The fungal lignin-degrading enzymes lignin peroxidase (LiP, E.C. 220.127.116.11), Mn-dependent peroxidase (MnP, E.C. 18.104.22.168), and phenol oxidase (laccase) (Lac, E.C. 22.214.171.124) can degrade or polymerize organic pollutants such as polychlorophenols, polycyclic aromatic hydrocarbons, and chlorinated hydrocarbons (Fernando and Aust, 1994; Hammel, 1989; Hirano et al., 2000; Levin et al., 2003;Lin et al., 1990; Lovley et al., 1994; Mohn and Tiedje, 1992; Reddyy et al., 1998). However, to maintain such fungal lignin-degrading enzymes at adequate levels for degradation or detoxification (bioremediation), appropriate additions of both microorganisms and nutrients are essential over long periods of time. Recently, phytoremediation technology has gained attention for its potential as an ecological remediation tool of contaminated soil and water, as plants can grow autotrophically. Establishment of effective phytoremediation technology is a suitable strategy for the long-term remediation of contaminated areas. Phytoremediation includes some processes based on the plant functions as follows; phytostabilization, which is accumulation of pollutants in the rhizosphere by absorption on the root surface, precipitation, and complexation of pollutants; rhizodegradation, which is degradation of pollutants by interaction with rhizosphere microorganisms; phytoaccumulation (phytoextraction), which is uptake and accumulation of pollutants by plants; phytodegradation (phytotransformation), which is uptake and degradation of pollutants by plants; and phytovolatilization, which is uptake and volatilization of pollutants by transpiration from contaminated area.To widely apply the benefit of phytoremediation, improvement and reinforcement of the abilities for uptake, accumulation and degradation of pollutants using genetic engineering are one of the important development subjects.
There have been many reports of phytoremediation using transgenic plants. For example, glutathione S transferase and cytochrome P450 expression showed high resistance to pesticides (Gullner et al., 2001; Doty et al., 2000), the overexpression of bacterial mercury reductase showed high resistance to organic mercury (Bizilly et al., 2003) and effective volatilization of ionic mercury (Haque et al., 2010), pentaerythritol tetranitrate reductase-expressing plants were able to degrade glycerol trinitrate and 2,4,6-trinitrotoluene (French et al., 1999), introduction of bacterial genes involved in polychlorinated biphenyl (PCB) degradation in plants showed removal of PCB from a contaminated area (Novakova et al., 2009), the bacterial arsenite S-adenosylmethyltransferase expression induced arsenic methylation and volatilization (Xiang-Yan et al., 2011), the expression of gamma-glutamylcysteine synthetase and the genes involved in phytochelatin synthesis in plant showed more resistance and accumulation of cadmium (Zhu et al, 1999, Wawrzyński et al, 2006 ), and the yeast metallothionein expressing tobacco showed effective copper uptake (Thomas et al, 2003).
Recently, attempts are carried out to enhance the environmental remediation in contaminated area by using appropriate genetically modified plants with usage of fungal peroxidases. This chapter mainly focused on the removal of bis-phenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane), which is one of the major chemicals used in plastics and resins and is well known to disrupt endocrine systems in humans and other animals, from contaminated areas with usage of transgenic technology. Although many organisms can degrade and metabolize BPA, which can lead to a reduction of the estrogenicity and toxicity of BPA (Kang et al., 2006), lignin-degrading basidiomycete fungi are particularly powerful degraders of organic pollutants including BPA. These fungi produce oxidative enzymes, such as LiP, Lac, and MnP, which can degrade and polymerize BPA both
2. LiP-expressing transgenic tobacco
cDNA (Accession no. AB158478.1) encoding LiP from the reverse transcription (RT) products of total RNA prepared from mycelia of
Integration of the cDNA into the genome of tobacco was confirmed by polymerase chain reaction (PCR) upon 10 independent transgenic lines. Two of the lines showed growth inhibition and thus were excluded from further analysis. Western blot analysis with root extracts of transgenic tobaccos and antiserum raised against LiP protein were performed to confirm the production of LiP protein in roots of transgenic lines.. To prepare the antiserum against LiP of
RB, Right border of T-DNA; N-pro, promoter region of nopaline synthase gene;
Lanes; 1, LiP transgenic line (FLP)-1; 2, FLP-2; 3, FLP-3; 4, FLP-4; 5, FLP-5; 6, FLP-8; 7, control plant.
