Pesticide Biochemistry and Physiology 83 (2005) 107–114

www.elsevier.com/locate/ypest

Cytochrome P-450 monooxygenase activity

in herbicide-resistant and -susceptible late watergrass
(Echinochloa phyllopogon)
Min-Soo Yun a,¤, Yasuhiro Yogo a,1, Reiichi Miura b, Yuji Yamasue b,
Albert J. Fischer c
a
Upland Weed Laboratory, Department of Field Environment, National Agricultural Research Center,
3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan
b
Laboratories of Agricultural Ecology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Kyoto, Japan
c
Weed Science Program, Department of Plant Sciences, University of California, Davis, CA 95616, USA

Received 28 January 2005; accepted 7 April 2005

Available online 31 May 2005

Abstract

Late watergrass is a major weed of rice in California, and several populations showed resistance to multiple herbi-
cides of diVering modes of action. Bispyribac-sodium, fenoxaprop-ethyl, and thiobencarb were used in this study as
inducers and substrates of cytochrome P-450 monooxygenases (P450s). To examine the role of P450s in herbicide
metabolism by a herbicide-resistant (R) and a herbicide-susceptible (S) late watergrass (Echinochloa phyllopogon) bio-
type, seedlings were grown in absence and presence of the above three herbicides as inducers. The eYcacy of the inducer
treatments was conWrmed by monitoring changes in P450 content and activity. Herbicide induction increased P450 con-
tent and activity in both R and S biotypes. This increase was higher in the R biotype. The induced P450s exhibited spec-
iWcity for the diVerent herbicide substrates. Our study suggests that induction of P450s is the mechanism involved in the
multiple-herbicide resistance observed in late watergrass.
 2005 Elsevier Inc. All rights reserved.

Keywords: Cytochrome P-450 monooxygenase; Herbicide-resistance; Late watergrass; Bispyribac-sodium; Fenoxaprop-ethyl; Thio-
bencarb

*
Corresponding author. Present address: Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural
Sciences, SE- 901 83 Umeå, Sweden. Fax: +46 90 786 8165.
E-mail address: minsoo.yun@genfys.slu.se (M.-S. Yun).
1
Present address: Organochemicals Group, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Iba-
raki 305-8604, Japan.

