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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
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Howard2,4,5, Stephen B Powles1 and Gavin R Flematti4
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1
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University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009,
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Australia
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Australian Herbicide Resistance Initiative, School of Agriculture and Environment,
Centre for Microscopy, Characterisation and Analysis, University of Western Australia, 35
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Stirling Highway, Crawley, Western Australia 6009, Australia
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3
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Crawley, Western Australia 6009, Australia
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4
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Crawley, Western Australia 6009, Australia
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*Corresponding author:
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School of Biological Sciences, University of Western Australia, 35 Stirling Highway,
School of Molecular Sciences, University of Western Australia, 35 Stirling Highway,
School of Chemistry,University of Leeds, Leeds, LS2 9JT, United Kingdom Phone +61 8 6488 1512 Email
[email protected] 18 19
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ABSTRACT:Synthetic auxin herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) are
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widely used for selective control of broadleaf weeds in cereals and transgenic crops.
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Although thetroublesome weed wild radish (Raphanusraphanistrum) has developed
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resistance to 2,4-D, no populations have yet displayed an enhanced capacity for metabolic
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detoxification of the herbicide, with both susceptible and resistant wild radish plants readily
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metabolizing 2,4-D. Using mass spectrometry and NMR, the major 2,4-D metabolite was
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identified as the glucose ester, and its structure confirmed by synthesis. As expected, both the
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endogenous and synthetic compounds retained auxin activity in a bioassay. The lack of
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detectable 2,4-D hydroxylation in wild radish, and the lability of the glucose ester, suggest
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that metabolic 2,4-D resistance is unlikely to develop in this species.
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KEYWORDS:2,4-dichlorophenoxyacetic acid; auxin; herbicide resistance; metabolism; wild
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radish (Raphanusraphanistrum)
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1. INTRODUCTION
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The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), which mimics the action of the
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key plant hormone indole-3-acetic acid,was discoveredover 70 years ago and was quickly
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adopted as a herbicide, a promotor of plant growth in tissue culture and an inhibitor of pre-
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harvest fruit drop.1 Although the use of 2,4-D as a herbicide in the United Stateshas declined
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from 18 million kg per year in the 1950s to its current level of around 1 million kgper year, it
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remains an essential component of selective dicotweed control in crops, turf areas and
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roadsidesworldwide.2The basis of selectivity of the synthetic auxins is mainly attributed to
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the greater capacity of monocotyledonous plants to metabolize these herbicides to inactive
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polar conjugates, but there is evidence that enhanced metabolism may not entirely account for
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the high levels of resistance seen in most grass species.3,4
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The major pathways of 2,4-D metabolism in several commercially important cereal and
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legume species were identified in the 1970s as (1) direct conjugation of glucose or amino
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acids (predominantly aspartate and glutamate) to the carboxyl group of the 2,4-D molecule
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via ester oramide linkages; and (2) hydroxylation of the phenol ring, followed by conjugation
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of a sugar (usually unidentified) through an ether linkage.5-14These conclusions were largely
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based on co-chromatography, whereradiolabeled metabolites extracted from [14C]-2,4-D-
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treated plants were chemically or enzymatically hydrolyzed and the retention times/Rf values
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of the hydrolysisproducts were compared with authentic standards.More recent studies have
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used liquid chromatography-mass spectrometry (LC-MS) to confirm the presence of 2,4-D
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amino acid conjugates15 and some sugar conjugates of 2,4-dichlorophenol16in crude plant
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extracts.
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The 2,4-D glucose ester (1-[(2,4-dichlorophenoxy)acetate]-β-D-glucopyranose) and most 2,4-
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D amino acid conjugates are readily cleaved back to the parent molecule by endogenous plant
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hydrolases and thus display auxin activity,9,15,17whilst ring-attached 2,4-D glycosides and
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their respective aglycones (predominantly 4-hydroxy-2,5-D and 4-hydroxy-2,3-D) are
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generally inactive.8Less commonly, 2,4-D has been found incorporated into larger inactive
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and/or
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triacylglycerols,19as well as lignin and oligopeptides,6 presumably as a mechanism to
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sequester 2,4-D in a low-toxicity form.
insoluble
molecules
such
as
3-(2,4-dichlorophenoxy)propionic
acid,18
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Wild radish (Raphanusraphanistrum), the major dicotyledonous weed of Western Australian
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cropping systems, is displaying increasing levels of resistance to 2,4-D.20 In previous work,
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13 populations were studied which showed no evidence of differential metabolism between
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susceptible and resistant plants.21,22 However, in this work, approximately 50% of applied
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2,4-D was converted to compounds which did not match known metabolites, and thus remain
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unidentified. As growers in Australia intensify their use of 2,4-D in response to resistance to
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other herbicides,20 the increased selection pressure could potentially result in the appearance
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of populations with a greater capacity to detoxify 2,4-D, as has occurred in corn poppy23 and
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nodding thistle.24Therefore, in this study we investigated the identity and herbicidal activity
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of the major 2,4-D metabolites produced in wild radish.
