Biotechnology Advances 25 (2007) 75 84

www.elsevier.com/locate/biotechadv

Research review paper

Herbicide resistance of transgenic rice plants expressing

human CYP1A1
Hiroyuki Kawahigashi a,, Sakiko Hirose a , Hideo Ohkawa b,1 , Yasunobu Ohkawa c
a

Plant Biotechnology Department, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan
b
Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe, Hyogo 657-8501, Japan
c
Agriculture, Forestry and Fisheries Research Council Secretariat, 1-2-1 Kasumigaseki Chiyodaku, Tokyo 100-8950, Japan
Received 21 August 2006; received in revised form 3 October 2006; accepted 12 October 2006
Available online 24 October 2006

Abstract
Cytochrome P450 monooxygenases (P450s) metabolize herbicides to produce mainly non-phytotoxic metabolites. Although
rice plants endogenously express multiple P450 enzymes, transgenic plants expressing other P450 isoforms might show improved
herbicide resistance or reduce herbicide residues. Mammalian P450s metabolizing xenobiotics are reported to show a broad and
overlapping substrate specificity towards lipophilic foreign chemicals, including herbicides. These P450s are ideal for enhancing
xenobiotic metabolism in plants. A human P450, CYP1A1, metabolizes various herbicides with different structures and modes of
herbicide action. We introduced human CYP1A1 into rice plants, and the transgenic rice plants showed broad cross-resistance
towards various herbicides and metabolized them. The introduced CYP1A1 enhanced the metabolism of chlorotoluron and
norflurazon. The herbicides were metabolized more rapidly in the transgenic rice plants than in non-transgenic controls. Transgenic
rice plants expressing P450 might be useful for reducing concentrations of various chemicals in the environment.
2006 Elsevier Inc. All rights reserved.
Keywords: Cytochrome P450 (CYP); Xenobiotic metabolism; Herbicide resistance; Atrazine; Chlorotoluron; Norflurazon; Phytoremediation;
Detoxification

Contents
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4.
5.
6.
7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plant and animal P450s for xenobiotic metabolism . . . . . . . . . . . . . .
Substrate specificity of CYP1A1 . . . . . . . . . . . . . . . . . . . . . . . .
Transgenic rice plants expressing CYP1A1 . . . . . . . . . . . . . . . . . .
Herbicide resistance of soil-grown transgenic rice plants expressing CYP1A1
Use of CYP1A1 rice for direct seeding . . . . . . . . . . . . . . . . . . . .
Use of transgenic rice plants for phytoremediation. . . . . . . . . . . . . . .

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Corresponding author. Tel.: +81 29 838 8374; fax: +81 29 838 8397.
E-mail address: shiwak@affrc.go.jp (H. Kawahigashi).
1
Present address: Faculty of Life Science and Biotechnology, Fukuyama University, Gakuencho 1, Fukuyama, Hiroshima, 729-0292, Japan.
0734-9750/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2006.10.002

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H. Kawahigashi et al. / Biotechnology Advances 25 (2007) 7584

