The metabolism of a novel herbicide ZJ0273 in oilseed rape and crickweed

Pesticide Biochemistry and Physiology 104 (2012) 44–49

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The metabolism of a novel herbicide ZJ0273 in oilseed rape and crickweed Ling Yue a,b, Wenyuan Qi b,⇑, Qingfu Ye a,⇑, Haiyan Wang a, Wei Wang a, Ailiang Han a a

Institute of Nuclear Agricultural Sciences, Key Laboratory of Nuclear Agricultural Sciences of Ministry of Agriculture and Zhejiang Province, Zhejiang University, Hangzhou, Zhejiang 310029, PR China b Crop Institute, Shanghai Academy of Agriculture Science, Shanghai 201403, PR China

a r t i c l e

i n f o

Article history: Received 28 August 2011 Accepted 27 June 2012 Available online 20 July 2012 Keywords: Metabolism Acetolactate synthase Herbicide ZJ0273 Crickweed Oilseed rape

a b s t r a c t Experiments were conducted to investigate the metabolism of a novel herbicide ZJ0273 in the seedlings of the susceptible crickweed and tolerant oilseed rape. Six metabolites of ZJ0273 were identified in crickweed seedlings, compared to 8 metabolites in oilseed rape. Among these metabolites, the metabolite M7, with the chemical name of 2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid, was found to be the most herbicidally effective molecule with significant inhibition to acetolactate synthase. The differences in the amount and rate of ZJ0273 transformation to M7, and its metabolic detoxification, contributed to the primary mechanism of herbicidal selectivity of ZJ0273. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction ZJ0273 (propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino)benzoate), is a novel broad-spectrum herbicide developed for pre- and post-emergence weed control primarily in oilseed rape fields [1]. At the field application rate of 45–60 g a.i. h1, effective control against many monocotyledonous and dicotyledonous weeds by ZJ0273 was reported with the efficacy over 80% [2]. The transformation, formation of extractable residue and bound residue, and mineralization of ZJ0273 in soils have been clarified [3–5]. The formed soil bound residue of ZJ0273 and its metabolites were found to be released under certain condition and resulted in significant inhibition on rice seedling [6]. The absorption, translocation and seed residues of ZJ0273 in oilseed rape have been investigated [7]. At the recommended application rates, only the parent compound ZJ0273 was found in the extractable residue in the oil rape seeds with a concentration of 0.09 mg/kg dry weight [7]. Previous studies demonstrated that ZJ0273 was a potential inhibitor of acetolactate synthase (ALS) under in vivo conditions while no inhibition was found under in vitro condition, suggesting that ZJ0273 was a herbicide precursor or pro-herbicide [8]. A pro-herbicide is usually inactive and must first be converted to the active molecules to impose the inhibitory effect on weeds [9]. Metabolism of herbicides in different plants is a major physiological factor for herbicide selectivity [10] Moreover, the bioactivation of pro-herbicides holds great potential for the development of ⇑ Corresponding authors. Fax: +86 571 86971423. E-mail addresses: [email protected] (W. Qi), [email protected] (Q. Ye). 0048-3575/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2012.06.010

new and safe selective herbicides [11]. The metabolism in oilseed rape is important for the safety assessment of ZJ0273, since oilseed rape is an important source of edible oil for human consumption. However, the metabolism of ZJ0273 in the susceptible weeds and the tolerant oilseed rape still remains unknown. Thus, in the current study, we explored the metabolism of ZJ0273 in the susceptible crickweed and the tolerant oilseed rape. 2. Materials and methods 2.1. Chemicals [B ring-4,6-14C]ZJ0273 (Fig. 1, radiochemical purity 99.4%; chemical purity 98.1%; specific activity 3.77  107 Bq/mmol), and [C ring-U-14C]ZJ0273 (Fig. 1, radio chemical purity 98.9%; chemical purity 98.1%; specific activity 3.74  107 Bq/mmol), the authentic standard of ZJ0273 and its metabolites, propyl 4-(N-(4,6-dimethoxypyrimidin-2-yl)-N-(2-hydroxybenzyl)amino)benzoate(M1), propyl 4-(N-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzyl)formamido)benzoate (M2), 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzamido)benzoic acid (M3), 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoic acid (M5), methyl 2-(4,6-dimethoxypyrimidin-2-yloxy)benzoate (M6), 2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid (M7) were synthesized by Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences [12,13]. The cocktail ingredients 2,5-diphenyloxazole (PPO) and 1,4-di[20 -(50 -phenyloxazolyl)]-benzene (POPOP) were both scintillation grade. Dimethylbenzene, dichloromethane, acetone, glycol-ether, and ethanolamine were analytical grade. Methanol, ethyl acetate,