To test the ability of BPA removal by LiP-expressing transgenic plants, we transferred two-month-old transgenic lines on MS medium (Murashige and Skoog, 1962) to fresh MS liquid medium containing 3 g/L of glucose and 100 μg/L of kanamycin. After one week of incubation at 25 C, BPA was added to the medium at the final concentration of 100 μM and the medium was hydroponically incubated for another week. The six LiP-expressing transgenic linesshowed 2- to 4-fold higher BPA removal ability than that of control plants during aqueous cultivation (Figure 3). LiP is a well-known enzyme that carries out direct and indirect oxidation of a number of environmental pollutants. Our confirmation that transgenic plants could express LiP in their roots and remove BPA will help us to establish improved methods for phytoremediation of contaminated environments.
The levels of BPA were analyzed by HPLC (λ=278 nm). The values shown are the average of results from three independent experiments. Lanes; 1, control; 2, FLP-1; 3, FLP-2; 4, FLP-3; 5, FLP-4; 6, FLP-5; 7, FLP-8. Error bars on the graph indicate standard deviations (
3. Lac-expressing transgenic tobacco
Lac is a member of the multicopper oxidase family found in a wide range of organisms such as animals, plants, bacteria, and fungi. The reduction of oxygen to water is accompanied by the oxidation of substrate by laccase.
cDNA encoding Lac (Accession no. D13372.1) from the reverse transcription products of total RNA prepared from mycelia of
cvL3, cDNA encoding Lac of
Concentrated 60 μg of crude extracellular protein was analyzed by IEF and active staining using 4-chloro-1-naphthol. Lanes, 1, Concentrated aqueous cultivation medium of
Two-month-old transgenic lines, which were incubated on MS medium, were transferred to fresh MS liquid medium and subjected to further incubation. After two weeks, to confirm the expression of Lac protein and secretion from the roots of each transgenic line into the rhizosphere, we concentrated the aqueous culture medium and analyzed it by iso-electric focusing electrophoresis (IEF) and active staining using 4-chloro-1-naphtol (Figure 5). Six independent transgenic lines apparently secreted active Lac protein into their rhizosphere, and we tested four of those to determine their ability to remove BPA. As described above, four independent transgenic lines were cultivated hydroponically. After one week of incubation, BPA was added to the medium at the final concentration of 100 μM and hydroponic incubation was done for another week. The ability to remove BPA of these Lac-expressing transgenic tobaccos was more than 5-fold that of the control line during hydroponic cultivation (Figure 6).
The levels of BPA were analyzed by HPLC (λ=278 nm). The results shown are the average of three independent experiments. Lanes; 1, control; 2, FL-4; 3, FL-9; 4, FL-20; 5, FL-22. Error bars on the graph indicate standard deviations (
All of these Lac-expressing transgenic tobaccos were somewhat shorter than control plants at the flowering stage, and most of the transgenic anthers failed to dehisce after blooming, while the anthers of control plants werenormally dehiscent (Figure 7). In addition, the nondehiscent anthers were brown in contrast to the greenish control lines. Brown pigmentation and rough epidermis were observed on the surface of transgenic anthers. Greater Lac activity was detected in the cell-free extracts of transgenic anthers than in the controls; however, there was no correlation with lignin contents in transgenic anthers (Figure 8). Histochemical analysis of anther tissues revealed apparent deformation of the stomium in transgenic plants (Figure 9). Beals reported that the stomium in anther tissue plays a crucial role in the dehiscence of anthers in tobacco (Beals, 1997), indicating that such deformation of stomium observed in the transgenic anther tissue might affect the appearance of the nondehiscent phenotype. The expression of Lac could promote the efficient removal of BPA, but it also influences some aspects of flower development.
