Studies of clomazone mode of action

Studies of clomazone mode of action

PESTICIDE Biochemistry & Physiology Pesticide Biochemistry and Physiology 85 (2006) 7–14 Studies of clomazone mode of ...

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PESTICIDE Biochemistry & Physiology

Pesticide Biochemistry and Physiology 85 (2006) 7–14

Studies of clomazone mode of action Yurdagul Ferhatoglu *, Michael Barrett Department of Agronomy, College of Agriculture, University of Kentucky, 105 Plant Science Building, Lexington, KY 40546-0312, USA Received 15 March 2005; accepted 11 October 2005 Available online 15 December 2005

Abstract Intact spinach chloroplasts were used to determine if clomazone, 5-OH clomazone, and/or 5-keto clomazone inhibited the chloroplastic isoprenoid pathway. When isopentenyl pyrophosphate was used as a precursor, neither clomazone nor the clomazone metabolites (5OH clomazone and 5-keto clomazone) inhibited the formation of products separated by HPLC in the organic phase. However, when pyruvate, a substrate for the first committed step of the pathway, was used as a precursor, both 5-keto clomazone and fosmidomycin reduced the formation of a non-polar product and increased the formation of a polar product in the organic phase. Only 5-keto clomazone, not 5-OH clomazone or clomazone, inhibited the formation of an additional product other than fosmidomycin in the aqueous phase from pyruvate incorporation. In an in vitro assay, 5-keto clomazone inhibited DXP synthase, the enzyme catalyzing the first committed step of the chloroplastic isoprenoid pathway. Therefore, our studies show that neither clomazone nor 5-OH clomazone inhibits the chloroplastic isoprenoid pathway, only 5-keto clomazone does.  2005 Elsevier Inc. All rights reserved. Keywords: Clomazone; 5-OH clomazone; 5-Keto clomazone; DXP synthase; Chloroplast; MEP pathway; IPP; Carotenoid; Chlorophyll

1. Introduction Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3isoxazolidinone), a soil-applied herbicide, was reported to interfere with chloroplast development and reduce or prevent accumulation of plastid pigments in susceptible species [1]. Clomazone treatment causes bleaching (white, yellow, or pale-green appearance) on plant seedlings, depending on the species and/or method and dose of treatment [2]. Clomazone inhibits the formation of chloroplastbound isoprenoids including photosynthetic pigments (phytol side chain of chlorophyll) [1–8], carotenoids [2,5– 9], electron carriers (plastoquinone) [5], tocopherol [6], and hormones (gibberellins) [4] in higher plants and in Scenedesmus acutus algae [10]. When clomazone was applied to etiolated plants, the initial effect upon placing the plants in the light is a retardation of chloroplast devel* Corresponding author. Present address: National Research Council Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Sask., Canada S7N 0W9. Fax: +1 306 975 4839. E-mail address: [email protected] (Y. Ferhatoglu).

0048-3575/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2005.10.002

opment. Disruption of chloroplast membranes occurred only after 12–24 h [11]. Clomazone had no effect on protochlorophyllide levels nor did it influence phototransformation of protochlorophyllide to chlorophyllide in both cowpea (Vigna unguiculata) [2] and pitted morningglory (Ipomoea lacunosa) [1]. However, the shibata shift, an in vivo spectral shift of protochlorophyllide absorption, is greatly slowed in both cowpea [11] and pitted morningglory [1]. Studies observed that the phytol pool is much lower in Scenedesmus cells treated with clomazone [10]. Retardation of longitudinal growth in both light and darkness by clomazone led to speculation that the herbicide inhibits synthesis of gibberellic acid. The growth-inhibiting effect of clomazone on pea [12] and corn (Zea mays) [4] seedlings can be partially reversed with exogenous gibberellic acid treatment. Herbicides blocking phytoene desaturase or later steps in the synthesis of carotenoids [13] cause abnormal accumulation of phytoene and/or phytofluene. Treatment of Scenedesmus algae [12], pitted morningglory [1], and cotton [9] with clomazone did not cause phytoene or phytofluene accumulation.


