Phytoremediation of Trichlorophenol by Phase II ... - ACS Publications

Mar 12, 2012 - Efficient phytoremediation of TCP is potentially feasible because it contains a hydroxyl group and is suitable for direct phase II meta...
0 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/est

Phytoremediation of Trichlorophenol by Phase II Metabolism in Transgenic Arabidopsis Overexpressing a Populus Glucosyltransferase Zhen-Hong Su,†,‡,§ Zhi-Sheng Xu,‡,§ Ri-He Peng,‡ Yong-Sheng Tian,‡ Wei Zhao,‡ Hong-Juan Han,‡ Quan-Hong Yao,‡ and Ai-Zhong Wu*,† †

College of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China

‡

ABSTRACT: Trichlorophenol (TCP) and its derivatives are introduced into the environment through numerous sources, including wood preservatives and biocides. Environmental contamination by TCPs is associated with human health risks, necessitating the development of cost-effective remediation techniques. Efficient phytoremediation of TCP is potentially feasible because it contains a hydroxyl group and is suitable for direct phase II metabolism. In this study, we present a system for TCP phytoremediation based on sugar conjugation by overexpressing a Populus putative UDP-glc-dependent glycosyltransferase (UGT). The enzyme PtUGT72B1 displayed the highest TCP-conjugating activity among all reported UGTs. Transgenic Arabidopsis demonstrated significantly enhanced tolerances to 2,4,5-TCP and 2,4,6-TCP. Transgenic plants also exhibited a strikingly higher capacity to remove TCP from their media. This work indicates that Populus UGT overexpression in Arabidopsis may be an efficient method for phytoremoval and degradation of TCP. Our findings have the potential to provide a suitable remediation strategy for sites contaminated by TCP.

■

INTRODUCTION Trichlorophenols (TCPs) comprise a large group of widely emitted industrial pollutants. Of the six isomers of TCP, the 2,4,5-isomer and the 2,4,6-isomer are considered priority pollutants.1,2 The 2,4,6-isomer is used extensively in wood preservatives, insecticides, and fungicides3−5 and 2,4,5-TCP has been used as a precursor for the synthesis of herbicides containing 2,4,5-trichlorophenoxyacetic acids.6,7 Trichlorophenols might be carcinogenic by inducing point mutations in the somatic genome.8 Due to their extensive use, these compounds are common pollutants of soils and fresh water.9 Although a small number of bacteria and fungi were found to be able to degrade TCP,10,11 the inoculation of microorganisms and nutrient applications in the environment are difficult. Removal of TCP from the environment is often accomplished by chemical treatments, such as adsorption over activated carbon, air stripping, chemical oxidation, solvent extraction, or incineration.12−14 However, the high cost and low efficiency of these processes limit their applicability. Plants are constantly exposed to synthetic compounds, such as pollutants and crop protection agents, and have developed diverse detoxification mechanisms.15,16 Plants transform xenobiotics by using a three-phase detoxification system: conversion, conjugation, and compartmentalization.17 In plants, the most commonly observed conjugation reaction is glycosylation, a © 2012 American Chemical Society

reaction catalyzed by GT1 family glycosyltransferases that are more normally engaged in secondary metabolism.18 Conjugation of 2,4,5-TCP in in vitro cultures of Lemna gibba (Lemnaceae) yielded β-glycosides that were progressively dehalogenated.19 Arabidopsis contains 107 GT1 family functional proteins,20 of which 44 proteins showed O-glucosyltransferase (OGT) activity toward TCP.21 This OGT was well represented in the large D, E, and L GT1 groups. On the basis of enzyme-specific activity, the E group enzymes were the most active, with AtUGT72B1 being 20 times more efficient than any other UGTs in conjugating TCP.21 Populus provides an attractive model system for studies of phytoremediation. For example, hybrid poplars (Populus spp.) could uptake and degrade trichloroethylene (TCE). The cells of poplar were able to metabolize TCE to produce trichloroethanol, and di- and trichloroacetic acid.22,23 Populus nigra cuttings in containers of sand with a nutrient solution were able to metabolize added polycyclic aromatic hydrocarbons (PAHs).24,25 Poplars are well suited for phytoremediation, but a careful study of genes contributing to the Received: Revised: Accepted: Published: 4016

October 25, 2011 February 27, 2012 March 12, 2012 March 12, 2012 dx.doi.org/10.1021/es203753b | Environ. Sci. Technol. 2012, 46, 4016−4024

