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Differential Oxidative Metabolism and 5-Ketoclomazone Accumulation Are Involved in Echinochloa phyllopogon Resistance to Clomazone1[C][W][OA] Hagai Yasuor*, Wei Zou, Vladimir V. Tolstikov, Ronald S. Tjeerdema, and Albert J. Fischer Weed Science Program, Department of Plant Sciences (H.Y., A.J.F.), Genome Center (W.Z., V.V.T.), and Department of Environmental Toxicology (R.S.T.), University of California, Davis, California 95616

Echinochloa phyllopogon (late watergrass) is a major weed of California rice (Oryza sativa) that has evolved cytochrome P450mediated metabolic resistance to different herbicides with multiple modes of action. E. phyllopogon populations from Sacramento Valley rice fields have also recently shown resistance to the herbicide clomazone. Clomazone is a proherbicide that must be metabolized to 5-ketoclomazone, which is the active compound that inhibits deoxyxylulose 5-phosphate synthase, a key enzyme of the nonmevalonate isoprenoid pathway. This study evaluated the differential clomazone metabolism within strains of the same species to investigate whether enhanced oxidative metabolism also confers clomazone resistance in E. phyllopogon. Using reverse-phase liquid chromatography-tandem mass spectrometry techniques in the multireaction monitoring mode, we elucidated that oxidative biotransformations are involved as a mechanism of clomazone resistance in this species. E. phyllopogon plants hydroxylated mostly the isoxazolidinone ring of clomazone, and clomazone hydroxylation activity was greater in resistant than in susceptible plants. The major clomazone metabolites resulted from monohydroxylation and dihydroxylation of the isoxazolidinone ring. Resistant plants accumulated 6- to 12-fold more of the monohydroxylated metabolite than susceptible plants, while susceptible plants accumulated 2.5-fold more of the phytotoxic metabolite of clomazone, 5-ketoclomazone. Our results demonstrate that oxidative metabolism endows multiple-herbicide-resistant E. phyllopogon with cross-resistance to clomazone through enhanced herbicide degradation and lower accumulation of the toxic metabolite in resistant versus susceptible plants.

Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl3-isoxazolidinone) has been used for the last 4 years in California to control Echinochloa phyllopogon, a major weed of rice (Oryza sativa), and other grass weeds in rice. To be active, clomazone must be metabolically converted to the active 5-ketoclomazone, which inhibits deoxyxylulose 5-phosphate (DXP) synthase, the first committed step of the nonmevalonate isoprenoid pathway, leading to the biosynthesis of isopentenyl pyrophosphate in plastids (Ferhatoglu and Barrett, 2006). This results in impaired chloroplast development and pigment loss in susceptible plants (Duke and Paul, 1986). When carotenoids are absent and plants are exposed to light, singlet oxygen and triplet chlorophyll degrade chlorophyll and initiate membrane 1

This work was supported by the California Rice Research Board and by a Pilot Project Grant provided by the Genome Center, University of California at Davis. * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hagai Yasuor ([email protected]). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.110.153296

lipid peroxidation (Hess, 2000). Clomazone is absorbed by roots and emerging shoots and is transported with the transpiration stream in the xylem (Senseman, 2007). Populations of this species have evolved resistance, after repeated herbicide use, to multiple herbicides from different chemical groups and with different modes of action (Fischer et al., 2000a). These include the lipid synthesis inhibitors molinate and thiobencarb; the acetyl-CoA carboxylase inhibitors fenoxapropethyl and cyhalofop; the acetolactate synthase inhibitors bispyribac-sodium, bensulfuron-methyl, and penoxsulam; and the DXP synthase inhibitor clomazone (Fischer et al., 2000a; Yasuor et al., 2008, 2009). Ratios of the GR50 (herbicide dose to inhibit growth by 50%) values of resistant to susceptible E. phyllopogon plants of approximately 2 indicated low levels of clomazone resistance (Yasuor et al., 2008). This resistance was not caused by differential uptake, translocation, or bioactivation of inactive clomazone to active 5-ketoclomazone (Yasuor et al., 2008). Based on inhibitor data, Fischer et al. (2000b) suggested that herbicide degradation mediated by cytochrome P450 monooxygenases (EC 1.14.14.1 [P450]) was a mechanism conferring E. phyllopogon resistance to multiple herbicides. Later, Yun et al. (2005) showed that the multipleherbicide-resistant E. phyllopogon biotype had greater P450 content and inducible monooxygenating activity toward these herbicides than a susceptible biotype.

