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Environ. Sci. Technol. 2005, 39, 7671-7677

Secretion of Bacterial Xenobiotic-Degrading Enzymes from Transgenic Plants by an Apoplastic Expressional System: An Applicability for Phytoremediation E. UCHIDA,† T. OUCHI,† Y. SUZUKI,‡ T. YOSHIDA,† H. HABE,† I. YAMAGUCHI,‡ T . O M O R I , †,§ A N D H . N O J I R I * ,† Biotechnology Research Center and Department of Applied Biological Chemistry, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

In search of an effective method for phytoremediation of wastewater contaminated with organic compounds, we investigated the application of an apoplastic expressional system that secretes useful bacterial enzymes from transgenic plants into hydroponic media through the addition of a targeting signal. We constructed transgenic Arabidopsis expressing the aromatic-cleaving extradiol dioxygenase (DbfB), which degrades 2,3-dihydroxybiphenyl (2,3-DHB), and transgenic tobacco expressing haloalkane dehalogenase (DhaA), which catalyzes hydrolytic dechlorination of 1-chlorobutane (1-CB). Although crude leaf extracts of transgenic plants expressing cytoplasm-targeted degradative enzymes showed higher activity than did those from transgenic plants expressing apoplast-targeted enzymes, the hydroponic media of the latter showed 23.2 times (DbfB) and 76.4 times (DhaA) higher activity than plants containing the cytoplasm-targeted enzymes. Addition of crystalline 2,3DHB to 100 mL of the hydroponic medium of transgenic or wild-type seedlings revealed that only medium from the transgenic Arabidopsis expressing apoplast-targeted DbfB showed rapid ring cleavage of 2,3-DHB. Transgenic tobacco expressing apoplast-targeted DhaA also resulted in the accumulation of the dehalogenation product 1-butanol in the hydroponic medium and showed a higher tolerance to 1-CB than wild-type or transgenic plants expressing cytoplasm-targeted DhaA. These results demonstrate the usefulness of the apoplastic expression of bacterial recombinant proteins in phytoremediation.

Introduction In the past century, the burden of man-made synthetic compounds in ecosystems has increased tremendously. The contamination of soil and water with persistent organic compounds has caused various environmental and human health problems. It is critical that the concentrations of such contaminants in the environment are reduced. Because * Corresponding author phone: 81-3-5841-3064; fax: 81-3-58418030; e-mail: [email protected]. † Biotechnology Research Center. ‡ Department of Applied Biological Chemistry. § Present address: Department of Industrial Chemistry, Shibaura Institute of Technology, 3-9-14 Shibaura, Minato-ku, Tokyo 1088548, Japan. 10.1021/es0506814 CCC: $30.25 Published on Web 08/27/2005