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H3CO

*

*

N

N

H3CO

OCH3

OCH3 N

N

O

O H N

* COOCH2CH2CH3

[B ring-4,6-14C]ZJ0273 Fig. 1. Structures of

14

H N COOCH2CH2CH3

[C ring-U-14C]ZJ0273

C radiolabelled ZJ0273 with asterisks marking the position of

and cyclohexane used in the chromatograph analysis were chromatographic grade. Scintillation cocktail A consisted of 5 g PPO, 0.5 g POPOP, 350 mL glycol-ether, and 650 mL dimethylbenzene. Scintillation cocktail B consisted of the following ingredients: 5 g PPO, 0.5 g POPOP, 175 mL ethanolamine, 225 mL glycol-ether, and 600 mL dimethylbenzene. 2.2. Seedling cultivation and treatment Crickweed (Malachium aquaticum L.) seedlings at the 2-leaf stage were transplanted from the field without a record of herbicide application to the plastic pots (width  length  depth, 32  45  25 cm) and cultivated under similar conditions. The oilseed rape (Brassica napus L.) seeds were sowed in the plastic pots after germination at 25 °C in Petri dishes containing filter papers moistened with distilled water. At the 5-leaf stage, ZJ0273 solution (containing 14C-labelled and non-labelled ZJ0273, 0.25% nonionic surfactant Tween-80) at the dosage of 12 mg ZJ0273 in 10 mL emulsion per pot was dabbed homogeneously at the leaves of the seedlings with the total radioactivity of 3.8  105 Bq ([B ring4,6-14C]ZJ0273 and [C ring-U-14C]ZJ0273) for oilseed rape and 3.0  105 Bq ([C ring-U-14C]ZJ0273) for crickweed. Prior to treatment, the soil surface of the pots were covered with filter papers to prevent the treatment solution dropping into the soil. After treatment, the seedlings were cultivated under simulated field conditions till harvest. During the experiment, no phytotoxic effect of ZJ0273 on the seedling of crickweed and oilseed rape was found. At 1, 2, 4, 8, and 12 days after treatment (DAT), three pots of seedlings were harvested. The seedlings were cut at the soil surface. The fresh weights of the seedlings of the oilseed rape and crickweed per pot were 350 ± 20 g. The leaves per pot were gently rinsed with acetone for 30 s for one leaf at a flow rate of 5 mL min1 using a Waters 600E pump (Waters Co., Massachusetts, USA) to remove the unabsorbed herbicide. The leaf washes were collected, combined, concentrated, and brought to 100 mL. An aliquot of 500 lL was transferred to a scintillation vial. After the acetone evaporated, 10 mL scintillation cocktail A was added, and the radioactivity was counted on a liquid scintillation spectrometer (LSC, Wallac 1414, Wallac, Finland) to determine the radioactivity of non-absorbed herbicide. In a preliminary experiment, leaves were spotted with 14C-ZJ0273 and sampled after 10 s to evaluate the efficiency of the leaf wash technique [14]. This leaf wash procedure removed about 98.5 ± 2.5% of the unabsorbed 14 C-ZJ0273. The treated seedlings were then stored in a freezer at 80 °C for further analysis. 2.3. The extraction and cleanup procedure of plant tissue samples The frozen seedlings of one individual pot were ground to a homogenized powder under liquid nitrogen using a mortar and pestle. The resulting powder was transferred to 100-mL capped

14

C.

