Identity and Activity of 2,4-Dichlorophenoxyacetic Acid Metabolites in Wild Radish (Raphanus raphanistrum)

Article abstract of DOI:10.1021/acs.jafc.8b05300

Synthetic auxin herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D), are widely used for selective control of broadleaf weeds in cereals and transgenic crops. Although the troublesome weed wild radish (Raphanus raphanistrum) has developed resistance to 2,4-D, no populations have yet displayed an enhanced capacity for metabolic detoxification of the herbicide, with both susceptible and resistant wild radish plants readily metabolizing 2,4-D. Using mass spectrometry and nuclear magnetic resonance, the major 2,4-D metabolite was identified as the glucose ester, and its structure was confirmed by synthesis. As expected, both the endogenous and synthetic compounds retained auxin activity in a bioassay. The lack of detectable 2,4-D hydroxylation in wild radish and the lability of the glucose ester suggest that metabolic 2,4-D resistance is unlikely to develop in this species.

Full text of DOI:10.1021/acs.jafc.8b05300

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Agricultural and Environmental Chemistry
Identity and activity of 2,4-D metabolites
in wild radish (Raphanus raphanistrum)
Danica Erin Goggin, Gareth L. Nealon, Gregory Cawthray, Adrian
Scaffidi, Mark Howard, Stephen Powles, and Gavin R. Flematti
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05300 • Publication Date (Web): 05 Dec 2018
Downloaded from http://pubs.acs.org on December 5, 2018
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Identity and activity of 2,4-D metabolites in wild radish (Raphanusraphanistrum)
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Danica E Goggin1*, Gareth L Nealon2, Gregory R Cawthray3, Adrian Scaffidi4, Mark J
Howard2,4,5, Stephen B Powles1 and Gavin R Flematti4
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1Australian Herbicide Resistance Initiative, School of Agriculture and Environment,
University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009,
Australia
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2Centre for Microscopy, Characterisation and Analysis, University of Western Australia, 35
Stirling Highway, Crawley, Western Australia 6009, Australia
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3School of Biological Sciences, University of Western Australia, 35 Stirling Highway,
Crawley, Western Australia 6009, Australia
4School of Molecular Sciences, University of Western Australia, 35 Stirling Highway,
Crawley, Western Australia 6009, Australia
5School of Chemistry,University of Leeds, Leeds, LS2 9JT, United Kingdom
*Corresponding author:
Phone +61 8 6488 1512
Email danica.goggin@uwa.edu.au
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ABSTRACT:Synthetic auxin herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) are
widely used for selective control of broadleaf weeds in cereals and transgenic crops.
Although thetroublesome weed wild radish (Raphanusraphanistrum) has developed
resistance to 2,4-D, no populations have yet displayed an enhanced capacity for metabolic
detoxification of the herbicide, with both susceptible and resistant wild radish plants readily
metabolizing 2,4-D. Using mass spectrometry and NMR, the major 2,4-D metabolite was
identified as the glucose ester, and its structure confirmed by synthesis. As expected, both the
endogenous and synthetic compounds retained auxin activity in a bioassay. The lack of
detectable 2,4-D hydroxylation in wild radish, and the lability of the glucose ester, suggest
that metabolic 2,4-D resistance is unlikely to develop in this species.
KEYWORDS:2,4-dichlorophenoxyacetic acid; auxin; herbicide resistance; metabolism; wild
radish (Raphanusraphanistrum)
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1. INTRODUCTION
The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), which mimics the action of the
key plant hormone indole-3-acetic acid,was discoveredover 70 years ago and was quickly
adopted as a herbicide, a promotor of plant growth in tissue culture and an inhibitor of pre-
harvest fruit drop.1 Although the use of 2,4-D as a herbicide in the United Stateshas declined
from 18 million kg per year in the 1950s to its current level of around 1 million kgper year, it
remains an essential component of selective dicotweed control in crops, turf areas and
roadsidesworldwide.2The basis of selectivity of the synthetic auxins is mainly attributed to
the greater capacity of monocotyledonous plants to metabolize these herbicides to inactive
polar conjugates, but there is evidence that enhanced metabolism may not entirely account for
the high levels of resistance seen in most grass species.3,4
The major pathways of 2,4-D metabolism in several commercially important cereal and
legume species were identified in the 1970s as (1) direct conjugation of glucose or amino
acids (predominantly aspartate and glutamate) to the carboxyl group of the 2,4-D molecule
via ester oramide linkages; and (2) hydroxylation of the phenol ring, followed by conjugation
of a sugar (usually unidentified) through an ether linkage.5-14These conclusions were largely
based on co-chromatography, whereradiolabeled metabolites extracted from [14C]-2,4-D-
treated plants were chemically or enzymatically hydrolyzed and the retention times/Rf values
of the hydrolysisproducts were compared with authentic standards.More recent studies have
used liquid chromatography-mass spectrometry (LC-MS) to confirm the presence of 2,4-D
amino acid conjugates15 and some sugar conjugates of 2,4-dichlorophenol16in crude plant
extracts.
