Available online at www.sciencedirect.com
Biochemical and Biophysical Research Communications 365 (2008) 453–458
Phytodegradation of organophosphorus compounds
by transgenic plants expressing a bacterial organophosphorus hydrolase
Xiaoxue Wang a , Ningfeng Wu a, *, Jun Guo a , Xiaoyu Chu a , Jian Tian a ,
Bin Yao b , Yunliu Fan a
a Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China
b Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China
Received 29 October 2007
Available online 26 November 2007
Organophosphorus (OP) compounds are widely used as pesticides in agriculture but cause broad-area environmental pollution. In this
work, we have expressed a bacterial organophosphorus hydrolase (OPH) gene in tobacco plants. An assay of enzyme activity showed
that transgenic plants could secrete OPH into the growth medium. The transgenic plants were resistant to methyl parathion (Mep),
an OP pesticide, as evidenced by a toxicity test showing that the transgenic plants produced greater shoot and root biomass than did
the wild-type plants. Furthermore, at 0.02% (v/v) Mep, the transgenic plants degraded more than 99% of Mep after 14 days of growth.
Our work indicates that transgenic plants expressing an OPH gene may provide a new strategy for decontaminating OP pollutants.
Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Phytodegradation; Organophosphorus compounds; Organophosphorus hydrolase; Transgenic plant; Bioremediation
Synthetic organophosphorus (OP) compounds are the
most widely used pesticides [1,2], and unacceptable levels
of environmental residues of these compounds have been
found in many countries worldwide [3–7]. Although most
OP compounds are not persistent, they still cause broadarea
pollution from continued use in agriculture and public
health . These pesticides are frequently not used as recommended
both in dosage and in spills, leading to an accumulation
of OP compounds in the environment.
Additionally, many of these compounds are mobile in the
environment, which may lead to diffuse contamination
; for example, traces of OP pesticides have been found
in hills where no previous application has occurred. Furthermore,
the accidental release or disposal of OP pesticides
has caused heavy pollution in certain areas. High
concentrations of the OP pesticide methyl parathion
(Mep) in soil are degraded to acceptable levels only after
many years .
* Corresponding author. Fax: +86 10 62136981.
E-mail address: email@example.com (N. Wu).
OP pesticides are acetylcholinesterase (AChE) inhibitors.
They are mostly liposoluble and pose a hazard to
humans through accumulation in the food chain from
the wide range of aquatic and terrestrial ecosystems contaminated
with OP pesticides. The loss of AChE results
in acetylcholine accumulation, which interferes with muscular
responses and produces serious symptoms in vital
organs, eventually leading to death. Therefore, it is
essential to decontaminate OP compounds in the environment.
However, although previous studies have
focused on persistent organic pollutants, little attention
has been paid to the remediation of OP pesticide
Some progress has been made on screening for OP
pesticides detoxifying bacterial strains and developing
organophosphorus hydrolases (OPHs; EC 126.96.36.199) [10–
16]. However, given the large scope of the untreated contamination
worldwide, chemical and enzyme preparation
approaches do not seem economical or effective. An
alternative approach, phytoremediation, has been
considered as a potentially low cost, very effective, and
0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
454 X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458
an environmentally friendly method; it may provide a
better option for dealing with such diffuse contamination.
Plants have a natural ability to metabolize OP compounds,
as reported in a previous phytoremediation
study of the use of the hydrophyte Typha latifolia to
remove Mep pollutants from water . Plants also prevent
contamination from spreading to other areas via
wind, rain or groundwater—an advantage of using phytoremediation
to address diffuse OP pollution at scattered
A substantial amount of work has been conducted to
increase the ability of plants to decontaminate areas that
have high concentrations of heavy-metal ions , organomercury
, and explosives (e.g. TNT and RDX) [20,21]
or chlorinated pesticides (e.g. atrazine) . The introduction
of a detoxification gene improves the tolerance of the
engineered plants to such toxic pollutants. To date, however,
few studies have addressed the production of transgenic
plants designed to degrade and detoxify OP
We have previously isolated an OP-degrading bacterial
strain (C2-1, Pseudomonas pseudoalcaligenes) from OP-polluted
soil . The gene ophc2, which encodes organophosphorus
hydrolase OPHC2, has been cloned (GenBank
Accession No. AJ605330)  and the enzyme has been
highly expressed in the yeast Pichia pastoris . In this
study, we introduced ophc2 into tobacco plants and investigated
whether these OPHC2-expressing transgenic plants
could degrade Mep in vitro and in vivo.