centrifuge tubes at 10 g powder per tube. The powder was then extracted with the following solutions (50 mL) in sequence by mixing on a shaker (G33, New Brunswick Scientific, New Jersey, USA) for 12 h each: 80% methanol, methanol, and ethyl acetate (twice). All the extracts from each step were filtered through the 10-layered gauze, and centrifuged at 4000 rpm (Universal 32, Hettich, Tuttlingen, Germany). The supernatant of each extract was collected and brought to a final volume of 500 mL. Three 0.5-mL aliquots from the final 500 mL extract of each step were transferred to 20-mL scintillation vials and the 14C radioactivity was determined by LSC after addition of 10 mL scintillation cocktail A. Analysis of radioactivity showed that all extractable 14C activity was removed at the end of the sequential extraction and the remaining 14C residue was defined as bound residue (BR). The 80% methanol extract was evaporated to remove methanol with a rotary evaporator at 40 °C (R-202, Shanghai Shensheng BioTech, Shanghai, China). The aqueous phase was adjusted to pH 3.0 with diluted HCl, partitioned three times with an equal volume of dichloromethane, and the aqueous layer (below background) was discarded. All the organic phases from the four-step extracts were combined and evaporated to near dryness under vacuum with a rotary evaporator. The residue was dissolved in 25 mL ethyl acetate and cyclohexane (1:1, v/v), and further condensed to 5 mL. After centrifugation at 18000 rpm for 30 min, the cleanup of the crude samples was carried out on the gel permeation chromatography (GPC) equipped with a 30  400 mm column packed with biobeads S-X3 (200–400 mesh, Bio-Rad, California, USA). The crude sample (2.5 mL  2) was loaded onto the GPC column and eluted with ethyl acetate and cyclohexane (1:1,v/v) at a flow rate of 2.0 mL min1 controlled using a Eyela ceramic pump (VSP-3050, Eyela, Tokyo, Japan). The eluted fractions were collected at 10 mL per tube. An aliquot of 0.5 mL eluant per tube was taken to determine the radioactivity on a ultra-low liquid scintillation spectrometer (ULLSS; Quantulus 1220, Perkin Elmer, Massachusetts, USA). All the radioactive fractions were combined and evaporated to near dryness under vacuum at 40 °C with a rotary evaporator. The residue was dissolved, brought to 5 mL with ethyl acetate and methanol (1:1, v/v) and injected onto a Florisil column (30  400 mm), followed by elution with ethyl acetate and methanol (1:1, v/v) at a flow rate of 2.0 mL min1. The eluted fractions were collected at 10 mL eluant per tube and 0.5 mL aliquots from each tube were taken to measure the radioactivity on the ULLSS. All the radioactive fractions were combined and evaporated to near dryness under vacuum at 40 °C with a rotary evaporator. The residue was then redissolved with 2 mL chromatographically pure methanol. The extracts were centrifuged 18,000 rpm (Universal 32, Hettich, Tuttlingen, Germany) for 15 min, filtered through a 0.22-lm pore size filter (Millipore, Billerica, Massachusetts, USA) and stored at 4 °C. The further separation and quantification of the above primary purified samples were conducted on a HPLC system coupled with LSC measurement. Aliquots of 20 lL sample were injected into the HPLC system equipped with a reversed-phase Diamonsil C18

