Xenobiotic responsiveness of Arabidopsis thaliana to a chemical series derived from a herbicide safener

Article abstract of DOI:10.1074/jbc.M111.252726

Plants respond to synthetic chemicals by eliciting a xenobiotic response (XR) that enhances the expression of detoxifying enzymes such as glutathione transferases (GSTs). In agrochemistry, the ability of safeners to induce an XR is used to increase herbicide detoxification in cereal crops. Based on the responsiveness of the model plant Arabidopsis thaliana to the rice safener fenclorim (4,6-dichloro-2-phenylpyrimidine), a series of related derivatives was prepared and tested for the ability to induce GSTs in cell suspension cultures. The XR in Arabidopsis could be divided into rapid and slow types depending on subtle variations in the reactivity (electrophilicity) and chemical structure of the derivatives. In a comparative microarray study, Arabidopsis cultures were treated with closely related compounds that elicited rapid (fenclorim) and slow (4-chloro-6-methyl-2-phenylpyrimidine) XRs. Both chemicals induced major changes in gene expression, including a coordinated suppression in cell wall biosynthesis and an up-regulation in detoxification pathways, whereas only fenclorim selectively induced sulfur and phenolic metabolism. These transcriptome studies suggested several linkages between the XR and oxidative and oxylipin signaling. Confirming links with abiotic stress signaling, suppression of glutathione content enhanced GST induction by fenclorim, whereas fatty acid desaturase mutants, which were unable to synthesize oxylipins, showed an attenuated XR. Examining the significance of these studies to agrochemistry, only those fenclorim derivatives that elicited a rapid XR proved effective in increasing herbicide tolerance (safening) in rice.

Full text of DOI:10.1074/jbc.M111.252726

Plant Biology:
Xenobiotic Responsiveness of Arabidopsis
thaliana to a Chemical Series Derived from
a Herbicide Safener
Mark Skipsey, Kathryn M. Knight, Melissa
Brazier-Hicks, David P. Dixon, Patrick G.
Steel and Robert Edwards
J. Biol. Chem. 2011, 286:32268-32276.
doi: 10.1074/jbc.M111.252726 originally published online July 21, 2011
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 37, pp. 32268–32276, September 16, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Xenobiotic Responsiveness of Arabidopsis thaliana to a
□
S
Chemical Series Derived from a Herbicide Safener*
Received for publication, April 21, 2011, and in revised form, July 21, 2011 Published, JBC Papers in Press, July 21, 2011, DOI 10.1074/jbc.M111.252726
Mark Skipsey‡1, Kathryn M. Knight§1,2, Melissa Brazier-Hicks‡1,3, David P. Dixon‡1, Patrick G. Steel§,
and Robert Edwards‡4
From the ‡School of Biological and Biomedical Sciences and the §Department of Chemistry, Durham University,
Durham DH1 3LE, United Kingdom
Plants respond to synthetic chemicals by eliciting a xenobiotic
response (XR) that enhances the expression of detoxifying
enzymes such as glutathione transferases (GSTs). In agrochem-
istry, the ability of safeners to induce an XR is used to increase
herbicide detoxification in cereal crops. Based on the respon-
siveness of the model plant Arabidopsis thaliana to the rice
safener fenclorim (4,6-dichloro-2-phenylpyrimidine), a series of
related derivatives was prepared and tested for the ability to
induce GSTs in cell suspension cultures. The XR in Arabidopsis
could be divided into rapid and slow types depending on subtle
variations in the reactivity (electrophilicity) and chemical struc-
ture of the derivatives. In a comparative microarray study, Ara-
bidopsis cultures were treated with closely related compounds
that elicited rapid (fenclorim) and slow (4-chloro-6-methyl-2-
phenylpyrimidine) XRs. Both chemicals induced major changes
in gene expression, including a coordinated suppression in cell
wall biosynthesis and an up-regulation in detoxification path-
ways, whereas only fenclorim selectively induced sulfur and
phenolic metabolism. These transcriptome studies suggested
several linkages between the XR and oxidative and oxylipin sig-
naling. Confirming links with abiotic stress signaling, suppres-
sion of glutathione content enhanced GST induction by fenclo-
rim, whereas fatty acid desaturase mutants, which were unable
to synthesize oxylipins, showed an attenuated XR. Examining
the significance of these studies to agrochemistry, only those
fenclorim derivatives that elicited a rapid XR proved effective in
increasing herbicide tolerance (safening) in rice.
elicitors released during infection (1). In addition, plants also
recognize and mount a specific stress response to a range of
synthetic compounds (xenobiotics) including drugs, pollutants,
and agrochemicals (2). This xenobiotic response (XR),5
involves the coordinated up-regulation of genes encoding a
group of proteins that detoxify foreign compounds, collectively
termed the xenome (3). The best known inducible xenome
components are the cytochrome P450 mixed function oxidases
(CYPs), family 1 glucosyltransferases, glutathione transferases
(GSTs), and ATP binding cassette transporter proteins (4–6).
Together, their enhanced expression allows plants to accelerate
the metabolism and sequestration of toxic chemicals. In agri-
culture, this response is exploited using safeners, a group of
crop protection agents that increase the rates of detoxification
of herbicides in cereal crops, thereby enhancing the selectivity
of graminicides used to control competing grass weeds (7).