8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

1. Introduction
Herbicides are laborsaving means of improving crop
yield and quality, because weed infestation reduces crop
yields and decreases market prices (Lockhart et al.,
1990). In addition, the proper use of herbicides
conserves soil and moisture, whereas mechanical weed
control results in loss of soil moisture and increased
erosion.
Herbicides are removed from the environment by
chemical degradation or degradation by bacteria and
plants. The metabolism of herbicides in higher plants
comprises three main phases-conversion (Phase I),
conjugation (Phase II), and compartmentalization (Phase
III) (Hatzios, 1997). In Phase I, hydrophobic herbicides
are converted to mainly less lipophilic metabolites
through oxidation, peroxidation, or reduction by cytochrome P450 monooxygenases (P450s). This conversion
usually produces less toxic metabolites, but some proherbicides are activated (Ohkawa et al., 1999; WerckReichhart et al., 2000). In Phase II, herbicides or their
Phase I metabolites are directly conjugated with glutathione, sugars, or amino acids to produce water-soluble
compounds that are easily eliminated. Finally, in Phase
III, conjugated metabolites are further converted to reduce
mobility, and then deposited in the vacuoles or cell walls
of plants (Hatzios, 1997).
Weeds have developed resistance to the majority of
known herbicide chemical classes and mechanisms of
action (Beckie et al., 2000; WeedScience.org, 2005).
Indeed, over 290 biotypes of herbicide-resistant weeds
have been reported in agricultural fields and gardens
worldwide (Beckie et al., 2000; WeedScience.org,
2005). This resistance can result from modification of
the target site or enhanced detoxification, as well as
alterations in uptake, translocation, and compartmentalization of the herbicide (Ohkawa et al., 1999).
The two primary mechanisms of tolerance in agricultural crops are modification of the target site and
development of enhanced detoxification (Putwain,
1990). Two enzyme systems play major roles in the
increased degradation of herbicides: P450s and glutathione S-transferases (Ohkawa et al., 1999). P450s
mainly catalyze monooxygenation of lipophilic, xenobiotic compounds, including herbicides. This enzyme

system is located in microsomes and comprises several

cytochrome P450 isoforms and a nonspecific NADPHcytochrome P450 oxidoreductase. In plants, most P450catalyzed reactions are hydroxylation reactions, but
oxidative dealkylation (Frear, 1995) and epoxidation
(Schuler, 1996) also occur. We focus here on our effort to
alter Phase I by introducing a P450 gene into plants.
Enhancement of enzymatic activity is a good way of
developing herbicide-resistant crops.
2. Plant and animal P450s for xenobiotic metabolism
Genome sequencing has revealed that cytochrome
P450s constitute the largest family of enzymatic proteins
in higher plants (Werck-Reichhart et al., 2002), with
over 1000 plant P450 sequences listed today (Nelson,
2004). The Arabidopsis genome houses 246 P450 genes
and 26 pseudogenes, and 356 P450 genes and 99 related
pseudogenes have been identified in Japonica and
Indica rices (Oryza sativa L.) Nelson et al., 2004). It
is supposed that some of the P450s are involved in
secondary metabolism and some are involved in herbicide tolerance.
In herbicide metabolism, many P450-dependent oxidations in plant microsomes have been reported,
including oxidation of chlorotoluron in maize (FonnePfister and Kreuz, 1990) and wheat (Mougin et al.,
1990); linuron in wheat (Frear, 1995) and maize
(Moreland et al., 1993); atrazine in tulip (Tulipa
generiana L.); and isoproturon in yam bean (Pachyrhizus erosus L. urban) (Belford et al., 2004). However,
molecular information on plant P450s related to
xenobiotic metabolism is limited. Only some herbicide-metabolizing P450 genes have been cloned and
characterized, such as CYP73A1 and CYP76B1 from
Jerusalem artichoke (Helianthus tuberosus) (Pierrel
et al., 1994; Robineau et al., 1998), CYP71A11 from
tobacco (Nicotiana tabacum) (Yamada et al., 2000), and
CYP71A10 from soybean (Glycine max) (Siminszky
et al., 1999). Sixteen cytochrome P450s presumably
involved in herbicide metabolism have been reported
(Fischer et al., 2001) in Lolium rigidum, which
developed cross-resistance to different classes of herbicides because of cytochrome P450-mediated detoxification (De Prado et al., 2005).

H. Kawahigashi et al. / Biotechnology Advances 25 (2007) 7584

In contrast, the enzymatic functions of mammalian

P450s have been well studied. Xenobiotic metabolizing
P450s are found at highest concentrations in the liver, the
mammalian detoxification center for both exogenous
and endogenous chemicals. A limited number of P450s
are primarily involved in xenobiotic metabolism. Human
P450s that metabolize xenobiotic chemicals belong
almost exclusively to the CYP1, CYP2, CYP3, and (to
a lesser degree) CYP4 families (Nebert and Russell,
2002). Among them, 11 P450s related to xenobiotic
chemical metabolism are involved in over 90% of P450dependent drug metabolism in the human liver (Funae
et al., 1998). Since a single P450 can metabolize a large
number of structurally diverse compounds, these
enzymes can collectively metabolize scores of chemicals
found in the diet, environment, and drugs. These P450s
have been reported to show broad, overlapping substrate

