Studies of clomazone mode of action

https://doi.org/10.1016/j.pestbp.2005.10.002Get rights and content

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 (5-OH 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.

Introduction

Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone), 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 chloroplast-bound isoprenoids including photosynthetic pigments (phytol side chain of chlorophyll) [1], [2], [3], [4], [5], [6], [7], [8], carotenoids [2], [5], [6], [7], [8], [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 development. 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.

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-hydroxy-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 Rodríguez-Concepción 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], [25], [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], [30], [31], [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], [34], [35] and reduces clomazone metabolism [35], [36], [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-3,5-isoxazolidinedione) 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.

Section snippets

Isolation of chloroplasts

Spinach [S. olerecea var. Bloomsdale (Turner Seed)] was grown in commercial potting mix (Carolina’s 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

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

Discussion

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

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.

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