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Further studies demonstrated that clomazone treatment inhibited synthesis of geranylgeranyl pyrophosphate (GGPP) in a cell-free spinach (Spinacia oleracea) preparation but there was an increase in isopentenyl pyrophosphate (IPP) levels [10]. It was hypothesized that IPP isomerase or a prenyl transferase is inhibited by clomazone. In studies with daffodil (Narcissus sp.) chromoplasts and mustard (Sinapis alba) etioplasts, clomazone did not inhibit the activity of IPP isomerase, prenyl transferases, or phytoene synthase, even at higher clomazone concentrations that cause loss of carotenoids in intact plants [14]. Weimer et al. [15] found that clomazone did not affect the synthesis of IPP from mevalonate or the conversion of IPP to geranylgeraniol derivatives (geraniol, farnesol, and phytol) in osmotically shocked spinach chloroplasts. Clomazone itself did not have any direct effect on IPP isomerase or the biosynthesis of GGPP [8,15,16]. In other studies, the sesquiterpenoid hemigossypol and the dimeric sesquiterpenoid gossypol accumulated in clomazone-treated cotton [9]. These compounds are derived from farnesyl pyrophosphate, the 15 carbon intermediate precursor of GGPP. Clomazone had no effect on the in vitro activity of hydroxy methylglutaryl-coenzyme A reductase (HMGR) [17], the enzyme that synthesizes mevalonate which is a precursor for IPP, but feedback-regulated HMGR was stimulated 4- to 7-fold by clomazone pretreatment of 5-day-old light-grown maize seedlings [17]. Due to lack of direct clomazone effect in previous studies, bioactivation of clomazone was suspected. The most significant evidence for bioactivation comes from studies where clomazone metabolites extracted from cotton (less clomazone-tolerant compared to soybean) cell cultures were toxic to velvetleaf (Albutilon theophrasti) (clomazone sensitive). However, clomazone metabolites extracted from soybean (clomazone-tolerant) cell cultures were not toxic to velvetleaf [5]. Additionally, clomazone metabolites from both cotton and soybean cell cultures were not toxic to soybean and there was no difference in the amount of clomazone metabolism between soybean and cotton cell cultures 48 h after treatment. This implies that the tolerance observed in soybean to clomazone is due to the metabolic detoxification of clomazone rather than lack of bioactivation. Later, clomazone and potential clomazone metabolites were evaluated for IPP isomerase and prenyl transferase activity. Clomazone and the clomazone metabolites 2-chloro-benzyl alcohol, 2-chloro-benzyl aldehyde, 2-chlorobenzoic acid, and 2-chloro-4-hydroxy benzoic acid did not have any effect on in vivo or in vitro extractible IPP isomerase and prenyl transferase in tomato (extremely sensitive) and tobacco (extremely clomazone-tolerant) cell suspension cultures or on light- or dark-grown tomato or pepper cotyledons [8]. At the time those studies were conducted, it was accepted that IPP, the common precursor of all isoprenoids, was synthesized through the well-known acetate/mevalonate pathway. In this pathway, three acetyl CoA form 3-hy-

droxy-3-methylglutaryl-CoA, and then mevalonate. Subsequent phosphorylation and decarboxylation of mevalonate yields IPP [18]. However, a second IPP biosynthetic pathway (originally named non-mevalonate pathway or recently renamed methylerythritol 4-phosphate (MEP) pathway), which proceeds from glyceraldehydef 3-phosphate (G3P) and pyruvate, rather than from mevalonate, was later discovered [18]. The first two intermediates of the MEP pathway are deoxyxylulose 5-phosphate (DXP) and MEP, which are formed by DXP synthase (DXS) and DXP reductoisomerase (DXR), respectively ([19,20], Fig. 1). The other enzymatic reactions of the MEP pathway have been reviewed in Rodrı´guez-Concepcio´n and Boronat [21] and in Dubey et al. [22]. Fosmidomycin, an anti-malaria drug [23], also inhibits the second step (DXR) in the MEP pathway [24–26]. This pathway produces isoprenoids used for carotenoid, phytol, plastoquinone-9, isoprene, mono- and diterpene (Ginko and Taxus) [27], and hormones (e.g., abscisic acid) [28] in the plastid [18,29]. The acetate/mevalonate pathway produces cytoplasmic sterols, sesquiterpenes, and triterpenoids [18,29]. Some exchange of IPP or a common down-stream intermediate also appears to take place between plastids and the cytoplasm (for review, see [18,23,29–32]). The failure to identify a site of action for clomazone in previous studies may be due to the focus on the inhibition of the cytoplasmic mevalonate pathway, rather than the plastidic MEP pathway. In fact, a metabolite of clomazone, rather than the parent clomazone molecule, may be the active inhibitor. This has led to the hypothesis that a clomazone metabolite is the active inhibitor. Previous data support the hypothesis of clomazone bioactivation in cotton [6]. Phorate protects cotton from bleaching [33–35] and reduces clomazone metabolism [35–37]. It has been previously shown that clomazone is converted to OH derivatives in soybean [38], which are prime candidates to be products of a cytochrome P450 reaction. Although the metabolic pathway of clomazone in cotton has not been published, the metabolic pathway in soybean suggests that a P450 enzyme could metabolize the herbicide in cotton. Phorate, a P450 inhibitor [39], reduced the formation of a specific metabolite which cochromatographed with a clomazone metabolite 5-OH clomazone (2-[(2-chlorophenyl)methyl]-5-hydroxy-4,4-dimethyl-3-isoxazolidinone) in corn microsomes [35,36]. Additionally 5-OH clomazone is thought to be metabolized into 5-keto clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3,5isoxazolidinedione) in soybean [38]. Oxidation of clomazone to 5-OH clomazone, and then subsequently to 5-keto clomazone, was also supported by the microbial metabolism of clomazone [40]. While these studies were underway, a report was published indicating that 5-keto clomazone inhibited DXP synthase [26]. Thus, in order to get some insight into the clomazone bioactivation and the mode of action of clomazone, the effect of clomazone, 5-OH clomazone, and/or 5-keto clomazone on the chloroplastic isoprenoid pathway was evaluated.