Fig. 1. Western blot analysis showing expression of OPHC2 in transgenic
tobacco plants. Molecular weight is given in kDa. a Positive control using
purified OPHC2 protein expressed in Pichia pastoris. b Total protein from
wild-type tobacco. c Total protein from transgenic tobacco lines. * Transgenic
lines expressed OPHC2.
Materials and methods
Construction of the OPHC2 expression vector. The OPHC2 expression
vector was constructed according to standard protocols . The gene
encoding OPHC2, ophc2, was amplified by PCR from the genomic DNA
of P. pseudoalcaligenes C2-1 using primer_F (TACGTA ACC ATG GCC
GCA CCG GCA CAA CAG AAG) and primer_R (GAGCTC TTT TTT
TTT TTT TTT TTT TTT TTT TTT TTT ATC AGC GGT CGC TAC
GGA TCG G). The underlined sequences correspond to SnaBI and SacI
sites, respectively. The PCR product was purified using the MinElute Ò
PCR purification kit (QIAGEN, USA), digested with SnaBI and SacI, and
directionally cloned into the SnaBI and SacI restriction sites of the vector
Teasy-ex, which contained a signal peptide sequence from the carrot
extensin gene [26,27]. The recombinant vector was designated pTeasyophc2.
The extensin signal sequence facilitated the extracellular expression
of OPHC2. The ophc2 with the extensin sequence was excised using SacI
and Xbal from pTeasy-ophc2 and ligated into pBI121. The final expression
cassette, which contained the cauliflower mosaic virus (CaMV) 35S
promoter, the extensin sequence, ophc2, and the nos termination sequence,
was designated pBI-E-ophc2.
Transformation of tobacco. The vector pBI-E-ophc2 was introduced
into Agrobacterium LBA4404 using electroporation (MicroPulser, Bio-
Rad, USA). The leaf disks of tobacco (Nicotiana tabacum cv. Xanthi) were
transformed according to the method of Horsch et al. . The selection
medium comprised Murashige–Skoog (MS) basic medium, 3% sucrose,
0.6% agar, 1 mg/l of 6-benzylaminopurine (6-BA), 0.1 mg/l of a-naphthaleneacetic
acid (NAA), 100 mg/l kanamycin (Km), and 500 mg/l of
Plant DNA isolation and PCR analysis. Plant genomic DNA was
prepared from leaves using the Phytopure kit (TIANGEN BIOTECH, Co.
LTD. Beijing, China). Primer 1 (GCC GCA CCG GCA CAA CAG
AAG) and primer 2 (TCA GCG GTC GCT ACG GAT CGG) were used
in this assay.
Western blot analysis. Transformed and untransformed leaves
(200 mg) from plants grown in a phytotron were ground in liquid nitrogen
and resuspended in 200 ll of protein extraction buffer (50 mM/l Tris–HCl,
pH 8.0, 0.02% NaN 3 , 100 lg/ml of phenylmethylsulfonylfluoride). Total
protein was extracted and quantified in the extracts for each sample.
Purified OPHC2 expressed in P. pastoris was used as a positive control
. Proteins of leaf extracts were subjected to SDS–PAGE through a 5%
polyacrylamide stacking gel above a 12% separating gel. After electrophoresis,
separated proteins on the gel were transferred electrophoretically
to polyvinylidene fluoride membranes (Millipore, USA) for Western blot
analysis. Immunodetection was achieved using a rabbit polyclonal antiserum
directed against OPHC2 as the primary antibody and peroxidaseconjugated
goat anti-rabbit IgG (Jackson ImmunoResearch, USA) as the
secondary antibody. Color development was performed in 100 ml TB
buffer (50-mM/l Tris–HCl, pH 7.6) containing 50 mg of 3,3 0 -diaminobenzidine
and 40 ll of 30% H 2 O 2.
Analysis of OPHC2 enzyme activity. Liquid MS growth medium
(2.5 ml) from plants grown for 10 days was concentrated to 100 ll using
10K Nanosep Ò Centrifugal Devices (PALL, USA). The 100 ll of concentrated
growth medium was added to 900 ll of assay buffer (50 mM/l
Tris–HCl, pH 8.0) containing 5 ll of 10 mg/ml Mep (99.9%) as substrate
and incubated at 37 °C for 20 min. The reaction was terminated by the
addition of 1 ml of 10% CCl 3 COOH (trichloroacetic acid), and 1 ml of
10% Na 2 CO 3 was added to display the color. Absorbance was measured at
Fig. 2. Hydrolysis of Mep by OPH.