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2. MATERIALS AND METHODS
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2.1 Chemicals and instrumentation. Technical-grade 2,4-D acid was a gift from Nufarm
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(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
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Cambridge Isotope Laboratories (Tewksbury, Massachusetts). All other chemicals were from
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C [U]-2,4-D from
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Sigma-Aldrich (Sydney, Australia), and solvents for extractions, synthesis and purifications
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were of high performance liquid chromatography (HPLC) grade, unless otherwise stated.
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HPLC was performed on an Agilent 1200 HPLC system equipped with a photodiode array
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detector and fraction collector. Plant extracts were separatedon a reversed phase Grace
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Apollo C18 HPLC column (250 mm long, 10 mm i.d., 5 μm particle size; Grace-Davison
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Discovery Sciences, Columbia, Maryland) with a 33 mm x 7 mm guard column of the same
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material.An in-line -RAM detector (IN/US Systems Inc, Pine Brook, New Jersey) was used
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to detect radioactivity in eluents from [14C]-2,4-D-containing extracts. HPLC-MSand high-
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resolution mass spectrometry (HRMS) were conducted using a Waters Alliance e2695 HPLC
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connected to a Waters LCT Premier XE time-of-flight (TOF) mass spectrometer with an
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electrospray ionization source (ESI) and direct injection valve. Ionization conditions were
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optimized using unlabeled 2,4-D in negative ionization mode. Cone and desolvation gas
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flows were set to 150 and 650 L h-1, respectively. The capillary voltage was set at 3 kV, cone
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voltage at 20 V with the source temperature at 80°C, and the desolvation temperature at
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350°C. HRMS calibration was achieved using leucine encephalin at 2 ng uL-1. HPLC-MS
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separations were achieved using a reversed phaseGrace AlltimaC18 HPLC column (250 mm
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long, 2.1 mm i.d., 5 μm particle size; Grace-Davison Discovery Sciences, Columbia,
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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,
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125.75 MHz)equipped with a 5 mm BBFO probe, or on a Bruker Avance IIIHD14.1 T
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spectrometer (1H at 600.13 MHz,
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(Bruker, Rheinstetten, Germany), with either deuteromethanol or deuteroacetoneas solvent.
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Chemical shifts are reported in ppm relative to the residual solvent signals (δ 3.31 and 2.05
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ppm for deuteromethanol and deuteroacetone respectively).
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C at
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C at 150.90 MHz) using a 1.7 mm TXI microprobe
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2.2 Plant material. Five of the wild radish (RaphanusraphanistrumL.; Brassicaceae)
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populations (R1, R2, R3, R6 and R8)characterized in previous work,22 which showed no
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differences in 2,4-D metabolism and retained most of the applied 2,4-D in the treated leaf,
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making for simpler processing of samples, were bulked together for this study. Seedlings
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were grown in potting mix (composted pine bark:riversand:peat moss, 2:1:1) and kept in a
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growth cabinet under a 12 h photoperiod of 200 µmol m-2 s-1white LED and incandescent
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light at day/night temperatures of 20/15°C. Plants were watered daily, and fertilized weekly
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with 1.5 g L-1commercial soluble fertilizer (Diamond Red: N 27%; P 5.7%; K 10.9% plus
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trace elements; Campbells Fertilisers, Laverton North, Australia). When seedlings had
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reached the 3-leaf stage, 2,4-Dwas applied as a 5 mM solution in 0.1% (v/v) Tween 20, with
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ten droplets of 1 µL placed on the first two leaves of each seedling. Plants were then returned
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to the growth cabinet and leaf tissue was harvested after 96 h. In order to identify HPLC
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fractions containing 2,4-D or its metabolites, and to pinpoint MS fragments originating from
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2,4-D, pilot studies were performed with (1) plants treated with 5 mMunlabeled 2,4-D plus 3
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kBq per leaf of [14C]-2,4-Dfor radio-HPLC analysis,and (2) plants treated with a 1:1 (5 mM
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total) mixture ofunlabeled2,4-D and [13C]-2,4-D for HPLC-MS analysis. To account for
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potential interfering signals from endogenous plant compounds, plants treated with 0.1%
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Tween 20 alone were extracted alongside the [13C]-2,4-D-treated plants. No signals in the m/z
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regions of 2,4-D or its metabolites were detected in the control plants (data not shown).
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2.3 Sample processing. Treated leaves were excised from seedlings and ground to powder in
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liquid nitrogen in a mortar and pestle. The time between excision and freezing was