 2005 American Chemical Society

physical and chemical treatments for large-scale remediation have high costs, bioremediation constitutes an intriguing alternative method. Although bacteria that degrade pollutants show good biodegradative abilities under laboratory conditions, they often compete poorly with indigenous microorganisms, resulting in a rapid decline in the biomass of the introduced bacteria. Recent attention has focused on phytoremediation, the use of green plants for in situ bioremediation. Plants have economic benefits, because they are a robust and renewable resource. Furthermore, they have an ability to extract compounds from the surrounding environment (1, 2). However, because the innate abilities of plants to biodegrade or biotransform various organic pollutants are lower than those of adapted bacteria and fungi, phytoremediation using engineered plant(s) expressing degradative enzymes from microorganisms may yield more useful systems for in situ bioremediation of xenobiotics. Several reports have been published that describe phytoremediation using transgenic plants. For example, French et al. (3) reported that transgenic tobacco expressing pentaerythritol tetranitrate reductase from Enterobacter cloacae strain PB2 had the ability to detoxify the explosive nitroglycerin. The same group also showed that transgenic tobacco expressing a nitroreductase from E. cloacae strain NCIMB10101 were able to tolerate and detoxify persistent 2,4,6-trinitrotoluene at contaminated sites (4). Transgenic tobacco expressing mammalian cytochrome P450 monooxygenase has been shown to harbor oxygenating activity for organic compounds, such as the herbicide chlortoluron, 7-ethoxycoumarin, benzo[a]pyrene, and halogenated hydrocarbons (5, 6). In addition, transgenic Arabidopsis expressing the bacterial mercury-processing enzymes organomercurial lyase (MerA) and mercuric reductase (MerB) have been shown to convert organomercury to the much less toxic mercury ion, and then to elemental mercury, which spontaneously volatilizes into the atmosphere (7). In addition, to increase the specific activity of MerB in plants, the enzyme was subcellularly targeted (8). To direct the desired proteins to be secreted into the cell wall or the apoplastic fraction, or to accumulate in the endoplasmic reticulum (ER), the proteins were modified by the addition of ER-targeting and ER-retention signal peptides. Borisjuk et al. (9), who termed this process “rhizosecretion,” argued its advantages in producing recombinant proteins, because of the ease of purifying the recombinant protein and the low risk of contamination by pathogenic viruses, which may be present in transgenic animals. The use of the secretion in phytoremediation has the strong advantage that the secreted enzyme has direct access to the pollutant chemicals. Recently, an extracellular fungal enzyme produced in tobacco, a laccase from Coriolus versicolor containing the original signal peptide, was successfully secreted into the rhizosphere (10). Although these reports discussed the secretion of enzymes into the rhizospere, an apoplastic expressional system had not been compared with a traditional cytoplasmic expressional system for usefulness in phytoremediation. In this study, we generated model plants that secrete bacterial organic-compound-degrading enzymes into the hydroponic media, owing to the addition of a plant secretion signal, and estimated their usefulness in the cleanup of organic compounds in water ecosystems. We employed two types of degradative enzymes that are involved in the detoxification of aromatic and aliphatic chemicals. The first is the meta-cleavage enzyme extradiol dioxygenase, or DbfB, which functions in the dibenzofuran degradation pathway VOL. 39, NO. 19, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Pathways of degradation of dibenzofuran by Terrabacter sp. strain DBF63 (A) (31) and of 1-chlorobutane by Rhodococcus sp. strain m15-3 (B) (13). Enzyme designations: DbfA, dibenzofuran 4,4a-dioxygenase; DbfB, 2,2′,3-trihydroxybiphenyl 1,2-dioxygenase; DbfC, 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid hydrolase; DhaA, haloalkane dehalogenase. in Terrabacter sp. strain DBF63 (Figure 1A) (11). This enzyme catalyzes the ring cleavage reaction that destroys the carbon skeleton of aromatic compounds. Meta-cleavage by DbfB of the catecholic ring of 2,3-dihydroxybiphenyl (2,3-DHB), a structural analogue of the initial oxygenation product of dibenzofuran, yields the yellow product 2-hydroxy-6-oxo6-phenylhexa-2,4-dienoate (HOPDA). Dihydroxybiphenyls, such as 2,3-DHB, are very toxic compounds for bacteria even after short incubation times, affecting cell viability (12). In addition, the production of a colored compound is highly useful in the detection of enzymatic activity in the plant and its hydroponic medium. We also used the 1-chlorobutane (1-CB)-degrading haloalkane dehalogenase (DhaA), isolated from Rhodococcus sp. strain m15-3 (formerly Corynebacterium sp. strain m15-3) (13, 14), which catalyzes the initial reaction in the 1-CB degradation pathway by this strain (Figure 1B). Most haloalkanes are toxic, persistent, and cause serious environmental problems (13). Since short-chain 1-halo-n-alkanes (C3-C8) are effectively hydrolyzed to nalcohol by DhaA, without requiring a cofactor, DhaA is appropriate for phytoremediation using the apoplastic expressional system. Therefore, we constructed transgenic plants expressing either apoplast- or cytoplasm-targeted DbfB or DhaA and compared the xenobiotic-degradation abilities of these plants.

Materials and Methods Construction of Binary Vectors for Plant Transformation. Polymerase chain reaction (PCR) was performed using Ex Taq HS DNA polymerase (TaKaRa Shuzo Co. Ltd., Kyoto, Japan), according to the manufacturer’s protocol. The dbfB gene was amplified by PCR using the plasmid pLM1 (11) as a template and pairings of the specific forward primers (cytoplasmic forward, 5′-GGATCC-GGAACC-ATG-GCT-AGTGGCGTAACCGAGCTC-3′; apoplastic forward, 5′-GGATCCATG-AGTGGCGTAACCGAGCTC-3′) with the reverse primer (5′-GCGGCCGC-GAACCCCGCCTTCGCTGC-3′). The artificial restriction sites (BamH I or Not I) that were introduced, the initiation codon, and a eukaryotic translation initiation signal sequence (ETS) (15) are indicated by underlining, bold, and italics, respectively. The PCR conditions used were an initial denaturation step at 95 °C for 2 min, 30 cycles of 30 s at 95 °C, 60 s at 50 °C, and 60 s at 72 °C, followed by 7 min at 72 °C. The nucleotide sequences of the resulting PCR products were confirmed to be correct, and the fragments were ligated into the pT7blue (R) vector to produce pT7C-dbfB (for cytoplasmic expression) and pT7A-dbfB (for apoplastic expression). The Not I-Hind III gene fragment from pRTRA15, which contains the c-myc tag, the KDEL (ER retention signal), 7672