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column (5-lm, 4.6  250 mm), a Waters 600 multi-solvent delivery unit and a Waters 996 photodiode array detector operating at 301 and 254 nm. The column temperature was maintained at 30 °C. The column was eluted with the mobile phase A (ddH2O with 0.1% acetic acid) and B (methanol with 0.1% acetic acid) in a gradient mode (%A/minutes: 80/0, 50/20, 30/50, 25/80, 10/90, 0/ 110, and 80/120) at a flow rate of 1 mL min1. The eluted solution was collected into 20 mL scintillation vials with 1.0 mL per vial. After addition of 10 mL of scintillation cocktail A, the 14C radioactivity was determined by ULLSC. Seven radioactive fractions were measured in the extracts of the crickweed, compared to 9 radioactive fractions in oilseed rape. To obtain adequate amounts of metabolites for the subsequent LC/MS/MS analysis, all the radioactive fractions were enriched through the HPLC system with the above method. In the LC/MS/MS analysis, the HPLC method was further optimized for each of the enriched metabolite (Table 1). During the whole process of analysis, the radioactivity of the fractions was monitored to discard the fractions with the radioactivity at or below the background. Three 1.0-g aliquots of the extracted solid residue after the fourstep extraction was converted to 14CO2 on a biological oxidizer (Sample Oxidizer 307, Perkin Elmer, Massachusetts, USA). The released 14CO2 was trapped in 15 mL of scintillation cocktail B. The 14 C radioactivity was measured by ULLSC and referred to as bound residue. 2.4. LC/MS/MS analysis To identify the structures of the metabolites, LC/ESI-MSn analyses were performed using an Agilent 1100 analytical HPLC system combined with a Bruker Esquire 3000plus ion trap mass spectrometer (Bruker–Franzen Analytik, Bremen, Germany) equipped with an electrospray ionization (ESI) source. The Agilent HPLC system was equipped with a G1312 Binpump, G1314A variable-wavelength detector, model 7725 injector with a 20-lL sample loop, an Agilent ChemStation data system and a reversed-phase Diamonsil C18 column (5-lm, 4.6  250 mm). The above optimized HPLC conditions were used for the analysis of the metabolites with the column temperature at 30 °C. The instrument control and data acquisition of ESI-MSn were performed with the Esquire 5.0 software. The ion source temperature was 250 °C, and needle voltage was set at 4.0 kV. Pure nitrogen was used as the drying and nebulizer gases at a flow rate of 10 L min1 and a back pressure of 30 psi. Helium was introduced into the trap with an estimated pressure of 6  106 mbar to improve trapping efficiency to act as the collision gas for the MSn data. The mass spectrometer was optimized in the collision energy range of 0.7–1.3 V. A positive full scan over the range of m/z 100–1000 was used in acquisition. The parent compound of ZJ0273 and its degradation products were identified by the co-chromatography with authentic standards, the mass spectrum and the radioactive data. Table 1 HPLC conditions for LC/MS/MS analysis.

3.1. Recovery of radioactivity The combined radioactivity in the washed liquid and absorbed ZJ0273 were calculated as the initially applied activity, which was used to estimate the overall radioactivity mass balance. The overall recovery for oilseed rape and crickweed was found to be >85% in all treatments. 3.2. Identification of the metabolites of ZJ0273 A total number of 7 and 9 radioactive fractions were detected in the extracts of the crickweed and oilseed rape, respectively. The retention time of parent compound M is 87 min and that of its metabolites M1, M2, M3, M4, M5, M6, M7, and M8 are 97, 71, 66, 58, 55, 48, 41, and 33 min under the HPLC conditions used, respectively. The parent compound was characterized with a mass spectrum including the parent ion m/z 424[M + H] and the ion fragments of 382, 364, 245 and 185 (Table 2). The retention time and the characterized mass spectrum of M were highly consistent with the authentic standard of ZJ0273, which confirmed that M was the parent compound ZJ0273. Although having the same molecular ion m/z 424[M + H] as M, M1 was different from M in the retention time and the main ion fragments. Based on the daughter ions of m/z 318 and 276, M1 was tentatively deduced to be propyl 4-(N-(4,6-dimethoxypyrimidin-2-yl)-N-(2-hydroxybenzyl)amino)benzoate, a Smiles rearrangement product of ZJ0273. The synthesized authentic standard yielded the same mass spectrum with M1, confirming M1 as the O–N-type smiles rearrangement product of ZJ0273. The Smiles rearrangement of ZJ0273 was first reported by Wang et al. [15]. Zhang et al. found that ZJ0273 was biotransformed to M1 through the O–N-type smiles rearrangement in the microsome of the etiolated wheat seedlings [16]. The ESI mass spectrum of M2 showed a quasi-molecular ion of m/z 452 and a daughter ion at m/z 424, along with the same daughter ions of the parent ZJ0273 (m/z 382, 364, 245 and 185) (Table 2), which indicated that M2 was probably a modified product from the parent ZJ0273. Based on the mass spectrum and the characteristic mass difference of 28 between M2 and the parent ZJ0273, M2 appeared to be a N-formylated product of ZJ0273. Thus, M2 was confirmed as propyl 4-(N-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzyl)formamido)benzoate by co-chromatography and fragmentation patterns of the authentic standard. The mass spectrum of M3 contained the molecular ion (m/z 396) and its main daughter ions at m/z 382, 364, 338, and 245 (Table 2). Based on HPLC co-chromatography with the authentic standard and fragmentation characteristics, M3 was identified as the product from the carbonylation on the benzyl carbon of

Table 2 Mass spectra of ZJ0273 and its major degradation products in plants.