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The 2,4-D glucose ester (1-[(2,4-dichlorophenoxy)acetate]-β-D-glucopyranose) and most 2,4-
D amino acid conjugates are readily cleaved back to the parent molecule by endogenous plant
hydrolases and thus display auxin activity,9,15,17whilst ring-attached 2,4-D glycosides and
their respective aglycones (predominantly 4-hydroxy-2,5-D and 4-hydroxy-2,3-D) are
generally inactive.8Less commonly, 2,4-D has been found incorporated into larger inactive
and/or insoluble molecules such as 3-(2,4-dichlorophenoxy)propionic acid,18
triacylglycerols,19as well as lignin and oligopeptides,6 presumably as a mechanism to
sequester 2,4-D in a low-toxicity form.
Wild radish (Raphanusraphanistrum), the major dicotyledonous weed of Western Australian
cropping systems, is displaying increasing levels of resistance to 2,4-D.20 In previous work,
13 populations were studied which showed no evidence of differential metabolism between
susceptible and resistant plants.21,22 However, in this work, approximately 50% of applied
2,4-D was converted to compounds which did not match known metabolites, and thus remain
unidentified. As growers in Australia intensify their use of 2,4-D in response to resistance to
other herbicides,20 the increased selection pressure could potentially result in the appearance
of populations with a greater capacity to detoxify 2,4-D, as has occurred in corn poppy23 and
nodding thistle.24Therefore, in this study we investigated the identity and herbicidal activity
of the major 2,4-D metabolites produced in wild radish.
2. MATERIALS AND METHODS
2.1 Chemicals and instrumentation. Technical-grade 2,4-D acid was a gift from Nufarm
(Laverton, Australia). Ring 14C [U]-2,4-D (specific activity 2.035 GBq mmol-1) was obtained
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from American Radiolabeled Chemicals(St Louis, Missouri), and ring C [U]-2,4-D from
Cambridge Isotope Laboratories (Tewksbury, Massachusetts). All other chemicals were from
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Sigma-Aldrich (Sydney, Australia), and solvents for extractions, synthesis and purifications
were of high performance liquid chromatography (HPLC) grade, unless otherwise stated.
HPLC was performed on an Agilent 1200 HPLC system equipped with a photodiode array
detector and fraction collector. Plant extracts were separatedon a reversed phase Grace
Apollo C18 HPLC column (250 mm long, 10 mm i.d., 5 μm particle size; Grace-Davison
Discovery Sciences, Columbia, Maryland) with a 33 mm x 7 mm guard column of the same
material.An in-line -RAM detector (IN/US Systems Inc, Pine Brook, New Jersey) was used
to detect radioactivity in eluents from [14C]-2,4-D-containing extracts. HPLC-MSand high-
resolution mass spectrometry (HRMS) were conducted using a Waters Alliance e2695 HPLC
connected to a Waters LCT Premier XE time-of-flight (TOF) mass spectrometer with an
electrospray ionization source (ESI) and direct injection valve. Ionization conditions were
optimized using unlabeled 2,4-D in negative ionization mode. Cone and desolvation gas
flows were set to 150 and 650 L h-1, respectively. The capillary voltage was set at 3 kV, cone
voltage at 20 V with the source temperature at 80°C, and the desolvation temperature at
350°C. HRMS calibration was achieved using leucine encephalin at 2 ng uL-1. HPLC-MS
separations were achieved using a reversed phaseGrace AlltimaC18 HPLC column (250 mm
long, 2.1 mm i.d., 5 μm particle size; Grace-Davison Discovery Sciences, Columbia,
Maryland) with a 7.5 mm x 2.1 mm guard column of the same material. NMR spectra were
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acquired at 298 K on a Bruker AvanceIIIHD11.4 Tspectrometer (1H at 500.10 MHz, C at
125.75 MHz)equipped with a 5 mm BBFO probe, or on a Bruker Avance IIIHD14.1 T
spectrometer (1H at 600.13 MHz, 13C at 150.90 MHz) using a 1.7 mm TXI microprobe
(Bruker, Rheinstetten, Germany), with either deuteromethanol or deuteroacetoneas solvent.