Plant PhysiologyÒ, May 2010, Vol. 153, pp. 319–326, www.plantphysiol.org Ó 2010 American Society of Plant Biologists

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Using P450 inhibitors and conducting metabolic profiling with [ 14C]penoxsulam, we recently demonstrated that herbicide-resistant E. phyllopogon was also cross-resistant to penoxsulam through enhanced P450-mediated detoxification ability (Yasuor et al., 2009). The Glc conjugate of the 2-chlorobenzyl alcohol is a major plant metabolite of clomazone in naturally resistant crops. It is produced by N-dealkylation that causes cleavage between the isoxazolidinone and aromatic rings (Norman et al., 1990; Wiemer et al., 1991; ElNaggar et al., 1992; Schocken, 1997). Other metabolites result from the monohydroxylation of clomazone on either the aromatic or the isoxazolidinone ring (ElNaggar et al., 1992; Fig. 1). Hydroxylation at the 5-methylene carbon and at the methyl group of the isoxazolidinone ring (yielding 5-hydroxyclomazone and hydroxymethylclomazone, respectively) and hydroxylation on the aromatic ring at the 3# carbon position (resulting in 3#-hydroxyclomazone) have been reported as major processes involved in the microbial biotransformation of clomazone (Liu et al., 1996; Fig. 1). Differential bioactivation of clomazone by metabolic conversion to its toxic form has also been suggested as a mechanism of selectivity among plants. Thus, Norman et al. (1990) concluded that soybean (Glycine max) tolerance to clomazone may involve

lower rates of bioactivation to 5-ketoclomazone than in cotton (Gossypium hirsutum). Therefore, measuring differential accumulation of the presumed active metabolite 5-ketoclomazone should allow differentiation of clomazone sensitivity levels among certain plants. The oxidated metabolic fate of clomazone in microorganisms and certain plants (ElNaggar et al., 1992; Liu et al., 1996; Siminszky, 2006) suggests P450 involvement. The inhibition of clomazone metabolism by P450 inhibitors to the active 5-ketoclomazone form was studied by Ferhatoglu et al. (2005). Blocking this bioactivation with P450-inhibiting organophosphate insecticides protected cotton and E. phyllopogon from clomazone damage (Culpepper et al., 2001; Ferhatoglu et al., 2005; Yasuor et al., 2008). Alternatively, inhibition of P450-mediated herbicide detoxification usually leads to increased herbicide toxicity (synergism). Thus, Fischer et al. (2000b) reduced or eliminated resistance to various herbicides by pretreating E. phyllopogon plants with P450 inhibitors, such as malathion and piperonylbutoxide. The use of P450 inhibitors has failed to clarify the role of enhanced metabolism in E. phyllopogon resistance to clomazone (Yasuor et al., 2008), presumably because the inhibitors used (1-aminobenzotriazole and disulfoton) lacked the ability to selectively inhibit P450 isozymes involved in

Figure 1. Proposed clomazone biotransformation routes in clomazoneresistant and -susceptible E. phyllopogon strains inferred from metabolite levels detected in plant tissues using RP-LCMS/MS. Roman numerals correspond to compounds detected in this study and listed in Table I. Although all metabolites were found in both strains, red and black arrows point at preferential metabolite accumulation by resistant and susceptible plants, respectively; the dotted arrow indicates a transformation (not numbered) not found in our experiments. Compounds VII, VIII, and IX result from hydroxylation in different unidentified positions of the aromatic ring. Structural information is based on analytical standards, the LightSight 2.0 metabolite identification algorithm, and according to ElNaggar et al. (1992), Liu et al. (1996), and TenBrook and Tjeerdema (2006). All transformations, except the hydrolytic conversion of compound II to X, are presumed to be P450 mediated. [See online article for color version of this figure.]