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and the cauliflower mosaic virus (CaMV) 35S terminator sequence (15), was used to add this gene fragment to the 3′ end of the dbfB gene modified for cytoplasmic expression. The KDEL was reported to stabilize the expressed enzymes in the cytoplasm (16). The plasmid pRTRA15 was kindly provided by Dr. Udo Conrad. To prepare the c-myc tag and the CaMV 35S terminator gene fragment for apoplastic expression, the c-myc tag or the CaMV 35S terminator gene fragment was separately amplified by PCR using pRTRA15 as template and the appropriate primer sets [for the c-myc tag gene, forward (5′-GCGGCCGCAGAACAAAAACTC-3′) and reverse (5′-GGGCTAGGATCCATTCAG-ATCCTCTTC-3′), and for the CaMV 35S terminator, forward (5′-CTGAATGGATCCTAGCCC-GATCCGTCGATAGAG-3′) and reverse (5′-AAGCTTGCATGCCTGCAGGTCATCGGA-3′)]. The complementary region used for the fusion of the two resulting PCR amplicons is indicated by italics. After the first PCR reaction, conducted using the above conditions, a second round of PCR was performed using a mixture of the first PCR amplicons and the c-myc tag forward and CaMV 35S terminator reverse primers, with the described above conditions. The nucleotide sequence of the resulting PCR fragment was confirmed as correct, and then the fragment was ligated into pT7blue (R) vector to produce pT7A-KDEL. To add a legumin B4 signal peptide (LeB4SP) gene fragment (17) at the 5′-end of the dbfB gene modified for apoplastic expression, we used the plasmid pBSSP, which contains the LeB4SP and same ETS as the cytoplasmic forward primer between the Xba I and BamH I sites of pBluescript II KS(-) (Stratagene, La Jolla, CA). The BamH I-Not I gene fragment from pT7C-dbfB and the Not I-Hind III gene fragment from pRTRA15 (c-myc tag, the KDEL, and CaMV 35S terminator) were simultaneously ligated into pBluescript II KS(-), and the resulting plasmids were named pBSC-dbfB (for cytoplasmic expression). On the other hand, the BamH I-Not I gene fragment from pT7AdbfB and the Not I-Hind III gene fragment from pT7A-KDEL (c-myc tag and CaMV 35S terminator) were simultaneously ligated into pBSSP, and the resulting plasmids were named pBSA-dbfB (for apoplastic expression). Xba I-Hind III gene fragments from pBSC-dbfB or pBSA-dbfB was excised [the Hind III site of the 3′ end of the CaMV 35S terminator was blunt-ended by treatment with the Klenow fragment (Roche Diagnostics GmbH, Mannheim, Germany)], and ligated between the Xba I and EcoR I sites of the plant transformation vector pBI121 (the EcoR I site was blunt-ended) (18) to produce pBIC-dbfB or pBIA-dbfB, respectively. The dhaA gene was fused into two cassettes, one for apoplastic expression in plants and the other for cytoplasmic expression. The primers used were as follows: cytoplasmic