Over the last 30 years a diverse range of safener chemistries
have been developed, with each compound used in partnership
with a herbicide for use in a specific crop (4, 8). Although safen-
ing activity toward herbicides is only observed in cereals and
some non-domesticated grasses (9), the ability of these com-
pounds to selectively induce xenome enzymes also extends to
dicotyledenous plants, such as poplar and Arabidopsis thaliana
(6, 10–12). In particular, the safener fenclorim (4,6-dichloro-2-
phenylpyrimidine; Fig. 1A), which is used in rice to increase
tolerance to chloroacetanilide herbicides, was found to be a
potent and selective inducer of GSTs in root and suspension
cultures of Arabidopsis (12). Based on the apparent conserva-
tion in safener recognition and xenome induction in Arabidop-
sis and cereals (10–12), the use of this model plant with all the
associated molecular genetic tools and available mutants offers
a powerful route to unraveling these hitherto intractable signal
transduction pathways associated with xenobiotic sensing.
Arabidopsis contains 54 GSTs, which based on sequence
identities can be divided into the phi, tau, lambda, theta, zeta,
tetrachlorohydroquinone dehalogenase and dehydroascorbate
reductase classes (13). Previous proteomic and transcriptomic
studies have shown that only a subset of these proteins are
induced by safeners in root and suspension cultures, notably
the xenobiotic-conjugating phi (F) GSTF8 and tau (U) GSTU19
and GSTU24 enzymes (10–12, 14). These GSTs are also known
Plants have a remarkable ability to elicit selective signaling
pathways after exposure to low molecular weight natural prod-
ucts. Such inducing agents include plant hormones, salicylate
and jasmonate derivatives, allelochemicals, and endogenous
* This work was supported by the Biotechnology and Biological Sciences
Research Council Grant BBD0056201 and a Biotechnology and Biological
Sciences Research Council research development fellowship (to R. E.).
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Tables 1–6 and Figs. 1 and 2.
The microarray data have been deposited at the NCBI gene expression and
hybridization array data repository, GEO accession number GSE28431.
1 These authors contributed equally to this work.
2 Present address: Croda Europe, Crop Care, Cowick Hall, Snaith, East York-
shire, DN14 9AA, UK.
3 Present address: Centre for Novel Agricultural Products, Department of Biol-
ogy, University of York, York, GL10 5YW, UK.
5 The abbreviations used are: XR, xenobiotic response; RXR, rapid XR; SXR,
slow XR; BSO, l-buthionine sulfoximine; CDNB, 1-chloro-2,4-dinitroben-
zene; CMPP, 4-chloro-6-methyl-2-phenylpyrimidine; CYP, cytochrome
P450 oxidoreductase; OPDA, 12-oxo-phytodienoic acid; Ph, phenyl.
4 To whom correspondence should be addressed: Food and Environment
Research Agency, Sand Hutton, York, YO41 1LZ, UK. Tel.: 44-1904-462415;
Fax: 44-1904-462111; E-mail: robert.edwards@fera.gsi.gov.uk.
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Xenobiotic Responsiveness in Arabidopsis
to be induced by a range of other chemical treatments (15), (Sigma). Total RNA was further purified using the RNeasy Midi
including exposure to natural product allelochemicals (5), kit (Qiagen, Crawley, West Sussex, UK) before submission to
xenobiotic pollutants (10), and copper salts (14). Similar the NASC Microarray service for probing against Affymetrix
responses have also been determined in cereal crops, although ATH1 Arabidopsis genome arrays. The raw microarray data
exposure to these nonspecific toxic chemicals does not result in were normalized to extract comparable probe intensities using
the enhanced herbicide tolerance observed with safeners (5, 6, the RMA algorithm in J-Express 2009 (molmine), giving 15,500
12). This observation suggests that the changes caused by gen- genes called as present. The triplicate results were further ana-
eral xenobiotics in plants must be distinguishable at a signaling lyzed using SAM Version 3.09 Excel plugin (22) to identify tran-
level from those caused by safeners such as fenclorim, which scripts showing statistically significant changes in abundance
cause no discernable phytotoxicity on application.
between treatments. The plugin default ␦ value was used, and
With an interest in investigating the chemical basis of the XR data were filtered for transcripts showing at least a 3-fold
in plants in greater detail, we have studied the selective induc- change in abundance. These cut-offs resulted in a calculated
tion of GSTs in Arabidopsis using a series of fenclorim deriva- expected false discovery rate of 0 in each case.
tives to investigate the chemical features that determine GST
Real-time PCR Analysis—Equal amounts of RNA, isolated
induction. We have then performed global transcriptome stud- from Arabidopsis using TRI-reagent (Sigma), were used to syn-
ies in Arabidopsis with closely related derivatives that elicit dif- thesize cDNA using Moloney murine leukemia virus reverse
ferent XRs to identify the associated changes in gene expression transcriptase (Promega) and an oligo-dT primer. For real-time
and potential metabolic pathways underpinning the different PCR, the primers were designed using Primer 3 so that each
responses. Finally, relating these studies in Arabidopsis back to primer pair spanned an intron, had an optimum annealing tem-
herbicide safening in cereals, we have then tested a subset of the perature of 60 °C, and produced a product sized between 199
fenclorim derivatives that elicit distinct XRs in Arabidopsis for and 219 bp. The housekeeping genes used as controls were
their ability to enhance herbicide tolerance in rice.