Transgenic and control tobaccos were cultivated at 24 C. a, Transgenic flower with nondehiscent anthers. b, Control flower with normal anthers. c, Stereomicroscopic view of a transgenic anther. d, Stereomicroscopic view of a normal anther.
Transgenic and control tobaccos were cultivated at 24 C. a, Laccase activity. Cell-free extracts were prepared from both transgenic and control anthers before they dehisced. Laccase activity was calculated using the extinction coefficient (6400 M-1cm-1) of oxidized guaiacol (λ=436nm), and activity was expressed as definitive units (1 unit = 1 mol guaiacol oxidized per min) (Eggert et al, 1996). b, Lignin content. Lignin was quantified by the Klason method. The results shown are the average of three independent experiments. Error bars on the graph indicate standard deviations (
Safranin-stained thin sections of a mature anther from a transgenic (a) and a control plant (b). ep, epidermis; st, stomium; en, endothecium cell.
4. MnP-expressing transgenic hybrid aspen
MnP is a heme peroxidase that can oxidize phenolic compounds in the presence of Mn (II) and hydrogen peroxide. Mn (II) is oxidized to Mn (III) by MnP; the resultant Mn (III) makes a chelating compound with an organic acid, and then organic compounds such as BPA are oxidized by the chelating compound. Previously, we isolated a cDNA (Accession no. AR429405) encoding MnP from
fmnp, cDNA encoding MnP of T. versicolor IFO1030 plus signal sequence. Other abbreviations are listed in Figure 1.
The levels of BPA were analyzed by HPLC (λ=278 nm). The values shown are the average of results from three independent experiments. Lanes; 1, control;2, FM-2; 3, FM-3; 4, FM-4; 5, FM-7. Error bars on the graph indicate standard deviations (N=3).
As described above, fungal peroxidase (LiP, Lac, and MnP)-expressing transgenic plants showed effective BPA removal ability, but no reaction products of BPA conversions by these fungal peroxidase-expressing transgenic plants were detected under our analytical conditions. The enzymatic reaction of fungal peroxidases is non-specific and free radical-based, so it is difficult to detect the reaction products. BPA might be degraded or polymerized, as reported in some previous studies of lignolytic enzymes (Hirano et al., 2000; Fukuda et al., 2001; Tsutsumi et al., 2001; Uchida et al., 2001). The increase of BPA removal efficiency by the fungal peroxidase expression in plants would contribute to the development of remediation systems for the cleanup of contaminated areas.
Plants can metabolize BPA. Cultured cells of plants were able to glucosylate BPA (Nakajima et al., 2002; Hamada et al., 2002), and, in seedlings, BPA was absorbed from roots and translocated to leaves after glucosylation (Nakajima et al., 2002). In addition, some glycosylated forms of BPA showed less estrogenic activity than that of non-glycosylated BPA (Morohoshi et al., 2003), and oxidative enzymes in plants such as peroxidases stimulated the degradation and polymerization of BPA (Sakuyama et al., 2003). Although the ability of plants to detoxify might be useful for remediation of soil and water contaminated with BPA, the expressions of fungal peroxidases in plants by genetic engineering, as reviewed above, reinforces their ability with respect to the detoxification of BPA. Furthermore it is worth noting that the MnP- and Lac-expressing transgenic plants could remove pentachlorophenol effectively from contaminated areas during hydroponic cultivation (Iimura et al., 2002; Sonoki et al., 2005). Plants could secrete Lac and generate Mn (III) in the rhizosphere, and then the Lac and Mn (III) might be able to affect hydrophobic substrates, such as pentachlorophenol, which is difficult for plant roots to absorb.
Plants producing fungal secretory peroxidases would provide us useful tools for the remediation of areas contaminated with environmental pollutants. Further studies on the effective expression and the secretion of introduced enzymes and the application with other substrates will play an important role in the development of phytoremediation technology.