77

specificity towards lipophilic xenobiotics, including

herbicides (Inui et al., 2001).
3. Substrate specificity of CYP1A1
P450s oxidize carbon and nitrogen, usually resulting in
the formation of a hydroxyl group and occasionally the
subsequent removal of alkyl and amino groups. Inui et al.
(2001), using recombinant yeast-expressing human
P450s, showed that 11 human P450s metabolized many
kinds of chemicals, including 27 herbicides, four
insecticides, and two industrial chemicals. Regarding
xenobiotic metabolism by CYP1A1 (EC 1.14.14.1),
human CYP1A1 metabolized 16 herbicides, including
triazines, ureas, and carbamates (Table 1) (Inui et al.,
2001). Not only pesticides, but also 757 chemicals are
reported in the Drug Metabolizing Database-system

Table 1
Herbicide resistance of CYP1A1 rice plants in germination test
HRAC
group a

A
C1

C2

F1
K1

K2

K3
L
N

Mode of action(Chemical family)

Inhibition of acetyl-CoA carboxylase

(Aryloxyphenoxypropionate)
Inhibition of photosynthesis
(Triazine)
(Triazine)
Inhibition of photosynthesis
(Urea)
(Urea)
(Urea)
Inhibition of carotenoid biosynthesis
(Pyridazinone)
Inhibition of microtubule assembly
(Phosphoroamidate)
(Dinitroaniline)
Inhibition of mitosis/microtubule
organization
(Carbamate)
Inhibition of VLCFAs
(Oxyacetamide)
Inhibition of cell wall synthesis
(Benzamide)
Inhibition of lipid synthesis
(Thiocarbamate)

Herbicide

CYP1A1
Yeast microsome

Transgenic rice

M b

%c

M d

10

88

0.2

Atrazine
Simazine

100
20

13
41

100
100

=
=

Chlorotoluron
Diuron
Methabenzthiazuron

100
100
100

84
99
46

100
150
200

++
+
=

Norflurazon

100

86

Amiprophos-methyl
Pendimethalin

200
100

9
66

2.5
20

++
++

Chlorpropham

100

27

7.5

++

Mefenacet

150

19

2.5

++

Isoxaben

10

11

100

2.5

Quizalofop-ethyl

Pyributicarb

1.25

Resistance e
++

++

VLCFA, very long chain fatty acid.

a
As defined in HRAC (http://www.plantprotection.org/HRAC/index.html).
b
Final concentration of herbicide used for HPLC analysis.
c
% degradation of herbicide in vitro; , not determined or less than 5% (Inui et al., 2001).
d
Final concentration of herbicide used for germination test.
e
++, transgenic plants grew well but control plants did not grow at all in the herbicide-containing medium; +, transgenic plants grew but were
retarded and control plants did not grow at all in the herbicide-containing medium; =, both transgenic and control plants grew well in the herbicidecontaining medium. In the case of norflurazon, ++ indicates that transgenic plants showed healthy green shoots, whereas the control plants showed
white shoots by inhibition of carotenoid synthesis.

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H. Kawahigashi et al. / Biotechnology Advances 25 (2007) 7584

Fig. 1. Chemical structures of atrazine, chlorotoluron, norflurazon, and mefenacet. Bold black arrows indicate the proposed sites of N-demethylation
or hydroxylation by CYP1A1 in a yeast microsome system in vitro (Inui et al., 2001).