GAPDH (At1g13440) and ubiquitin-conjugating enzyme 21
(At5g25760). Real time PCR was performed in a Rotorgene 3000
(Qiagen) using SYBRꢀ Green JumpStartTM Taq ReadyMixTM
EXPERIMENTAL PROCEDURES
Chemicals—Pretilachlor was obtained from Greyhound/ (Sigma). Analysis was carried out using rotor gene 6.0 software by
Chem Service, whereas fenclorim and its derivatives were syn- comparative quantification with expression of the gene of interest
thesized as detailed in the supplemental Methods using pub- normalized against the mean of the housekeeping genes GAPDH
lished methods where appropriate (16–20). In each case, or ubiquitin-conjugating enzyme 21 (both gave similar results).
compound identities were confirmed by mass spectrometry Primer sequences can be found in supplemental Table 1.
(MS) and NMR.
GST Activity Analysis—GST activity toward 1-chloro-2,4-
Chemical Treatments of Plant Tissues—The Arabidopsis dinitrobenzene (CDNB) was determined as described (23).
fad3-2/fad7-2/fad8 triple knock-out line was obtained from Crude plant protein was extracted in 2 vol/wt of 50 mM Tris-Cl,
John Browse (Washington State University). For the cell sus- pH 7.5, 1 mM DTT, and desalted using Zeba desalting columns
pension studies, Arabidopsis Col-0 cultures were grown in the (Pierce) before activity assays.
dark in MS medium and used 5 days after subculturing (21).
RESULTS
Root cultures were grown in the dark in Gamborg’s B5 medium
and used 14 days after initiation (21). For studies with whole
GST Induction in Arabidopsis on Treatment with Xenobiotics—
plants, Arabidopsis was grown in greenhouse conditions, with To first define differences in the XR to chemicals that show
16 h of light using supplementary lighting. Excised rosette safening in cereals with those that are general xenobiotics, Ara-
leaves were then floated on treatment solutions diluted in bidopsis cell cultures were treated with fenclorim and the non-
water. For the studies with cultures and leaves, the inducing specific electrophilic chemical CDNB. Although CDNB is a
chemical treatments were prepared as 100 mM stocks in ace- well known inducer of GSTs in plants, its use does not enhance
tone and added to the medium as a 1:1000 dilution. Control herbicide tolerance (12, 15). To define biomarkers for the XR in
treatments consisted of 0.1% v/v acetone. For thiol depletion Arabidopsis, the cells were assayed for the accumulation of
studies, Arabidopsis root cultures were treated with 1 mM l-bu- GSTF8, GSTU19, and GSTU24 transcripts, which are classi-
thionine sulfoximine (BSO) 5 days before standard chemical cally associated with the XR in this species. In addition, tran-
treatment. For herbicide safening trials, rice seedlings (Oryza scripts encoding the lambda enzyme GSTL1 were included, as
sativa ssp. japonica cv. Nipponbare) were germinated and this gene is also known to be responsive to a wide range of
grown in magenta vessels (Sigma) on 0.3% agar containing pre- chemicals and abiotic stress treatments (15). In all cases tran-
tilachlor (10 ␮M) and safener treatments (1 or 10 ␮M). In each script abundance was determined over the 60-min period
case, the chemical treatments were added to the molten agar in immediately post-treatment (Fig. 1B). After a 20-min treatment
a total volume of 2% v/v acetone. Safening activity was assessed with fenclorim, GSTF8, GSTU19, and GSTU24 transcripts
by measuring the protective effect on shoot and root growth. began to accumulate, whereas their levels were unaffected by
All treatments were performed in biological triplicate and exposure to CDNB over the full 60-min period. GSTL1 tran-
assayed in duplicate.
scripts were not induced by either treatment over the 60-min
RNA Microarray Analysis—Safener compounds were added period even though this gene is known to be induced by chem-
to Arabidopsis root cultures for 4 and 24 h periods before RNA icals over longer exposure periods (15). This simple experiment
extraction from biological triplicate samples using Tri reagent demonstrated that the xenobiotic responses could be divided
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Xenobiotic Responsiveness in Arabidopsis
FIGURE1. Theglutathionylationandxenomeinducingactivityoffenclorimin Arabidopsis. A, onenteringplantcellsthesafenerundergoesGST-mediated
glutathionylation. B, shown is induction of GSTU19, GSTU24, GSTF8, and GSTL1 transcripts by fenclorim and CDNB in Arabidopsis cell suspension cultures (f,
fenclorim; Œ, CDNB; ꢁ, acetone) over 60 min post-treatment. C, the induction of GSTU19 and GSTU24 was also determined in different Arabidopsis plant tissues
after 60 min along with GST activity toward CDNB 24 h post-treatment.
into rapid and slow XR types as invoked by fenclorim and tions at the 2-position (4–7, 19) introducing steric variation.
CDNB, respectively. The induction of GSTU19 and GSTU24 The core structure was also modified through the generation of
mRNAs by fenclorim was then monitored in other Arabidopsis isosteric analogues such as phenyl pyridine (20) and biphenyls
tissues, namely root cultures and plant leaves (Fig. 1C). In all (21 and 22) in which the pattern of electrophilic centers and
cases induction of the GST genes was observed, confirming that steric bulk are retained but with differing reactivities and bind-
all these Arabidopsis tissue types were responsive to the ing requirements (Table 1). This series also encompassed a
safener. The induction observed in the suspension and root known metabolite of fenclorim (12) formed in Arabidopsis and
cultures was greater than that determined in the intact leaves. rice plants treated with the safener in vivo, which was known to
Based on previous metabolism studies, it appeared most likely be an inducer of GSTs and to undergo glutathionylation despite
that this difference in response was due to the very rapid uptake having a reduced electrophilicity compared with the parent
of the safener in plant cultures as compared with the foliage safener (20). Each compound was tested for its ability to
(20).