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3 Acetyl CoA




G3P + Pyruvate DXS

5-keto clomazone

1-Deoxy-D-Xylulose-5-P (DXP)

HMG Mevalonate



2-C-Methyl-D-erythritol-4-P (MEP)






GPP (C10)

IPP Isoprene Monoterpenes

+2 IPP

FPP (C15) Sesquiterpenes 2X

GGPP (C20)


Sterols Triterpenes

Diterpenes (phytol)

Norflurazone +5IPP Carotenoids (C40)


Plastoquinone-9 (C45)

Ubiquinone Q-9 + Q-10

Fig. 1. Cytoplasmic and plastidic isoprenoid pathway. Taken from Lichtenthaler [50]. Scheme is updated by the addition of multiple enzymatic reactions as indicated by *.

2. Materials and methods 2.1. Isolation of chloroplasts Spinach [S. olerecea var. Bloomsdale (Turner Seed)] was grown in commercial potting mix (CarolinaÕs Choice from Carolina Soil) with a daylength of 14 h and temperatures of 25 C (day) and 21 C (night) in a greenhouse and watered and fertilized as needed. When seedlings were 5–15 days old, intact chloroplasts were isolated from fresh young spinach leaves with a one-step Percoll gradient [41]. The extraction medium included 330 mM sorbitol, 50 mM Tricine–KOH (pH 7.9), 2 mM EDTA, and 1 mM MgCl2. The leaves were ground with a Polytron homogenizer and the homogenate was filtered through three layers of washed cheesecloth and two layers of Miracloth. The filtrate was layered on top of 4–6 ml of 40% Percoll underlay solution prepared with 330 mM sorbitol, 50 mM Tricine–KOH (pH 7.9). The preparation was then centrifuged at 2500g for 5 min in a swinging bucket centrifuge (Sorvall HB-4). The upper layer was aspirated and the pellet was resus-

pended with 300 ll of resuspension buffer [50 mM Hepes (pH 7.9), 1 mM DTT, and 300 mM sorbitol]. All isolation steps were performed at 4 C. 2.2. Inhibition of isoprenoid biosynthesis in intact spinach chloroplasts Chloroplasts (equivalent to 0.4 mg of protein) were incubated in a total volume of 0.5 ml hypotonic incubation buffer [42] containing 100 mM Hepes (pH 8), 2 mM MnCl2, 2 mM NADP, 20 lM FAD, 5 mM NAF, 6 mM ATP, 1 mM NADPH, and either 16.8 lM [1-14C]isopentenyl pyrophosphate (IPP, ammonium salt; specific activity 57.5 lCi lmol 1, Amersham Biosciences) or 114 lM [2-14C]pyruvate (sodium salt, specific activity 17.5 lCi lmol 1, Perkin-Elmer Life Sciences) at 30 C for 1 h. A 330 mM sorbitol concentration for IPP and a 0.165 mM sorbitol concentration for pyruvate incubation provided hypotonic conditions. The incubations were placed under growth lights (46 lE m 2 s 1 PAR) and agitated gently (30 cycle min 1). Following the incubation period, the