0048-3575/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.pestbp.2005.04.002
108 M.-S. Yun et al. / Pesticide Biochemistry and Physiology 83 (2005) 107–114
1. Introduction
Late watergrass [Echinochloa phyllopogon
(Stapf) Kossenko] is a noxious weed of rice in the
Sacramento Valley of California. In 1997, late
watergrass biotypes putatively resistant to multi-
ple herbicides, namely, bispyribac-sodium, fenox-
aprop-ethyl, molinate, and thiobencarb, were
found in rice Welds scattered throughout the Sac-
ramento Valley [1,2]. It was suggested that these
biotypes originated from a single biotype,
because of their similarities in morphological and
ampliWed fragment-length polymorphism
(AFLP) traits [3].
Bispyribac-sodium, a pyrimidinyl carboxy her-
bicide, is eVective to control many annual and
perennial grasses, sedges, and broad-leaved weeds
in rice Welds. The mode of action has been consid-
ered as inhibition of acetolactate synthase (ALS,2
also known as acetohydroxyacid synthase or
AHAS; EC 4.1.3.18) in the biosynthetic pathway
of three branched-chain amino acids [4]. Resis-
tance to bispyribac-sodium had already been
detected in some late watergrass populations
before this herbicide had ever been used commer-
cially in California [1,2]. Fenoxaprop-ethyl is
used as postemergence herbicide to control vari-
ous annual grass weeds in rice and other crops [5].
This herbicide is an aryloxyphenoxy propionate
that inhibits acetyl coenzyme A carboxylase
(ACCase; EC 6.4.1.2) [6] and was introduced to
California in the early 1990s to control Echino-
chloa spp. and Leptochloa fascicularis (Lam.) A.
Gray in rice. Molinate and thiobencarb are thioc-
arbamate herbicides widely used for weed control
in rice, and were introduced to California as early
postemergence herbicides in the 1960s and early
1980s, respectively [7].
Plants can metabolize foreign compounds,
such as herbicides, and thus reduce the amounts
of these xenobiotics that can reach their action
2
Abbreviations used: ABT, aminobenzotriazol; ACCase, ace-
tyl coenzyme A carboxylase; AFLP, ampliWed fragment-length
polymorphisms; ALS, acetolactate synthase; CO, carbon mon-
oxide; DMSO, dimethyl sulfoxide; GST, glutathione transferas-
es; HSD, honestly signiWcant diVerence; LSD, least signiWcant
diVerence; P450, cytochrome P-450 monooxygenase; PBO, pi-
peronyl butoxide; R, herbicide-resistant; S, herbicide-susceptible.
sites. There are two phases in xenobiotic detoxiW-
cation. The Wrst phase involves oxidative reac-
tions, such as hydroxylation, and the second
phase involves conjugation with glutathione or
other compounds [8]. The cytochrome P-450
monooxygenases (P450s) are membrane-bound
and heme–iron containing enzymes that use
NADPH + H+ as a co-substrate by forming a
complex with a cytochrome P-450 reductase. In
plants, P450s participate in secondary metabo-
lism [9]. P450s are also well known as key
enzymes in phase I metabolism of xenobiotics,
and have been implicated in the initial detoxiWca-
tion of herbicides [9,10]. For example, thiocarba-
mates such as molinate and thiobencarb are
initially metabolized through typical monooxida-
tion reactions in plants, as generally observed for
other carbamate esters, i.e., thiol sulfur oxidation
to the corresponding sulfoxide [11,12]. It was also
known that thiobencarb is metabolically detoxi-
Wed by N-demethylation to form a demethyl
derivative and by hydroxylation of a phenyl ring
[13].
Through diVerent substrate speciWcities, P450
isozymes contribute to herbicide selectivity
between weeds and crops, and in some cases
confer herbicide resistance to weed biotypes
[14–18]. P450s are implicated in metabolism-
based resistance to multiple herbicides in grass
weeds such as blackgrass (Alopecurus myosuro-
ides Huds.) [19]. A blackgrass biotype of a
population collected from Peldon in the United
Kingdom, which showed multiple-resistance to
phenylurea and aryloxyphenoxypropionate
herbicides, exhibited higher P450 activities than
a herbicide-susceptible biotype [20]. It was also
suggested from a study with P450 inhibitors
that P450-mediated herbicide degradation
was involved in the multiple-herbicide resistance
of the Californian late watergrass [2]. In the
present study, we used microsomal fractions