increase GSTU19 and GSTU24 transcript abundance over 60
Screening of a Fenclorim Analog Series for Their Associated min and enhance GST enzyme activity toward CDNB over a
XRs in Arabidopsis—Having established the relative rates of 24-h period. The latter screen was included as a classic measure
enhancement of GSTU19 and GSTU24 transcripts induction as of the XR in Arabidopsis and other plants (10, 12, 15, 20, 24).
a biomarker of the type of XR, a series of fenclorim derivatives
This screen confirmed that the xenobiotic responses in Ara-
were prepared and tested for their ability to induce GST tran- bidopsis could be divided into a slow (S) SXR and a rapid (R)
scripts in suspension cultures. The synthetic strategy adopted RXR. In the SXR invoked by compounds 5-15, 17, 19, 20, and
to generate the fenclorim series was based on varying the reac- 22, GST enzyme activity was significantly enhanced over 24 h,
tivity and molecular size of the safener (Table 1). Thus, the without a rapid (within 60 min) induction of the respective
electrophilicity of the substituted pyrimidine ring was altered transcripts. In the RXR shown with compounds 1–4, 16, and
by varying potential leaving groups (1–3, 8–17), with substitu- 18, the enzyme enhancement at 24 h was associated with rapid
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Xenobiotic Responsiveness in Arabidopsis
TABLE 1
classic RXR inducer, whereas 4-chloro-6-methyl-2-phenylpy-
rimidine (CMPP; 10) was chosen as a very closely related deriv-
ative of the parent safener that did not induce an RXR. After
treatment with the chemicals, Arabidopsis cell cultures were
harvested at 4 and 24 h for transcriptome analysis. These time
points were chosen to distinguish between the RXR and SXR
but also to allow enough time at the early time point to allow
both sensitive detection of early events and at 24 h to allow
secondary events to be monitored.
A series of fenclorim derivatives was prepared and assayed for the
ability to induce GSTs in suspension cultures of Arabidopsis
Each compound was administered at 100 ␮M, and the -fold enhancement in GST-
mediated conjugation of CDNB was determined after 24 h as compared with the
enzyme activities determined in acetone-treated cultures of Arabidopsis (1.11 Ϯ
0.10 nanotiatals mgϪ1). As compared with controls, the induction of GST activity
for Arabidopsis cell cultures treated with 100 ␮M CDNB was measured at 1.34 Ϯ
0.18 nanotiatals mgϪ1. In Arabidopsis the induction of GSTU19 and GSTU24 tran-
scripts was measured over 60 min by quantitative PCR using ubiquitin-conjugating
enzyme 24 as a control. Results shown are the means of three determinations Ϯ S.D.
Comparing the microarray data for responses 4 h after treat-
ment with fenclorim or CMPP, it was clear that as compared
with the controls, both chemical treatments had a marked
effect on transcription. In each case, large numbers of mRNAs
were induced, some very strongly, whereas a smaller number of
transcripts were down-regulated. Comparison of the two treat-
ments (Fig. 2) showed a strong positive linear correlation (R2 ϭ
0.73) between the two responses with respect to the entire tran-
scriptome. Overall, fenclorim appeared a slightly better modu-
lator of transcript abundance than CMPP, giving on average a
10% higher response. Treatments with both fenclorim and
CMPP rapidly and strongly induced many genes involved in
xenobiotic detoxification including GSTs, glucosyltransferases,
CYPs, and ATP binding cassettes (Fig. 2; supplemental Tables
2–4). In particular, GSTU24 transcripts were up-regulated
56-fold by CMPP and 168-fold by fenclorim in the microarrays,
confirming the relative strength of the inductions observed in
quantitative PCR studies. After the 4-h treatment of the 100
genes most strongly up-regulated by fenclorim, 6 were GSTs, 12
were glucosyltransferases, 7 were CYPs, 14 were transporters,
transcriptional GST activation. Compound 21 did not induce and 15 were redox-catalyzing enzymes, with these xenome
any response and, therefore, exhibited neither a SXR nor a RXR. components representing half of the most responsive tran-
The results suggested that the observed differences in the XRs scripts. In contrast, a large number of cell wall-modifying gene
invoked were dependent on both the electrophilicity and the transcripts were down-regulated by both fenclorim and CMPP
size of the derivative. Based on the SXR determined on treat- treatment, indicating a rapid reduction in the expression of
ment with compounds 8–11, it was concluded that two good genes associated with cell growth. In addition to the common-
leaving groups are required to induce the RXR. Moreover, to ality of responses to the two chemical treatments, it was also
elicit an RXR, the pyrimidine ring had to be sufficiently electron clear that a subset of genes showed a strong deviation from this
deficient, as shown by the activity of 1–4, 16, and 18 as com- correlation, and these differences were studied in more detail.