Y. Ferhatoglu, M. Barrett / Pesticide Biochemistry and Physiology 85 (2006) 7–14

mixture was extracted twice with 1 ml of chloroform– MeOH (2:1, v/v) and the organic and aqueous layers separated. The organic phase was dried under N2 and then redissolved with 0.1 ml of ACN–MeOH–2-propanol (85:10:5, v/v/v). Radioactivity associated with both the organic phase and the aqueous phase was quantified by liquid scintillation spectrophotometry. Radioactive compounds in the organic phase were separated and quantified using a HPLC apparatus equipped with a Partisil 5 ODS-2 (5U; 4.6 · 250 mm) column and eluted with 100% ACN– MeOH–2-propanol (85:10:5, v/v) at a flow rate of 1 ml min 1 [1]. This HPLC system is suitable for the separation of various carotenes, including their cis/trans isomers and of different xanthophylls [43]. Radioactive compounds in the aqueous phase were separated with an ion pair HPLC system using an Alltech Adsorbosphere HS column (5U; 250 · 4.6 mm, Alltech Associates) [44]. The aqueous components were eluted using a ternary mobile phase consisting of 10 mM tetran-butyl ammonium acetate (TBAA) in water (solvent A), 10 mM TBAA in 70% (v/v) methanol (solvent B), and 70% methanol (solvent C). The elution program consisted of isocratic flow of 100% solvent A for 10 min, a linear gradient to 80% solvent B:20% solvent A over the next 65 min, a linear gradient to 100% solvent C over the next 10 min, and an isocratic flow of 100% solvent C for the next 10 min with a flow rate of 0.8 ml min 1. This was followed by a linear gradient to 100% solvent A for the next 5 min and an isocratic flow of 100% solvent A for another 20 min. The total elution time was 125 min. Clomazone and clomazone metabolites (5-OH clomazone, 5-keto clomazone), and herbicide norflurazone (4-chloro-5-methylamino-2-[3-(trifluoromethyl)phenyl]-3(2H)pyridazinone), or antibiotic fosmidomycin (Echelon Biosciences), all at 20 lM, were preincubated in the reaction mixture for 15 min before addition of [1-14C]IPP or [2-14C]pyruvate. Inhibitors, other than fosmidomycin, were added in ethanol to a final ethanol concentration of 0.1% (v/v). The control treatment had ethanol added to the same concentration. The fosmidomycin treatment did not contain ethanol and a treatment without ethanol was used as the fosmidomycin control. 2.3. Inhibition of heterologously expressed Catharanthus roseus DXP synthase activity Escherichia coli expressing C. roseus DXP synthase clone (XL1-blue/pTCRDXS) [42] was provided by Dr. Marc Clastre of the Laboratoire de Biologie Moleculaire et Biochimie Vegetale, Faculte de Pharmacie, 31, avenue Monge, 37200 Tours, France. Bacterial cultures were grown at 37 C for 6 h in Luria–Bertani medium containing 50 lg ml 1 ampicillin before induction with isopropylthiogalactoside (IPTG, 0.5 mM) at 25 C overnight. Following induction, cells were harvested, and a crude enzyme extract was prepared and used to measure DXP synthase activity as described by Chahed et al. [45]. DXP

synthase assays were performed with the extracts from induced E. coli cells both expressing and not expressing (vector without insert) C. roseus DXP synthase. The assay system for DXP synthase consisted of 50 mM Tris–HCl (pH 7.6), 1 mM DTT, 5 mM MgCl2,, 0.5 mM thiamine-diphosphate, 5 mM D-G3P, 5 mM [2-14C]pyruvic acid (37 kBq), and enzyme extract (equivalent to 0.5 mg of protein) in a total volume of 100 ll. G3P was omitted from the control treatments. The mixture was incubated for 2 h at



CPM Phytoene

5-OH clomazone

retention time (min)

5-Keto clomazon

CPM Clomazone

retention time (min)

Fig. 2. HPLC chromatographs of the organic phase from [1-C14]IPP-fed spinach chloroplasts treated with norflurazone, 5-OH clomazone, 5-keto clomazone, and clomazone.

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37 C and then the reaction was stopped by heating the mixture at 80 C for 3 min. After centrifugation at 12,000g for 5 min, 14C-products in the supernatant were quantified with an ion pair HPLC system as described for the chloroplast assays. Enzyme activity was expressed as pmol of deoxyxylulose synthesized per min per mg of protein. 5-OH clomazone, 5-keto clomazone, and clomazone treatment solutions were prepared in ethanol and added to the assays to a final concentration of 20 lM. Final ethanol concentration was 0.5% (v/v) volume in all treatments including the control. Assays were preincubated for 15 min before addition of substrates ([1-14C]IPP or [2-14C]pyruvate). Inhibition of C. roseus DXP synthase activity was determined by comparing the product formed and not formed from non-expressing cells. Solvent concentration had no effect on the product formation. DXP synthase enzyme activity was calculated in pmol min 1 mg 1 protein. 2.4. Statistics Both intact chloroplast feeding studies and DXP synthase inhibition assays were repeated at least two times and each treatment was replicated three times. The accompanying chromatographs are representations of at least two biological replicates and three treatment replicates.

Control 6.3 12.4


3.8 8.7


retention time (min)

Fig. 3. HPLC chromatograph of the organic phase from [2-C14]pyruvatefed spinach chloroplasts untreated (control).