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Oxidative Metabolism, 5-Ketoclomazone, and Clomazone Resistance

between resistant and susceptible E. phyllopogon plants were characterized and quantified.

bioactivation or in the detoxification of clomazone or, alternatively, perhaps P450 enzymes were not involved. Cytochrome P450 inhibitors are known to differ in isozyme specificity (Werck-Reichhart et al., 2000). Therefore, more detailed studies on clomazone metabolism are needed to elucidate if enhanced P450-mediated oxidative detoxification contributes toward clomazone resistance in multiple-herbicide-resistant E. phyllopogon biotypes. In addition to P450-related biotransformation and conjugation with sugars, clomazone can also be detoxified in plants via conjugation with glutathione (Norman et al., 1990; Vencill et al., 1990a, 1990b; Wiemer et al., 1991; ElNaggar et al., 1992). To date, no natural alteration of the target site of clomazone (DXP synthase) has been reported in plants, although the possibility of endowing clomazone resistance through overproduction of DXP synthase was demonstrated by expressing this enzyme under the control of the cauliflower mosaic virus 35S promoter in transgenic Arabidopsis (Arabidopsis thaliana; CarreteroPaulet et al., 2006). Mass spectrometry (MS) can be useful in the structural elucidation of clomazone biotransformation products, since clomazone and its major metabolites have a chlorine atom in their structures, which provide characteristic mass-to-charge ratio (m/z) +2 isotope peaks of single chlorine-containing fragments (Liu et al., 1996). We evaluate here differential clomazone metabolism within plants of the same species by comparing the clomazone metabolite profile of multiple-herbicide (including clomazone)-resistant and -susceptible E. phyllopogon. The procedure involved using reverse-phase liquid chromatography-tandem mass spectrometry (RP-LC-MS/MS) analysis in the multireaction monitoring (MRM) mode to identify the biotransformation products. Thus, differences in clomazone metabolism

RESULTS AND DISCUSSION

Differential clomazone metabolism was investigated in E. phyllopogon strains that were either resistant or susceptible to herbicides for grass control in rice, including clomazone. Using the MRM-LC-MS/MS acquisition mode, we screened for a wide range of possible clomazone metabolites suggested by the biotransformation data set and the predictive algorithm of the LightSight software and also for known clomazone biotransformation products (ElNaggar et al., 1992; Ferhatoglu et al., 2005; TenBrook and Tjeerdema, 2006) and in microorganisms (Liu et al., 1996). Thus, a group of mostly oxidative (phase I) and conjugation (phase II) clomazone transition products was identified for screening. A survey scanning conducted in MRM mode for these specific compounds yielded a clomazone degradation profile in plants and in their hydroponic growth medium. Growth medium samples were assayed to establish if certain metabolites detected in clomazone-treated plants could have originated from clomazone degradation in the hydroponic solution prior to plant uptake. Compounds in Table I correspond to clomazone (I) and all its metabolites (II– XIII) identified in plant extracts. The first value in the MRM characterization corresponds to the target ion identified in the first MS/MS quadrupole (Q1) and represents a biotransformation product of the parent clomazone (m/z 240), and the second value is for a specific associated fragment ion identified with the third quadrupole (Q3) from the MS/MS spectrum of the target ion. A principal component analysis of all identified metabolites clearly showed that clomazone was metabolized differently between resistant and

Table I. Abundance (peak area) of clomazone and metabolites in tissue extracts of herbicide-susceptible and -resistant E. phyllopogon plants 48 and 96 h after hydroponic clomazone (50 mM) treatment, MRM transitions, and retention times (RT) were obtained using RP-LC-MS/MS analysis Statistical analysis was conducted on ln-transformed data in order to normalize variances; transformed means are presented for convenience. Within rows, means accompanied by the same letter do not differ according to Tukey’s honestly significant difference test with P = 0.05. No.

MRM Transition m/z

min

I II III IV V VI VII VIII IX X XI XII XIII

240/125 254/125 256/125

10.20 10.40 8.04 9.13 9.01 9.10 9.40 8.22 7.93 8.62 8.08 8.04 8.08

256/141

272/125 268/125 320/125


RT

Susceptible

Resistant

48 h

96 h

17.78 a 13.90 b 13.32 d 11.32 a 12.30 b 13.93 c 12.93 ab 14.52 a 13.98 a 14.81 bc 11.11 d 8.96 d 7.36 c

17.75 a 14.30 a 14.68 c 11.93 a 12.37 b 14.84 b 13.07 a 14.77 a 14.52 a 15.04 a 12.83 c 10.10 c 9.03 b