Chemical shifts are reported in ppm relative to the residual solvent signals (δ 3.31 and 2.05
ppm for deuteromethanol and deuteroacetone respectively).
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2.2 Plant material. Five of the wild radish (RaphanusraphanistrumL.; Brassicaceae)
populations (R1, R2, R3, R6 and R8)characterized in previous work,22 which showed no
differences in 2,4-D metabolism and retained most of the applied 2,4-D in the treated leaf,
making for simpler processing of samples, were bulked together for this study. Seedlings
were grown in potting mix (composted pine bark:riversand:peat moss, 2:1:1) and kept in a
growth cabinet under a 12 h photoperiod of 200 µmol m-2 s-1white LED and incandescent
light at day/night temperatures of 20/15°C. Plants were watered daily, and fertilized weekly
with 1.5 g L-1commercial soluble fertilizer (Diamond Red: N 27%; P 5.7%; K 10.9% plus
trace elements; Campbells Fertilisers, Laverton North, Australia). When seedlings had
reached the 3-leaf stage, 2,4-Dwas applied as a 5 mM solution in 0.1% (v/v) Tween 20, with
ten droplets of 1 µL placed on the first two leaves of each seedling. Plants were then returned
to the growth cabinet and leaf tissue was harvested after 96 h. In order to identify HPLC
fractions containing 2,4-D or its metabolites, and to pinpoint MS fragments originating from
2,4-D, pilot studies were performed with (1) plants treated with 5 mMunlabeled 2,4-D plus 3
kBq per leaf of [14C]-2,4-Dfor radio-HPLC analysis,and (2) plants treated with a 1:1 (5 mM
total) mixture ofunlabeled2,4-D and [13C]-2,4-D for HPLC-MS analysis. To account for
potential interfering signals from endogenous plant compounds, plants treated with 0.1%
Tween 20 alone were extracted alongside the [13C]-2,4-D-treated plants. No signals in the m/z
regions of 2,4-D or its metabolites were detected in the control plants (data not shown).
2.3 Sample processing. Treated leaves were excised from seedlings and ground to powder in
liquid nitrogen in a mortar and pestle. The time between excision and freezing was <1 min
for the pilot studies using labeled 2,4-D, and <5 min for the preparative studies (leaves were
kept on ice during this time). The frozen powder was ground further in cold 100% methanol19
and filtered through pre-wetted Miracloth. The residue was extracted three more times in
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fresh methanol and all filtered extracts were combined and clarified by centrifugation (2000 g
for 10 min at 4°C). The extract was concentrated to near-drynessin a rotary evaporator at
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36°C and reconstituted in a small volume of aqueous methanol (1:1, v/v). Recovery of C
from extracts of plants treated with [14C]-2,4-D was >80% as measured by liquid scintillation
counting. Approximately 10 treated leaves per experiment were used for the pilot studies with
labeled 2,4-D, and 80 – 100 treated leaves for each partial purification and subsequent
structural analysis of metabolites from unlabeled 2,4-D.
2.4 Sample fractionation and MS. An extract from [14C]-2,4-D-treated plants was separated
by HPLC using a linear gradient of 10 – 100% (v/v) acetonitrile in water containing 0.1%
(v/v) formic acid over 40 min at 24°C, at a flow rate of 4 mL min-1, and the retention times of
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fractions containing a C signal were noted. Parent 2,4-D eluted at 24.3 min, matching the
retention time of an authentic [14C]-2,4-D standard, along with a major metabolite
(designated metabolite 1) at 18.1 min, a minor metabolite (designated metabolite 2) at 19.5
min, and a second minor metabolite (designated metabolite 3) at 22.3 min (Figure 1).