3. Results 3.1. Inhibition of isoprenoid biosynthesis in intact chloroplasts Since spinach is sensitive to clomazone [5] and isolation of intact chloroplasts from spinach is established, the effect of the clomazone and the selected metabolites on the chloroplastic isoprenoids from pyruvate and IPP was addressed by using spinach chloroplasts. 14C-products formed from feeding [1-14C]IPP to intact spinach chloroplasts and partitioned into the organic phase were separated by HPLC (Fig. 2). Clomazone, and the clomazone metabolites 5-OH clomazone and 5-keto clomazone, did not affect production of these products. Only the peak eluting at 31 min in the control samples shifted to 34 min with 5-OH clomazone treatment. The peak eluting at 12 min cochromatographed with an authentic standard of eluted lutein. The other peaks were not identified. A new peak eluting at 26 min and not present in the control sample was detected in the organic phase from the norflurazone treatment. This peak cochromatographed with the phytoene standard. Accumulation of phytoene [46] demonstrated active carotenoid biosynthesis from IPP in the chloroplasts. Five 14C-products from the organic phase from 14 [2- C]pyruvate-fed chloroplasts were separated by HPLC (Fig. 3). Neither norflurazone nor clomazone and 5-OH clomazone affected the amount of radioactivity found in these peaks (Table 1). However, 5-keto clomazone and fosmidomycin increased the formation of the product eluting at 5–8 min and inhibited the formation of the product eluting at 12.4 min. Further studies with 5-keto clomazone showed that inhibition of product eluting at 12.4 min peak was dose-dependent (Fig. 4). No product formed from [2-14C]pyruvate feeding in initial experiments using a 330 mM sorbitol concentration in the reaction buffer. In later studies, using 0.165 mM sorbitol, chloroplasts fed pyruvate formed nine 14C-products which partitioned into the aqueous phase (Fig. 5). 5-Keto clomazone prevented production of the compounds eluting

Table 1 Distribution of 14C associated peaks in organic phase from [2-C14]pyruvate-fed chloroplasts treated with norflurazone, 5-OH clomazone, 5-keto clomazone, clomazone, and fosmidomycin Treatment Retention times (min):

Distribution of %


C (% of total peaks)a,b





Control +Norflurazone +5-OH clomazone +5-keto clomazone +Clomazone

16 ± 4 15 ± 2 12 ± 2 14 ± 1 16 ± 7

44 ± 1 47 ± 6 49 ± 4 65 ± 3 45 ± 2

a a a b a

10 ± 2 9±3 6±2 8±1 7±3

24 ± 3 24 ± 3 25 ± 3 6±6 22 ± 1

Control +Fosmidomycin

13 ± 6 15 ± 2

47 ± 5 a 61 ± b

9±1 7±2


21.6 a a a b a

3±3 6±2 8±2 9±4 11 ± 3

25 ± 5 a 8±3 b

7±2 9±3

Means ± SD. Means within the same column followed by different letters are significantly different at P < 0.05, according to FisherÕs LSD (Analyse-it Software, Leeds, UK). b

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% Incorporation of Total Peaks



Table 2 Inhibition of C. roseus DXP synthase activity by 5-keto clomazone


DXP synthasea

5-Keto treatment (lM)

DXP synthase activity (pmol min 1 mg 1 protein)

Inhibition (% of untreated control)

+ +

0 20 0 20

60 ± 18 50 ± 13 1700 ± 105 900 ± 53*

— 17 — 47

10 8 6 4 2 0 0


1.5 15 150 1500 15000 5-keto clomazone concentration (nM)

Fig. 4. Effect of 5-keto clomazone on the radioactivity associated with the peak eluting at 12.4 min from [2-C14]pyruvate-fed spinach chloroplasts. Results are means ± SD.



48.6 30.2

63.4 54.4


67.9 69.9 66.3 78



a , Extracts from E. coli cells transformed with vector minus the DXP synthsase gene from C. roseus. +, Extracts from E. coli cells transformed with vector plus the DXP synthase gene from C. roseus. * Significant difference by ANOVA (P < 0.05) compared to the untreated chloroplast within same column.

one inhibited formation of an additional product other than fosmidomycin in the pathway. 3.2. Inhibition of heterologous expressed C. roseus DXP synthase activity DXP synthase activity in crude extracts from E. coli expressing the DXP synthase gene from C. roseus was inhibited 47% by 20 lM 5-keto clomazone (Table 2). The inhibition of DXP synthase was more severe with the plant DXP synthase as the bacterial DXP synthase activity was only inhibited 17% (Table 2). Neither clomazone (20 lM) nor 5-OH clomazone (20 lM) inhibited the DXP synthase activity (data not shown). 4. Discussion


CPM 63.2 54.2


3.8 29.9


5-Keto clomazone 48.3

63.4 54.3 79.3

retention time (min)

Fig. 5. HPLC chromatographs of the aqueous phase from [2-14C]pyruvate-fed chloroplasts treated with fosmidomycin and 5-keto clomazone.