isolated from herbicide-resistant (R) and -suscep-
tible (S) late watergrass plants to determine
P450 content and activity. A representative of
each herbicide group against which R late
watergrass exhibits resistance in California
rice Welds was used as P450 inducer and sub-
strate.
 M.-S. Yun et al. / Pesticide Biochemistry and Physiology 83 (2005) 107–114
2. Materials and methods
2.1. Chemicals
Technical-grade herbicides were used for all
assays. Bispyribac-sodium {sodium 2,6-bis[(4,6-
dimethoxypyrimidin-2-yl)oxy]benzoate} was pro-
vided by Kumiai Chemical Industry3; fenoxaprop-
ethyl {ethyl(§)-2-[4-(6-chloro-1,3-benzoxazol-2-yl-
oxy)phenoxy]propionate} and thiobencarb (S-p-
chlorobenzyl diethylthiocarbamate) were obtained
commercially from Wako Pure Chemical Industries.4
2.2. Plant material and herbicide treatment
Two inbred strains of late watergrass, R (511)
and S (401), were used in the present experiment.
These strains originated from accessions collected
in 1997 in rice Welds of the Sacramento Valley in
California. They represent strains derived after two
successive selWng cycles from a multiple-herbicide-
resistant and a susceptible biotype, respectively.
Plant morphology and AFLP Wngerprints of these
strains were described in detail elsewhere [1,3].
Seeds of these two strains were germinated in an
incubator at 30 °C in the dark for 24 h. Emerged
seedlings were transferred to plastic pots and
grown in aqueous calcium sulfate solution (1 mM).
The pots were covered with aluminum foil and
placed in the dark at 30 °C. After 6 days, etiolated
shoots were excised and used for microsome isola-
tion [14,17]. Prior to excision, plants were subirri-
gated for 24 h with 1
M bispyribac-sodium,
fenoxaprop-ethyl, or thiobencarb.
2.3. Microsome isolation
Since P450s are mainly found in microsomal
fractions [21], microsomes were isolated from
entire shoots [22]. Approximately 10–15 g of
pooled shoots were harvested from R and S plants
and homogenized at 4 °C in sodium phosphate
3
Life Science Research Institute, Kumiai Chemical Industry,
3360 Kamo, Kikugawa-cho, Ogasa-gun, Shizuoka 439-0031,
Japan.
4 5
Wako Pure Chemical Industries, 1-2, Doshomachi 3-
Chome, Chuo-Ku, Osaka 540-8605, Japan.
109
buVer (100 mM; pH 8.0) containing -mercap-
toethanol (14 mM), EDTA (10 mM), and ascorbate
(40 mM) [18,22]. Shoots were ground in liquid
nitrogen using mortar and pestle. The crude
extract was Wltered through four layers of cheese-
cloth to remove tissue debris and then centrifuged
at 10,000g for 20 min. The resulting supernatant
was centrifuged at 100,000g for 90 min. The Wnal
pellet was re-suspended in sodium phosphate
buVer (100 mM; pH 8.0) containing glycerol
(250 ml L¡1) prior to use.
2.4. P450 content
Amounts of P450s in the microsomal fractions
were quantiWed using reduced carbon monoxide
(CO) diVerence spectroscopy [23,24]. An aliquot
(1 ml) of a microsomal preparation (2 mg
protein ml¡1) was placed in a 10 ml test tube. The
preparation was reduced with 3 mg solid sodium
dithionite. Standard CO gas (99.9% purity) was
lightly bubbled in the test tube for 30 s. The Wnal
solution was transferred to a 96-well microplate.
Absorbance was then measured from 350 to
550 nm using a microplate reader (SPECTRAmax
190, Nihon Molecular Devices5). P450 content was
calculated as nmol mg¡1 microsomal protein using
the extinction coeYcient of 91 mM¡1 cm¡1 and the
value of absorbance at 450–490 nm [21,25]. The
enzyme solution was assayed for protein contents
by the method of Bradford [26] using bovine serum
albumin as a standard. Linear regression of a stan-
dard curve was used to determine the concentra-
tion of protein in samples prepared from the
enzyme fractions.
2.5. P450 monooxygenase activity
All assays were done in a total volume of 500
l
in a 1.5 ml microtube. The assay mixture contained
sodium phosphate buVer (100 mM; pH 8.0), micro-
somal protein (1 mg ml¡1 assay solution), and
NADPH (1.0 mM). Bispyribac-sodium (0.1 mM as
Wnal concentration), fenoxaprop-ethyl (0.25 mM),
or thiobencarb (0.25 mM) was individually dis-
Nihon Molecular Devices, 3-21 Kanda Nishiki-cho, Chi-
yoda-ku, Tokyo 100-0054, Japan.
110 M.-S. Yun et al. / Pesticide Biochemistry and Physiology 83 (2005) 107–114