X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458 455
410 nm. The enzyme activity could be calculated based on the amount of
the hydrolysis product, p-nitrophenol. One unit of OPH activity is defined
as the amount of enzyme that liberated 1 lM ofp-nitrophenol per minute
at 37 °C.
Mep degradation assay. Five microliters of Mep (commercial product,
50% Mep emulsifiable concentrates) was added to 30 ml of MS medium
with 0.5% agar in the flasks, under aseptic conditions, shaking to uniformity.
Wild-type and transgenic tobacco seedlings were transferred into
the flasks, grown for 14 days in 12-h day/12-h night light cycles at 25 °Cin
the growth chamber. Three flasks that did not contain plants were used as
the control. After 14 days, Mep from the medium and plants was
extracted. The extraction procedure was modified according to the
methods described in internal standard and by Steinwandter . A 2-ml
mixture of each extraction was evaporated to dryness using nitrogen gas
and suspended in 1 ml of dichloromethane for quantification. Mep residue
was quantified by gas chromatography (Agilent Technologies, 6890N;
detector: flame photometric detector, detector temperature 250 °C; HP-5
5% phenyl methyl siloxane column; capillary columns: 30.0 m · 320 lm
· 0.25 lm nominal; inlets heater: 250 °C; initial oven temperature: 70 °C
for 1 min, then increased at a rate of 20 °C/min to 180 °C, held for 2 min
and ramped again at 10 °C/min to 250 °C, then held for 5 min; N 2 flow:
1.2 ml/min; split ratio: 5:1). The standard Mep solution was 100 lg/ml
(China Standards GSBG23010-92).
Construction and selection of ophc2-transgenic plant lines
The transformation produced 101 independent T0-generation
kanamycin-resistant plants that were selected for
PCR analysis was used to determine which lines contained
the ophc2 transgene. Of the 101 lines, 80 were shown
to have the transgene, with DNA from wild-type tobacco
showing no PCR product (data not shown).
Western blot analysis was undertaken on the transgenic
plants to determine whether OPHC2 was expressed.
Twenty-four of the 60 randomly chosen transgenic plants
expressed OPHC2. Part of Western blot analysis result
was showed on Fig. 1.
OPHC2 enzymatic activity study in transgenic tobacco
OPH activity was assayed in vitro to assess the secretion
and enzymatic activity of the transgene product. Under
aseptic conditions, transgenic and wild-type seedlings
(seedlings with identical biomass, root or shoot size, and
leaf amounts were chosen) were allowed to grow in liquid
MS medium for 10 days, after which the medium was collected
and concentrated 25-fold. The OPH activity of the
concentrated medium was then determined, using Mep as
the substrate. OPH can hydrolyze Mep at its phosphoester
bond, releasing p-nitrophenol (Fig. 2) and generating a yellow
product that can be quantified with a spectrophotometer
at 410 nm. Table 1 shows the enzymatic activity of
OPHC2 detected in the growth medium of different transgenic
plants. The transgenic line 4–91 showed the highest
enzymatic activity and was used in the subsequent experiments.
Furthermore, T1- and T2-generation transgenic
In vitro OPHC2 enzyme activity a
Wild-type · 10 3 U/mL Transgenic tobacco lines · 10 3 U/mL
4–14 4–23 4–32 4–38 4–43 4–45 4–56 4–67 4–80 4–90 4–91 4–97 4–98 4–99 4–100
0.37 ± 0.51 6.28 ± 0.62 24.49 ± 1.30 14.77 ± 0.35 15.11 ± 0.62 14.9 ± 3.16 0.08 ± 0.45 17.39 ± 3.21 14.70 ± 1.51 17.90 ± 3.60 8.20 ± 0.78 25.82 ± 1.47 18.17 ± 0.40 18.84 ± 1.52 1.88 ± 1.38 18.01 ± 3.64
Transgenic and wild-type seedlings were grown aseptically in MS liquid medium for 10 days. Hundred microliter of concentrated growth medium was used as the enzyme solution for activity determination. Results shown are means and standard deviation of
456 X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458
tobacco plants grew normally and still showed OPHC2
enzymatic activity (data not shown).