HPLC conditions

M M1 M2 M3 M4 M5 M6 M7 M8

3. Results and discussion

Elution

Detection wavelength (nm)

Injection (lL)

35%A + 65%B 35%A + 65%B 50%A + 50%B 50%A + 50%B 55%A + 45%B 50%A + 50%B 50%A + 50%B 70% A + 30%B 70%A + 30%B

301 301 301 301 301 254 254 254 254

20 20 20 20 20 20 20 20 20

Phase A: ddH2O with 0.1% acetic acid; Phase B: acetonitrile with 0.1% acetic acid.

Metabolites

Quasi-molecular

MS/MS daughter ions of the quasi-molecular ion (m/z)

M M1 M2 M3 M4 M5 M6 M7 M8

424 424 452 396 440 382 291 277 425

382,364,245,185 318,276 424,382,364,245,185 382,364,338,245 422,382,364,245 364,338,245 259 259,139 411,407,263,245,185

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de-esterified ZJ0273, with the chemical name of 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzamido)benzoic acid. The molecular ion with m/z of 440 produced 3 characteristic daughter ions (382, 364, 245) as ZJ0273 and an ion with m/z of 422 through dehydration from the quasi-molecule ion (Table 2). The radioactivity analysis confirmed that this molecule was a metabolite of the parent compound ZJ0273. The mass difference of 16 between this metabolite M4 and the parent compound ZJ0273 suggested that M4 is a hydroxylated product of ZJ0273. However, no further valuable information on fragmentation pattern was available for the identification of the structure of M4. The mass spectrum of M5 contained a parent ion at m/z of 382. The MS–MS decomposition of the quasi-molecular ion produced 3 major daughter ions with m/z of 364, 338 and 245 (Table 2). Compared with the parent compound ZJ0273, similar fragmentation patterns in the mass spectrum and the mass difference of 42 indicated that M5 was a depropylated product of ZJ0273. The HPLC retention time and the characteristic mass spectrum of M5 were coincident with the authentic standard, which further confirmed that M5 was 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoic acid. Therefore, M5 was a product of the depropylation of ZJ0273 through the ester hydrolysis catalyzed probably by an esterase enzyme. Earlier studies have shown that pesticides often decompose through ester hydrolysis that is commonly catalyzed by esterases and to a much lesser extent by lipases and proteases [11]. Metabolite M6 was characterized with the parent ion at m/z 291 [M + H]. However, only one characteristic daughter ion at m/z at 258.9 was detected. According to the nitrogen rule, an organic compound with an odd-numbered molecular weight contains an odd number of nitrogen atoms [17]. Therefore, it could be deduced that the structure of M6 only contains 2 nitrogen atoms, which coincided with the feature of the pyrimidine ring of ZJ0273. Based on this information, along with the radioactivity analysis and the mass spectra information, M6 appeared to be methyl 2-(4,6dimethoxypyrimidin-2-yloxy)benzoate. The standard of M6 was synthesized for co-chromatographic analysis. The retention time and the mass spectra confirmed that M6 was methyl 2-(4,6dimethoxypyrimidin-2-yloxy)benzoate. The quasi-molecular ion of M7 appeared at m/z 277 and its decomposition led to 2 major daughter ions at m/z 259 and 139 (Table 2). Based on the radioactivity analysis and the nitrogen rule, the structure of M7 was characterized with the pyrimidine ring and the radiolabelled benzene ring. On the basis of the above information, M7 was identified as 2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid. The comparison of the retention time and fragmentation patterns between M7 and the standard verified that M7 was 2(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid. The mass spectrum of M8 showed a quasi-molecular ion at m/z 425 and 5 characteristic fragment ions at m/z 411, 407, 263, 245 and 185 (Table 2). From the mass difference of 14 and 18, the daughter ions at m/z 411 and 407 were likely the demethylated product and the dehydrated product, respectively. On the basis of the nitrogen rule, the even molecular weight of M8 indicated that M8 contained 2 nitrogen atoms, consistent with the feature of the pyrimidine ring. The radioactivity analysis showed that M8 likely contained the radiolabelled benzene ring. Therefore, M8 was tentatively identified as a conjugated product of a ZJ0273 decomposition product, with the structure containing only the pyrimidine ring and benzene ring. Among the fragment ions, the m/z 263 ion most probably indicated the decomposition product from ZJ0273, which was previously also identified in the soil degradation of ZJ0273, with the chemical name of 2-(4-hydroxy-6-methoxypyrimidin-2yloxy) benzoic acid. The m/z 245 and 185 ions were the characteristic daughter ions. Many studies have shown that pesticides, along with their metabolites, could conjugate with glucose, cellulose,

