured continuously. The potting soil was obtained from our experimental base, located in Fushan, Chengmai County, Hainan Province, China. Surface soil (0 to 15 cm) was collected, air-dried, and ground to pass a 2-mm sieve. Nitrogen, phosphorous, and potassium (N/P/K=1:1:1) were added to soils as base fertilizers. Identifying the Transgenic Lines The leaves of the transgenic plants were collected, and genomic DNA was extracted. PCR was performed by using primers specific to merB1 (PF: 5′-ATGGACAAGACTATTTATTCCA AAA-3′ and PR: 5′-TACTGGGCTTTCCTCGCAGTCCTCT-3′). If one 0.6-kb band could be amplified, the plant had been transformed successfully. To determine whether all the foreign genes could be expressed normally in the transgenic plant, total RNA was extracted from the leaves of the transgenic plants with a Takara MiniBEST Plant RNA Extraction Kit (Takara, Dalian, China). The cDNA was synthesized with a RevertAid First Strand cDNA synthesis kit (Fermentas, http://www.thermoscientificbio.com/fermentas). MerB1, merB2, merT, merP, and ppk were amplified from the cDNA of Ntk. MerB1, merB2, merT, merP, and pcs were amplified from the cDNA of Ntp. The primers used in these PCR reactions are shown in Table 1. Mercury Accumulation Measurements in Plants and Soils The mercury contents of different tissues were measured according to a previously published method [17]. Immediately prior to flowering, the tobacco leaves or roots were harvested and washed thoroughly in distilled water. The shoots and roots were separated and immersed directly in liquid nitrogen. The frozen plants were dried using a freeze-dryer, and the dry weight was determined. After being ground to a fine powder under liquid nitrogen, the samples Table 1 Primers used to identify transgenic tobacco
Primer name
sequence
PTF
5′-ATGTCTGAACCACAAAACGGGC-3′
PTR PPF
5′-ATAGAAAAATGGAACGACATAG-3′ 5′-ATGAAGAAACTGTTTGCCTC-3′
PPR
5′-CTTCTTCAGCTCAGATGGGT-3′
PB1F
5′-ACAAGACTATTTATTCCAAAA-3′
PB1R
5′-ACATGACAGCAAAACGAACAA-3′
PB2F
5′-ATGAAGCTCGCCCCATATATT-3′
PB2R
5′-CGATTAAACTCCTGGCCCAAG-3′
PPKF
5′-ATGGGTCAGGAAAAGTTATATATCG-3′
PPKR PPCSF
5′-ATATCGACAAAGAACTCAGTAACCG-3′ 5′-ATTGTGGTTTGGCTAGCCTTTCTAT-3′
PPCSR
5′-CCTTGTTATCCTTGCATTTCTTGAG-3′
Appl Biochem Biotechnol
(three replicates of each treatment) were acid digested by step-wise additions of 70 % (v/v) nitric acid, 30 % (v/v) hydrogen peroxide, and concentrated HCl. The mercury content was then determined via flameless cold-vapor atomic adsorption spectrometry with an atomic mercury analyzer (RA-2A, Nippon Instruments, Tokyo, Japan). To measure the mercury content in the soil, soil samples were digested with 4 mol/L nitric acid. The other steps were the same as that for measuring the mercury content in plant samples. Mercury Resistance Measurements in Plants The tobacco seeds were surface-sterilized by shaking them in 10 % hydrogen peroxide for 10 min. They were then washed five times in sterile water. Subsequently, the tobacco seeds were germinated on an MS agar medium for 3 weeks. Some of the seedlings were then transferred to an MS liquid medium containing 10 μmol/L mercuric chloride or methylmercury chloride and cultured for 1 week. The remaining seedlings were transferred to an MS liquid medium without mercury to act as controls. The fresh wet weights of the seedlings were measured. The other method for measuring plant mercury tolerance was performed as follows. The surface-sterilized tobacco seeds were germinated on MS agar medium for 3 weeks. The seedlings were then transferred to soil (50/50 sand and potting soil) in a greenhouse at 22 °C with 16 h of light. All pots were watered twice per week with half-strength Hoagland’s solution. Immediately prior to flowering, the plant heights and fresh wet weights of the aboveground tissues were measured. All measurements were performed for three replicates, and the average values were used. Mercury-Transfer Capacity Measurement in Plants The soil was obtained from our experimental base, located in Fushan, Chengmai County, Hainan Province, China. Surface soil (0 to 15 cm) was collected, air-dried, and ground to pass a 2-mm sieve. Nitrogen, phosphorous, and potassium (N/P/K=1:1:1) were added to the soils as base fertilizers. Mercury chloride was added as a powder to the soil at final concentration of 21 mg/kg. Approximately 50 kg of such soil was transferred into 50-cm diameter 50-cm high plastic pots. For organic mercury treatment, methylmercury chloride was added to the soil at final concentration of 19 mg/kg. Approximately 50 kg of such soil was transferred into 50-cm diameter 50-cm high plastic pots. The tobacco seeds were germinated in vermiculite at a nursery site in a greenhouse at 22 °C with 16 h of light. After 1 month, each seedling was transferred to an individual 7-cm-diameter 13-cm-high plastic cup containing vermiculite. After 1 month, each seedling was transferred to an individual 50-cm diameter 50-cm high plastic pot containing soil treated with mercury chloride or methylmercury chloride. The pots were placed in greenhouse at 22 °C with 16 h of light. The soil was watered once every 2 weeks. Immediately before flowering, the tobacco plants were removed from the pots. The mercury content in the soil was measured as described above.