This work was supported in part byGrant-in-Aid for Scientific Research A (no. 21248037) from Japan Society for the Promotion of Science (JSPS) and Hirosaki University Grant for Exploratory Research by Young Scientists.
Beals T. P. Goldberg R. 1997A novel cell ablation strategy blocks tobacco anther dehiscence. Vol. 9, 9 1527 1545, 1040-4651
Bizilly S. P. Kim T. Kandasamy M. K. Meagher R. 2003Subcellular targeting of methylmercury lyase enhances its specific activity for organic mercury detoxification in plants. 131 2 463 471, 0032-0889
Brasileiro A. C. Leple J. C. Muzzin J. Ounnoughi D. Michel M. F. Jouanin 1991An alternative approach for gene transfer in trees using wild-type strains.Plant Mol. Biol. 17 3 441 452, 0167-4412
Doty S. L. Shang T. Q. Wilson A. M. Tangen J. Westergreen A. D. Newman L. A. Strand S. E. Gordon M. 2000Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1. Vo. 97, 12 6287 6291, 0027-8424
Eggert C. Temp U. Eriksson K. 1996The ligninolytic system of the white rot fungus : purification and characterization of the laccase. Appl. Environ. Microbiol. 62 4 1151 1158, 0099-2240
Fernando T. Aust S. 1994Biodegradation of toxic chemicals by white-rot fungi, In:Chaudhry, G. R., 386 402, Chapman & Hall, 978-0-41262-290-8London.
Fukuda T. Uchida H. Takashima Y. Uwajima T. Kawabata T. Suzuki 2001Degradation of bisphenol A by purified laccase from . Biochem, Biophys. Res. Commun. 284 3 704 706, 0000-6291x.
Gullner G. Komives T. Rennenberg H. 2001Enhanced tolerance of transgenic poplar plants overexpressing gamma-glutamylcysteine synthetase towards chloroacetanilide herbicides. 52 358 971 979, 0022-0957
Hamada H. Tomi R. Asdada Y. Furuya T. 2002Phytoremediation of bisphenol A by cultured suspension cells of -regioselective hydroxylation and glycosylation. Tetrahedron Lett., 43 22 4087 4089, 0040-4039
Hammel K. 1989Organopollutant degradation by ligninolytic fungi. 11 11 776 777, 0141-0229
Haque S. Zeyaullh M. Nabi G. Srivastava P. S. Ali A. 2010Transgenic tobacco plant expressing environmental gene for enhanced volatilization of ionic mercury. J. Microbiol. Biotechnol. 20 5 917 924, 1017-7825
Hirano T. Honda Y. Watanabe T. Kuwahara M. 2000Degradation of bisphenol A by the lignin-degrading enzymes, manganese peroxidase, produced by the white-rot basidomycete, . Biosci. Biotechnol. Biochem. 64 9 1958 1962, 0916-8451
Iimura Y. Ikeda S. Sonoki T. Hayakawa T. Kajita S. Kimbara K. Tatsumi K. Katayama Y. 2002, Expression of a gene for Mn-peroxidase from in transgenic tobacco generates potential tools for phytoremediation. Appl. Microbiol. Biotechnol. 59 2-3, 246 251, 0175-7598
Kajita S. Honaga F. Uesugi M. Iimura Y. Masai E. Kawai S. Fukuda M. Morohoshi N. Katayama Y. 2004, Generation of transgenic hybrid aspen that express a bacterial gene for feruloyl-CoA hydratase/lyase (FerB), which is involved in lignin degradation in SYK-6. J. Wood Sci. 50 3 275 280, 1435-0211
Kang J. H. Katayama Y. Kondo F. 2006Biodegradation or metabolism of bisphenol A: from microorganisms to mammals. Vol. 217, 2-3, 81 90, 0003-0483X.