hemicellulose, lignin, glutathione, among others [9,11,18,19]. Among these conjugations, glycosides, sulfates, and catabolites of glutathione conjugations were most frequently reported and b-D-glucosides were the most common form of conjugate reported in plants (18). Bispyribac, a pyrimidinyl carboxy herbicide, was metabolized mainly by O-demethylation and 5-hydroxylation of a pyrimidine ring, followed by glucose conjugation [20]. Based on the above knowledge, along with the mass difference of 162 between M8 and 2-(4-hydroxy-6-methoxypyrimidin-2-yloxy) benzoic acid, M8 appeared to be a conjugated product of 2-(4-hydroxy-6-methoxypyrimidin-2-yloxy) benzoic acid with glucose. The neutral loss of 162 Da corresponding to losing a hexose residue lead to the result that the relevant fragment ion was not detected in the LC–MS2 data. On the basis of the identified metabolites, the metabolic pathway of ZJ0273 in the seedlings of the tested plants were proposed as in Fig. 2. Transformations labeled with ‘‘OR’’ were observed only in the oilseed rape. 3.3. The metabolism dynamics of ZJ0273 The absorption of ZJ0273 in oilseed rape and crickweed was estimated as the total radioactivity of extracted ZJ0273, the metabolites, and non-extractable residues. No significant difference was found in the absorption of ZJ0273 between oilseed rape and crickweed 1 DAT. However, the absorption in the oilseed rape 12 DAT reached 17.9% of the applied amount, which was greater than that in crickweed (13.8%) (Fig. 3). The extracted fractions showed a similar pattern as the total absorption in both plants, reaching 17.31% and 12.32% of the applied amount 12 DAT for the oilseed rape and crickweed, respectively. The bound residue reached a similar amount of 1.18% and 1.26% of the applied amount in the oilseed rape and crickweed, respectively, which indicated that conjugation

H3CO

OCH3 N O

N

H N

HO

OCH3

COOPr-n

OR

O

N OR

COOH

COOPr-n

M2 OCH3 N

N O

H N

O

COOH

M3

OCH3

H3 CO

OCH3 N O CHO

M4 (Unknown)

H N

H3CO

N O

OCH3

H3CO OR

N

N

Conjugated product

O

COOCH3 M6

H3CO N

M5

N

M1

N

N

N

N

COOPr-n ZJ0273 H3CO

OCH3

H3CO

N

COOH M7

M8

Fig. 2. The metabolic pathway of ZJ0273 in the plants.

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L. Yue et al. / Pesticide Biochemistry and Physiology 104 (2012) 44–49

Percentage in the applied (%)

30

Oilseed rape Crickweed

25

20

15

10

0

2

4

6

8

10

12

Days after treatment (d) Fig. 3. Total absorption of ZJ0273 in oilseed rape and crickweed.