forward, 5′-GGATCC-GGAACC-ATG-GCT-TCAGAAATCGGTACAGGCTTCCCC-3′; apoplastic forward, 5′-GGATCC-ATGTCAGAAATCGGTACAGGCTTCCCC-3′; and reverse, 5′GCGGCCGC -GAGTGCGGGGAGCCAGCGCGCGATCTC-3′. The introduced restriction sites are underlined and the ETS (15) is italicized. The PCR conditions used were an initial denaturation step at 95 °C for 2 min, 30 cycles of 30 s at 95 °C, 30 s at 65 °C, and 60 s at 72 °C, and a final extension step for 7 min at 72 °C. The plant transformation vectors were constructed as for pBIC-dbfB or pBIC-dbfB and are designated pBIC-dhaA or pBIA-dhaA, respectively. Plant Transformation. The T-DNAs of two dbfB constructs were introduced into Arabidopsis (Arabidopsis thaliana ecotype Columbia), and the T-DNAs of the two dhaA constructs were introduced into tobacco (Nicotiana tabacum L. cv. Petit Havana SR1), using Agrobacterium (Agrobacterium tumefaciens GV3101)-mediated transformation (19, 20). Western Blot Analysis. Western blot analysis was performed using the ECL Plus Western Blotting Detection System (Amersham Pharmacia Biotech UK Ltd., Little Chalfont, Buckinghamshire, England), according to the manufacturer’s instructions. Crude leaf extracts were prepared by grinding leaf segments in liquid nitrogen and suspending the powder in 50 mM Tris-HCl (pH 8.0) containing 200 mM NaCl, 5 mM EDTA, and 0.1% Tween 20. The protein concentrations of the extracts were determined using a protein assay kit (BioRad Laboratories, Richmond, CA) with bovine serum albumin as the standard. Protein samples containing equal amounts of crude protein were separated by 12% SDS-PAGE and electrophoretically transferred onto Trans-Blot Transfer Medium Nitrocellulose membrane (Bio-Rad Laboratories) using the electrophoresis power supply EPS 600 (Amersham Pharmacia Biotech UK Ltd.). DbfB and DhaA were detected immunologically using an anti-c-myc-tag monoclonal antibody, produced by mouse hybridoma cells as the primary antibody (21), and a horseradish-peroxidase-labeled antibody as the secondary antibody. The DbfB signals were visualized through chemiluminescence produced by the degradation of 3,3-diaminobenzizine. The DhaA signals were visualized using the LAS-1000 Plus luminescent image analyzer (Fuji Photo Film Co. Ltd., Tokyo, Japan). The DbfB and DhaA bands on the Western blots were quantitated using Image Gauge software (Fuji Photo Film Co. Ltd., Tokyo, Japan). Detection of Enzymatic Activities in Crude Leaf Extracts. The DbfB activity toward 2,3-DHB in crude leaf extracts was assayed by measuring the increase in absorbance at 434 nm for HOPDA according to the previously described method (22). One unit of activity was defined as the amount of enzyme required to form 1 µmol of HOPDA per minute. The DhaA activity toward 1-CB in crude leaf extracts was determined by the release of chloride ions in a colorimetric assay. Crude leaf extracts were buffer-exchanged into 50 mM sodium phosphate (pH 7.5) by ultrafiltration using a Centriprep-10 unit (Millipore, Bedford, MA) to remove the chloride ions derived from the plants. The protein concentrations of the concentrated and buffer-exchanged crude leaf extracts were determined as described above. The assay was performed at 30 °C using 50 mM sodium phosphate (pH 7.5) containing 10 mM 1-CB. The production of chloride ion was detected spectrophotometrically at 460 nm with mercuric thiocyanate and ferric ammonium sulfate (23). One unit of activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol chloride per minute. Detection of Enzymatic Activities in Hydroponic Media. Ten 7-day-old transgenic Arabidopsis seedlings expressing the DbfB were transferred to 300-mL flasks containing 100 mL of sterile Murashige and Skoog (MS) medium and grown for 14 days in darkness at 25 °C, with reciprocal shaking at 120 rpm. After 2 weeks, the DbfB activity in the hydroponic medium, instead of crude leaf extracts, was determined as described above.

Ten 7-day-old transgenic and wild-type tobacco seedlings were transferred to a 300-mL flask containing 100 mL of sterile MS medium and grown for 10 days in continuous light at 25 °C. The hydroponic medium was then recovered and buffer-exchanged into 50 mM sodium phosphate (pH 7.5) by ultrafiltration using a Centriprep-10 unit to remove the chloride ions derived from the plants. The protein concentrations of the concentrated and buffer-exchanged hydroponic media (0.5 µg total soluble protein/µL), instead of crude leaf extracts, were determined as described above. The DhaA activity in the hydroponic medium was determined as described above. Quantification of 1-Butanol. The 1-butanol formed by DhaA-expressing plants was quantitated using gas chromatography-mass spectrometry (GC-MS) (model JMS-Automass 150 GC-MS system; JEOL, Ltd., Tokyo, Japan) fitted with a fused silica chemically bonded capillary column (TCWAX; 0.25 mm i.d. × 30 m, 0.5 µm film thickness; GL Sciences Inc., Tokyo, Japan). Ten 7-day-old transgenic tobacco seedlings expressing DhaA were transferred to 500-mL flasks containing 200 mL of sterile MS medium and grown for 10 days under continuous light at 25 °C. After 10 days, 10 mM 1-CB was added to the medium and the flasks were incubated under the same conditions. Twenty-milliliter samples of the media were collected at various times (10, 20, or 40 min, or 1, 2.5, 5, 10, 15, or 25 h). After the addition of tert-pentyl alcohol as an internal standard, the samples of hydroponic medium were extracted three times with 10 mL of n-hexane, and the extracts were dried over anhydrous sodium sulfate. The samples were then injected into the column at 40 °C, and the column temperature was increased at 5 °C min-1 to 150 °C. The head pressure of the helium carrier gas was 65 kPa. Toxicity Experiments. Ten 7-day-old wild-type and transgenic seedlings were transferred to 300-mL flasks containing 100 mL of sterile MS medium and grown for 10 days in continuous light at 25 °C. 1-CB was then added to the hydroponic medium to final concentrations of 0, 8, 12, or 16 mM, and the flasks were incubated for 6 days more under the same conditions. The effects on growth induced after 6 days of exposure were estimated by determining the fresh weight of the plant.