(http://jbic1.jbic.or.jp/ec3/biodb/, accessed 31 May 2005)

to be metabolized by CYP1A1.
Atrazine is metabolized by CYP1A1 in vitro in a
stepwise N-dealkylation to produce dealkylated metabolites (Fig. 1) (Wu et al., 1998; Inui et al., 2001).
Similarly, simazine is metabolized to N-dealkylated
metabolites by CYP1A1 in vitro. In plants, N-demethylation is supposedly metabolized by endogenous P450s.
N-dealkylation of a single side-chain results in partial
loss of phytotoxicity, and that of both side-chains causes
complete loss of phytotoxicity (Shimabukuro et al.,
1973).
Chlorotoluron is metabolized through N-demethylation and ring methyl hydroxylation by CYP1A1 in yeast
expression systems (Inui et al., 2001). Chlorotoluron is
metabolized by plants as well. The N-demethylated
metabolites are partly phytotoxic, and the N-demethylation pathway is favored by susceptible blackgrasses
(Alopecurus myosuriodes), wild oats (Avena fatua), and
Lolium perenne (Ryan et al., 1981; Hyde et al., 1996). In
tolerant plants, such as wheat (Triticum aestivum) and
barley (Hordeum vulgare), chlorotoluron is metabolized
through ring methyl hydroxylation.
Norflurazon (Fig. 1) is metabolized to N-demethylated metabolites by CYP1A1 expressed in recombinant
yeast (Inui et al., 2001). In cranberries (Vaccinium
macrocarpon), norflurazon is metabolized to an Ndemethylated analog that is biologically inactive

(Yachlich et al., 1974). Mefenacet is metabolized though

ring-hydroxylation as well as N-demethylation in the
yeast expression system (Inui et al., 2001).
4. Transgenic rice plants expressing CYP1A1
Rice is the major crop in East Asia, with an annual
production of approximately 608 million tons (International Rice Research Institute, 2005). The projected
population increase was about 27% in 2025 compared
with 2000, resulting in an estimated population of 7.9
billion in 2025. The world's most populous countriessuch as Bangladesh, China, Egypt, India, and Indonesiaconsume about 85% of the world's crop, and rice is the
staple grain for these people (Smil, 2004). It is predicted
that the demand for rice will grow by 25% in the next
25 years (Smil, 2004). To satisfy these demands,
increased crop yields, improvement in nutritional
quality, and reductions in post-harvest losses are needed.
The evolution of herbicide-resistant weeds is one of the
major problems associated with the use of herbicides. To
increase crop yields, the introduction in plants of P450s
involved in xenobiotic metabolism is considered to be a
useful technique for producing crops with cross-resistance
to various herbicides (Ohkawa et al., 1999). Transgenic
plants harboring such mammalian P450 genes are
expected to be resistant to herbicides and be able to
clean up residual agrochemicals (Ohkawa et al., 1999).

H. Kawahigashi et al. / Biotechnology Advances 25 (2007) 7584

79

Fig. 2. T-DNA region of pIES1A1 plasmid used to express the human CYP1A1 gene. RB, right border; LB, left border; NOS, nopaline synthase
promoter; NT, nopaline synthase terminator; NPTII, neomycin phosphotransferase II; 35S, cauliflower mosaic virus (CaMV) 35S promoter; E7,
CaMV 35S promoter with seven-enhancer region (-290 to -90) from CaMV 35S promoter; AMV-5UTR, alfalfa mosaic virus 5-untranslated region;
HPT, hygromycin B phosphotransferase.

Thus, we introduced a human gene for CYP1A1, a

cytochrome P450 monooxygenase that metabolizes
xenobiotic chemicals, into Oryza sativa cv. Nipponbare
by Agrobacterium-mediated transformation (Fig. 2). We
demonstrate here that transgenic rice expressing human
CYP1A1 under the control of modified CaMV 35S
showed strong herbicide resistance resulting from the
detoxification of several types of herbicides by CYP1A1.
Integration of CYP1A1 was confirmed by Southern
blotting, and expression of CYP1A1 was confirmed by
Western blot analysis.
When compared with non-transgenic Nipponbare
plants in a greenhouse, the CYP1A1 rice plants showed
normal morphology, including normal height, leaf color,
flowering time, fertility, and seed size. The CYP1A1
rice plants were physiologically the same as nontransgenic Nipponbare, except for the feature derived
from the introduced CYP1A1 gene. In contrast, it is
reported that negative effects of rabbit CYP2C14 were
observed in the growth of the transgenic tobacco plants.