Synthesised, [14C]-labeled versions of potential metabolites were not used for radio-HPLC.
The experiment was repeated with extracts from plants treated witha1:1 mixture
ofunlabeled2,4-D and [13C]-2,4-D, and 4-mL fractions were collected from the column.
Fractions corresponding to the retention times of parent 2,4-D and metabolites 1, 2 and 3
were evaporated to dryness under a stream of nitrogen, resuspended in a small volume of
acetonitrile, and injected into theHPLC-mass spectrometer operating in both positive and
negative electrospray ionization mode.Separation was achieved using a linear gradient of 10
– 100% (v/v) acetonitrile in water containing 0.1% (v/v) formic acid over 40 min at a flow
rate of 0.3 mL min-1.Mass ions and fragments with characteristic m/z differences of 6 (the
difference between 2,4-D and ring-labeled[13C]-2,4-D) plus the 2 m/z differences caused by
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natural chlorine isotopes, were identified in the mass spectra and high resolution
measurements were conducted.Tentative molecular ions [M-H]-and solvent adducts were
identified containing the isotopic signature and were present at m/z 219 (parent 2,4-D), 427
(formate adduct of metabolite 1), 334 (metabolite 2) and 335(metabolite 3). High resolution
mass spectra(ESI -ve) of metabolite 1as the formate adduct [M+CHOO]- found m/z 427.0189
(C15H17O10Cl2 requires 427.0199). Metabolite 2 [M-H]-gavem/z 333.9870 (C12H10NO6Cl2
requires 333.9885),while metabolite 3 [M-H]- gavem/z 334.9734(closest logical match
suggested C12H9O7Cl2; requires 334.9725).
The major metabolite observed, metabolite 1,was purified from extracts of leaves treated with
unlabeled 2,4-D by C18 silica (50 g, Grace-Vydac reversed phase C18, 40-63 µm, p/n
5134095, Hesperian, California) filtration eluted with 100% water (200 mL), followed by 3:2
methanol:water (200 mL) and methanol (200 mL). The 3:2 methanol:water fraction was
evaporated under reduced pressure and separated usingsemi-preparative HPLCas above. The
fraction containing metabolite 1 eluted at ca. 19 min, as confirmed by direct injection into the
mass spectrometer, and this was purified further on the same column, eluting with 1:1
methanol:water(containing 0.1% formic acid) at 3.5 mL min-1,to provide the purified
compound eluting at 23.5 min (Supporting Information, SI Figure S1). This sample was
subjected to 1Hand 2D NMR analysis.1H NMR (600 MHz, (CD3)2CO) δ 3.36 (dd, J = 9.0, 8.2
Hz, 1H), 3.41-3.45(m, 2H), 3.50 (dd, J = 9.0, 9.0 Hz, 1H), 3.69 (dd, J = 11.9, 4.3 Hz, 1H),
3.81 (dd, J = 11.9, 2.1 Hz, 1H), 4.96 (s, 2H), 5.61 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 8.9 Hz,
1H), 7.31 (dd, J = 8.9, 2.6 Hz, 1H), 7.48 (d, J = 2.6, 1H) (Supporting Information, SI Figure
S2).