Assessment of Mep toxicity in an ophc2-transgenic tobacco
A toxicity study was conducted to determine the effect of
Mep on the growth of both wild-type and transgenic line
4–91 seedlings. Identically sized cuttings of wild-type and
transgenic plantlet were transferred into sterile flasks containing
solid MS medium with 0, 0.01%, 0.02%, 0.03% or
0.04% (v/v) Mep (commercial product, 50% Mep emulsifiable
concentrates). After 14 days of culture, the transgenic
4–91 plants could tolerate 0.02% Mep and showed no signs
of phytotoxicity. At 0.03% Mep, line 4–91 plants kept
growing, with a slightly stunted root and fewer root hairs.
At 0.04% Mep, the radicles of line 4–91 plants were swollen,
with no secondary roots and no visible root hairs. By
contrast, the wild-type seedlings displayed symptoms at
0.02% Mep that were similar to those observed in the
0.04% Mep-treated transgene plants (Fig. 3). Growth after
14 days in the presence of various concentrations of Mep
was also quantified as wet plant weight for both the wildtype
and the transgenic line 4–91 (Table 2).
There was a negligible difference in the root growth of
4–91 seedlings on 0%, 0.01%, and 0.02% (v/v) Mep medium,
the amount of roots was approximately 100 (Fig. 3A), and
the wet weight gain was 6.5-fold for each concentration
(Table 2). At a concentration of 0.01% Mep, wild-type
tobacco plants exhibited phytotoxicity in the form of
fewer root branches (50–60), reduced root length, and
darkened leaves compared with plants not exposed to Mep
(Fig. 3B and D), and the wet weight gain was nearly 3-fold
(Table 2). At 0.02% Mep, growth of the wild-type tobacco
was very stunted—less than 20 small, bare root branches
were produced, the shoot and root length (about 2.3 cm
and 1.5 cm respectively) were both markedly reduced compared
with 4–91 (about 3.2 cm and 2.8 cm, respectively)
(Fig. 3E), and the wet weight gain was only 2-fold (Table
2). No difference was observed between line 4–91 and wildtype
plants grown in medium without Mep, which indicates
that the ophc2 insertion had no deleterious effect on the
plants (Fig. 3C). Wild-type plants grown in medium without
Mep appeared healthy, indicating that the toxic effects
observed were caused solely by Mep.
Mep degradation in seedlings and growth medium
To assess whether transgenic plants expressing
OPHC2 can degrade environmental OP toxins, we tested
the ability of the transgenic line 4–91 plants to degrade
Mep. After 14 days of growth on solid MS medium
Fig. 3. Growth of transgenic line 4–91 and wild-type tobacco plants on Mep-containing medium 14 days after transplantation. Each sample was tested in
triplicate. (A, B) Root morphology in the presence of 0%, 0.01% or 0.02% (v/v) Mep. (C–E) Plant morphology difference between 4–91 and wild-type: (C)
medium without Mep, (D) medium with 0.01% (v/v) Mep, (E) medium with 0.02% (v/v) Mep.
Seeding weight gain following Mep exposure a
Mep % (v/v) Wild-type Line 4–91
Weight before Mep (g) Weight after Mep (g) Weight gain (g) Weight before Mep (g) Weight after Mep (g) Weight gain (g)
0 0.60 ± 0.01 4.47 ± 0.02 3.87 ± 0.03 0.56 ± 0.03 4.24 ± 0.54 3.67 ± 0.55
0.01 0.63 ± 0.04 2.48 ± 0.44 1.85 ± 0.40 0.62 ± 0.04 4.53 ± 0.31 3.59 ± 0.40
0.02 0.63 ± 0.08 1.78 ± 0.20 1.15 ± 0.26 0.64 ± 0.05 4.00 ± 0.60 3.36 ± 0.56
a The identically sized cuttings of wild-type and transgenic line 4–91 plantlet were transferred into sterile flasks containing solid MS medium with 0,
0.01%, 0.02% (v/v) Mep (commercial product, containing 50% Mep). After 14 days of growth, wet weights were measured to establish plant biomass gain,
which was used as an indicator of phytotoxicity. Results shown are means and standard deviation of three replicate flasks.