Results Tobacco Transgenic Lines Ntk and Ntp Were Developed As described in the BMaterials and Methods,^ the fusion genes merT-merP-merB1-merB2-ppk (Fig. S1) and merT-merP-merB1-merB2-pcs1 (Fig. S2) were constructed. The genes were fused with the LP4/2A linker (Figs. S1 and S2). The fusion genes were then cloned into
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plasmid vector pRI101-AN (Takara) (Figs. 1 and 2). The resulting plasmids were named p(merT-merP-merB1-merB2-ppk) and p(merT-merP-merB1-merB2-pcs1). The tobacco transformed with p(merT-merP-merB1-merB2-ppk) was named Ntk (Fig. 3), and the tobacco transformed with p(merT-merP-merB1-merB2-pcs1) was named Ntp (Fig. 4). For each vector, 50 seedlings were selected from the root medium containing kanamycin (50 mg/L) and cultured in soil. Approximately two thirds of the Ntk plants and approximately half of the Ntp plants were clearly shorter than the wild type. Ten Ntp plants and 15 Ntk plants flowered and set seeds normally. Ntk-7 and Ntp-36, two transgenic lines that can set seeds normally and had plant heights similar to that of the wild type, were selected for the following experiments. To identify whether foreign genes were expressed in these two transgenic lines, PCRs were performed from cDNA that was re-transcribed from the total RNA isolated from the transformed seedlings. The results showed that all the foreign genes can be amplified in the selected plants, and there was no corresponding band when the PCR was performed on the wild-type cDNA (Figs. 5 and 6). Thus, all the foreign genes could be expressed successfully in the selected transgenic plants. The Transgenic Lines Had Mercury Tolerance Levels That Were Significantly Higher than That of the Wild Type The results showed that the phenotypes of Ntk-7, Ntp-36, and the wild type were similar when grown in soil without mercury (data not shown). However, when they were grown in soil containing 100 μmol/L mercuric chloride, the plant heights and the fresh weights of the wild type were significantly lower than those of the transgenic lines (Figs. 7 and 8). The phenotypes of Ntk-7 and Ntp-36 were similar when grown in soil containing 100 μmol/L mercuric chloride (Figs. 7 and 8), demonstrating that they had similar mercury tolerances. To explore the transgenic lines’ mercury tolerance, their seeds were germinated on MS agar medium for
Fig. 1 The gene clusters were purified from a 0.8 % agarose gel. M1 is a λ-Hind III DNA marker (Takara, Dalian, China). M2 is a DL2000 DNA marker (Takara). Lane 1 is merT-merP-merB1-merB2-ppk. Lane 2 is merT-merP-merB1-merB2-pcs1
Appl Biochem Biotechnol Fig. 2 The original pRI101-AN vector was digested with Not I. M2 is a λ-Hind III DNA marker (Takara). Lane 1 is the original pRI101-AN vector as digested with Not I
Fig. 3 Ntk seedlings grown on a root medium containing kanamycin (50 mg/L)
Appl Biochem Biotechnol
Fig. 4 Ntp seedlings grown on a root medium containing kanamycin (50 mg/L)
3 weeks. Some of the seedlings were transferred to an MS liquid medium containing 10 μmol/ L mercuric chloride or 10 μmol/L methylmercury chloride and cultured for 1 week. The remaining seedlings were transferred to an MS liquid medium without mercury as controls. The fresh wet weights of the seedlings were measured. The results showed that the fresh wet weights of the wild type, Ntp-36, and Ntk-7 cultured in an MS liquid medium without mercury were similar (Fig. 9). However, for seedlings cultured in medium containing 10 μmol/L mercuric chloride, the fresh wet weight of the wild type was significantly lower than the
Fig. 5 Identifying the expressions of foreign genes in the transgenic Ntk-7 line. M is the DNA marker DM10000 (CWBIO, Beijing, China). Lanes 1 and 2 show the PCR results for primers PTF and PTR. Lanes 3 and 4 show the PCR results for primers PPF and PPR. Lanes 5 and 6 show the PCR results for primers PB1F and PB1R. Lanes 7 and 8 show the PCR results for primers PB2F and PB2R. Lanes 9 and 10 show the PCR results for primers PPPKF and PPPKR. Lanes 1, 3, 5, 7, and 9 show the PCR results for the cDNA that was re-transcribed from the total RNA isolated from the transgenic line Ntk-7. Lanes 2, 4, 6, 8, and 10 show the PCR results for the cDNA that was re-transcribed from the total RNA isolated from the wild-type tobacco