Environmental Science & Technology

Article

GV3101 by electroporation. Arabidopsis thaliana (ecotype Colubia) was transformed by the floral dip method.29 Plants were grown on Murashige and Skoog medium30 for 15 d, and transferred into the pots filled with a 9:3:1 mixture of vermiculite/peat moss/perlite in a controlled environmental chamber at 22 °C under a 16 h/8 h (light/dark) photoperiod at a daylight intensity of 100 Em2− s−1. To compare the activity of PtUGT72B1 and AtUGT72B1 in plants, the plant expression vector of AtUGT72B1 was also constructed and transformed to A. thaliana. Plants were self-crossed and the homozygous T3 generations on MS plates containing 25 mg/L hygromycin were selected for experiment. Reverse Transcription−PCR Analysis. Total RNA was extracted with the multisource total RNA miniprep kit (Axygen, Union City, CA, USA) according to the manufacturer’s instruction and treated with DNase I (Promega, Madison, WI, USA) to remove genomic DNA. The first strand of cDNA was synthesized using 5 μg of total RNA as a template with the Reverse Transcription System (Promega) in a 20-μL reaction volume. A 257 bp fragment of PtUGT72B1 gene was amplified using a forward primer 5′-GTCGCTAATG CCACCTACTT-3′ and a reverse primer 5′-ATCCATGCGTTCATCTTCTG-3′. A same long fragment of AtUGT72B1 gene was amplified with primers: 5′-GAATTCGAC TCTAGAGAGTG-3′ and 5′-CATCAT CCTTCAACACCCTA-3′. The PCR reaction was carried out in 27 cycles of 40 s at 94 °C, 30 s at 50 °C, 20 s at 72 °C, and a final extension at 72 °C for 5 min. The A. thaliana actin gene (AtAc2, accession NM112764) was used as an internal control. The PCR products were separated on 2% agarose gel and quantified using a Model Gel Doc 1000 (Bio-Rad, USA). The DNA intensity ratio of the PtUGT72B1 gene to AtAc2 was determined with a Shine Tech Gel Analyzer (Shanghai Shine Science of Technology Co., Ltd., China) to evaluate the expression level of the PtUGT72B1 gene. Pichia and Plant Resistance Assay. To assay yeast TCP resistance, the P. pastoris strain GS115 transformants (transfected with linearized pPIC9K or pPIC-UGT) were grown in BMGY medium. After induction with 1% methanol for 24 h, the yeast cells were diluted to 103 cells/μL and 0.5−10 μL of cells was transferred to fresh BMMY medium supplemented with 20 mg/L 2,4,6-TCP or 9 mg/L 2,4,5-TCP. To assay plant TCP resistance, seeds were germinated on agar on half-strength MS agar plates containing either 6 mg/L 2,4,6-TCP or 2 mg/L 2,4,5-TCP predissolved in methanol.31 About 30 seeds of each line were placed on plates under aseptic conditions. Plants were grown vertically for 2 weeks. The taproot length and fresh weight of approximately 10 seedlings were measured for each treatment. Enzyme Extraction and Data Assay. For extraction of expressed PtUGT72B1 from yeast, 50 mL of the cell suspension was centrifuged and then resuspended in 1 mL of breaking buffer (50 mM Tris-HCl, pH 7.4). An equal volume of acid-washed glass beads (0.5 mm) was added to the resuspended cells, and the mixture was vortexed for 30 s and incubated on ice for 30 s. This step was repeated 10 times. The total protein was obtained from the lysed cells by centrifugation at 4 °C at 12 000g. The pellets were resuspended in 1 mL of EBuffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0, 1 mM DTT). The total protein content of the extract was determined according to the method of Bradford.32 The supernatants were separated on 12% SDS-PAGE gels and visualized with Coomassie Brilliant Blue R-250. Every band was quantified with a Shine Tech Gel Analyzer (Shanghai Shine Science of

phytoremediation capacity is necessary to fully realize the potential of poplar-based degradation of environmental toxins. In this paper, we described a GT72B1-like glycosyltransferase (Genbank XP_002320190) in Populus. Our aims were (1) to obtain gene function information and compare the enzyme activity in Populus and Arabidopsis and (2) determine whether the novel glycosyltransferase from Populus could be used to degrade TCPs more effectively in transgenic plants than natural system.

■

MATERIALS AND METHODS Design and Chemical Synthesis of PtUGT72B1. According to the amino acid sequence (GenBank Accession XP_002320190), the putative glycosyltransferases gene PtUGT72B1 from Populus trichocarpa was synthesized by successive polymerase chain reaction (PCR).26,27 To improve the efficiency of gene transcription and RNA stability, the G+C and A+T contents in PtUGT72B1 gene were balanced and predicted hairpin structures and motifs containing over 6 consecutive A/Ts were eliminated by using degenerate codons. PCR was carried out with 1.5 pmol of oligonucleotides PtUGT2 to PtUGT35 for 25 cycles with 2.5 U pyrobest polymerase (TaKaRa, Dalian, China). The conditions of this PCR-mediated assembly were 30 s at 94 °C, 30 s at 45 °C, and 30 s at 72 °C for each cycle followed by an additional 10 min at 72 °C to ensure complete extension for all PCR reactions. For amplification of PCR products, 1 μL of the assembled mixture was used as the template with the oligonucletides PtUGT1 and PtUGT36 as primers, and performed at 94 °C for 1 min for the first cycle and then 30 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, followed by an additional 10 min at 72 °C. PtUGT72B1 Gene Expression in Pichia pastoris. The synthesized gene PtUGT72B1 was digested with BamHI and Sac I and then inserted between the BamHI/Sac I sites of the modified pPIC9K vector. The restriction endonuclease sites for BamHI and Sac I were added in the multiple cloning site and the α-factor signal sequence was deleted in the modified pPIC9K vector. The recombinant plasmid pPIC-UGT was linearized with BglII and inserted into Pichia pastoris strain GS115 cells by electroporation.28 To compare the enzyme characteristics, the yeast-expression vector of Arabidopsis UGT72B1 (AtUGT72B1) was also constructed using the same method as above. The cells were plated on histidinedeficient SD medium and incubated at 30 °C for 3 d. The transformants were cultured in YPD medium for 2 d, and then the DNA of all cells was extracted. The PtUGT72B1 gene was examined with the primers PtUGT 1 and 36. For large-scale protein production, the positive colonies were cultivated in 50 mL of BMGY (2% peptone, 1% yeast extract, 1.34% YNB, 0.4 μg/mL biotin, 1% glycerol) at 30 °C with constant shaking at 200 rpm for 48 h until the OD600 reached 1.0. The cells were harvested in sterile centrifuge bottles by centrifuging at 3000g for 5 min at room temperature. To induce expression, the cell pellet was resuspended in an equal volume of BMMY (2% peptone, 1% yeast extract, 1.34% YNB, 0.4 μg/mL biotin, 1% glycerol, 1% methanol). To maintain induction, methanol was added to the culture to a final concentration of 1% every 24 h. Generation of Transgenic Plants with the Synthesized PtUGT72B1 Gene. To generate CaMV35S: PtUGT72B1, the synthesized PtUGT72B1 gene was digested with BamHI and Sac I and inserted into the binary vector pCAMBIA-1301 with the hygromycin gene as the genetic selection marker. The construct was introduced into Agrobacterium tumefaciens 4017