pared with 20 and 22. In addition, evidence for a steric require- Statistical analysis of the 4-h microarray data were used to
ment could be identified, in that modulating the bulk of the extract transcripts showing significant modulation by (a) fen-
2-phenyl substituent (5–7 and 19) resulted in the modified fen- clorim versus carrier, (b) CMPP versus carrier, and (c) fenclorim
clorim losing RXR activity, suggesting a need for more specific versus CMPP (supplemental Tables 2–4). A number of genes
binding requirements in safener recognition. This notion was specific for the RXR induced by fenclorim were linked to a
reinforced through the comparison of compounds 1 and 18, in sulfur starvation response (25). These included thioglucosi-
which the relative position of the nitrogen atom between Cl and dase, a high affinity sulfate transporter, storage proteins,
Ph-substituted carbons was conserved, whereas varying the 5Ј-adenylyl phosphosulfate reductase, sulfite reductase, and
second, ring-activating, nitrogen atom did not cause any signif- 5-adenylyl sulfate reductase. Similarly, phenolic secondary
icant loss in RXR activity. Finally, although the pattern of elec- metabolism appeared to be differentially up-regulated by fen-
trophilic sites, leaving groups, and C-2 substituent size is main- clorim as compared with CMPP, with multiple genes encoding
tained in 21, this compound was ineffective in promoting any phenylalanine ammonium lyase, 4-coumarate-CoA ligase,
XR.
caffeoyl-CoA methyltransferase, N-hydroxycinnamoyl benzo-
Microarray Study with Compounds Invoking Rapid and Slow yltransferase, and a range of CYPs all induced. These RXR-
Xenobiotic Responses—Having identified two classes of specific transcriptional responses closely match the changes in
response, a more detailed study of the differences between RXR metabolism observed during safening in cereals, which include
and SXR was performed. A microarray study was carried out to increased levels of glutathione (GSH) and flavonoids (26–28).
determine whether compounds invoking an RXR in Arabidop-
After 24 h of treatment, it was anticipated that the SXR would
sis induced different subsets of genes to closely related chemi- be fully deployed and effectively more closely match the RXR.
cals that only caused an SXR. Fenclorim (1) was selected as the However, compared with the 4-h results, the transcriptome
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Xenobiotic Responsiveness in Arabidopsis
FIGURE 2. Cluster plot showing the correlation between -fold induction over solvent control of transcripts after 4 h of treatment with either fenclorim
or CMPP, derived from averages of triplicate microarray analyses. Each microarray probe is represented by a black point, whereas genes associated with
xenobiotic detoxification (GSTs, glucosyltransferases, CYPs, and the ATP binding cassette transporter PDR12) are shown as red dots with the most highly
induced transcripts labeled. CMPP and fenclorim responses are highly correlated, and both induce substantial changes in the transcriptome, with xenobiotic
detoxifying genes overrepresented among the most highly induced transcripts.
responses to fenclorim and CMPP were more divergent after
24 h. Although a positive linear correlation remained between
the two treatments, it was much reduced (R2 ϭ 0.52). Many of
the genes that were strongly up-regulated by both treatments at
4 h had declined significantly in abundance in the fenclorim-
treated cells by 24 h. Of the 200 genes most highly up-regulated
after 4 h by both treatments, by 24-h transcript abundance was
decreased by 59% in the cultures exposed to fenclorim, whereas
for CMPP this decrease was only 18% (Fig. 3). Thus, not only
were the RXR transcripts more rapidly induced by fenclorim,
but their induction was also more transient as compared with
the SXR invoked by CMPP.
The XR and Glutathione Metabolism—Reasoning that the
XR in plants must be related to “natural” abiotic or biotic stress
FIGURE 3. Box and whisker plot showing transcript abundance changes
at 4 and 24 h for the 200 transcripts most highly induced by both fenclo-
rim and CMPP after 4 h. The median line is shown, with boxes indicating the
25th and 75th percentiles, and whiskers indicating the total range of changes.
Although both chemicals caused a major enhancement of these transcripts, this
induction was much more transient in the case of fenclorim (Fen) treatment.
responses, the microarray data were interrogated for patterns
of response that would suggest coordinated biochemical
changes associated with defense. The clear link between the
RXR response and activation of sulfur-starvation pathways
coupled with the known glutathione-mediated metabolism of
fenclorim (20) suggested that changes in GSH metabolism CDNB that caused the greater loss only elicited an SXR (Fig.
could play an active role in the safener response. Similarly, 1B). To further probe the link between GSH content and the
changes in GSH content and the relative abundance of its oxi- XR, the selective glutathione synthetase inhibitor BSO was used
dized disulfide derivative GSSG have been found to be involved to deplete the thiol pool in Arabidopsis root cultures (Table 2).
in redox stress signaling in a number of plants (29). Both CDNB The BSO treatment resulted in a reduction of GSH content to
and fenclorim are known to be rapidly glutathionylated when barely detectable levels and caused a basal elevation in GSTU19
fed to Arabidopsis cells and, therefore, have the potential to and GSTU24 transcripts in all cultures examined. When the
elicit their XR through a rapid perturbation of thiol homeosta- GSH-depleted cells were treated with CDNB or fenclorim, sim-
sis (12, 15). To determine the effect of adding these xenobiotics, ilar XRs were determined with either chemical treatment. Low-
the GSH pool was determined 60 min after dosing Arabidopsis ering the GSH content, therefore, appeared to abolish the dif-
root cultures with either 100 ␮M fenclorim or 100 ␮M CDNB. ferential sensitivity of the cells to fenclorim as compared with
These studies showed that whereas fenclorim reduced GSH CDNB observed in the earlier studies (Fig. 1B), showing the
content by 40%, CDNB treatment led to a 93% depletion in the availability of the thiol modulated the XR.
thiol (Table 2). From this result it was concluded that although
The RXR and Oxylipin Signaling—Comparative analysis of
GSH depletion was a common feature in both responses, the the microarray data with other transcriptome experiments
RXR could not be explained by the scale of the perturbation, as suggested a strong similarity in the XRs promoted by both
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Xenobiotic Responsiveness in Arabidopsis
TABLE 2
The effect of depleting cellular glutathione on GSTU19 and GSTU24 induction in Arabidopsis root cultures
Glutathione content was determined after a 60-min exposure to 100 ␮M fenclorim or CDNB after a 5-day pretreatment with (ϩ) or without (Ϫ) the glutathione synthesis
inhibitor BSO. Results shown are the means of three determinations Ϯ S.D.