Results Secretion of a Meta-Cleavage Enzyme by Transgenic Arabidopsis. We constructed transgenic Arabidopsis that constitutively expresses apoplast- or cytoplasm-targeted DbfB from Terrabacter sp. strain DBF63 (11). These modified enzymes were expressed in Escherichia coli and confirmed to exhibit meta-cleavage activity similar to that of the intact DbfB (data not shown). T1 plants were selected, and the T2 and T3 generations were obtained by self-fertilization. To check the translation of the transgene, Western blot analysis was performed using crude leaf extracts prepared from T3 plants. The amounts of cytoplasm-targeted DbfB in the transgenic lines were generally higher than those of apoplasttargeted DbfB (data not shown). Consequently, we isolated eight and 10 independent transgenic lines expressing apoplast- and cytoplasm-targeted DbfB, respectively, and used them in further investigations. Detection of DbfB Activity in Crude Leaf Extracts and Hydroponic Medium. To determine whether the enzymes expressed in the transgenic plants were active, crude leaf extracts were prepared and assayed for meta-cleavage activity toward 2,3-DHB. The specific activities detected in the transgenic lines are shown in Figure 2A. The average specific activity in the 10 transgenic lines expressing cytoplasmtargeted DbfB was 0.636 U/mg (0.212-1.54 U/mg), that in the eight transgenic lines expressing apoplast-targeted DbfB was 0.341 U/mg (0.0945-0.687 U/mg), and no DbfB activity was detected in wild-type plants. Although the DbfB activities VOL. 39, NO. 19, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Detection of DbfB activities in crude leaf extracts and hydroponic media of transgenic Arabidopsis expressing in the apoplast (lines a1-a8) or the cytoplasm (lines c9-c18), and the wild-type plants (w). The DbfB activity was assayed by measuring the increase in absorbance at 434 nm for the yellow meta-cleavage product (HOPDA) with a spectrophotometer. The activities are shown as the means and standard deviations of measurements from three independent experiments. Panel A shows meta-cleavage activities for 2,3-dihydroxybiphenyl (2,3-DHB) in crude leaf extracts of transgenic Arabidopsis expressing DbfB. Panel B shows metacleavage activities for 2,3-DHB in hydroponic media of transgenic Arabidopsis expressing DbfB. N.D., not determined. Panel C shows the initial increase of HOPDA in the hydroponic media of transgenic plants expressing DbfB. detected in the respective lines differed, the average in the transgenic lines expressing apoplast-targeted DbfB was about half that in the transgenic lines expressing cytoplasm-targeted DbfB. However, this tendency roughly corresponded to the 7674

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level of expression of DbfB, since the values resulting from division of the specific activity in each line (a1, a3, a6, c12, c15, and c16) by the level of expression of DbfB against line c12 were nearly the same (c12, c15, and c16, 0.99-1.54; a1, a3, and a6, 1.21-1.45). To confirm that the active DbfB was secreted into the hydroponic medium from the transgenic lines, we determined the DbfB activities in the hydroponic media. Clear activity was detected in the hydroponic media of the apoplasttargeted DbfB, whereas only negligible activity was observed in those of the cytoplasm-targeted DbfB (Figure 2B). The average activities were 86.7 mU/mL in the eight transgenic lines expressing apoplast-targeted DbfB (19.5-295 mU/mL) and 3.75 mU/mL in the nine transgenic lines expressing cytoplasm-targeted DbfB (1.14-6.41 mU/mL). No DbfB activity was detected in the hydroponic medium of wildtype plants. This result clearly demonstrated that the apoplast-targeted DbfB secreted as an active form into the hydroponic medium from the transgenic plants. Next, after the addition of 18 mg of crystalline 2,3-DHB to 100 mL of the hydroponic medium containing seedlings of the transgenic lines a3 expressing apoplast-targeted DbfB or c12 expressing cytoplasm-targeted DbfB, the accumulation of the yellow compound HOPDA produced by DbfB in the hydroponic medium of each line was compared. As shown in Figure 2C, rapid accumulation of HOPDA was observed in the hydroponic medium of the transgenic line a3. In contrast, HOPDA accumulated slowly in the hydroponic medium of line c12. The initial velocities of the production of HOPDA by the transgenic plants were 5.18 U/g fw for line a3 and 0.434 U/g fw for line c12. Transgenic Tobacco Showing Dehalogenase Activity. Transgenic tobacco expressing DhaA in the apoplastic space or the cytoplasm was generated. We confirmed that the modifications to the coding sequence had negligible effects on the DhaA activity in E. coli cells (data not shown). Western blot analysis was performed to evaluate the levels of transgene product using crude leaf extracts prepared from T1 plants in which the transgene had been detected by PCR and Southern blot analysis (data not shown). Although transgenic lines expressing DhaA in the apoplastic space or the cytoplasm showed the expression of DhaA, the cytoplasm-targeted DhaA showed significantly higher DhaA signal than the apoplasttargeted DhaA (data not shown). In contrast, no DhaA protein was detected in the wild-type extracts (data not shown). We randomly chose 12 transgenic lines expressing apoplasttargeted DhaA (lines A1-A12) and five transgenic lines expressing cytoplasm-targeted DhaA (lines C14-C18) from the T1 lines for further investigation. To determine whether DhaA was expressed in the active form in the transgenic tobacco, crude leaf extracts were prepared and assayed for their dehalogenase activities toward 1-CB. The transgenic lines expressing cytoplasm-targeted DhaA generally showed stronger DhaA activity than the transgenic lines expressing apoplast-targeted DhaA (Figure 3A). The average specific activities were 10.9 mU/mg in the five transgenic lines expressing cytoplasm-targeted DhaA (8.89-14.5 mU/mg) and 1.42 mU/mg in the 12 transgenic lines expressing apoplast-targeted DhaA (0.240-3.62 mU/ mg). As compared to the line C16 in Western blot analysis, the lines A3 and A4 had signals of only 2.23% and 8.26%, respectively (data not shown), and the enzymatic activities of the lines A3 and A4 were 20.6% and 25.0% of the average activity in C16 (Figure 3A). No significant DhaA activity was detected in crude leaf extracts from wild-type plants (Figure 3A, line W). Accordingly, the two lines A3 and A4 and the line C16 were chosen for further investigations. Because further investigations needed many seeds of transgenic lines, the T2 generations of these lines were obtained by self-fertilization. To confirm whether DhaA was secreted in the active form into the hydroponic medium, the dehalogenase activities of