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2.5 Synthesis of 1-[(2,4-dichlorophenoxy)acetate]-β-D-glucopyranose. Diethyl
azodicarboxylate (220 μL, 1.4 mmol) was added to glucose (160 mg, 0.9 mmol), 2,4-
dichlorophenoxyacetic acid (200 mg, 0.9 mmol) and triphenylphosphine (240 mg, 0.9 mmol)
in dimethylformamide (5mL) at 0ºC. The reaction was slowly warmed to room temperature
and allowed to stand overnight. The reaction was concentrated under reduced pressure and
subjected to flash chromatography (1:19 methanol:ethyl acetate) to give a mixture of α and β
anomers (1.3:1 ratio) as a colorless gum (258 mg, 76%). To isolate exclusively the β-anomer,
a small sample of the above mixture was separated using semi-preparative HPLCas above,
except thatseparation was achieved using a flow rate of 4 mLmin-1 and a mobile phase of a
1:3 mixture of acetonitrile:water. The β-anomer was isolated as a colorless gum.1H NMR
(500 MHz, (CD3)2CO) δ 3.37 (dd, J = 9.0, 8.2 Hz, 1H), 3.42-3.47 (m, 2H), 3.51 (dd, J = 9.0,
9.0 Hz, 1H), 3.70 (dd, J = 11.9, 4.7 Hz, 1H), 3.82 (dd, J = 11.9, 2.1 Hz, 1H), 4.96 (s, 2H),
5.62 (d, J = 8.1 Hz, 1H), 7.11 (d, J = 8.9 Hz, 1H), 7.31 (dd, J = 8.9, 2.6 Hz, 1H), 7.48 (d, J =
2.6, 1H) (Supporting Information, SI Figure S3);13C NMR (125.8 MHz, (CD3)2CO) δ 62.4,
66.4, 71.0, 73.9, 77.7, 78.6, 96.0, 116.1, 124.2, 126.9, 128.7, 130.6, 153.6, 167.8 (Supporting
Information, SI Figure S4); HRMS (ESI,-ve) [M+CHOO]-m/z
=
427.0188
(C15H17O10Cl2requires 427.0199).
2.6 Preparative thin-layer chromatography. Approximately 30 leaves treated with
unlabeled 2,4-D, or control leaves treated with 0.1% (v/v) Tween 20 alone, were
homogenised in methanol as above and partitioned against diethyl ether and then 1-butanol21
to separate the polar metabolites 1 and 2 from the less-polar metabolite 3 and parent 2,4-D.
Concentrated fractionswere resuspended in 100% methanol and applied to 10 cm x 10 cm
aluminum-backed silica gelthin-layer chromatography (TLC) plates (Sigma-Aldrich, Sydney,
Australia; two plates per fraction) which were developed in toluene:2-butanone:acetic acid
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(45:55:3).19Regions corresponding to the Rf values of parent 2,4-D (0.5), metabolite 3(0.32)
and combined metabolites 1+2 (0.0 – 0.1, overlapping), as identified in previous experiments
using [14C]-2,4-D-treated plants,21 were scraped off the plates and extracted three times in
methanol. The supernatants were placed into pre-weighed tubes, evaporated to dryness and
stored at -80°C until used in bioassays. Two independent experiments were performed.
2.7 Bioassay for auxin activity. The average proportion of recovered 2,4-D equivalents in
each TLC band was determined in previous experiments with[14C]-2,4-D to be: parent 2,4-D:
47%; metabolites 1+2: 34%;metabolite 3: 19%.22 Therefore, based on these proportions and
an assumed post-TLC recovery of 50% of applied 2,4-D (also calculated from previous [14C]-
2,4-D experiments), the preparative TLC bands extracted above were resuspended in 100%
ethanol in volumes adjusted so that each fraction contained the same theoretical concentration
(3.2 mM) of 2,4-D equivalents. The untreated control fractions were resuspended to the same
mass of solids per L as the corresponding treated fractions, to account for possible growth
inhibition by endogenous plant compounds present in the fraction.
A dose-response experiment was conducted using authentic 2,4-D, synthesized 2,4-D glucose
ester,or TLC fractions from untreated and 2,4-D-treated plants,alldiluted in 0.6% (w/v) agar
in 90mm-diameter petri dishes.21 Authentic 2,4-D was used at concentrations of 0, 10-12, 10-
11, 10-10, 10-9, 10-8, 10-7, 5x10-7 and 10-6 M, and 2,4-D glucose ester and the TLC fractions at
0, 10-8, 10-7, 5x10-7 and 10-6 M 2,4-D equivalents (concentrations were estimated in the case
of the TLC fractions). The radicle length of 3-day-old seedlings from awell-characterized 2,4-
D-susceptible population21was measured before placing seedlings on treatment agar, and
again after 7 d. There were three replicates of each treatment, with three seedlings per
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replicate. The experiment was performed twice, using the two sets of TLC extracts, and the
results combined.