The plants showed phenotypic changes, notably a

tendency of rapid senescence (Saito et al., 1991).
In germination tests, the CYP1A1 rice plants showed
high resistance to 10 out of 13 herbicides belonging to
different chemical families (Kawahigashi et al., 2003).
These were aryloxyphenoxypropionate (quizalofopethyl), benzamide (isoxaben), carbamate (chlorpropham), dinitroaniline (pendimethalin), oxyacetamide
(mefenacet), phosphoamidate (amiprophos-methyl),
pyridazinone (norflurazon), thiocarbamate (pyributicarb), and urea (chlorotoluron and diuron) (Fig. 3,
Table 1). Most of the tested herbicides could inhibit the
growth of the control plants at concentrations of several
micromoles-similar to those used in the field-with the
exception of the photosynthesis-inhibiting herbicides.
The acetyl-CoA carboxylase-inhibiting herbicide quizalofop-ethyl, and the very long-chain fatty acid (VLCFA)
synthesis-inhibiting herbicide mefenacet, inhibited the germination of Nipponbare, but had little effect on the growth
of CYP1A1 rice plants. CYP1A1 rice showed resistance to

Fig. 3. Phytotoxicity of various herbicides toward transgenic rice plants expressing CYP1A1. CYP1A1 rice plants showed normal growth, but nontransgenic rice plants did not. In the case of norflurazon, non-transgenic rice plants showed white shoots because of inhibition of carotenoid synthesis.
Germination tests were carried out in test tubes (diameter, 2.5 cm; height, 15 cm) each with 10 mL of MS solid medium (Murashige and Skoog, 1962)
containing 7.5 M chlorpropham, 2.5 M mefenacet, 20 M pendimethalin, or 0.5 M norflurazon. Four seeds of transgenic and non-transgenic
(control) rice plants were embedded in the medium and cultured at 27 C for 7 to 14 days under 16 h of light (photon flux density 40 mol m 2 s 1).
Lane C, Nipponbare without herbicide (control); lane N, Nipponbare with herbicide; lanes 1A1, CYP1A1 rice plants with herbicide.

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H. Kawahigashi et al. / Biotechnology Advances 25 (2007) 7584

Fig. 4. Herbicide resistance of CYP1A1 rice plants to chlorotoluron, diuron, and methabenzthiazuron. CYP1A1 rice plants grew well, but nontransgenic rice plants were severely damaged by the photosynthesis-inhibiting herbicides. Five 7-day-old plants were transplanted to glass pots
(diameter, 9 cm; height, 19 cm) with 500 mL water and 500 g of Kumiai-Ryujyou-Baido K soil (Kureha Chemical, Tokyo, Japan). Transgenic plants
and non-transgenic Nipponbare plants were grown in a greenhouse at 28 C during the day and 25 C at night for 2 weeks. We then added each
herbicide into the water. Growth was noted after 2 weeks. (A) Resistance to chlorotoluron; 3.9 mg chlorotoluron (twice that used in cornfields) was
applied. (B) Resistance to diuron; 2.1 mg diuron (twice that used in fruit tree orchards) was applied. (C) Resistance to methabenzthiazuron; 3.7 mg
methabenzthiazuron (twice that used in cornfields) was applied.

norflurazon, which caused bleaching of the shoots of

Nipponbare by the inhibition of carotenoid synthesis.
The microtubule assembly/organization-inhibiting
herbicides amiprophos-methyl, chlorpropham, and pendimethalin inhibited the growth of Nipponbare, but not
the growth of CYP1A1 rice plants. The cell wall
synthesis-inhibiting herbicide isoxaben and the unknown function herbicide pyributicarb inhibited the
root growth of Nipponbare, but CYP1A1 rice plants had
roots and grew better than Nipponbare.
CYP1A1 rice plants seemed to metabolize the herbicides during and after germination, keeping the concentration of the herbicide in plant tissues under the lethal
threshold. Therefore, the CYP1A1 rice plants could
metabolize a broad spectrum of herbicides and showed
cross-resistance to several herbicides with different
chemical structures and different modes of action.
5. Herbicide resistance of soil-grown transgenic rice
plants expressing CYP1A1
Non-transgenic rice plants grew well in germination
testing in the presence of the photosynthesis-inhibiting
herbicides atrazine, simazine, and methabenzthiazuron.
These herbicides did not affect the germination of rice
plants (even non-transgenic rice plants) under germination test conditions. With the other photosynthesisinhibiting herbicides (chlorotoluron and diuron) about
100 M of herbicide was needed to inhibit the growth of
control Nipponbare plants in germination tests. This is a