FIGURE 3. Detection of DhaA activities in crude leaf extracts and hydroponic media of transgenic tobacco expressing in the apoplast (lines A1-A12) or the cytoplasm (lines C14-C18), and the wild-type plants (W). The activities are shown as the means and standard deviations of measurements from three independent experiments. Panel A shows the DhaA activities toward 1-CB in crude leaf extracts of the transgenic and wild-type lines. The DhaA activity in crude leaf extracts was determined by the release of chloride ions in a colorimetric assay. Panel B shows the biodegradation of 1-CB in the hydroponic media of each line. Specific DhaA activity in the concentrated hydroponc medium was detected using a colorimetric assay. Panel C shows the change over time in the specific DhaA activity in the hydroponic medium. Panel D shows the time course of accumulation of the dehalogenation product 1-butanol in the hydroponic medium. The 1-butanol formed in the hydroponic medium was quantified by GC-MS. the hydroponic media were assayed. As shown in Figure 3B, dehalogenase activities were clearly detected in hydroponic media from seedlings of the lines A3 (16.2 ( 2.0 mU/mg) and A4 (20.9 ( 1.6 mU/mg), whereas hydroponic media from the line C16 and the wild type showed negligible activity (0.3 ( 0.3 and 0.2 ( 0.2 mU/mg, respectively). Although the crude leaf extracts of the line C16 showed stronger DhaA activity than did those of lines A3 and A4 (Figure 3A), the hydroponic medium of the transgenic lines expressing apoplast-targeted DhaA accumulated the DhaA enzyme in the active form. Next, to investigate the time-dependent manner of the DhaA secretion into the hydroponic medium, we periodically measured the DhaA activity in the hydroponic medium after beginning the incubation (2, 4, 8, 13, and 22 h) of transgenic plants (lines A3, A4, and C16) in freshly prepared media. As shown in Figure 3C, the DhaA activities in the hydroponic medium of lines A3 and A4 increased linearly within the first 10 h of incubation, after which the activity stabilized. In contrast, the dehalogenase activity in the hydroponic medium of transgenic line C16 was negligible throughout the incubation. To confirm that dehalogenation of 1-CB occurred, the hydroponic media were analyzed by GC-MS for the dehalogenation product 1-butanol. As shown in Figure 3D, rapid accumulation of 1-butanol was observed in the hydroponic medium of the line A4, whereas the accumulation of 1-butanol could be detected 60 min later in the line C16, eventually accumulating to a greater amount than in the line A4 medium.

TABLE 1. Fresh Weight of Wild-Type and Transgenic Seedlings after 6 days of Exposure to 1-CBa wild type 1-CB (mM) 0 8 12 16

weight (g)

line C16 TI (%)

2.44 ( 0.44 100 1.45 ( 0.34 59.3 0.99 ( 0.24 40.4 0.17 ( 0.03 7.1

weight (g)

line A4 TI (%)

2.43 ( 0.62 100 1.26 ( 0.65 51.8 0.10 ( 0.04 4.06 0.08 ( 0.03 3.21

weight (g)

TI (%)

2.26 ( 0.33 100 2.23 ( 0.54 98.3 2.01 ( 0.57 88.7 0.14 ( 0.05 6.2

a The results quoted are the means of measurements from eight individual seedlings, and the tolerance index (TI ) fresh weight of 1-CBtreated seedlings/fresh weight of untreated seedlings × 100) was calculated for wild-type and transgenic seedlings at each 1-CB concentration tested.