solved in dimethyl sulfoxide (DMSO, Wnal concen-
tration: 1.0 ml L¡1) and added to the assay mixture
as a substrate. This concentration of DMSO did
not aVect the enzyme activity. The reaction was ini-
tiated by addition of 50
l NADPH (10 mM). A
treatment without NADPH was included as a con-
trol. Solutions were incubated at 30 °C for 60 min
and the reaction was terminated by adjusting the
pH to 3.0 with 75
l phosphoric acid (1.4 M). The
solution was partitioned with ethyl acetate
(2 £ 1.0 ml). The ethyl acetate fractions were com-
bined, dried under nitrogen, and then dissolved in
methanol (1.0 ml). Substrates were separated by
high performance liquid chromatography (HPLC;
Shimadzu LC-10Ai, Shimadzu6) with a Nucleosil
5C18 column (4.6 mm £ 250 mm). The solvent gra-
dient was formed through an initial proportion of
80% solvent A (0.5% phosphoric acid in water) to
20% solvent B (0.5% phosphoric acid in acetoni-
trile). After 20 min, the solvent gradient had
reached 80% B. The Xow rate of the solvent was
kept constant at 1.0 ml min¡1. All solvents were
HPLC grade and were degassed before use.
Twenty microliter assay solution was injected.
Substrates except bispyribac-sodium (246 nm)
were detected at a wavelength of 240 nm. All sub-
strates were quantiWed by peak areas using a UV
detector (Shimadzu SPD-10A UV-VIS detector,
Shimadzu6). The peaks were identiWed by matching
their retention times with analytical standards of
each substrate. Hydroxylation activity was calcu-
lated as speciWc activity (pmol mg¡1 microsomal
protein min¡1).
2.6. Statistical analysis
Preliminary trials were performed with a single
or two replications for the microsomal fraction-
ation and also for quantitative analyses of P450
content and activity. The experiment was repeated
with reproducible eVects, and data presented here
are for R and S etiolated plants, with or without
herbicide pretreatment, for which P450 content
and activity was determined with three replicated
measurements. For each pretreatment, paired R
6 statistically diVerent according to the Tukey–Kramer honestly
Shimadzu, 1 Nishinokyo, Kuwabara-cho, Nakagyo-ku,
Kyoto 604-8511, Japan.
and S mean values were compared using standard
errors and Fisher’s protected least signiWcant
diVerence (LSD) test. Means of either R or S plants
across pretreatments were compared using the
Tukey–Kramer honestly signiWcant diVerence
(HSD) test. The type I error rate was set at 0.05 for
all statistical tests, and the SPSS (Ver. 8.0J) soft-
ware was used.
3. Results and discussion
The P450 contents in R and S microsomal frac-
tions determined using spectroscopy at 450–
490 nm under reduced carbon monoxide [23,24] are
shown in Table 1. These fractions were isolated
from plants pretreated with either bispyribac-
sodium, fenoxaprop-ethyl or thiobencarb. The R
biotype exhibits resistance to these herbicides in
the Weld, while the S biotype is killed by the indi-
vidual herbicides at recommended doses [1]. The
microsomal fraction prepared from R plants not
pretreated with any herbicide contained
3.12 nmol mg¡1 protein, which is signiWcantly
larger than the 2.95 nmol mg¡1 protein found in S
plants. When plants were pretreated with either
bispyribac-sodium, fenoxaprop-ethyl or thioben-
carb, both R and S plants increased their micro-
somal P450 content compared to non-pretreated
Table 1
P450 monooxygenase contents in microsomal fractions isolated
from shoots of R and S late watergrass plants pretreated with
either bispyribac-sodium, fenoxaprop-ethyl, or thiobencarb
Herbicide P450 content (nmol mg¡1 protein)
pretreatmenta (§SE)b
R plants S plants
None ¤
3.12 § 0.04a 2.95 § 0.03a
Bispyribac-sodium¤ 5.82 § 0.06c 4.10 § 0.01b
Fenoxaprop-ethyl¤ 4.61 § 0.12b 4.03 § 0.11b
Thiobencarb¤ 5.53 § 0.05c 5.14 § 0.10c
a
Plants were pretreated with a 1
M concentration of each
herbicide for 24 h prior sampling for assay. An asterisk (¤) indi-
cates statistical diVerence between a pair of R and S means
within a row according to Fisher’s protected least signiWcant
diVerence (LSD) test at P < 0.05.
b
Within each column, means followed by diVerent letters are
signiWcant diVerence (HSD) test at P < 0.05.
 M.-S. Yun et al. / Pesticide Biochemistry and Physiology 83 (2005) 107–114 111