48 h

96 h

17.85 a 13.90 b 15.84 b 12.20 a 13.50 a 14.75 b 12.09 c 13.45 c 13.31 b 15.03 ab 13.25 b 11.24 b 9.52 b

17.83 a 13.47 c 16.40 a 12.26 a 13.19 a 15.35 a 12.32 c 13.92 b 13.26 b 14.61 c 14.42 a 11.87 a 10.53 a

ln peak area

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susceptible plants and that metabolites in plant extracts were different from those in the growth medium (Fig. 2A). Three principal components explained 53%, 17%, and 16% of the variability. Using the first and third principal components, we identified three major groupings corresponding to metabolites in resistant plants, susceptible plants, and those found in the growth medium (Fig. 2A). The clomazone parent compound peak was removed from the analysis to facilitate cluster separations, since treated plants were continuously exposed to clomazone throughout the assays and metabolic profiles always included similar and large amounts of parent clomazone. Clomazone Metabolism in E. phyllopogon

According to the principal component analysis, differences in the overall clomazone metabolite profile between resistant and susceptible plants were already evident by 2 d after clomazone was added to the growth medium and became even more distinct by 4 d (Fig. 2A). The contribution of specific compounds toward this metabolite profile differentiation between resistant and susceptible plants was characterized (Fig. 2B). As would also happen in nonsterile rice paddies, where clomazone is applied as a granular formulation onto the soil surface, root uptake of certain metabolites that were in the growth medium could have conceivably contributed to their presence in plant tissues (Supplemental Files S2 and S3).

Major Metabolites Differentiate Clomazone-Resistant from -Susceptible Plants

The qualitative composition of the clomazone metabolite profile was similar in both strains, but certain major clomazone metabolites were more abundant in plant tissues and accumulated at higher rates in resistant than in susceptible plants (P , 0.05; Table I). The data suggest that hydroxylation of the isoxazolidinone moiety of the clomazone molecule is a major mechanism of enhanced clomazone detoxification by resistant E. phyllopogon plants (Fig. 1). Compound III was the most abundant clomazone metabolite in resistant plants. They accumulated 12 and six times more of this compound than susceptible plants by 48 and 96 h, respectively, after clomazone addition (Table I, Fig. 3). LC-MS/MS structure elucidation suggested that compound III results from the monohydroxylation of the clomazone isoxazolidinone moiety (Fig. 1). It is presumably the hydroxymethylclomazone identified by ElNaggar et al. (1992) and Liu et al. (1996) as a soybean and soil microbial metabolite of clomazone. Although it shares the same mass fragmentation, higher polarity differentiates compound III from 5-hydroxyclomazone (IV), which was the most common clomazone degradation product found in soybean by ElNaggar et al. (1992). Another major metabolite (XI) resulted from a clomazone transformation involving a mass gain of 32 (Table I), suggesting a dihydroxylation in the isoxazolidinone moiety of the parent compound (Fig. 1). Resis-

Figure 2. Differential clomazone metabolism in resistant and susceptible E. phyllopogon plants and in their growth media according to a principal component analysis of the clomazone metabolite profile. A, Score plot where two subclusters (dotted lines) within each major cluster separated metabolism after 48 and 96 h of incubation; a third group in the growth medium cluster corresponds to metabolism in the absence of resistant (red open symbols) or susceptible (blue open symbols) plant roots. Each data point corresponds to one replicate sample. B, Loading plot demonstrating the contribution of individual clomazone metabolites found in plant extracts (Roman numerals correspond to compounds listed in Table I) to the clustering shown in A. The length and direction of each vector indicates the strength and type (positive or negative) of the correlation between specific metabolites and the principal components. [See online article for color version of this figure.] 322


Oxidative Metabolism, 5-Ketoclomazone, and Clomazone Resistance Figure 3. Clomazone metabolites from herbicideresistant (R) and herbicide-susceptible (S) E. phyllopogon plant extracts resolved by RP-LC-MS/MS analysis conducted at 48 and 96 h (expanded graphs) after hydroponic treatment with 50 mM clomazone. Roman numerals correspond to compounds listed in Table I and Figure 1. The major clomazone (I) transformation products detected consisted of hydroxylations of the isoxazolidinone moiety of the clomazone molecule (III and XI), hydroxylations on the aromatic ring moiety (VI, VIII, and IX), an unidentified transition (XII), and 5-ketoclomazone, which was found either as an isoxazolidinone ring oxidation product (II) or as a presumed open ring derivative (X). Both ketoclomazone forms were more abundant in susceptible than in resistant plant extracts. [See online article for color version of this figure.]