Effects on Growth of Wild-Type and Transgenic Plants in 1-CB-Containing Medium. The toxicity of 1-CB toward the growth of seedlings of the lines C16, A4, and wild type was determined. The results are summarized in Table 1. Seedlings of the line A4 appeared to be resistant to higher concentrations of 1-CB (under 12 mM), as they exhibited no significant changes in growth. However, 16 mM of 1-CB severely inhibited the growth of the line A4 seedlings. In contrast, exposure of the wild type and the line C16 to 8 or 12 mM 1-CB clearly inhibited their growth. Seedlings of the line C16 were somewhat more sensitive to 1-CB than wildVOL. 39, NO. 19, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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type seedlings. None of the lines grew on medium containing greater than 16 mM 1-CB.

Discussion In this study, we generated transgenic Arabidopsis and tobacco expressing apoplast- and cytoplasm-targeted bacterial xenobiotic degradative enzymes and estimated the effects of apoplast targeting on in planta xenobiotic degradation and resistance. In both Arabidopsis expressing DbfB and tobacco expressing DhaA, crude leaf extracts of transgenic plants expressing cytoplasmic enzymes showed higher xenobiotic-degrading activity than did leaves expressing apoplast-targeted enzymes. The expression levels of cytoplasm-targeted DhaA and DbfB in the crude leaf extracts were both higher than those of the apoplast-targeted enzymes. However, the bacterial xenobiotic enzymes were successfully secreted as the active form into the hydroponic medium only from transgenic seedlings expressing the apoplast-targeted enzymes and were able to catalyze the degradation of a xenobiotic in the hydroponic medium. Although in Western blot analysis DhaA signals detected in crude leaf extracts of transgenic lines A3 and A4 were much weaker (2.23% and 8.26%) than that in line C16, which expresses DhaA cytoplasmically, crude leaf extracts of lines A3 and A4 showed 20.6% and 25.0% of the activity of line C16 (Figure 3A). These results suggest that the DhaA targeted into the apoplastic space via the secretory pathway was derivatized and became less reactive to the antiserum used in Western blot analysis. Core and terminal glycosylations of translocated proteins are known to occur in the ER and the Golgi bodies, respectively. Since the 3′-terminus of the c-myc tag followed by a BamH I site is translated as AsnGly-Ser, it is possible that an N-linked sugar chain was linked to the last amino acid (Asn) of the c-myc tag site in plants. This possibility may show that fortuitous modifications must be considered when transgenic plants express ER- or apoplast-targeted proteins through the plant secretory pathway. We showed the high activity in the hydroponic medium of transgenic plants expressing apoplast-targeted DbfB or DhaA (Figures 2B,C and 3B,C) and the tendency of the activity of apoplast-targeted DbfB or DhaA in the hydroponic medium roughly corresponded to that of the activity observed in crude leaf extracts. If higher levels of expression of xenobiotic enzymes in the apoplastic space can be achieved, we should be able to generate transgenic plants with highly improved biochemical properties including, notably, resistance to and degradation of their respective xenobiotics. Periodic monitoring of the dehalogenase activity in the hydroponic medium of the transgenic plants expressing apoplast-targeted DhaA showed that the activity reached the maximum level after 10 hours of incubation without shaking (Figure 3C). It is possible that the transgenic plants expressing apoplast-targeted DhaA secrete recombinant protein continuously, with the amount of the DhaA secreted from transgenic plants roughly equaling the amount of enzyme that was being inactivated after 10 h of incubation. The instability of DhaA in the hydroponic medium is supported by the results shown in Figure 3D, in that the rate of production of 1-butanol at the later times was lower in the line A4 than in the line C16. One of the factors that inactivate the secreted DhaA is the environmental pH. Yokota et al. (14) reported that DhaA is stable in the pH range 7.5-8.5, but the pH of the hydroponic medium in our experiments was 5.8. In addition, the protein secreted from the roots may face proteolytic degradation in the hydroponic medium, as suggested in experiments in which a monoclonal antibody was rhizosecreted into the hydroponic medium (24). Considering that accumulation of the intact antibody was maximal at 7-11 days after the start of incubation (24), the times required to reach equilibrium are highly dependent 7676