Levin L. Viale A. Forchiassin A. 2003, Degradation of organic pollutants by the white rot basidiomycete 52 1 1 5, 0964-8305
Liang X. W. Dron M. Schmid J. Dixon R. A. Lamb C. 1989, Dixon, R. A., Lamb, C. J. 1989. Developmental and environmental regulation of a phenylalanine ammonia-lyase-beta-glucronidase gene fusion in transgenic tobacco plants. Vo. 86, 23 9284 9288, 0027-8424
Lin J. E. Wang H. Y. Hickey R. 1990Degradation kinetics of pentachlorophenol by . Biotech. Bioeng. 35 11 1125 1134, 0006-3592
Lovley D. R. Woodward J. C. Chapelle F. 1994Characterization of the mnp2 gene encoding manganese peroxidase isozyme 2 from the basidomycete . GeneVol. 142, 231 235, 0378-1119
Mohn W. W. Tiedje J. 1992Microbial reductive dehalogenation. 56 3 482 507, 1098-5557
Morohoshi K. Shiraishi F. Oshima Y. Koda T. Nakajima N. Edmonds J. S. Morita M. 2003Synthesis and estrogenic activity of bisphenol A mono- and Di-beta-D-glucopyranosides, plant metabolites of bisphenol A. 22 10 2275 2279, 0730-7268
Murashige T. Skoog F. 1962A revised medium for the rapid growth and bioassay with tobacco tissue cultures. 15 473 497, 0032-0889
Nakajima N. Ohshima Y. Serizawa S. Kouda T. Edmonds J. S. Shiraishi F. Aono M. Kubo A. Tamaoki M. Saji H. Morita M. 2002Processing of bisphenol A by plant tissues: glucosylation by cultured BY-2 cells and glucosylation/translocation by plants of . Plant Cell Physiol. 43 9 1036 1042, 0032-0781
Novakova M. Mackova M. Chrastilova Z. Viktorova J. Szekeres M. Demnerova K. Macek T 2009Cloning the bacterial gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation. Biotechnol. Bioeng. 102 1 29 37, 0006-3592
Reddyy G. V. B. Gelpke M. D. S. Gold M. 1998Degradation of 2,4,6-trichlorophenol by : Involvement of reductive dechlorination. J. Bacteriol. 180 19 5159 5164, 0021-9193
Sakuyama H. Endo Y. Fujimoto K. Hatano . 2003, Oxidative degradation of alkylphenols by horseradish peroxidase. 96 3 227 231, 1389-1723
Sonoki T. Kajita S. Ikeda S. Uesugi M. Tatsumi K. Katayama Y. Iimura Y. 2005, Transgenic tobacco expressing fungal laccase promotes the detoxification of environmental pollutants. 67 1 138 142, 0175-7598
Thomas J. C. Davies E. C. Malick F. K. Endreszi C. Williams C. R. Abbas M. Petrella S. Swisher K. Perron M. Edwards R. Osenkowski P. Urbanczyk N. Wiesend W. N. Murray K. 2003, Yeast metallothionein in transgenic tobacco promotes copper uptake from contaminated soils. Biotechnol. Prog. 19 2 273 280, Online 1520-6033
Tsutsumi Y. Haneda T. Nishida T. 2001, Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin degrading basidomycetes. Vol. 42, 3 271 276, 0045-6535
Uchida H. Fukuda T. Miyamoto H. Kawabata T. Suzuki M. Uwajima T. 2001Polymerization of bisphenol A by purified laccase from . Biochem. Biophys. Res. Commun. 287 2 355 358, 0000-6291X.
Wawrzyński A. Kopera E. Wawrzyńska A. Kamińska J. Bal W. Sirko A. 2006Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants. J. Exp. Bot. 57 10 2173 2182, 0022-0957
Xiang-Yan M. Jie Q. Li-Hong W. Gui-Lan D. Guo-Xin S. Hui-Lan W. Cheng-Cai C. Hong-Qing L. Barry P. R. Yong-Guan Z. 2011Arsenic biotransformation and volatilization in transgenic rice. Vol. 191, 1 49 56, 0002-8646X.
Zhu Y. L. Pilon-Smits E. A. Tarun A. S. Weber S. U. Jouanin L. Terry N. 1999Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine synthetase. 121 4 1169 1178, 0032-0889