2.8 Statistical analysis. Root elongation at each concentration of ʻtreatmentʼ TLC fractions
(i.e. those extracted from 2,4-D-treated plants) was expressed as a percentage of root
elongation at the corresponding concentration of ʻcontrolʼ TLC fractions (i.e. those extracted
from untreated plants) in order to account for potentialinhibitory effects of the endogenous
plant compounds co-migrating with the 2,4-D metabolites on TLC.Dose-response curveswere
created using the drc package in R,25 in which the data were fitted to a three-parameter log-
logistic model with the following equation:
푑
푦 =
1 + exp(푏 log푥 − log푒 )
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where d is the upper limit of the root elongation data, b is the slope of the curve, x is the
herbicide dose and e is the dose at which root elongation was inhibited by 50% (otherwise
known as the ED50).26 The 2,4-Ddose-response equation was used to calculate the
concentration of 2,4-D equivalents in the TLC fractions (x) based on their inhibition of root
elongation (y), and the ED50 values for the TLC fractions were subsequently obtained using
the drc package in R.
3. RESULTS
3.1 Identification of 2,4-D metabolites. Treatment of plants with [14C]-2,4-D and
subsequent extraction and separation by HPLC showed one major (metabolite 1) and two
minor (metabolites 2 and 3) radiolabeledmetabolites of 2,4-D (Figure1). To determine the
mass of these metabolites, the experiment was repeated with an extract from plants treated
with a stable isotope-labeledmixture(1:1 [12C]:[13C]-2,4-D),using the same method and
instrument. Fractions with elution times corresponding to those of the original
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radioactivefractions were then collected and analysed by HPLC-MS, looking for a specific
isotope pattern separated by 6 mass units.The MS showed an isotopic series of ions in
negative electrospray ionization corresponding to metabolite 1 atm/z 417, 427, 495 and 526
(Figure 2). For the minor metabolites 2 and 3, only minor isotopic signals were observed at
m/z 334 and 335 respectively (Figure 2).
The major metabolite 1 at first did not match any known metabolites based on high-resolution
MS measurements, and therefore isolation was attempted.Minor metabolite 2had an accurate
mass in good agreement with the common aspartic acid conjugate of 2,4-D,15while metabolite
3 had a mass that also did not match any known 2,4-D metabolites. The identities of
metabolites2 and 3 were not investigated further due to their abundance being too low for
isolation.
3.2 Isolation of metabolite 1. For isolation of metabolite 1, unlabeled2,4-D was applied to
plants in a scaled-up experiment. Plant metabolites were extracted with methanol and the
crude extract was pre-filtered through C18-silica with solvent mixturesconsisting of increasing
concentrations of methanol and water. The 3:2 (v/v)methanol:waterfraction was separated
using the same C18 semi-preparativeHPLC method as above to provide a fraction containing
metabolite 1, as determined by direct injection into the mass spectrometer. This fraction was
purified further with semi-preparative HPLC using 1:1 (v/v) methanol:water(containing 0.1%
formic acid) to provide a sufficiently pure sample for NMR analysis. The NMR spectra
indicated all the features of 2,4-D were present, along with a sugar moiety that was inferred
to be attached to the carboxylate group. This was supported by a heteronuclear multiple bond
correlation (HMBC) of the anomeric proton to the carbonyl group of the carboxylate group of
2,4-D (Figure 3). This structural assignment agreed with a quasi-molecular ion [M+CHOO-]-
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observed at m/z 427, but previous TLC experiments21and the observation of higher m/z values
for this metabolite initially raised some doubt. It was possible that the glucose
wassulfated16or had another conjugate which was NMR silent. However, to our knowledge
no NMR spectrahave been reported for the 2,4-D glucose conjugate which could be used to
verify the structure. Previous identification in the literature has been based on -glucosidase
digestionof water-soluble[14C]-labeled metabolites,followed bychromatographicanalysis of
the aglycones.7,8,12Hence, to unambiguously confirm the identity of metabolite 1 as the
glucose ester of 2,4-D, a synthesis was undertaken.