at 66.3, 67.9, and 69.9 min (Fig. 5). No inhibition of these peaks was seen in the 5-OH clomazone or clomazone treatments (data not shown). Fosmidomycin inhibited formation of the products eluting at 66.3 and 69.9 min but not the product eluting at 67.9 min. As fosmidomycin inhibits the second step of the chloroplastic isoprenoid biosynthetic pathway, DXP reductoisomerase [47] and 5-keto clomaz-

The herbicide clomazone and its metabolite 5-OH clomazone, that typically bleach sensitive plants like cotton ([48], unpublished data), did not inhibit the MEP pathway in spinach chloroplasts in our studies. However, 5-keto clomazone, which is proposed to originate from 5-OH clomazone in soybean [39], inhibited the MEP pathway between pyruvate, G3P, and IPP in this study. This had been suggested in an earlier study by Zeidler et al. [49] which showed that 5-keto clomazone inhibited the production of isoprene in different plant species. Our studies with C. roseus DXP synthase confirm the results of a previous study with Chlamydomonas [26], which showed inhibition of DXP synthase with 5-keto clomazone. Inhibition of chlorophyll and carotenoids formation by 10 5 M clomazone and 5-keto clomazone was reversed by 5 mM DXP in barley [49]. Our studies also show that neither clomazone nor 5OH clomazone inhibits the synthesis of a chloroplastic isoprenoid, or DXP synthase in the MEP pathway, as was shown in an earlier study [26], only 5-keto clomazone does. These results also support the involvement of ER-bound P450 enzymes in the bioactivation of clomazone to 5-OH clomazone [35] because clomazone itself was not active in chloroplasts, and suggest that 5-keto

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clomazone is not synthesized within the chloroplasts from 5-OH clomazone or directly from clomazone. Therefore, our studies support the toxicity and bleaching observed in susceptible plants treated with clomazone are due to the metabolic activation of clomazone to 5-OH clomazone, and subsequently to 5-keto clomazone, the active toxicant. Acknowledgments The authors thank Dr. Marc Clastre from the Laboratoire de Biologie Moleculaire et Biochimie Vegetale, Faculte de Pharmacie for providing the construct expressing C. roseus DXP synthase (XL1-blue/TCRDXS). This work was supported by the Higher Education Council of Turkey (YOK) fellowship to Y. Ferhatoglu and FMC Corp. grant. References [1] S.O. Duke, W.H. Kenyon, R.N. Paul, FMC 57020 effects on chloroplast development in pitted morningglory (Ipomea lacunose) cotyledons, Weed Sci. 33 (1985) 786–794. [2] S.O. Duke, W.H. Kenyon, Effects of dimethazone (FMC 57020) on chloroplast development. II. Pigment synthesis and photosynthetic function in cowpea (Vigna unguiculata L.) primary leaves, Pestic. Biochem. Physiol. 25 (1986) 11–17. [3] A.J. Goetz, D.E. Westberg, T.L. Lavy, R.L. Oliver, Uptake, translocation, and metabolism of clomazone in soybeans (Glycine max), sicklepod (Cassica obtusifulia), common cocklebur (Xanthium strumanium) and velvetleaf (Abutilon theophrasti). Thesis. Department of Agronomy, University of Arkansas, Fayetteville, AR 72703, 1988. [4] W.K. Vencill, K.K. Hatzios, H.P. Wilson, Growth and physiological responses of normal, dwarf, and albino corn (Zea mays L.) to clomazone treatment, Pestic. Biochem. Physiol. 35 (1989) 81–88. [5] M.A. Norman, R.A. Liebl, J.M. Widholm, Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures, Plant Physiol. 94 (1990) 704–709. [6] M.A. Norman, R.A. Liebl, J.M. Widholm, Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotropic cell suspension cultures, Plant Physiol. 92 (1990) 777–784. [7] J.E. Scott, L.A. Weston, Cole crop (Brassica oleracea) tolerance to clomazone, Weed Sci. 40 (1992) 7–11. [8] J.E. Scott, L.A. Weston, J. Chappell, K. Hanley, Effects of clomazone on IPP isomerase and prenyl transferase activities in cell suspension cultures and cotyledons of solanaceous species, Weed Sci. 42 (1994) 509–516. [9] S.O. Duke, R.N. Paul, J.M. Becerril, J.H. Schmidt, Clomazone causes accumulation of sesquiterpenoids in cotton (Gossypium hirsutum L.), Weed Sci. 39 (1991) 339–346. [10] G. Sandman, P. Boger, Interconvension of prenyl pyrophosphates and subsequent reactions in the presence of FMC 57020, Z. Naturforsch. C 42 (1987) 803–807. [11] S.O. Duke, R.N. Paul, Effect of dimethazone (FMC-57020) on chloroplast development. 1. Ultrastructural effects in cowpa (Vigna unguiculata L.) primary leaves, Pestic. Biochem. Physiol. 25 (1986) 1–10. [12] G. Sandman, P. Boger, Interference of dimethazone with formation of terpenoid compounds, Z. Naturforsch. C 41 (1986) 729–732. [13] S.M. Ridley, Carotenoids in herbicide action in carotenoid chemistry and biochemistry, in: C. Britton, T.W. Goodwin (Eds.), IUPAC Carotenoid Chemistry and Biochemistry, Pergamon Press, Oxford, UK, 1982, pp. 353–369. [14] M. Lutzov, P. Beyer, H. Kleining, The herbicide Command does not inhibit the prenyl diphosphate-forming enzymes in plastids, Z. Naturforsch. C 45 (1990) 856–858.