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Fig. 6 Identifying the expressions of foreign genes in the Ntp-36 transgenic line. M is DNA marker DM10000 (CWBIO). Lanes 1 and 2 show the PCR results for primers PTF and PTR. Lanes 3 and 4 show the PCR results for primers PPF and PPR. Lanes 5 and 6 show the PCR results for primers PB1F and PB1R. Lanes 7 and 8 show the PCR results for primers PB2F and PB2R. Lanes 9 and 10 show the PCR results for primers PPCSF and PPCS R. Lanes 1, 3, 5, 7, and 9 show the PCR results for the cDNA that was re-transcribed from the total RNA isolated from the Ntp-36 transgenic line. Lanes 2, 4, 6, 8, and 10 show the PCR results for the cDNA that was retranscribed from the total RNA isolated from the wild-type tobacco
weights of Ntk-7 and Ntp-36 (Fig. 9). There was no definite difference between the fresh wet weight of Ntk-7 and that of Ntp-36 (Fig. 9). For the seedlings cultured in the medium containing 10 μmol/L methylmercury chloride, the fresh wet weight of the plants can be ordered from lowest to highest as follows: wild type, Ntk-7, and Ntp-36 (Fig. 10). The fresh wet weights of Ntp-36 cultured in the medium containing 10 μmol/L mercuric chloride or methylmercury chloride were similar to those of Ntp-36 cultured in medium without mercury (Figs. 9 and 10). These findings demonstrate that the transgenic tobacco had a higher mercury tolerance than the wild type. The mercury tolerance of plants can be ordered from lowest to highest as follows: wild type, Ntk-7, and Ntp-36. The Mercury Accumulation in Ntp-36 Was Significantly Greater than the Accumulation in the Wild Type and Ntk-7 Immediately prior to flowering, the mercury contents of the tobacco strains grown in soil containing 100 μmol/L mercuric chloride were measured. The results showed that the mercury accumulation was much greater in the transgenic lines than in the wild type (Fig. 11). For example, the mercury content in the roots of the wild type was approximately 80 μg/g, and the values of Ntk-7 and Ntp-36 were 213 and 251 μg/g, respectively (Fig. 11). Furthermore, the mercury accumulation in Ntp-36 was significantly higher than that in Ntk-7. The mercury content in the Ntk-7 roots was much higher than that in the leaves (Figs. 11 and 12). This trend was also observed in the wild type and Ntp-36 (Figs. 11 and 12). These findings demonstrate that most mercury accumulated in the plant roots. Only a small portion of the mercury was transferred to the aboveground tissues. The mercury-accumulation ability of Ntp-36 was
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Fig. 7 The fresh wet weights of plants grown in soil containing 100 μmol/L mercuric chloride compared to the controls (no mercury). The data were collected after the plants flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
greater than that of Ntk-7. Thus, the transgenic plants can accumulate much more mercury than the wild type. Ntp-36 and Ntk-7 Can Take Up Organic Mercury from the Soil in Addition to Inorganic Mercury To identify whether merB1 and merB2 played roles in the transgenic lines, the mercury content of plants grown in soil containing methylmercury chloride was also measured. The results showed that transgenic plants can accumulate much more mercury from soil containing organic mercury than the wild type (Figs. 13 and 14). For example, the mercury content of the roots from the wild type grown in soil containing 100 μmol/L methylmercury chloride was approximately 75 μg/g, and the values of Ntk-7 and Ntp-36 were 202 and 248 μg/g, respectively (Fig. 13). Although the corresponding data in leaves were lower than the concentrations in the roots, the mercury contents in the leaves of the transgenic tobacco were
Fig. 8 The heights of plants grown in soil containing 100 μmol/L mercuric chloride compared to the controls (no mercury). The data were collected after the plants flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
Appl Biochem Biotechnol
Fig. 9 The fresh wet weights of seedlings cultured in medium containing 10 μmol/L mercuric chloride compared to the controls (no mercury). The data were collected 10 days after the seedlings were transferred to liquid medium with 10 μmol/L mercuric chloride. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp36, respectively