dx.doi.org/10.1021/es203753b | Environ. Sci. Technol. 2012, 46, 4016−4024

Environmental Science & Technology

Article

Technology Co., Ltd.). The structure of PtUGT72B1 was modeled using the Swiss-model (http://swissmodel.expasy. org/) referring the AtUGT72B1 (PDB 2VCE) and drawn with Swiss-pdb viewer.33 For extraction of Arabidopsis crude protein, fresh leaves tissue (0.1 g) was homogenized in 1.6 mL of chilled 50 mM Tris-HCl (pH7.4) using a chilled pestle and mortar kept in an ice bath. The homogenate was filtered with two layers of muslin and centrifuged at 13000g for 30 min in a refrigerated centrifuge at 4 °C. Crude enzyme was quantified by Coomassie Brilliant Blue G250 and used for the in vitro activity assay. The glucosyltransferase assay was performed as described by Mok and co-workers with modifications.34 The assay mix (200 μL) contained 1 or 2 μg of recombinant protein, 50 mM TrisHCl (pH 7.0), 5 mM UDP-glucose, and 2,4,5-TCP. The reaction was carried out at 30 °C for 30 min, and was stopped by the addition of 20 μL of trichloroacetic acid (240 mg/mL), quick-frozen, and stored at −20 °C before HPLC analysis. Reverse-phase HPLC was performed using an Agilent 1100 HPLC system (Agilent Technologies, CA, USA) and a Columbus 5-μm C18 column (250 × 4.60 mm, Phenomenex). Elutes were monitored by absorbance at 205 nm, and the peaks were identified by their retention times. To determine the Km and Vmax, different substrate standard concentrations ranging 25, 37.5, 50, 62.5, 75, 87.5, and 100 μM were used to evaluate the reaction rate. Kinetic parameters were determined by the Lineweaver−Burk method. The specific enzyme activity was expressed as nanomoles of TCP glucosylated per second (nanokatal) per 1 μg of protein. Measurements of TCP and Glucosylated-TCP by HPLC and GC-MS. Transgenic UGT and ET plant seeds were surface-sterilized and germinated in plates for a week. About 20 seedlings were transferred to MS liquid medium containing TCP and grown for 2 weeks under rotary shaking at 120 rpm and 23 °C. The liquid samples were used directly to measure TCP by reverse-phase HPLC. Glucosylated TCP in medium were separated from their aglycone by a linear gradient of 20% to 70% acetonitrile in H2O (all solution contained 0.1% trifluoroacetic acid) at 1 mL/min over 20 min and monitored at 205 nm. A 10-μL aliquot of each sample was injected onto the column. The seedling samples were extracted with 80% methanol and analyzed with an Agilent LC/MSD SL system (Agilent Technologies, CA, USA). Glucosylated TCP in seedling were separated from their aglycone by a linear gradient of 10% to 80% methanol in H2O at 0.5 mL/min over 30 min and monitored at 205 nm. Gas chromatography−mass spectrometry (GC-MS) was performed with APCI ionization source on 3000 V, 5 L/min drying gas flow, 60 psi nebulizer pressure, and 350 °C drying gas temperature and vaporizer temperature.

Figure 1. Expression of the putative glycosyltransferases gene PtUGT72B1 from Populus trichocarpa (XP_002320190) in Pichia Pastoria. (A) The UGT was analyzed on a 12% (w/v) polyacrylamide gel and visualized with Coomassie Brilliant Blue staining. (B) and (C) Yeast cells that overexpressed the product of PtUGT72B1 exhibited significant resistance to 2,4,5-TCP (B) and 2,4,6-TCP. The yeast cells were diluted to 103 cells/μL and 2−10 μL of cells was added to the BMMY plate supplemented with 20 mg/L 2,4,6-TCP or 9 mg/L 2,4,5TCP. CK, yeast transformants with empty vector pPIC9K.

■

RESULTS Synthesis of the PtUGT72B1 Gene from Populus trichocarpa. We synthesized a putative glycosyltransferases gene, PtUGT72B1, on the basis of the encoding amino acid of the wild type gene from Populus trichocarpa (GenBank Accession XP_002320190). BLAST search showed that the synthesized gene was 81.9% identical to the wild type. The value of A+T content in the synthesized gene was about 50%, slightly less than the wild type (53.1%) due to the removal of twelve motifs containing over 6 consecutive A/Ts and two hairpin structures.