[15] M.R. Weimer, N.E. Balke, D.D. Buhler, Herbicide clomazone does not inhibit in-vitro geranylgeranyl synthesis from mevanolate, Plant Physiol. 98 (1992) 427–432. [16] R. Croteau, Clomazone does not inhibit the conversion of isopentenyl pyrophosphate to geranyl, farnesyl, or geranyl geranyl pyrophosphate in-vitro, Plant Physiol. 98 (1992) 1515–1517. [17] W. Ji, K.K. Hatzios, Regulation of HMG-CoA reductase in corn by selected herbicides, Proc. South. Weed Sci. 43 (1990) 350–354. [18] H.K. Lichtenthaler, The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 47–65. [19] S. Takahashi, T. Kuzuyama, H. Watanabe, H. Seto, A 1-deoxy-Dxylulose 5-phosphate reductoisomerase catalyzing the formation of 2C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis, Proc. Natl. Acad. Sci. USA 95 (1998) 9879–9884. [20] B.M. Lange, R. Croteau, Isoprenoid biosynthesis via a mevalonateindependent pathway in plants: cloning and heterolous expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from peppermint, Arch. Biochem. Biophys. 365 (1999) 170–174. [21] M. Rodrı´guez-Concepcio´n, A. Boronat, Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics, Plant Physiol. 130 (2002) 1079–1089. [22] V.S. Dubey, R. Bhalla, R. Luthra, An overview of the nonmevalonate pathway for terpenoid biosynthesis in plants, J. Biosci. 28 (2003) 637–646. [23] H. Jomaa, J. Wiesner, S. Sanderbrand, B. Altincicek, C. Weidemeyer, M. Hintz, I. Turbacova, M. Eberl, J. Zeidler, H.K. Lichtenthaler, Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs, Science 285 (1999) 1573–1576. [24] J.G. Zeidler, J. Schwender, C. Muller, J. Wiesner, C. Weidemeyer, E. Beck, H. Jomaa, H.K. Lichtenthaler, Inhibition of the non-mevalonate 1-deoxy-D-xylulose-5-phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin, Z. Naturforsch. C 53 (1998) 980–986. [25] J. Schwender, C. Muller, J. Zeidler, H.K. Lichtenthaler, Cloning and heterologous expression of a cDNA encoding 1-deoxy-D-xylulose-5phosphate reductoisomerase of Arabidopsis thaliana, FEBS Lett. 455 (1999) 140–144. [26] C. Mueller, J. Schwender, J. Zeidler, H.K. Lichtenthaler, Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis, Biochem. Soc. Trans. 28 (2000) 792–793. [27] W. Eisenreich, B. Menhard, P.J. Hylands, M.H. Zenk, A. Bacher, Studies on the biosynthesis of taxol: the taxene carbon skeleton is not of mavalonoid origin, Proc. Natl. Acad. Sci. USA 93 (1996) 6431–6436. [28] B.V. Milborrow, H.S. Lee, Endogenous biosynthesis precursors of (+)-abscissic acid. VI. Carotenoids and ABA are formed by the ‘‘nonmevalonate’’ triose-pyruvate pathway in chloroplasts, Aust. J. Plant Physiol. 25 (1998) 507–512. [29] H.K. Lichtenthaler, J. Schwender, A. Disch, M. Rohmer, Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate independent pathway, FEBS Lett. 400 (1997) 271–274. [30] M. Rohmer, Isoprenoid biosynthesis via the mevalonate-independent route, a novel target for antibacterial drugs, Prog. Drug Res. 50 (1998) 135–154. [31] H. Kasahara, K. Takei, N. Ueda, S. Hishiyama, T. Yamaya, Y. Kamiya, S. Yamaguchi, H. Sakakibara, Distinct isoprenoid origins of cis- and trans-zeatin biosyntheses in Arabidopsis, J. Biol. Chem. 279 (2004) 14049–140054. [32] V.M. Sponsel, The deoxyxylulose phosphate pathway for the biosynthesis of plastidic isoprenoids: early days in our understanding of the early stages of gibberellin biosynthesis, J. Plant Growth Regul. 20 (2001) 332–345. [33] M.A. Harrison, R.M. Hayes, Utilization of command with selected in-furrow insecticides in no-till and conventional cotton, Proc. South Weed Sci. Soc. 41 (1992) 44–48.