GSTU19 relative
GSTU24 relative
Treatment
؎BSO
GSH
nmol g
transcript abundance
transcript abundance
Ϫ1
Control
Fenclorim
CDNB
Ϫ
ϩ
12.4 Ϯ 1.9
1.0 Ϯ 0.2
5.0 Ϯ 0.6
1.0 Ϯ 0.4
0.2 Ϯ 0.0
15.5 Ϯ 0.7
Ϫ
ϩ
7.4 Ϯ 0.7
0.1 Ϯ 0.1
5.3 Ϯ 1.1
23.7 Ϯ 3.1
9.5 Ϯ 0.7
2.5 Ϯ 0.7
7.3 Ϯ 1.9
33.2 Ϯ 7.7
Ϫ
ϩ
0.8 Ϯ 0.6
8.0 Ϯ 1.1
0.0 Ϯ 0.0
31.5 Ϯ 7.9
fenclorim and CMPP treatments with that determined in
mixotrophic Arabidopsis cell cultures exposed to the phyto-
prostane PPA1 (30). PPA1 and a group of related compounds
termed oxylipins are a group of reactive electrophilic metabo-
lites oxidatively generated from endogenous unsaturated fatty
acids during plant wounding (30). Comparison of microarray
data for fenclorim and CMPP treatments with that for a 4-h
treatment of cell cultures with the phytoprostane (30) showed a
highly significant overlap between the responses to these treat-
ments. For example, a global comparison of gene induction by
fenclorim compared with gene induction by PPA1 (supplemen-
tal Fig. 2) showed a strong positive correlation (r ϭ 0.49), with a
p Ͻ 0.0001, being a correlation due solely to chance. Of the 50
most PPA1-inducible genes, 27 of these were also induced at
TABLE 3
Effect of linolenic acid content in Arabidopsis root cultures on RXR, as
measured by induction of the GST transcripts GSTU19 and GSTU24 1 h
after treatment with either fenclorim (RXR inducer) or acetone
(control)
Wild-type plants have high levels of linolenic acid, whereas fad3-2/fad7-2/fad8
mutants accumulate linoleic acid instead of linolenic acid. Results shown are the
means of three determinations Ϯ S.D.
Relative transcript abundance
Plant
Wild type
Treatment
GSTU19
GSTU24
Acetone
1.0 Ϯ 0.3
1.0 Ϯ 0.7
Fenclorim
5.7 Ϯ 1.3
24.2 Ϯ 7.8
fad3-2/fad7-2/fad8
Acetone
1.2 Ϯ 0.2
1.7 Ϯ 0.7
2.6 Ϯ 0.2
Fenclorim
7.7 Ϯ 2.3
least 2-fold by fenclorim. Conversely, of the 50 most fenclorim- potential for xenobiotic conjugating GSTs to show similar
inducible genes, 39 were induced at least 2-fold by PPA1. detoxifying activities toward oxylipins. As such, GSTs could
Intriguingly, the xenome enzymes CYP710A1, CYP71A12, and share a common function in modulating the availability of elec-
CYP81F2 were strongly differentially induced by fenclorim, trophilic signaling agents of both synthetic and natural origins.
whereas nine heat shock protein transcripts were at least eight- Previous studies have shown that OPDA can be conjugated by
fold more induced by PPA1 as compared with fenclorim. At the Arabidopsis enzymes GSTU6, GSTU10, GSTU17, GSTU19
least some of the differences in gene induction between the two and GSTU25 (33) and GSTF8 (30). By testing further members
treatments will be due to differences in the experimental sys- of the Arabidopsis superfamily, we demonstrated that in total
tems, with the remaining strong similarities in transcriptome 11 GSTs can catalyze the glutathionylation of oxylipins in Ara-
responses pointing to fenclorim and PPA1 inducing very similar bidopsis (supplemental Table 5). Intriguingly, while examining
responses. However, the response to PPA1 in cell cultures had further functional links between fenclorim and oxylipin detox-
already been shown to be similar to the response of Arabidopsis ification, we also demonstrated that the parent safener 1 and
seedlings to treatment either with phytoprostanes or the oxyli- compounds 2, 3, and 4, which all elicit an RXR, lead to an
pin 12-oxo-phytodienoic acid (OPDA) (30), confirming the via- inhibition of the GSTU19-catalyzed conjugation of OPDA
bility of test platforms when comparing plants with cultured (supplemental Table 6).