Expression of PtUGT72B1 in Pichia pastoris. The recombinant pPIC-UGT plasmid was transformed and targeted into the Pichia pastoris GS115 genome. PCR was used to tag the positive strain for insertion of the recombinant PtUGT72B1. Twenty-five positive colonies were selected for further induction with methanol in shaker flasks to screen the expression strain by SDS-PAGE. The recombinant PtUGT72B1 was about 53 kDa. Determined by the Shine Tech Gel Analyzer, the maximal content of recombinant 4018

dx.doi.org/10.1021/es203753b | Environ. Sci. Technol. 2012, 46, 4016−4024

Environmental Science & Technology

Article

Figure 2. Analysis of PtUGT72B1 enzyme properties. (A) HPLC analysis of 2,4,5-TCP glucoconjugates by PtUGT72B1. The reaction mixes contained 2,4,5-TCP incubated with PtUGT72B1 for 0 min (top) and 30 min (bottom). (B) Comparison of kinetics of the PtUGT72B1 and AtUGT72B1 expressed in Arabidopsis. The activity of glycosyltransferase at various concentrations of 2,4,5-TCP was assayed at 30 °C in saturating concentrations of UDP-glucose (5 mM). The Km values for 2,4,5-TCP were determined by fitting the data to v = Vmax * [S]/(Km + [S]), where v is the velocity of the reaction (expressed in nkat/mg), Vmax is the maximum velocity, S is the concentration of substrate, and Km is the Michaelis constant.

Table 1. Kinetic Data for TCP as a Substrate of UGTsa

a

UGT

Km (μM)

Kcat (s−1)

Kcat/Km (mM−1 s−1)

PtUGT72B1 AtUGT72B1

42.7 ± 4.7 81.7 ± 6.3

6.7 ± 0.3 4.4 ± 0.5

156.9 53.8

after addition of UDP-glucose, indicting that PtUGT72B1 can recognize the hydroxyl groups on 2,4,5-TCP and glucosylate the substrate like the proto-enzyme AtUGT72B1. As expected, one single HPLC peak corresponding to O-glucosylated TCP was identified after incubation (Figure 2A). To compare the kinetic properties, PtUGT72B1 and AtUGT72B1 were overexpressed in P. pastoris and extracted for activity assay. Under the conditions of the HPLC assay, PtUGT72B1 transferred Glc from UDPGlc to 2,4,5-TCP with a Km of 42.7 μM, which was approximately half of the Km of AtUGT72B1 (Figure 2B). In addition, PtUGT72B1 had a higher Kcat and Kcat/Km value than the wild type enzyme AtUGT72B1 (Table 1), indicating that PtUGT72B1 was more efficient at glucosylating TCP. Structures Analysis of PtUGT72B1. Sequence analysis showed that the two enzymes shared only 65% amino acid sequence identity. In spite of the low amino acid sequence identity, their secondary and tertiary structures were highly conserved (Figure 3A and 3B). As in AtUGT72B1, the crystal structure of PtUGT72B1 revealed that the N-terminal domain and the C-terminal domain pack very tightly to form a narrow cleft constituting the substrate pocket. The substrate pocket

Mean values from three independent experiments ±SD are shown.

PtUGT72B1 reached more than 50% of the total protein after 72 h induction (Figure1A). Both 2,4,5-TCP and 2,4,6-TCP are highly toxic. Several UDP-glc-dependent glycosyltransferases (UGTs) in plant have been shown to have O-glucosyltransferase (OGT) activity toward TCP. Therefore, overexpression of a plant UGT in yeast may confer resistance to TCP. The growth of yeast cells transformed with control pPIC9K plasmid was inhibited by 20 mg/L 2,4,6-TCP or 9 mg/L 2,4,5-TCP. When yeast was transformed with the pPIC-UGT plasmid, however, the PtUGT72B1-yeast exhibited less growth inhibition in the presence of the same toxin concentrations (Figure 1B and 1C). Enzyme Properties of PtUGT72B1. To investigate the potential of PtUGT72B1 for glucosylating and detoxifying TCP, glucosyltransferase preparations from yeast culture medium were examined. The UGT consumed TCP quickly 4019

dx.doi.org/10.1021/es203753b | Environ. Sci. Technol. 2012, 46, 4016−4024

Environmental Science & Technology

Article

Figure 3. continued providing the sugar donor interactions are shown in green and the sugar acceptor interactions are shown in magenta. The proposed residues that form the sugar acceptor pocket are indicated in blue.

Figure 4. Expression levels of PtUGT72B1 and O-glucosylation activities in transgenic Arabidopsis. (A) RT-PCR amplified PtUGT72B1 fragment from the transgenic plants of empty vector ET and UGT lines (PtUGT2, PtUGT5, PtUGT9, and AtUGT6) with AtAC2 as a reference. (B) Activities of glycosyltransferase in the ET and UGT plants were shown by the reduction of TCP.

contained a sugar donor and an acceptor region. The sugar donor mainly interacted with the PSPG motif from the Cterminal domain. Of the 44 amino acids in the PSPG motif, only 6 amino acids were different between these UGTs, and the residues interacting with the sugar donor were identical (Figure 3A). The sugar acceptor pocket is formed mostly by N-terminal residues. Residues from the loops N1, N2, N4, partial N3, N5, and some C-terminal loops (loop C1 and loop C5) form the sugar acceptor pocket. Residues reported to form part of the acceptor pocket were also identical between the wild type and synthesized UGT (Figure 3A and 3B). The similarity in secondary and tertiary structures indicated that PtUGT72B1 might glucosylate the same substrates as AtUGT72B1. There are two regions that did show significant difference between PtUGT72B1 and AtUGT72B1: the N2 loop and N3− N3α loop region. In the N2 loop of PtUGT72B1, the amino acid D46 replaced the E45 in AtUGT72B1, and the basic amino acid R85 was substituted by the nonpolar amino acid L86 in N3α. Construction of Transgenic Arabidopsis. To efficiently express the UGT in plants, the modified PtUGT72B1 gene was inserted into the binary vector pCAMBIA-1301 under the control of the cauliflower mosaic virus 35S promoter, and introduced into Arabidopsis via Agrobacterium-mediated transformation. Plants transformed with AtUGT72B1 expression vector and empty vector were also carried out as controls. Twelve PtUGT72B1 transgenic (UGT) plants (T1) were