3.4 Synthesis of 2,4-D glucose ester. The synthesis of 2,4-D glucose ester was achieved by a
Mitsunobu reaction between 2,4-D and glucose (Figure 4). The reaction yielded the β-anomer
as the major isomer which was isolated by semi-preparative HPLC. The synthesized ester
provided NMR spectroscopic data (Figure 5),along with HPLC retention time and mass
spectrometric data, that correlatedperfectly with that of metabolite 1 obtained from 2,4-D
treated plants, thus confirming the identity of this metabolite as the 2,4-D glucose ester.
3.5 Auxin activity of 2,4-D metabolites. In terms of inhibition of seedling root elongation,
the ED50 value for authentic 2,4-D was 1.02 nM, which was significantly (P< 0.05) lower
than that for the synthesized 2,4-D glucose ester (14.7 nM) (Figure6a). The parent 2,4-D and
metabolite 1+2 bands extracted from TLC plates both caused increasing inhibition of root
elongation with increasing concentration (Figure6b, c).The ED50 values for extracted parent
2,4-D and metabolite 1+2 were calculated at 1.27 and 1.45 nM, respectively, which were
significantly lower than that of synthesized 2,4-D glucose ester but not different from that of
authentic 2,4-D. The TLC fraction corresponding to metabolite 3 caused a relatively
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consistent inhibition of root elongation at all concentrations used and thus the data could not
be fitted to a dose-response curve (Figure6d).
4. DISCUSSION
This study has identified the glucose ester as the major metabolite of 2,4-D in wild radish, a
prominent weed species. This is, to our knowledge, the first reported NMR analysis of the
2,4-D glucose ester isolated from a biological extract and confirmed by organic
synthesis.Very little work has been done on 2,4-D metabolism in the Brassicaceae family,
and no study has detected the glucose ester.In an investigation targeting hydroxylated 2,4-D
metabolites, trace levels of 4-OH-2,5-D werefound in 2,4-D-treated wild mustard (Brassica
kaber).27In a study focused on the potential for amino acid conjugation of 2,4-D in
Arabidopsis thaliana, small amounts of the aspartyl and glutamyl2,4-D amides were
isolated.15Wild radish may also produce low levels of the aspartyl amide of 2,4-D, based on
the mass spectral characteristics of a minor metabolite examined in the current study. The
more comprehensive surveys of 2,4-D metabolism in non-cruciferous species have detected
trace or low amounts of the glucose ester in dicots,10,11andhigh levels in some grasses.9,28
As expected,9both laboratory-synthesized 2,4-D glucose ester and the partially-purified wild
radish TLC fraction containing this molecule displayed auxin activity.The fact that the ED50
of the TLC fraction was 10-fold lower than that of the synthesized glucose ester (and almost
identical to that of parent 2,4-D)suggests that the ester in the TLC fraction was hydrolyzed
back to 2,4-D during extraction from the acidic silica of the TLC plate, in line with the high
acid- and base-lability demonstrated in previous studies.21,28The TLC fraction containing the
unidentified minor metabolite 3 caused a relatively consistent, concentration-independent
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level of root growth inhibition in the bioassay, suggesting that the plant may hydrolyze this
metabolite to an auxin-active form at very slow rates.
In terms of 2,4-D resistance evolution in weeds, it appears that even the most-resistant wild
radish populations so far characterized have little to no capacity for metabolic detoxification
of 2,4-D.22 In particular, there is no chemical, structural (current study)or
pharmacological21evidence for the presence of low-toxicity, ring-hydroxylated 2,4-D
metabolites. Therefore, it can be surmised that repeated selection of wild radish with 2,4-D in
the field has so far not resulted in increased expression/activity of potential 2,4-D-
hydroxylating enzymes such as cytochrome P450 monooxygenase29 or iron (II)/-
ketoglutarate dependent hydroxylase30(see also Supporting Information, SI Table S1).The
enzyme responsible for the observed reversible conjugation of glucose with 2,4-D is likely to
be a UDP-glycosyltransferase (UGT). A few members of the large UGT superfamily have
been identified as having activity against 2,4-D, but at much lower levels (1 – 15%) than
against the natural auxins indole-3-acetic acid and indole-3-butyric acid.31,32,33 A cursory
search of the gene expression data generated in a transcriptomic study on the response of wild
radish to 2,4-Dat 2, 8 and 24 h after application22 revealed consistent up-regulation of five
UGT genesin both resistant and susceptible plants (Supporting Information, SI Table S1).