also significantly higher than that of the wild type (Fig. 14). These findings demonstrated that merB1 and merB2 played roles in the transgenic plants. Organic mercury can be reduced into mercuric ions in the transgenic plants. The mercury accumulation in the root of Ntp-36 plants grown in soil containing 100 μmol/L methylmercury chloride was significantly higher than that found in the roots of Ntk-7 with the same treatment (Fig. 13). Similar results were also obtained from the leaves of Ntp-36 and Ntk-7 (Fig. 14). These findings demonstrated that Ntp36 was superior to Ntk-7 in the removal of organic mercury from soil. Ntp-36′ Mercury-Transfer Efficiency Was Higher than Those of Ntk-7 and Wild Type The results showed that after tobacco plants were grown, the mercury content in the soil in which the wild-type seedlings were grown was slightly lower than that in which no plant was
Fig. 10 The fresh wet weights of seedlings cultured in medium containing 10 μmol/L methylmercury chloride compared to the controls (no mercury). The data were collected 10 days after the seedlings were transferred to a liquid medium with 10 μmol/L methylmercury chloride. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
Appl Biochem Biotechnol
Fig. 11 The mercury contents in the roots of plants grown in soil containing 100 μmol/L mercuric chloride. The data were collected immediately before the tobacco flowered. Items 1, 2, and 3 correspond to the wild type, Ntk7, and Ntp-36, respectively
grown (Table 2). The mercury transfer ratio (MTR) of the wild-type tobacco grown in soil appended with mercury chloride was only approximately 2.38 % (Table 2). In the soil appended with methylmercury chloride, the MTR of the wild type was also only approximately 2.22 % (Table 2). The MTR of Ntk-7 for mercury chloride was approximately 14.95 % (Table 2). Although this value was higher than that of the wild type, it is lower than that of Ntp36 (Table 2). For plants grown in soil containing methylmercury chloride, the order of MTRs from the highest to the lowest was Ntp-36, Ntk-7, and wild type (Table 2). This demonstrated that Ntp-36 had the best potential for mercury removal from soil.
Discussion Tobacco is an ideal plant for removing mercury from polluted soil. However, although several engineered tobaccos have been constructed, they cannot be used for practical applications [6, 11–13]. Some transgenic plants release elemental mercury directly into the air [6, 11, 12], which can cause public anxiety. Additionally, the mercury accumulation of some transgenic plants is worse than expected [13]. The ideal engineered tobacco should at a minimum meet the following two criteria: The tobacco should be able to (1) take up organic mercury and
Fig. 12 The mercury contents in the shoots of plants grown in soil containing 100 μmol/L mercuric chloride. The data were collected immediately before the tobacco flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
Appl Biochem Biotechnol
Fig. 13 The mercury contents in the roots of tobacco strains grown in soil containing 100 μmol/L methylmercury chloride. The data were collected immediately before the tobacco flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
mercuric ions from the soil and (2) store mercury in the plant without discharging it into the environment. In the cells of the two transgenic lines developed for this paper (Ntk-7 and Ntp36), merT and merP enhance the uptake of mercuric ion from the soil by plants [9]. Organic mercury can be reduced by merB1 and merB2 [9]. Polyphosphate, which was synthesized by ppk, binds with the mercuric ions and form chelatins [13]. The toxicity of Hg2+-chelatins was lower than that of Hg2+. Phytochelatins, which were encoded by pcs1, can also bind with mercuric ions and form chelatins [14]. Hg2+-phytochelatins can be transferred into the vacuole, further reducing the mercury toxicity [18]. The mercury resistance of Ntk-7 and Ntp-36 was much higher than that of the wild type with regard to both organic mercury and mercuric ions. The transformed plants grown in soil containing mercury accumulated significantly more mercury than the wild type grown in soil with the same treatment. The transformed plants can remove more mercury from the mercury-polluted soil. Thus, these engineered plants have good prospects for use in removing mercury from the soil. The engineering of metabolic pathways in plants has often required the expression of more than one gene [19]. The use of linker peptides to construct fusion genes is considered to be a good method for multiple gene transformation [15]. LP4/2A is a hybrid linker that contains the first amino acids of LP4 and 20 amino acids from 2A [20]. After translation, the amino acid residues of the linker will be removed from the mature protein by protease in cytoplasm [15].