was unlikely the basis of the different selectivity of ZJ0273 between oilseed rape and crickweed. In the extracts of oilseed rape and crickweed, the fraction of the parent ZJ0273 was far greater than that of its metabolites (Tables 3 and 4). One day after treatment, 14% of absorbed ZJ0273 was metabolized in the crickweed, compared to 9% in the oilseed rape. About 23% of absorbed ZJ0273 was decomposed in crickweed 12 DAT, compared to a lower percentage of 16.7% in oilseed rape. As a pro-herbicide, the decomposition rate of the parent plays a very important role in the herbicidal effect and selectivity. Preliminary experiments showed that metabolite M1, a product from the O–N type rearrangement, had no herbicidal activity (data not shown). Thus, the rearrangement of ZJ0273 could be considered as a detoxification step. However, greater or similar levels of M1 were found in crickweed than in the oilseed rape throughout

the experiment, suggesting that the formation of M1 was not the basis of the different susceptibility between the oilseed rape and crickweed. The level of M2 generally decreased with time and was not detected in crickweed 12 DAT, probably owing to its rapid transformation to further products. However, on the whole, the content of M2 in the oilseed rape increased with time and reached 0.61% of the absorbed amount, which indicated slower transformation of M2 to other intermediates. Metabolite M3 was not detected at the early time points after treatment in crickweed seedlings. However, the fraction of M3 reached a maximum of 3.26% of the absorbed amount in crickweed 8 DAT and then decreased to 2.52% 12 DAT. In the oilseed rape seedlings, M3 was not detected 1 DAT, and the fraction then increased slightly to 1.01% of the absorbed amount 12 DAT. In the seedlings of the oilseed rape, the level of M4 increased with time and reached 1.88% of the absorbed amount 12 DAT. However, M4 was not detected in crickweed throughout the experiment. These results probably demonstrated a different metabolic pathway of ZJ0273 in crickweed, as compared to the oilseed rape. The content of M5 increased with time and reached 0.76% of the absorbed amount in the oilseed rape seedling 12 DAT. At the early time points after treatment, M5 was not detected in crickweed, probably owing to the quick transformation of M5 to its further metabolites. However, the content of M5 in crickweed increased to 4.55% of the absorbed amount 12 DAT. In general, the fraction of M6 increased with time in the oilseed rape, reaching 4.85% of the absorbed radioactivity. However, no M6 was detected in the crickweed seedlings throughout the experiment. This observation again indicated that the metabolic pathway of ZJ0273 in crickweed was different from that in the oilseed rape. The formation of M6 from M7 may be considered a detoxification pathway as M6 is not an effective ALS inhibitor (data not shown). As shown in Table 3, ZJ0273 was transformed to M7 quickly in crickweed and the content of M7 reached 5.07% of the absorbed amount 1 DAT., the amount of M7 increased further to 10.41% of

Table 3 Dynamics of metabolites of ZJ0273 in crickweed seedlings. Metabolites

M M1 M2 M3 M5 M7 M8

Days after treatment 1

2

4

8

12

86.48 ± 0.58 5.39 ± 0.10b1 2.24 ± 0.21c1 Nd Nd 5.07 ± 0.07 0.82 ± 0.01

83.87 ± 0.46a 4.99 ± 0.24b1 2.24 ± 0.16c1 Nd Nd 7.22 ± 0.70d 2.81 ± 0.09e

82.92 ± 0.74a 4.88 ± 0.28b1 1.75 ± 0.15c2 Nd Nd 7.04 ± 0.17d 2.76 ± 0.22e

80.75 ± 1.49a 2.02 ± 0.23b2 1.57 ± 0.32c2 3.26 ± 0.26 Nd 8.69 ± 0.38 3.33 ± 0.39e

77.58 ± 0.96 2.36 ± 0.20b2 Nd 2.52 ± 0.24 4.55 ± 0.15 10.41 ± 0.46 2.22 ± 0.07

Nd: not detected. Values are percent of absorbed ZJ0273. The comparison was made among days after treatment of each compound. Data followed by the same letter are not significantly different according to ANOVA (P 6 0.05).