Table 2
P450 monooxygenase activities in microsomal fractions isolated from shoots of R and S late watergrass plants pretreated with either
bispyribac-sodium, fenoxaprop-ethyl, or thiobencarb
Herbicide pretreatmenta Substrateb P450 activityc (pmol mg¡1 protein min¡1) (§SE)
R plants S plants
None Bispyribac-sodium 0.0a 0.0a
Fenoxaprop-ethyl 0.0a 0.0a
Thiobencarb 0.0a 0.0a
Bispyribac-sodium Bispyribac-sodium¤ 33.3 § 0.5b 11.0 § 0. 2a
Fenoxaprop-ethyl 0.0a 0.0a
Thiobencarb 0.0a 0.0a
Fenoxaprop-ethyl Bispyribac-sodium 0.0a 0.0a
Fenoxaprop-ethyl¤ 246.7 § 5.7d 143.3 § 14.9b
Thiobencarb¤ 37.5 § 1.1b 22.7 § 5.3a
Thiobencarb Bispyribac-sodium 0.0a 0.0a
Fenoxaprop-ethyl 0.0a 0.0a
Thiobencarb¤ 297.1 § 5.5e 245.2 § 2.6c
a
Plants were pretreated with a 1
M concentration of each herbicide for 24 h prior sampling for assay.
b
Bispyribac-sodium, fenoxaprop-ethyl, and thiobencarb were added to the assay medium as hydroxylation substrates at concentra-
tions of 0.1, 0.25, and 0.25 mM, respectively. An asterisk (¤) indicates statistical diVerence between a pair of R and S means within a row
according to Fisher’s protected least signiWcant diVerence (LSD) test at P < 0.05.
c
Within each column, means followed by diVerent letters are statistically diVerent according to the Tukey–Kramer honestly signiW-
cant diVerence (HSD) test at P < 0.05.