cells. To further test the potential involvement of oxylipins in
Correlation between the XR in Arabidopsis and Physiological
the XR, root cultures from wild type plants and mutants Safening in Rice—To test whether or not the type of XR in
(fad3Ϫ2/fad7-2/fad8) defective in forming the oxylipin pre- Arabidopsis correlated with safening, the ability of the fenclo-
cursor linolenic acid were treated with either fenclorim or rim series to protect rice from herbicides was determined. Rice
acetone carrier. Mass spectrophotometric analysis (31) of seedlings were germinated on agar containing the chloroacet-
the fatty acid content of wild type and mutant plantlets con- anilide herbicide pretilachlor in the presence and absence of
firmed that unlike wild type plants, the mutant contained no members of the fenclorim chemical series. Two concentrations
detectable linolenic (18:3) or 16:3 fatty acids incorporated of safener were employed corresponding to a low (1 ␮M) and
into lipids, with linoleic (18:2) and 16:2 fatty acids dominat- high (10 ␮M) treatment rate. In each case, herbicidal activity
ing, as expected (32). This mutant line is, therefore, unable to was assessed by determining root and shoot elongation relative
synthesize OPDA but could form other dienoic acid-derived to untreated controls (supplemental Fig. 1). When exposed to
oxylipins. On treatment with fenclorim, the suppression in 10 ␮M pretilachlor, rice seedling growth was strongly arrested,
induction of GSTs showed that the RXR was markedly with this effect largely reversed in the presence of 1 ␮M fenclo-
depressed in the fad3-2/fad7-2/fad8 plants (Table 3), con- rim (Fig. 4). A strong protective effect at this lower concentra-
sistent with a link between the RXR response and endoge- tion (1 ␮M) of safener was also observed with the dibromo- (2)
nous oxylipin signaling.
and difluoro- (3) fenclorim derivatives. Only at the higher con-
In a further examination of the link between safener- and centration of 10 ␮M did compounds 4, 9, 12 (Fig. 4), and 18 also
oxylipin-mediated signaling and metabolism, we also tested the gave significant protection, whereas10 and 16 were inactive.
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Xenobiotic Responsiveness in Arabidopsis
chemistry required in the generation of the probe resulting in
the loss of the RXR. Similarly, other attempts to identify plant
proteins that bind to safeners have proved inconclusive. In
studies with the radiolabeled safener dichlormid (N,N-diallyl-
2,2-dichloroacetamide), an apparently selective binding inter-
action to a methyltransferase of unknown function was deter-
mined in maize seedlings (8). However, the functional
significance of this binding to eliciting safening in maize was
not determined.
FIGURE 4. Safening activity of fenclorim in rice. When exposed to the her-
bicide pretilachlor, the normal stunting of growth of rice seedlings was ame-
liorated by applications of safener-active compounds such as fenclorim (1),
whereas closely related compounds such as 12 fail to protect the plants. Lane
1, acetone control; lane 2, 10 ␮M pretilachlor; lane 3, 1 ␮M safener and 10 ␮M
pretilachlor; lane 4, 1 ␮M safener. A, safener treatment fenclorim (1). B, safener
treatment 4-chloro-6-(methylthio)-2-phenylpyrimidine (12).
In addition to the differential speed at which GST transcripts
were induced in the RXR and SXR, the studies with fenclorim
and CMPP also demonstrated that these two closely related
compounds elicited subtly different effects on the Arabidopsis
transcriptome. Much of this difference was observed in the
kinetics of the respective responses. This highlights the impor-
tance of the timing of sampling in xenobiotic treatment studies,
with our results suggesting that assaying at either a single time
point or at time points much later than 4 h after treatment
would overlook the differences in gene expression induced by a
safener as compared with those determined by chemicals
inducing an SXR. Overall, the profile of transcripts induced by
fenclorim showed considerable overlap with those induced by
These experiments showed a good correlation between an RXR
response in Arabidopsis and safening in rice, with five of the
RXR-activating compounds also having safening activity in
rice. However, the correlation between the responses in the two
plant species was not perfect, as two compounds (9, 12) that
showed some safening activity in the cereal only invoked an
SXR in Arabidopsis.
DISCUSSION
Our results demonstrate that plants can respond to xenobi- other electrophilic chemicals, such as the toxic allelochemical
otics by either eliciting a rapid, or slow, XR. In the case of fen- benzoxazolin-2(3H)-one (5), or the reactive B1-type phytopros-
clorim, the ability to induce an RXR was found to be surpris- tanes, such as PPA1, released on plant wounding (40). Of the 50
ingly sensitive to changes in both the electrophilicity of the most benzoxazolin-2(3H)-one-inducible genes, 44 were also
pyrimidine ring (suggestive of an ability to selectively alkylate induced by at least 2-fold by fenclorim and 34 at least 2-fold by
soft nucleophilic groups such as cysteinyl residues) and to PPA1. Collectively these results suggest that different electro-
minor variations in the phenyl ring substituent. Such structure philes lead to essentially similar transcriptional modulation but
activity relationships are typically demonstrated in protein- over different timescales. We postulate that general reactive
based recognition systems. In terms of xenobiotic recognition, electrophiles, such as CDNB, elicit an SXR after causing cellular
such systems are not unprecedented in mammals and fungi, toxicity due to the alkylation of sensitive proteins and DNA.
with the receptor protein releasing transcription factors after The resulting disruption in metabolism then leads to the release
selective alkylation with electrophiles. For example, in mam- of reactive endogenous signaling molecules, which on selective
mals the keap1 protein sequesters the transcription factor Nrf2 recognition, activate a protective XR. In contrast, electrophiles,
in an inactive form in the cytoplasm (34). In the presence of including safeners, which mimic these endogenous stress sig-
stress stimuli such as electrophilic agents, cysteinyl residues on naling molecules, directly activate this protective receptor sys-
keap1 that interact with Nrf2 are modified, leading to a reduc- tem and induce a relatively large but short-lived signaling
tion in binding affinity and the release of the transcription fac- response that activates cellular defenses before the xenobiotic
tor. Nrf2 then translocates to the nucleus, where it binds to causing extensive damage. Intriguingly, the chemical depletion
regulatory antioxidant responsive elements, which lead to the of GSH content in the Arabidopsis cells led to an enhanced
transcription of genes encoding phase II detoxifying enzymes sensitivity in the XR to both CDNB and fenclorim treatments.