much higher concentration than that used in the field.

Even under these herbicide conditions, CYP1A1 rice
plants grew vigorously.
When these herbicides were applied to rice plants
grown in soil, CYP1A1 rice plants showed strong
resistance but non-transgenic rice plants were severely
damaged by inhibition of photosynthesis. Control
Nipponbare plants did not grow in the presence of
chlorotoluron (36.7 M), diuron (18 M), or methabenzthiazuron (33.4 M) (Fig. 4). The dosages of these
herbicides were twice that used in the field (Tomlin,
2000; Nouyaku Hand Book Hensyu Iinkai, 2001). At the
germination stage both non-transgenic and CYP1A1 rice
plants could survive, possibly because they gained
nutrition from the seed or because photosynthesis was
not as severely damaged under the light conditions of the
culture room; the amount of active oxygen produced
may not have been enough to kill the plants.
The observed herbicide resistance of the CYP1A1 rice
plants was consistent with the in vitro catalysis of these
herbicides by recombinant yeast microsomes expressing
human CYP1A1 (Table 1) (Inui et al., 2001). Thus, the
CYP1A1 plants presumably metabolized these herbicides
when fully grown as adult plants. TLC data showed that
chlorotoluron and norflurazon were metabolized quickly
in the transgenic rice plants (Kawahigashi et al., 2003).
Therefore, the CYP1A1 rice plants showed enhanced
metabolism towards these herbicides by detoxifying them
to produce the same intermediate metabolites as those
produced by the recombinant yeast expression system.

H. Kawahigashi et al. / Biotechnology Advances 25 (2007) 7584

Similar results of herbicide resistance have been

observed in transgenic tobacco and potato expressing
CYP1A1. Transgenic tobacco plants expressing the rat
CYP1A1 gene metabolize the herbicide chlorotoluron
through N-demethylation and ring-methyl hydroxylation
(Shiota et al., 1994, 2000). In addition, transgenic potato
plants (Solanum tuberosum) expressing rat or human
CYP1A1 show resistance toward the herbicides chlorotoluron, atrazine, and diuron (Inui et al., 1998, 1999) when
these are sprayed onto the plants. The transgenic potato
plants also showed a broad herbicide resistance. Therefore,
the introduced CYP1A1 worked effectively in adding
herbicide resistance to both monocot and dicot plants by
detoxifying the herbicides.
The evolution of herbicide-resistant weeds is a big
problem in the use of herbicides. Several strategies are
used to prevent or reduce herbicide resistance in weed
populations. To avoid the appearance of resistant weeds,
the use of several herbicides in rotation or in a mixture
has been proposed as the most practical approach to
prevent or delay the appearance of resistance, because
the repeated use of a single herbicide for a long time
tends to promote the growth of weeds that are resistant
(Putwain, 1990). Wider cross-resistance to herbicides
having different modes of action and different chemical
structures seems to be a special feature of transgenic
plants expressing mammalian P450 genes. This crossresistance of transgenic plants expressing CYP1A1
should prove useful in preventing the development of
herbicide resistance in weeds, because the use of several
herbicides in rotation would not harm the crop.
6. Use of CYP1A1 rice for direct seeding
Herbicide tolerance during germination should be
important for weed control in rice fields, especially with
direct-seeding systems. In transplanting-cultivation of
rice seedlings, water standing in the paddies prevents the
germination of many weeds; as a result, few major weeds
are present. However, in direct-seeding systems without
water cover, the germinating rice must compete with
many kinds of weeds. As shown in Fig. 5A, when rice
seeds were seeded in glass pots with mefenacet under
simulated field conditions, the CYP1A1 rice plants were
germinated healthily but Nipponbare did not germinate.
Other herbicides such as diuron and norflurazon can be
utilized as well. Transgenic rice with cross-resistance to
various types of herbicides should be an ideal plant for
weed control with herbicide mixtures, especially in
direct seeding.
In some cases, pro-herbicides are metabolized to active
compounds by P450s. CYP1A1 metabolizes the benzofu-