Table 4 Dynamics of metabolites of ZJ0273 in oilseed rape seedlings. Metabolites

M M1 M2 M3 M4 M5 M6 M7 M8

Days after treatment 1

2

4

8

12

91.49 ± 0.97a 0.99 ± 0.14b1 0.38 ± 0.06c1 Nd 0.78 ± 0.05 0.20 ± 0.02 1.89 ± 0.07 1.64 ± 0.30 2.99 ± 0.16

90.93 ± 0.75a 0.93 ± 0.11b1 0.39 ± 0.03c1 0.50 ± 0.02d 0.65 ± 0.02 0.47 ± 0.02f 3.59 ± 0.24g1 0.42 ± 0.04 1.43 ± 0.15

89.13 ± 0.83a 1.63 ± 0.08b2 0.53 ± 0.05c2 0.58 ± 0.03d 1.02 ± 0.10e 0.46 ± 0.02f 3.55 ± 0.51g1 0.56 ± 0.04 1.84 ± 0.23

87.34 ± 0.82 1.77 ± 0.28b2 0.58 ± 0.04c2 0.70 ± 0.02 1.09 ± 0.12e 0.52 ± 0.04f 4.70 ± 0.13g2 0.77 ± 0.05 2.12 ± 0.08

83.87 ± 0.56 1.93 ± 0.12b2 0.61 ± 0.02c2 1.01 ± 0.08 1.88 ± 0.21 0.76 ± 0.04 4.85 ± 0.09g2 2.26 ± 0.25 2.89 ± 0.10

Nd: not detected. Values are percent of absorbed ZJ0273. The comparison was made among days after treatment of each compound. Data followed by the same letter are not significantly different according to ANOVA (P 6 0.05).

L. Yue et al. / Pesticide Biochemistry and Physiology 104 (2012) 44–49

the absorbed amount 12 DAT, which was significantly greater than that in the oilseed rape seedlings (2.35%, Table 4). The levels of M7 in crickweed were higher than that for any of the other metabolites. M7 was degraded to its further metabolite which would conjugate with the plant matrix molecules to form M8. The formation of M8 may be considered a detoxification pathway for ZJ0273 as M8 was herbicidally inactive (data not shown). The content of M8 increased with time after a decrease at 2 DAT and reached 2.89% of the absorbed amount 12 DAT in the oilseed rape, which was slightly greater than that (2.22%) in the seedlings of crickweed. The metabolites in the susceptible crickweed were far more important than that in the tolerant oilseed rape in the elucidation of the herbicidal mechanism of ZJ0273. Among the metabolites of ZJ0273 in crickweed, M1 and M8 may be assumed inert and thus do not contribute to the herbicide susceptibility. In addition, the fast transformation of M2 to its further metabolites was a good indication that M2 was not an important herbicidal metabolite of ZJ0273. Moreover, M2 was not detected 12 DAT when the apparent injury of ZJ0273 on the susceptible plants was observed. On the other hand, our previous study showed that M7 was much more effective for inhibiting susceptible rice seedlings than either M3 or M5 [6]. Nezu et al. also demonstrated that M7 imposed severe inhibition on the ALS of the etiolated pea shoots, with the IC50 at 0.39 lM, compared to a far weaker inhibition of M6 on ALS with the IC50 at 100 lM [21]. Therefore, it is reasonable to consider M7 as the major herbicidal metabolite from ZJ0273, which contributed to the inhibition of the susceptible plants. Metabolite M6 is not an ALS inhibitor. Therefore, the formation of M6 in oilseed rape but not in crickweed may account for the difference in selectivity of ZJ0273 between oilseed rape and crickweed. This result provided a useful knowledge for the modification of molecular of ZJ0273 to attain a more effective and selective herbicide. Based on the metabolic pathway and the patterns of metabolite accumulation and dissipation, it may be further concluded that the difference in the amount and rate of ZJ0273 transformation to M7 was the primary mechanism for the different susceptibility between crickweed and the tolerant oilseed rape. 4. Conclusions The metabolism of ZJ0273 in oilseed rape and crickweed in the current study was of great importance in the elucidation of the mechanism of the new herbicide ZJ0273. M7, 2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid, was identified as the major herbicidal metabolite from ZJ0273, which revealed the major herbicidal mechanism of ZJ0273. The metabolism of ZJ0273 in the tolerant oilseed rape was different from that in the susceptible crickweed, which constituted the selectivity of ZJ0273. Acknowledgments The research was funded by the grant ‘‘Application of nuclear technique in low carbon and high efficient agriculture’’ from the

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Chinese Ministry of Agriculture (Grant No. 201103007), National Natural Science Foundation (Grant No. 20977076), and the project from Science and Technology Department of Zhejiang Province (Grant No. 2010R50033).

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