and antioxidant stress proteins (35). To date, no ortholog of the This raises the possibility that under normal conditions GSH
keap1 receptor protein has been identified in plants. Alterna- affords protection to electrophile-sensitive protein thiols,
tively, several enzymes involved in signaling events are also thereby preventing alkylation by CDNB, but that these groups
known to have their activity regulated by selective alkylation. remain sensitive to fenclorim modification, perhaps through a
For example, the active site cysteinyl residues of phosphatases selective association of the safener at a ligand binding site prox-
involved in the regulation of protein phosphorylation can be imal to key cysteinyl residues.
inactivated by alkylation (36). Such selective enzyme inhibition
Following the hypothesis that safeners elicit endogenous
by xenobiotics has also been demonstrated in plants with stress signaling pathways, it was of interest that the XR of Ara-
enzymes of primary metabolism, including S-formylgluta- bidopsis was sensitive to perturbations in fatty acid desatura-
thione hydrolase (37) and ribulose-bisphosphate carboxylase/ tion (Table 3). As treatment of Arabidopsis with OPDA or phy-
oxygenase (38). In the current study we attempted to identify a toprostanes gives a response very similar to that observed with
fenclorim-binding protein using “click”-based approaches fenclorim (41), we speculate that the safener must be interact-
using the safener derivatized with an azido function (39). These ing with the signaling invoked by oxylipins, with these metab-
studies were unable to demonstrate any specific binding, olites in turn derived from unsaturated fatty acids. Whereas
although as our structure activity studies evolved it became reactive oxylipin derivatives such as OPDA are derived from
clear that this was due to the minor modification in fenclorim linolenic acid by the concerted action of lipoxygenases, allene
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Xenobiotic Responsiveness in Arabidopsis
oxide synthases, and allene oxide cyclase (42), the phytopro- between the RXR and agronomically useful herbicide safening
stanes are derived from the spontaneous oxidation of linolenic from SXR and the more commonly encountered general xeno-
acid and to a lesser extent linoleic acid (43). Many of these biotic response.
signaling-active derivatives are unstable and will react with
Acknowledgment—We are grateful to Lesley Edwards for technical
assistance.
GSH and potentially protein-sulfhydryl groups over time (41).
The fact that a reduction in unsaturated fatty acids attenuates
the RXR invoked by fenclorim in Arabidopsis suggests that the
safener must act either in parallel or upstream of oxylipin sig-
naling, potentially through regulating the availability of these
endogenous molecules. However, to date we have been unable
to determine any major effects of fenclorim on OPDA or phy-
toprostane metabolism in planta, suggesting that this safener-
mediated regulation involves a minor subset of these com-
pounds, or operates in a restricted spatiotemporal manner.
Although a link between safening with oxylipin signaling has
been proposed (7), a causative unifying mechanism of action is
yet to be determined. Based on the observations from the cur-
rent study, we can propose three potential mechanisms
whereby xenobiotics could interact with endogenous stress rec-
ognition pathways to elicit an RXR. First, fenclorim or a rapidly
formed downstream metabolite could selectively bind and acti-
vate a signaling protein that normally binds to and thus senses
oxylipins, in both cases presumably through modification of a
reactive cysteine residue. Recent studies have shown that oxy-
lipins selectively alkylate and modify the function of a number
of redox-sensitive cysteinyl-bearing proteins, some of which
are implicated in signaling (41). Second, fenclorim (or a down-
stream metabolite) could activate a minor release of oxylipins
leading to signal elicitation. A candidate enzyme for such bio-
activation would be a lipase with many plants including Arabi-
dopsis, accumulating relatively large amounts of esterified
OPDA and phytoprostanes in lipids (44). If the safeners were to
cause a selective release of such preformed stores through the
up-regulation of hydrolytic enzymes, this would potentially
lead to an RXR. Third, fenclorim (or a downstream metabolite)
could prevent the metabolic deactivation of oxylipins, leading
to their transient accumulation and resulting signal initiation.
The widespread ability of GSTs to catalyze oxylipin glutathio-
nylation (supplemental Table 5) coupled with their consistent
sensitivity to inhibition by glutathionylated fenclorim and cer-
tain related compounds (supplemental Table 6) suggests that
fenclorim treatment could transiently increase free oxylipin
levels through inhibition of their enzyme-mediated glutathio-
nylation. However, inconsistencies in the inhibitory versus
safening activity of other compounds in the series do not sup-
port a simple link between interfering with GST activity and
disrupting oxylipin metabolism (supplemental Table 6). Fur-
ther studies are now required to establish how safeners inter-
cede in oxylipin turnover and signaling. In view of the lack of
discernable disruption in total oxylipin content on safening,
one promising area may be to study the effect of fenclorim on
the intracellular disposition of these endogenous signals
between the cytosol, vacuole, and peroxisomes as mediated by
ATP binding cassette transporters (45, 46). Although our stud-
ies raise additional questions as to how xenobiotics can selec-
tively intercede in intracellular stress signaling pathways, the
use of the fenclorim derivatives in defining structure activity
relationships clearly demonstrates the subtle distinctions
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