Y. Ferhatoglu, M. Barrett / Pesticide Biochemistry and Physiology 85 (2006) 7–14

[34] R.M. Hayes, P.A. Bradley, G.L. Lentz, Insect control and cotton injury with clomazone-insecticide combinations, Proc. South Weed Sci. Soc. 46 (1993) 33–36. [35] Y. Ferhatoglu, M. Barrett, S. Avduishko, The basis for the safening of clomazone by phorate insecticide in cotton and inhibitors of cytochrome P450s, Pestic. Biochem. Physiol. 81 (2005) 59–70. [36] Y. Ferhatoglu, S. Avduishko, M. Barrett, Phorate inhibition of clomazone metabolism, North Central Weed Sci. Soc. Am. Abstr. (2000). [37] A.S. Culpepper, A.C. York, J.L. Marth, F.T. Corbin, Effect of insecticide on clomazone absorption, translocation, and metabolism in cotton, Weed Sci. 49 (2001) 613–616. [38] S.F. ElNaggar, R.W. Creekmore, M.J. Schocken, R.T. Rosen, R.A. Robinson, Metabolism of clomazone herbicide in soybean, J. Agric. Food Chem. 40 (1992) 880–883. [39] R.J. Baerg, M. Barrett, N. Polge, Insecticide and insecticide interactions with cytochrome P450 mediated activities in maize, Pestic. Biochem. Physiol. 55 (1996) 10–20. [40] S.Y. Liu, M. Shocken, J.P.N. Rosazza, Microbial transcformation of clomazone, J. Agric. Food Chem. 44 (1996) 313–319. [41] W.R. Mills, K.W. Joy, A rapid method for isolation of purified physiological active chloroplasts, used to study intracellular distribution of amino acids in pea leaves, Planta 148 (1980) 75–83. [42] M. Fellermeier, K. Kis, S. Sagner, U. Maier, A. Bacher, M.H. Zenk, Cell free conversion of 1-deoxy-D-xylulose 5-phosphate and 2-C-methyl-D-erythritol 4-phosphate into carotene in higher plants and its inhibition by fosmidomycin, Tetrahedron Lett. 40 (1999) 2743–2746.

[43] S. Ernst, G. sandman, Poly-cis carotene pathway in the Scenedesmus mutant C-6D, Arch. Microbiol. 150 (1988) 590–594. [44] D. McCaskill, R. Croteau, Procedures for the isolation and quantification of the intermediates of the mevalonic acid pathway, Anal. Biochem. 215 (1993) 142–149. [45] K. Chahed, K.A. Oudin, N. GuivarcÕh, J.-C. Chenieux, M. Rideau, M. Clastre, 1-Deoxy-D-xylulose 5-phosphate synthase from periwinkle: cDNA identification and induced gene expression in terpenoid indole alkaloid-producing cells, Plant Physiol. Biochem. 38 (2000) 559–662. [46] M.P. Mayer, D.L. Bartlett, P. Beyer, H. Kleining, The in vitro mode of action of bleaching herbicides on the desaturation of 15-cisphytoene and ciscarotene in isolated daffodil chromoplasts, Pestic. Biochem. Physiol. 34 (1989) 11–117. [47] J. Schwender, C. Mueller, J. Zeidler, H.K. Lichtenthaler, Cloning and heterologous expression of a cDNA encoding 1-deoxy-D-xylulose-5phosphate reductoisomerase of Arabidopsis thaliana, FEBS Lett. 455 (1999) 140–144. [48] J.H. Chang, M.J. Konz, E.A. Aly, R.E. Sticker, K.R. Wilson, N.E. Krog, P.E. Diockinson, 3-Isoxazolidinones and related compounds a new class of herbicides, in: D.R. Baker, J.G. Fenyes, W.K. Moberg, B. Cross (Eds.), Synthesis and Chemistry of Agrochemicals, ACS Symposium Series 355, American Chemical Society, Washington, DC, 1987, pp. 10–23. [49] J. Zeidler, J. Schwender, C. Mueller, H.K. Lichtenthaler, The nonmevalonate isoprenoid biosynthesis of plants as a test system for drugs against malaria and pathogenic bacteria, Biochem. Soc. Trans. 28 (2000) 796–798. [50] H.K. Lichtenthaler, Isopredonid biosynthesis: enzymes, genes and inhibitors, Biochem. Soc. Trans. 28 (2000) 785–789.