Figure 3. Comparative analyses of the structures of PtUGT72B1 and AtUGT72B1. (A) Amino acid sequence alignment of the PtUGT72B1 and AtUGT72B1. The PSPG motif that forms the sugar donor pocket is underlined and 10 conserved sugar donor interacting residues are marked with an asterisk. The loops close to the sugar acceptor pocket are shown in gray and the residues forming the pocket are double underlined. The interdomain linker region between N-terminal and Cterminal domain is highlighted with a dotted line. (B) Crystal structure showing the 3D folding of elements of secondary structure with αhelices shown in red and β-strands shown in cyan. The amino acids 4020

dx.doi.org/10.1021/es203753b | Environ. Sci. Technol. 2012, 46, 4016−4024

Environmental Science & Technology

Article

UGT5 compared to UGT9 (relative to the reference gene AtAc2). The glucosyltransferase activities in the UGT and ET lines were assayed with crude enzyme extracts from their seedlings. The activity was expressed as the amount of TCP glucosylated per milligram of protein. All three PtUGT72B1 lines showed higher UGT activity than AtUGT72B1 lines during the incubation. Noticeably different glucosyltransferase activities were also observed in the UGT lines that were well correlated with the transcription levels. A much lower UGT activity was measured in enzyme extracts prepared from ET plants (Figure 4B), indicating that endogenous glucosyltransferase activity made only a small contribution to the total UGT activity of transgenic Arabidopsis plants. Enhanced Tolerances of UGT Plants to TCP on Plates. Thirty seeds from each ET and UGT line were sown on halfstrength MS agar plates containing 2 mg/L 2,4,5-TCP or 6 mg/ L 2,4,6-TCP and grown vertically for 2 weeks. The PtUGT, AtUGT, and ET plants grew at about the same rate in control media (Figure 5A), but the PtUGT transgenic plants showed higher tolerances than AtUGT and ET plants in the medium containing 2 mg/L 2,4,5-TCP, with greener and broader leaves, as well as higher growth rates (Figure 5A). The growth of the AtUGT plants was retarded a little; with their average fresh weight being about 85% that of the PtUGT plants (Figure 5B). At 6 mg/L 2,4,6-TCP, the growth of all plants was severely inhibited, but PtUGT plants exhibited less growth suppression than AtUGT and ET. Efficient root formation is a crucial characteristic of transgenic plants designed for potential application in phytoremediation. PtUGT, AtUGT, and ET plants developed similar root systems with extensive secondary root branches and root hairs on the control medium without TCP, with the longest reaching about 4.1 cm. On the medium containing 2 mg/L 2,4,5-TCP for 2 weeks, PtUGT plants exhibited only slight phytotoxic effects, producing more root hairs and showing a normal shoot development, while ET seedlings exhibited a stunted development, producing shorter roots and fewer root hairs. The average root length of ET plants was about 43% shorter than PtUGT5 plants in 2 mg/L 2,4,5-TCP. The roots of AtUGT plants were shorter and with less hairs than those of PtUGT plants. At 6 mg/L 2,4,6-TCP, the root development of UGT plants was seriously affected; they lost geotropism and could not produce secondary roots. However, the ET seedlings displayed more serious symptoms, as the average root length of ET plants was about 53% that of PtUGT5 plants (Figure 5A and C). Higher TCP Removal Efficiencies of UGT Plants. After 2-week incubation, a significant decrease of both 2,4,5-TCP and 2,4,6-TCP concentration was detected in the media containing the PtUGT plants (Figure 6A and B). Indeed, PtUGT plants generally showed 3−5 times higher degradation rates than ET plants (Figure 6C). However, no detectable amount of glucosylated TCP was found in either media. Figure 6D shows the traces of extracts from seedling harvested at 2 weeks following treatment with 2,4,5-TCP. GC-MS analysis revealed that detectable novel compounds accumulated in the extracts of PtUGT plants. The peak at the time of 18.692 on the chromatogram was confirmed to be TCP-O-glucoside (Figure 6D).

Figure 5. Enhanced TCP tolerances of PtUGT72B1 transgenic plants on plates. (A) Control transgenic plants ET and UGT transgenic plants germinated and grown vertically for 2 weeks on half-strength MS agar plates containing 2 mg/L 2,4,5-TCP and 6 mg/L 2,4,6-TCP. (B) Shoot fresh weight of 2-week-old transgenic plants grown on halfstrength MS agar plates containing 2 mg/L 2,4,5-TCP and 6 mg/L 2,4,6-TCP. (C) Root length of 2-week-old transgenic plants grown on half-strength MS agar plates containing 2 mg/L 2,4,5-TCP and 6 mg/ L 2,4,6-TCP.

initially identified by PCR from 16 putative transgenic plants regenerated on half-strength Murashige and Skoog (MS) agar plates containing hygromycin. All the transgenic plants were identical to wild type plants in phenotype when grown either on half-strength MS agar plates or in soil in the growth room, suggesting that the insertion of the PtUGT72B1 gene in these plants caused no visible morphological effects. Stable Expression of the PtUGT72B1 in Transgenic Arabidopsis. The homozygous transgenic lines expressing the PtUGT72B1 gene were selected from the T3 plants using hygromycin selection and RT-PCR. A RT-PCR product of approximately 250 bp corresponding to the 3′ domain of the PtUGT72B1 coding sequence was detected in all three UGT lines analyzed, whereas no such PCR product was amplified in empty vector transgenic (ET) plants (Figure 4A). Relatively higher expression levels were detected in plant lines UGT2 and