The most 2,4-D-responsive of these, UGT74E2, is known to have low but measurable activity
against 2,4-D.32 Thus, UGT74E2 could potentially be responsible for glucose conjugation of
2,4-D in wild radish, but this needs to be confirmed experimentally.It is more difficult to
pinpoint a potential candidate for the 2,4-D-glucose hydrolase from the existing
transcriptomic data, as its expression may not have been affected by application of free 2,4-D
within the 24-h time-frame of the transcriptomic experiment.
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Metabolic detoxification of 2,4-D is likely to be a rare (at best) resistance mechanism in wild
radish, based on the findings that (1) there is no differential metabolism of this herbicide
across the 13 independently-evolved populations studied so far,22 and (2) the major
metabolite, 2,4-D glucose ester, retains auxin activity.Therefore, there is little opportunity to
manage 2,4-D resistance in wild radish with the use of chemical inhibitors of 2,4-D-
metabolizing enzymes. It is likely that in most of the populations studied to date, as-yet-
unidentified alterations in auxin perception and/or signaling are responsible for resistance,22
which will probably require biological or genetic solutions rather than chemical methods of
resistance amelioration.
ASSOCIATED CONTENT
Supporting Information
(1) HPLC chromatograms showing the semi-preparative HPLC separation of metabolite 1
1
from wild radish extracts (Figure S1); (2) Full H NMR spectrum of isolated metabolite 1
1
(Figure S2); (3) H NMR spectrum of synthetic 2,4-D-glucose ester (Figure S3); (4) 13C
NMR spectrum of synthetic 2,4-D glucose ester (Figure S4); (5)Expression of potential 2,4-
D-metabolizing enzymes in 2,4-D-treated wild radish plants (Table S1).
ACKNOWLEDGEMENTS
This work was funded by the Australian Research Council (grant LP150100161). We
acknowledge the facilities and the scientific and technical assistance of the Australian
Microscopy & Microanalysis Research Facility at the Centre for Microscopy,
Characterisation & Analysis, The University of Western Australia; a facility funded by the
University andthe State and Commonwealth Governments. We thank Roberto Busi (UWA)
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for statistical advice,Chad Sayer (Nufarm, Australia) for helpful discussions and two
anonymous reviewers for their suggestions on improving the manuscript.
The authors declare no competing financial interest.
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FIGURE CAPTIONS
Figure1.HPLC chromatogram of a leaf extract from wild radish plants treated with [14C]-2,4-
14
D. Only C-labeled compounds are registered by the -RAM detector. The diagonal line
shows the concentration of acetonitrile in the mobile phase over time.
Figure 2. Mass spectra of the 2,4-D metabolites 1, 2, 3 and of parent 2,4-D isolated from 1:1
[12C]:[13C]-2,4-D-treated wild radish plants.
Figure 3. Selected region of the H-13C HMBC of metabolite 1 showing the existence of
1
correlations between the anomeric proton and the carbonyl group of the ester, and the
methylene bridge protons with the same ester and the chlorinated aromatic ring.
Figure4. Synthesis of 1-[(2,4-dichlorophenoxy)acetate]-β-D-glucopyranose from 2,4-
dichlorophenoxyacetic acid and -D-glucose.
Figure 5. Overlaid 1H NMR spectra of the isolated metabolite 1 (top) and the synthetic 2,4-D
glucose ester (bottom).
Figure6. Response of seedling roots to 2,4-D and its metabolites. Preparative TLC was used
to partially purify 2,4-D and its metabolites from extracts of 2,4-D-treated leaves, and their
effect on seedling root elongation was measured. Dose-response curves are shown for (a)
authentic 2,4-D and synthesised 2,4-D glucose ester, (b) TLC parent 2,4-D band; (c) TLC
metabolite 1+2 band; (d) TLC metabolite 3 band. Concentrations of 2,4-D equivalents in the
TLC bands were calculated from the dose-response curve equation of authentic 2,4-D. Values
are means ± SE, with data pooled from two independent experiments (n = 3 for each).
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Figure 1.
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Figure 3.
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Figure 4.
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Figure 5.
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