X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458 457
(0.5% agar) with 0.02% Mep, the concentration of Mep
residue in the medium and plant material was then measured
using gas chromatography. The total absolute
quantity of Mep reduction was 99.6% in the flasks of
line 4–91 plants and 27% in the flasks of wild-type
plants, compared with the flasks without plants
The absolute quantity of Mep in the tissue of wild-type
plants was 163-fold that of the tissue of line 4–91 plants
(Fig. 4B). This indicates either higher degradation or lower
absorption of Mep by the 4–91 line. However, Mep residue
extracted from the MS medium exhibited an even higher
(173-fold) absolute quantity of Mep in the wild-type
growth flask (Fig. 4B), so we suggest that the lower level
of Mep residue in the transgenic plant flask was due to
the increased degradation of Mep. Mep residue in line
4–91 plant material extract was 0.16 ± 0.01 lg/g plant
material. However, it was 16.74 ± 2.33 lg/g plant material
in the wild-type extract. The growth medium of wild-type
plants contained 68.94 ± 4.55 lg Mep/g MS medium,
which was much higher than that of the transgenic line
4–91 (0.39 ± 0.01 lg Mep/g MS medium).
absolute quantity of Mep(ug)
absolute quantity of Mep (ug)
blank flask WT 4-91
Total extraction from the whole plant and its medium
WT plant 4-91 plant WT medium 4-91 medium
Extraction from plant and growth medium
Fig. 4. Extraction of Mep from plant tissue and growth medium after 14
days of culture in MS medium with 0.5% agar containing 0.02% (v/v)
Mep. Results shown are means and standard deviation of three replicate
A wide range of OP compounds present in pesticides
and chemical warfare agents can be degraded by OPHs
because many OP compounds—aside from displaced
groups—have analogous structures. We selected Mep as
a test substrate for OPHC2 degradation of OP pesticides
in this study.
Many investigations have focused on the bioremediation
of persistent organic pollutants, but few have been
conducted for OP compounds. Our approach shows that
an OPH, OPHC2, identified in bacteria isolated from a
polluted site can be redeployed in plants to increase the
ability of the plants to degrade Mep. In the analysis of
Mep degradation in both seedlings and growth media,
there was a reduction of total Mep residue in the flasks
containing wild-type tobacco compared with the flask
without any plants. This could be because of the natural
metabolism of Mep by tobacco plants. However, it is not
enough to reduce the amount of OP pesticide residue to
acceptable levels. Total Mep residue in wild-type plant
flasks still remained 73% (on average) of that of the control
flask. And the amount of Mep residue was much
higher in the medium than in the plants themselves, which
indicated that limited Mep was absorbed by the plants at
high Mep concentrations. Furthermore, because of the
low metabolism efficiency for the absorbed Mep, there
was still a high level of Mep in the tissue of wild-type
plants. The transgenic line 4–91 plants were able to
degrade Mep to a much lower level, which we expect to
be sufficient to decontaminate environmental OP pollution.
In addition, effective degradation of Mep in the
transgenic plant tissue can keep the plants healthy, which
is essential for phytoremediation.
Pollution caused by OP pesticides is wide ranging; most
OP pesticides are directly liberated by spraying, and a perceptible
amount is released through evaporation from
water surfaces, leaves and soil , causing air pollution
and long-range impacts via atmospheric transport. However,
there is currently no economical way to block the diffusion
of OP in the environment. The improved
degradation of OP pesticides by transgenic plants highlights
a new way to combat such diffuse contamination.
In contrast to previous studies that aimed to increase the
removal rate of pollutants, our results make possible a substantial
increase in OP degradation in the environment.
Because of its constitutive extracellular expression, OPHC2
was detected in both the transgenic plant tissue and the rhizosphere.
With the continued release of OPHC2 by the
expanded root system of plants, OP compounds diffused
in soils or water could be detoxified. Combined with an
OP pollution study, our investigation takes a step towards
determining whether OPHC2 secretion by transgenic plant
leaves can counteract the contaminating effects of OP-polluted
rain, fog or snow. Further studies should be conducted
to investigate how ophc2-transgenic plants work
in natural conditions.
458 X. Wang et al. / Biochemical and Biophysical Research Communications 365 (2008) 453–458
We thank Sheng Gao for gas chromatography analysis.
This work was supported by the National Natural Science
Foundation of China (Grant No. 30470031).
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