■

DISCUSSION Plants normally metabolize xenobiotic pollutants in three sequential steps: phase I−III. Phase I involves conversion of xenobiotic compounds to more polar, chemically active 4021

dx.doi.org/10.1021/es203753b | Environ. Sci. Technol. 2012, 46, 4016−4024

Environmental Science & Technology

Article

Figure 6. Removal of TCP in the media by plants. (A) HPLC analyses of 2,4,6-TCP in the media. After seedlings were grown in half-strength MS liquid media containing 6 mg/L 2,4,6-TCP for 2 weeks, the concentrations of 2,4,6-TCP in ET (top) and PtUGT2 (bottom) media were determined by HPLC. (B) 2,4,5-TCP removal in the media. ET, top; PtUGT2, bottom. (C) TCP degradation rates after 2-week incubation. TCP degradation was calculated from the remaining TCP in the medium, and degradation rates were expressed as mg/g fresh weight. (D) LC/MSD trace of the extracts from the transgenic line PtUGT2 (bottom) and the control line ET (top) grown in media containing 2,4,5-TCP.

to catalyze the glucosylation of TCP when expressed as a recombinant enzyme in yeast. A comparative study of PtUGT72B1 with AtUGT72B1 was also performed. Yeast and plants transformed with PtUGT72B1 showed greater TCP resistance compared to AtUGT72B1 (Figure 1B and C, and Figure 5). Kinetic analysis of the PtUGT72B1 indicated that it had higher activities in conjugating TCP than AtUGT72B1 (Figure 2C). Although the amino acid sequence of the Populus glucosyltransferase had only 65% identity with AtUGT72B1, the similar structure of PtUGT72B1 proved that both glycosyltransferases belong to the same group. Both PtUGT72B1 and AtUGT72B1 have highly conserved residues within the 44 amino acid PSPG motif that interacts with the UDP-sugar.39,40 The residues forming the sugar acceptor pocket were also similar in both glycosyltransferases even

compounds by the addition or exposure of functional groups (e.g., hydroxyl or carboxyl). Phase II involves conjugation of xenobiotic metabolites of phase I to an endogenous hydrophilic molecule such as sugars and glutathione to form less toxic products. Then the conjugated xenobiotics are compartmentalized in vacuoles or bound to cell wall components such as lignin or hemicellulose in phase III.35,36 Trichlorophenols are suitable for direct phase II metabolism due to hydroxyl group in the ring. It was reported that Arabidopsis plants can metabolize 2,4,5TCP by O-glucosylation.37 As an excellent glucose acceptor, TCP can be glycosylated by several UDP-glc-dependent glucosyltransferases (UGTs) in Arabidopsis. Among all UGTs, UGT72B1 was shown to be more efficient than any other UGT in conjugating TCPs.21,38 In this present study, a UGT72B1like glycosyltransferase PtUGT72B1 in Populus was confirmed 4022

dx.doi.org/10.1021/es203753b | Environ. Sci. Technol. 2012, 46, 4016−4024

Environmental Science & Technology

Article

Author Contributions

though these residues were contained in less conserved loop regions. As shown in AtUGT72B1, the sugar acceptor TCP was enclosed in an entirely buried and compact hydrophobic site by Ile-86, Leu-118, Phe-119, Phe-148, Leu-183, and Leu-197, with only one hydrophilic function residue, Glu-83. This residue caps the acceptor site on the solvent side and presents a potential gateway to ligand entry and departure.21 All these residues were conserved in PtUGT72B1. The conserved residues let PtUGT72B1 interact with the same sugar donor and sugar acceptor as AtUGT72B1. A comparison of the position of these residues near the sugar acceptor pocket showed that two residues, D46 and L86, were different within the loops N2 and N3 (Nα3), respectively. Analyses of the VvGT1 and AtUGT72B1 crystal structures with the acceptors kaempferol and TCP, respectively, show that the residues within the sugar acceptor pocket were positioned too far from the acceptor to enable direct interactions. The acceptors are positioned within the compact acceptor pocket by hydrophobic interactions alone as no hydrogen bonds can be formed between the acceptor and the amino acid residues facing the active site of the enzyme.41,42 Based on these results, we speculate that the small amino acid D48 and the hydrophobic amino acid L86 allow greater TCP stabilization in the acceptor position by hydrophobic interactions than E47 and R85. The stabilization of the acceptor position as well as the acceptor in the acceptor binding pocket are highly important for activity, thus presenting a possible explanation for the greater activity of PtUGT72B1. Although PtUGT transgenic plants can remove TCP effectively from medium in a lab, further study on their remediation ability in natural environment condition should be done. The plants that mediate the site cleanup have to be where the pollutant is and required to be capable of acting on it. Therefore, the soil properties, toxicity level, and climate are very important for phytoremediation efficiency. Chlorophenols are strongly adsorbed by humic acid.43 The pH and the organic content of the soil is the most important factor controlling sorption. For example, pentachlorophenol (PCP) can bind strongly to soil and the rate of PCP dissipation is related to temperature, aeration, and organic matter, partly dependent on cation exchange capacity and pH.44 In a preliminary experiment, we found that higher content of peat moss in culture medium significantly impacted TCP uptake. The TCP removal ability of PtUGT transgenic plants was still stronger than that of wild type plants in this condition (data not shown). Our next target is to obtain more information on TCP degradation mechanisms of transgenic plants in soil and build up soil remediation technology. In conclusion, PtUGT72B1 transgenic Arabidopsis seedlings were able to glycosylate TCP. They exhibited better growth in medium containing 2,4,5-TCP and 2,4,6-TCP compared to the wild type plants. We further demonstrated that the overexpression of glycosyltransferases in the transgenic lines was correlated to plant TCP resistance and to the decreased level of TCP in the medium. Transgenic plants overexpressing PtUGT72B1 gene may provide a generally applicable strategy for bioremediation of TCP in soil.