tant plants accumulated from eight to five times more of this metabolite than susceptible plants by 48 and 96 h after clomazone addition, respectively (Table I; Fig. 3). To the best of our knowledge, this is the first report of a possible dihydroxylation of the isoxazolidinone ring of clomazone. There are other dihydroxylations at the aromatic ring (Fig. 1; Liu et al., 1996; TenBrook and Tjeerdema, 2006). No such metabolites were found in our plant extract or growth medium assays. The only quantitative difference in growth medium assays was the greater (P , 0.05) amount of a isoxazolidinone ring hydroxylate (V) in the solution incubated with resistant E. phyllopogon roots, which paralleled the 2- to 3-fold accumulation of this metabolite in resistant versus susceptible plant extracts (Table I; Fig. 1). Since the seeds had been sterilized prior to incubation and seedlings were grown in an initially axenic growth medium common to both resistant and susceptible plants, compound V could be a product of differential plant hydroxylation that was released to a significant extent by roots into the growth medium. Resistant plant roots could also have induced greater proliferation of eventual microbial contaminants producing this metabolite and releasing it for plant uptake. Compared with susceptible plants, resistant plants accumulated greater amounts (approximately 2-fold) of an aromatic ring hydroxylate (VI) with a mass shift and a retention time matching those of the 3#-hydroxyclomazone standard (Table I; Fig. 1). Compound VI is likely a microbial metabolite, since it was one of the most abundant metabolites in the growth medium incubated without plant roots (for data on growth medium metabolites, see Supplemental Files S2 and S3). Assuming similar root uptake rates by these strains (Yasuor et al., 2008), Plant Physiol. Vol. 153, 2010

greater detection of compound VI in resistant plants should result from differential hydroxylation rates between resistant and susceptible plants. In addition to the major metabolites discussed so far, certain low-abundance unidentified isoxazolidinone ring transformations were also preferentially detected in resistant plant extracts. Metabolite XII accumulated approximately 10- and 6-fold more in resistant than in susceptible plants by 48 and 96 h after clomazone addition, respectively (Table I; Fig. 3) and could correspond to the addition of either a CO group (Holcˇapek et al., 2008) or two ketone groups to the isoxazolidinone ring. These structures do not fit other known plant or microbial clomazone biotransformations (ElNaggar et al., 1992; Liu et al., 1996). The only significant evidence of phase II metabolism of clomazone was metabolite XIII, which was four to nine times (48 and 96 h) more abundant in resistant versus susceptible plant extracts (Table I). Its MRM signal fits that of a Glc conjugate of 2-chlorobenzyl alcohol (Liu et al., 1996; Holcˇapek et al., 2008), a plant metabolite of clomazone (Norman et al., 1990; Wiemer et al., 1991; ElNaggar et al., 1992). Although not all metabolites could be fully structurally characterized, the principal component analysis suggests high correlation among these isoxazolidinone ring alterations discussed (III, XI, XII, and XIII; Fig. 2B), which could thus conceivably belong to a common biotransformation pathway. Susceptible Plants Accumulate 5-Ketoclomazone

Other studies (ElNaggar et al., 1992; Ferhatoglu and Barrett, 2006; Yasuor et al., 2008) have consistently suggested that clomazone toxicity is due to its oxida323