Fig. 14 The mercury contents in the shoots of tobacco strains grown in soil containing 100 μmol/L methylmercury chloride. The data were collected immediately before the tobacco flowered. Items 1, 2, and 3 correspond to the wild type, Ntk-7, and Ntp-36, respectively
Appl Biochem Biotechnol
Table 2 Transgenic plants’ mercury-transfer efficiency
Treatment
Initial Hg in soil (mg/kg)
Final Hg in soil (mg/kg)
Mercury transfer ration (%)
WT (HgC12)
21
20.5
WT (CH3HgC1)
19
18.6
2.38 2.22
Ntk-7 (HgC12)
21
17.9
14.95
Ntk-7 (CH3HgC1)
19
17.4
8.42
Ntp-36 (HgC12)
21
17.3
17.62
Ntp-36 (CH3HgC1)
19
15.1
21.58
We found that all the foreign genes in the transgenic plants are well expressed. The expression level of one gene was not significantly different from those of other genes in the cluster, and all genes in the clusters can be expressed simultaneously. These findings demonstrated that the strategy used in this paper for multiple gene transference was successful. The phenotypes of the transgenic lines grown in soil containing mercury or cultured in media containing mercury also confirmed this conclusion. The ppk gene was originally isolated from Enterobacter aerogenes [21]; thus, it is a foreign gene for tobacco. Although polyphosphate kinase (ppk) can catalyze the synthesis of polyphosphate in the plant’s cytoplasm, there might not be any proteins responsible for transporting polyphosphate across the tonoplast in plant cells. Thus, polyphosphate-Hg2+ chelatins might be locked in the cytoplasm. Although the toxicity of polyphosphate-Hg2+ was lower than that of mercury ions, the mercury in these chelatins can still harm the organelles in the cytoplasm. Phytochelatins are native proteins for plants. Therefore, certain proteins located within the tonoplast must be present to transfer phytochelatin-Hg2+ across the vacuolar membrane. Recently, Atabcc1/atabcc2 was cloned and considered to be the candidate genes responsible for transferring phytochelatin-Hg2+ across the tonoplast [18]. With aid of these genes, phytochelatin-Hg2+ chelatins can be transferred into the vacuole and stored there. Because there are no important organelles in the vacuole and the bioactivity of this organelle is very low, the phytochelatin-Hg2+ inside the vacuole will not significantly harm the plant cell. Cells containing phytochelatin-Hg2+ had greater chances of survival than those containing polyphosphate-Hg2+. Plants transformed with pcs1 were more likely to survive and can accumulate more mercury than those transformed with ppk. Our experiments showed that although the mercury resistance of Ntk-7 grown in soil containing mercuric ions was similar to that of Ntp-36 grown in similar soil, the mercury resistance of Ntp-36 grown in soil containing methylmercury chloride was significantly higher than that of Ntk-7 grown under the same conditions. Additionally, the MTR of Ntp-36 for methylmercury chloride was approximately 21.58 %, while that value of Ntk-7 was only approximately 8.42 %. For removing mercury chloride, the MTR of Ntp-36 was also higher than that of Ntk-7. Thus, the mercury removal efficiency of Ntp-36 was better than that of Ntk-7. In our experiments, we found that although the transgenic tobacco plants accumulated much more mercury than the wild type, most of the mercury accumulated in the roots. Similar results were also found in the wild-type plants. But, it is difficult to manually remove tobacco roots from soil. Furthermore, more than 70 % of the biomass of the tobacco plant is in aboveground tissues. If the mercury in the roots cannot be transferred to the aboveground tissues, the utility of these transgenic plants to farmers will be debatable. The approaches needed to overcome this defect and capitalize on the advantages of transgenic plants require further studies.
Appl Biochem Biotechnol Acknowledgments This work was supported by the National Science Foundation of China (No. 41201309).
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