81

Fig. 5. Germination testing of soil-grown CYP1A1 rice plants with

mefenacet and benfuresate. (A) CYP1A1 rice plants showed normal
growth, but non-transgenic rice plants were killed by mefenacet just
after germination. (B) In contrast, CYP1A1 rice plants did not grow
with benfuresate, but non-transgenic rice plants showed normal
growth. Rice seeds were seeded into glass pots (diameter, 9 cm; height,
19 cm) with 500 mL water and 500 g of Kumiai-Ryujyou-Baido K soil.
At the same time, (A) 25.4 mg of HINOCHLOA (Bayer CropScience
Japan, Tokyo, Japan), which contained 1 mg of mefenacet, or (B)
0.34 mg of benfuresate was applied to the pots. The dosage was the
same as that used in rice fields in Japan to prevent weed growth. The
rice seedlings were grown in a greenhouse at 28 C during the day and
25 C at night for 2 weeks.

ran herbicides benfuresate and ethofumesate, which inhibit

lipid synthesis (Inui et al., 2001). However, CYP1A1 rice
plants showed susceptibility to ethofumesate and benfuresate. Both herbicides inhibited the germination of
transgenic rice plants at a concentration of 2.0 M in the
culture medium, whereas control Nipponbare plants
showed normal growth because the metabolites were
more phytotoxic than the parent compounds (Kawahigashi
et al., 2002). As shown in Fig. 5B, when rice seeds were
seeded in glass pots with benfuresate under simulated field
conditions, non-transgenic rice plants were germinated
healthily but the CYP1A1 rice plants did not germinate. If
the CYP1A1 rice plants are either accidentally or purposely

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H. Kawahigashi et al. / Biotechnology Advances 25 (2007) 7584

mixed with non-transgenic rice plants in the fields, the

combination of CYP1A1 rice plants and ethofumesate or
benfuresate may be useful as a negative selection for the
first step of large-scale screening of the transgenic plants.
If the introduced genes are to escape to the environment
by cross-pollination with wild species native to South East
Asia, a multiple problem may arise. The hybrids would be
susceptible to these herbicides and the use of ethofumesate
or benfuresate could be one method to detect the escape of
CYP1A1 gene. However, it is necessary to examine the
possibility of the cross-pollination between wild species
and the transgenic plants and the susceptibility of possible
transgenic wild plants towards herbicides.
7. Use of transgenic rice plants for phytoremediation
CYP1A1 rice plants showed broad herbicide resistance and also showed the ability to reduce the
concentration of herbicide in the culture medium. The
results of TLC analysis have revealed that the amounts of
chlorotoluron, norflurazon, and atrazine decreased in
CYP1A1 rice plants and in the medium of CYP1A1 rice
faster than with non-transgenic Nipponbare (Kawahigashi et al., 2003). Both the CYP1A1 plants and the nontransgenic control plants metabolized these herbicides
and reduced their residual amounts, but CYP1A1 plants
decreased the amount of residual chlorotoluron in the
culture medium 3.3 times more, and of norflurazon 2.6
times more, during 6 days of incubation (Fig. 6).