■

§

These authors contributed equally to the data reported in this article. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The research was supported by the National Natural Science Foundation (31071486), International Scientific and Technological Cooperation (2010DFA62320), 863 Programs (2006AA06Z358), and the key project fund of Shanghai Municipal Committee of Agriculture (2009-6-4, 2011-1-8).

■

REFERENCES

(1) Sittig, M. Handbook of Toxic and Hazardous Chemicals; Noyes Publications: Park Ridge, NJ, 1981. (2) Agency for Toxic Substances and Disease Registry (ATSDR). Toxicological profile for chlorophenols, 1999; http://www.atsdr.cdc.gov/ toxprofiles/tp107.html 4/21/09. (3) Aranda, C.; Godoy, F.; Becerra, J.; Barra, R.; Martínez, P. Aerobic secondary utilization of a non-growth and inhibitory substrate 2,4,6trichlorophenol by Sphingopyxis chilensis S37 and Sphingopyxis-like strain S32. Biodegradation 2003, 14, 265−274. (4) Sánchez, M.; Vásquez, M.; González, B. A previously unexposed forest soil microbial community degrades high levels of the pollutant 2,4,6-trichlorophenol. Appl. Environ. Microbiol. 2004, 70, 7567−7570. (5) Xun, L.; Webster, C. A monooxygenase catalyzes sequential dechlorinations of 2,4,6-trichlorophenol by oxidative and hydrolytic reactions. J. Biol. Chem. 2004, 279, 6696−6700. (6) Karns, S.; Duttagupta, S.; Chakrabarty, A. M. Regulation of 2,4,5trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100. Appl. Environ. Microbiol. 1983, 46, 1182−1186. (7) Hübner, A.; Clyde, E.; Danganan, C. E.; Xun, L.; Chakrabarty, A. M.; Hendrickson, W. Genes for 2,4,5-Trichlorophenoxyacetic acid metabolism in Burkholderia cepacia AC1100: Characterization of the tftC and tftD genes and locations of the tft operons on multiple replicons. Appl. Environ. Microbiol. 1998, 64, 2086−2093. (8) Wang, J.; Hegemann, W. Microbial dehalogenation of trichlorophenol by a bacterial consortium: Characterization and mechanism. Sci. China 2003, 46, 207−215. (9) Aguayo, J.; Barra, R.; Becerra, J.; Martĭnez, M. Degradation of 2,4,6-tribromophenol and 2,4,6-trichlorophenol by aerobic heterotrophic bacteria present in psychrophilic lakes. World J. Microbiol. Biotechnol. 2009, 25, 553−560. (10) Joshi, D. K.; Gold, M. H. Degradation of 2,4,5-Trichlorophenol by the Lignin-Degrading Basidiomycete Phanerochaete chrysosponium. Appl. Environ. Microbiol. 1993, 59, 1779−1785. (11) Pringer, G.; Bhattacharya, S. K. Toxicity and fate of 2, 4, 6 Trichlorophenol in anaerobic acidogenic system. Water Res. 1999, 33, 2674−2682. (12) Ganigar, R.; Rytwo, G.; Gonen, Y.; Radiana, A.; Mishael, Y. G. Polymer−clay nanocomposites for the removal of trichlorophenol and trinitrophenol from water. Appl. Clay Sci. 2010, 49, 311−316. (13) Gaya, U. I.; Abdullah, A. H.; Hussein, M. Z.; Zainal, Z. Photocatalytic removal of 2,4,6-trichlorophenol from water exploiting commercial ZnO powder. Desalination 2010, 263, 176−182. (14) Jiang, Z.; Liu, X.; Bu, J. Removal of 2,4,6-Trichlorophenol by Iron and Manganese Oxides/Granular Activated Carbon with H2O2. Adv. Mater. Res. 2011, 154−155, 28−33. (15) Zhang, Q.; Xu, F. X.; Lambert, K. N.; Riechers, D. E. Safeners coordinately induce the expression of multiple proteins and MRP transcripts involved in herbicide metabolism and detoxification in Triticum tauschii seedling tissues. Proteomics 2007, 7, 1261−1278. (16) Peng, R. H.; Xu, R. R.; Fu, X. Y.; Xiong, A. S.; Zhao, W.; Tian, Y. S.; Zhu, B.; Jin, X. F.; Chen, C.; Han, H. J.; Yao, Q. H. Microarray analysis of the phytoremediation and phytosensing of occupational toxicant naphthalene. J. Hazard. Mater. 2011, 189, 19−26.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-21-62203180; fax: +86-21-62203180; e-mail: [email protected]. 4023