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on the proteins. For efficient phytoremediation using the apoplastic expressional system, it will be necessary to search for optimal conditions for retaining the active forms of the apoplast-targeted enzymes. Enzymes that are active as a single protein are evidently more suitable for applications using apoplatic expressional system than multi-subunit enzymes. DhaA has been determined to be a monomeric enzyme, based on gel-filtration chromatography (14). However, many meta-cleavage enzymes have been reported to be active as multimeric proteins (25). Although the quaternary structure of DbfB has not been determined experimentally, it is possible that, if the protein is multimeric, the subunits would have successfully assembled to form the accurate quaternary structure after the translocation of the protein from the cytoplasm into the ER. The enzyme could then show activity in the ER, the apoplast, and the hydroponic medium. In fact, the light and heavy chains of murine immunoglobulin have been shown to assemble before the secretion of the assembled protein into the apoplastic space (26, 27). However, if weak protein interactions (i.e., the interaction between electron transport proteins) are necessary for activity, the enzymes may not be able to function in the apoplast or hydroponic medium, because of dilution of the required components. In addition, the binding of prosthetic group(s) that are required to constitute a holoenzyme may be detrimental to the secretion of the active form of the enzyme. In this study, the Fe2+ ion must have been successfully incorporated into the apoenzyme, because DbfB requires Fe2+ in its active center. Since required prosthetic groups may not be incorporated into the apoenzyme in the secretory pathway, the types of prosthetic groups that can be incorporated, resulting in retention of activity after secretion into the apoplastic space, should be investigated. In this study, we constructed transgenic plants that target DhaA to the apoplastic or the cytoplasmic space. In transgenic plants expressing DhaA cytoplasmically, although no DhaA activity was detected in the hydroponic media, the hydroponic medium contained a higher amount of 1-butanol, the product of 1-CB (Figure 3B-D). These results clearly suggest that not only 1-CB but also 1-butanol were readily translocated through the cell membrane by passive transport. This is consistent with the fact that the detection of 1-butanol in the hydroponic medium of the transgenic line expressing cytoplasm-targeted DhaA was delayed by 60 min. However, the transgenic line expressing apoplast-targeted DbfB caused rhizospheric degradation of 2,3-DHB, which has lower solubility in water than 1-CB and 1-butanol (Figure 2C), that was 11.9 times more rapid than in the transgenic line expressing cytoplasm-targeted DbfB. These results suggest that the apoplastic expressional system is more suitable for the degradation of highly hydrophobic pollutants than the cytoplasmic expressional system. Because most persistent and toxic organic compounds are hydrophobic, show low solubility in water, and adsorb easily to hydroponic materials such as humus and the surface of roots, plants may take up very little of these compounds (28). If cytoplasmic recombinant enzymes cannot come in contact with their substrates, apoplastic expressional system constitutes a useful alternative methodology for cleaning up contaminated sites. As shown in Table 1, the seedlings expressing apoplasttargeted DhaA were more tolerant to 1-CB than wild-type plants and seedlings expressing cytoplasm-targeted DhaA. Because the transgenic plants expressing the apoplasttargeted DhaA secrete the active form of DhaA into the medium, it is possible that some portion of the 1-CB was converted to the less toxic 1-butanol. As plant cells naturally contain alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities, 1-butanol could be oxidized to 1-butanoic acid via 1-butanal by successive reactions catalyzed by ADH and ALDH. 1-Butanal has been shown to cause

DNA strand breaks, in other words, carcinogenic properties (29). However, Bucher et al. (30) have reported that tobacco leaves have very limited activity with respect to such alcohol metabolic pathways. Therefore, the low amounts of 1-butanol that penetrate gradually into plant tissues might not result in the accumulation of 1-butanal at toxic levels. In contrast, the more severe damage observed in the transgenic seedlings expressing cytoplasm-targeted DhaA implies the possibility that a large amount of 1-butanal is produced in the cytoplasm of these plants and shows toxicity to plant cells. As a result, transgenic seedlings expressing cytoplasm-targeted DhaA might be somewhat more sensitive to 1-CB than wild-type seedlings. Although investigation of the 1-butanol metabolism by wild-type and transgenic plants will be necessary to confirm the above possibility, this result indicates that phytoremediation using apoplastic expressional system is more appropriate than the cytoplasmic expressional system, when the intermediary metabolites of the contaminant targeted for degradation by recombinant enzymes accumulate and are harmful to the plant. In conclusion, bacterial xenobiotic-degrading enzymes were shown to be expressed in the apoplastic space and secreted into the hydroponic medium, when the transgenic plants were grown in liquid culture. The secreted enzymes retained their respective activities. This study has proven that the apoplastic expressional system can be used for xenobiotic degradation, by employing transgenic plants as continuous suppliers of bacterial xenobiotic-degrading enzymes into the rhizosphere. Although the rate of production of apoplast-targeted xenobiotic-degrading enzymes should be improved, and the usefulness of apoplastic expressional system determined for various types of enzymes, this system could be a useful new technology for phytoremediation.

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Received for review April 9, 2005. Revised manuscript received July 4, 2005. Accepted July 25, 2005. ES0506814 VOL. 39, NO. 19, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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