Similarly, cultured tobacco cells transformed with

CYP1A1 or CYP1A2 show high metabolic activity
towards atrazine to produce N-dealkylated metabolites.
They metabolize atrazine faster than non-transformed
tobacco cells (Bode et al., 2004).
Phytoremediation is the use of plants and plant growth
as a technique for detoxifying environmental polluted
soils, sediments, and aquatic sites contaminated with
organic and inorganic pollutants (Salt et al., 1998).
Phytoremediation costs much less than physical and
chemical remediation treatments and has proven to be a
sustainable technology for bioremediation (Salt et al.,
1995, 1998).
According to Schnoor et al. (1995), phytoremediation is best suited for sites with shallow contamination
(b 5 m depth), moderately hydrophobic pollutants
(logKow = 0.53), short-chain aliphatic chemicals, and
excess nutrients. Most pesticides are moderately hydrophobic, so phytoremediation is one possible method of
removing pesticides from contaminated water and soil.
However, field trials have suggested that the rate of
contaminant removal using conventional plants is
insufficient (Goel et al., 1997). In plants, pollutants can
be remediated through several biochemical processes
adsorption, transport, and translation; hyperaccumulation; or transformation and mineralizationthat protect
the plants themselves from toxic organic foreign
chemicals. Over-expression of endogenous plant genes
or transgenic expression of bacterial or animal genes is

Fig. 6. Time course of metabolism of chlorotoluron (A) and norflurazon (B) by control (Nipponbare) and CYP1A1 transgenic rice plants. CYP1A1
rice plants decreased the concentrations of both herbicides in the culture medium quicker than did the non-transgenic Nipponbare rice plants. Rice
seeds were embedded in MS solid medium and incubated at 27 C for 6 days under 16 h of light (photon flux density 40 mol m 2 s 1). Individual 6day-old plants were transferred to 3 mL of Hyponex 5-10-5 (Hyponex, Osaka, Japan) solution containing 40 000 dpm 14C-chlorotoluron or 14Cnorflurazon at 10 M in a test tube (diameter, 2.5 cm; height, 15 cm). The plants were incubated under 24 h of light (40 mol m 2 s 1), and the culture
medium was sampled on days 1, 3, and 6 of incubation. The culture medium was dried and dissolved in 90% methanol. Then 2000-dpm aliquots of
culture medium were applied to the lanes of a silica gel 60F254 thin-layer chromatography (TLC) plate (Merck, Darmstadt, Germany). The developing
solvents were chloroform:ethanol (9:1, v/v) for chlorotoluron and dichloromethane:methanol (98:2, v/v) for norflurazon. Radioactivity was measured
in an FLA-2000 Bio-Imaging Analyzer (Fuji Photo Film Co. Ltd., Tokyo, Japan). These values are the averages of three independent experiments.

H. Kawahigashi et al. / Biotechnology Advances 25 (2007) 7584

required to significantly increase the remediation ability

of plants (Hooker and Skeen, 1999, Doty et al., 2000;
Dietz and Schnoor, 2001; Linacre et al., 2003).
Rice is a good candidate for phytoremediation because it grows in paddy fields and can remove contaminants from stream water. Application of CYP1A1 to
transgenic plants may prove useful for phytoremediation. Pesticides have been developed to be less toxic to
the environment, but there remains the possibility of
non-target effects of pesticides on the environment as a
result of agricultural runoff (Cooper, 1993; Relyea,
2005). We expect that CYP1A1 rice will also prove
useful in degrading and thus decreasing the environmental loads of herbicides, insecticides, industrial
chemicals, and pollutants in paddy fields and connected
water streams.
8. Concluding remarks
CYP1A1 rice plants showed broad herbicide resistance towards various herbicides and will also prove
useful in degrading, and thus decreasing the environmental load of herbicides in paddy fields. CYP1A1
showed broad and overlapping substrate specificity
towards foreign chemicals with different chemical
structures, and thus it is necessary to study further the
substrate specificity of the introduced CYP1A1 towards
xenobiotics and secondary metabolites in plants (e.g.
regarding metabolic activation to toxic or genotoxic
products). Further experiments on environmental safety
assessment are also needed before such transgenic plants
can be put to practical use. In future, transgenic plants
expressing P450s should be good not only for developing herbicide-resistant rice but also for reducing the
environmental impacts of agrochemicals.
Acknowledgement
This study was supported by the Bio-oriented Technology
Research Advancement Institution (BRAIN), Tokyo, Japan.
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