dx.doi.org/10.1021/es203753b | Environ. Sci. Technol. 2012, 46, 4016−4024

Environmental Science & Technology

Article

(17) Eapen, S.; Singh, S.; D’Souza, S. F. Advances in development of transgenic plants for remediation of xenobiotic pollutants. Biotechnol. Adv. 2007, 25, 442−451. (18) Jones, P.; Vogt, T. Glycosyltransferases in secondary plant metabolism: Tranquilizers and stimulant controllers. Planta 2001, 213, 164−174. (19) Ensley, H. E.; Sharma, H. A.; Barber, J. T.; Polito, M. A. Metabolism of chlorinated phenols by Lemma gibba Duckweed. In Phytoremediation of Soil and Water Contaminants; Kruger, E. L., Anderson, T. A., Coats, J. R., Eds.; American Chemical Society: Washington, DC, 1997; pp 238−253. (20) Li, Y.; Baldauf, S.; Lim, E.; Bowles, D. J. Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana. J. Biol. Chem. 2001, 276, 4338−4343. (21) Brazier-Hicks, M.; Edwards, R. Functional importance of the family 1 glucosyltransferase UGT72B11 in the metabolism of xenobiotics in Arabidopsis thaliana. Plant J. 2005, 42, 556−566. (22) Newman, L.; Strand, S.; Choe, N.; Duffy, J.; Ekuan, G.; Ruszaj, M.; Shurtleff, B. B.; Wilmoth, J.; Heilman, P.; Gordon, M. P. Uptake and biotransformation of trichloroethylene by hybrid poplars. Environ. Sci. Technol. 1997, 31, 1062−1067. (23) Gordon, M. P.; Choe, N.; Duffy, J.; Ekuan, G.; Heilman, P.; Muiznieks, I.; Ruszaj, M.; Shurtleff, B. B.; Strand, S. E.; Wilmoth, J. Phytoremediation of trichloroethylene with hybrid poplars. Environ. Health Perspect. 1998, 106, 1001−1004. (24) Ballach, H. J.; Kuhn, A.; Wittig, R. Biodegradation of anthracene in the roots and growth substrate of poplar cuttings. Environ. Sci. Pollut. Res. 2003, 10, 308−316. (25) Kuhn, A.; Ballach, H. J.; Wittig, R. Studies in the biodegradation of 5 PAHs (phenanthrene, pyrene, fluoranthene, chrysene and benzo(a)pyrene) in the presence of rooted poplar cuttings. Environ. Sci. Pollut. Res. 2004, 11, 22−32. (26) Xiong, A. S.; Yao, Q. H.; Peng, R. H.; Li, X.; Fan, H. Q.; Cheng, Z. M.; Li, Y. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequence. Nucl. Acids Res. 2004, 32, e98. (27) Xiong, A. S.; Yao, Q. H.; Peng, R. H.; Duan, H.; Li, X.; Fan, H. Q.; Cheng, Z. M.; Li, Y. PCR-based accurate synthesis of long DNA sequences. Nat. Protoc. 2006, 1, 791−797. (28) Wu, S.; Letchworth, G. J. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. BioTechniques. 2004, 36, 152−154. (29) Zhang, X.; Henriques, R.; Lin, S. S.; Niu, Q. W.; Chua, N. H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 2006, 1, 641−646. (30) Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962, 15, 473−497. (31) Wang, G. D.; Li, Q. J.; Luo, B.; Chen, X. Y. Ex planta phytoremediation of trichlorophenol and phenolic allelochemicals via an engineered secretory laccase. Nat. Biotechnol. 2004, 22, 893−897. (32) Bradford, M. M. A rapid and sensitive method for the quantitation of miaogram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (33) Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. The swiss-model workspace: A web-based environment for protein structure homology modelling. Bioinformatics 2006, 22, 195−201. (34) Dixon, S. C.; Martin, R. C.; Mok, M,C.; Shaw, G.; Mok, D. W. S. Zeatin glycosylation enzymes in Phaseolus: Isolation of O-glucosyltransferase from P. vulgaris. Plant. Physiol. 1989, 90, 1316−1321. (35) Coleman, J. O. D.; Blake-Kalff, M. A. A.; Emyr-Davis, T. G. Detoxification of xenobiotics by plants: Chemical modification and vacuolar compartmentalisation. Trends Plant Sci. 1997, 2, 144−151. (36) Dietz, A. C.; Schnoor, J. L. Advances in phytoremediation. Environ. Health Perspect. 2001, 109, 163−168. (37) Messner, B.; Thulke, O.; Schaffner, A. R. Arabidopsis glucosyltransferases with activities toward both endogenous and xenobiotic substrates. Planta 2003, 217, 138−146.

(38) Brazier-Hicks, M.; Offen, W. A.; Gershater, M. C.; Revett, T. J.; Lim, E. K.; Bowles, D. J.; Davies, G. J.; Edwards, R. Characterization and engineering of the bifunctional N- and O-glucosyltransferase involved in xenobiotic metabolism in plants. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 20238−20243. (39) Hughes, J.; Hughes, M. A. Multiple secondary plant product UDP-glucose glucosyltransferase genes expressed in cassava (Manihot esculenta Crantz) cotyledons. DNA Seq. 1994, 5, 41−49. (40) Paquette, S.; Møller, B. L.; Bak, S. On the origin of family 1 plant glycosyltransferases. Phytochemistry 2003, 62, 399−413. (41) Imberty, A.; Wimmerova, M.; Koca, J.; Breton, C. Molecular modeling of glycosyltransferases. Methods Mol. Biol. 2006, 347, 145− 156. (42) Osmani, S. A.; Bak, S.; Møller, B. L. Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling. Phytochemistry 2009, 70, 325−347. (43) Gribble, G. W. Amazing organohalogens. Am. Sci. 2004, 92, 342−349. (44) Warrington, P. D. The fate of chlorophenols in the environment. In Ambient Water Quality Guidelines for Chlorophenols; Warrington, P.D., Eds.; BC Environment, Water Management Branch, 1997.

4024

dx.doi.org/10.1021/es203753b | Environ. Sci. Technol. 2012, 46, 4016−4024