plants. But, this increase in P450 content was larger
for R plants in all cases. The higher contents of
inducible P450 in the R biotype in response to spe-
ciWc herbicide pretreatments suggest a correspon-
dence with its superior ability to withstand toxicity
from these herbicides.
The speciWc activities of P450s from R and S
microsomal fractions, as determined by the degra-
dation rates of the herbicides used as substrate, are
shown in Table 2. We assumed that this activity
corresponded to P450 monooxygenase hydroxyl-
ation of the herbicide molecule, since it was deter-
mined in the presence of NADPH. No P450
activity was observed in microsomal fractions
from plants not pretreated with herbicide, but her-
bicide pretreatment induced P450 activity in both
R and S microsomal fractions. When plants were
pretreated with bispyribac-sodium, the microsomal
fractions exhibited P450 activity only against this
herbicide, and the R biotype exhibited higher P450
activity than the S biotype. Likewise, P450 activity
on fenoxaprop-ethyl was observed only when
plants had been pretreated with this herbicide.
However, P450 activity on thiobencarb was
induced by pretreatment with thiobencarb and
with fenoxaprop-ethyl, although the level of activ-
ity on thiobencarb induced by fenoxaprop-ethyl
was substantially lower than that observed when
thiobencarb itself was used as the inducer. In all
cases, pretreatment-induced P450 activity was
higher in R than in S microsomal fractions. The
higher ability of induced R plants to utilize as sub-
strates for degradation the herbicides with which
they had been pretreated reXects the higher P450
content observed in the induced R plants (Table 1).
These results suggest that R late watergrass has
inducible P450s responsible for enhanced hydrox-
ylation rates of bispyribac-sodium, fenoxaprop-
ethyl, and thiobencarb, which reXect the observed
resistance to these herbicides. Although pretreat-
ment with fenoxaprop-ethyl increased the ability
of R and S plants to hydroxylate both fenoxaprop-
ethyl and thiobencarb, P450 induction was herbi-
cide-speciWc in all other cases (Table 2). This sug-
gests that induction of P450 isozymes with
diVerent substrate speciWcity is most likely the
mechanism involved in the multiple-herbicide
resistance observed in late watergrass.
Several possible mechanisms need to be consid-
ered when investigating resistance to herbicides in
weeds. Resistant weed biotypes may contain an
action site with low herbicide aYnity or may have
112 M.-S. Yun et al. / Pesticide Biochemistry and Physiology 83 (2005) 107–114
evolved a high ability to detoxify herbicides. In our
earlier study with R and S late watergrass diVering
in their sensitivity to the above herbicides [1], we
demonstrated that resistance to bispyribac-sodium
did not involve an altered herbicide-binding site on
the ALS, but instead, a mechanism of enhanced
degradation that was possibly P450 mediated [2].
The P450 involvement was indirectly postulated
because resistance to bispyribac-sodium was abol-
ished by a simultaneous application of either piper-
onyl butoxide (PBO) or malathion, which are
known P450 inhibitors. Similarly, resistance to thio-
bencarb in hydroponically grown R late watergrass
was overcome by adding the P450 inhibitor amin-
obenzotriazol (ABT) to the growing medium (A.
Fischer 2004, personal communication). An
increase in P450-dependent metabolism was Wrst
demonstrated in resistant Lolium rigidum Gaudin
biotypes from Australia and blackgrass biotypes
that appeared in Europe [27,28]. Blackgrass resis-
tance to ACCase-inhibitors, including fenoxaprop-
ethyl, can involve an insensitive ACCase. However,
blackgrass populations in the United Kingdom are
resistant to these herbicides through enhanced met-
abolic detoxiWcation. In vivo metabolism studies
have established the role of P450s in the enzymatic
detoxiWcation of diclofop-methyl and fenoxaprop-
ethyl in wheat and barley [8,29]. In the present
study, an R late watergrass biotype with multiple-
herbicide resistance to bispyribac-sodium, fenoxa-
prop-ethyl, and thiobencarb exhibited higher P450
hydroxylation activity toward these herbicides than
an S biotype. Therefore, the results presented here
directly prove our hypothesis of P450 involvement
as a mechanism for resistance in late watergrass.
Our study also demonstrates that induction of
P450 isozymes and their mediated herbicide
hydroxylation resulted from herbicide application.
Furthermore, the R biotype has the ability to
induce the isozymes that allow resistance to bis-
pyribac-sodium. Resistance to bispyribac-sodium
was detected in the R biotype before this herbicide
had ever been used in California [1]. The induced
P450 isozymes diVered in substrate speciWcity and
were not able to display measurable activity
towards all herbicides. This metabolic versatility
also appears in the S biotype tested here, but at
lower levels. Resistance in late watergrass seems to
be a long-term creeping process by which these
plants could have accumulated mutations, gene
ampliWcations, or duplications conferring higher
herbicide detoxiWcation ability [30]. Evidently
plants have an enormous diversity of P450 genes
[31–33], given the chemical diversity of secondary
metabolites and the many P450 genes involved in
their biosynthesis [34]. However, there is yet no evi-
dence that P450 genes are ampliWed by herbicide
treatment. Further studies are needed to clarify
how many and which P450 isozymes are induced
by speciWc herbicide treatments, and what is their
mode of inheritance. The involvement of other
mechanisms in the observed resistance cannot be
discarded. Several studies have demonstrated the
role of glutathione transferases (GST) in the detox-
iWcation of fenoxaprop via glutathione conjuga-
tion in other species [8,35,36]. This mechanism was
also implicated in the resistance of blackgrass in
Europe to multiple herbicides, including fenoxa-
prop, and it has even been postulated that GST
and P450 xenobiotic detoxiWcation systems can be
coordinately regulated [37]. Therefore, further
studies should investigate the role of GST activity
in the resistance to fenoxaprop in resistant popula-
tions of late watergrass.
Acknowledgments
We are grateful to Dr. T. Shinohara of Kumiai
Chemical Industry Co., Ltd. for the gift of bispyri-
bac-sodium and their helpful suggestions.
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