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tive conversion to the toxic 5-ketoclomazone (II; Fig. 1). In our study, susceptible plants accumulated 2.5fold more 5-ketoclomazone than resistant plants by 96 h after clomazone addition (Table I; Fig. 3). The vector for compound II in Figure 2B further associates 5-ketoclomazone preferentially with the metabolite profile of susceptible plants. Clomazone bioactivation by conversion into 5-ketoclomazone occurs via the intermediate 5-hydroxyclomazone (IV; Fig. 1; ElNaggar et al., 1992; Liu et al., 1996). Similar amounts of 5-hydroxyclomazone appeared in both strains (IV; Table I), consistent with earlier evidence of similar clomazone bioactivation in resistant and susceptible E. phyllopogon (Yasuor et al., 2008). Therefore, since we have already shown that resistant E. phyllopogon plants are less affected by direct 5-ketoclomazone applications than susceptible plants (Yasuor et al., 2008), the lower abundance of 5-ketoclomazone by 96 h in the resistant plants of this study (II; Table I) must result from an enhanced ability to detoxify this compound compared with susceptible plants. It could also be hypothesized that resistant plants preferentially convert 5-hydroxyclomazone into the dihydroxy derivative rather than into 5-ketoclomazone compared with susceptible plants (Fig. 1). An open ring derivative of 5-ketoclomazone resulting from esterase hydrolysis of the isoxazolidinone ring had been suggested as an intermediate product occurring during the biotransformation of clomazone by soybeans (ElNaggar et al., 1992). The greater accumulation in susceptible plants by 96 h of metabolite X (Table I), whose MRM signal matches that of a 5-ketoclomazone open ring derivative (Fig. 1), parallels the greater accumulation of the toxic 5-ketoclomazone in those plants (Table I). Oxidative Metabolism and Clomazone Resistance

The major clomazone metabolites discussed in the preceding sections are almost all products of oxidative biotransformations, such as those typically associated with P450 metabolism in plants (Werck-Reichhart et al., 2000; Siminszky, 2006). Our earlier studies demonstrated that resistance to thiocarbamate herbicides and to acetolactate synthase- and acetyl-CoA carboxylaseinhibiting herbicides in E. phyllopogon from California rice fields was due to enhanced P450 activity (Fischer et al., 2000b; Yun et al., 2005; Ruiz-Santaella et al., 2006; Yasuor et al., 2009). By working with an herbicideresistant E. phyllopogon strain with known enhanced inducible P450 activity (Yun et al., 2005) and using mass spectrometry techniques, we were able to reveal that oxidative and presumably P450-mediated biotransformations were involved as a mechanism of clomazone resistance. The phase I metabolic profiling obtained allowed for a clear differentiation between clomazone metabolism in plants and in the growth medium and also provided strong evidence of enhanced oxidative clomazone metabolism in resistant versus susceptible plants. The P450-mediated resis324

tance to various other herbicides that has already been documented for this same resistant E. phyllopogon strain is conferred by different inducible isozymes (Yun et al., 2005). The complex network of several oxidative biotransformation steps conferring clomazone resistance to E. phyllopogon could result from the participation of different P450 isozymes and several genes (Werck-Reichhart et al., 2000). Low-level field resistance to clomazone could gradually increase in E. phyllopogon populations if recurrent suboptimal clomazone use allows the accumulation of genes for partial resistance (Powles and Neve, 2005; for review, see Gressel, 2002). SUMMARY AND CONCLUSION

Clomazone-resistant and -susceptible E. phyllopogon plants differ in their ability to detoxify clomazone. Resistant plants have a greater hydroxylation activity yielding a monohydroxylated derivative (III; Table I) as a major metabolite, which was detected at 6- to 12-fold lower amounts in susceptible plants. Susceptible plants accumulated 2.5-fold more of the toxic 5-ketoclomazone metabolite. These differences in clomazone metabolism between resistant and susceptible plants may explain the failure to control E. phyllopogon with clomazone in the field. Therefore, we demonstrated that enhanced oxidative herbicide metabolism, presumably cytochrome P450 mediated, has endowed multiple-herbicide-resistant E. phyllopogon plants with cross-resistance to clomazone. Our study also provides direct evidence associating the accumulation of 5-ketoclomazone with increased clomazone toxicity. The mechanism of clomazone resistance we elucidate here is part of a complex multifactorial suite of enhanced mechanisms whereby P450 herbicide metabolism in conjunction with enzymatic conjugation and photooxidative damage mitigation endow resistance to a wide array of herbicides to the E. phyllopogon accessions (Yun et al., 2005; Ruiz Santaella et al., 2006; Yasuor et al., 2008). MATERIALS AND METHODS Plant Material and Growing Conditions Two inbred Echinochloa phyllopogon strains were used in this study. These strains were either resistant or susceptible to the herbicides currently used for grass control in rice (Oryza sativa), including clomazone (Yasuor et al., 2008). They originated from accessions collected in 1997 in rice fields of the Sacramento Valley in California, where E. phyllopogon has evolved resistance to multiple herbicides (Fischer et al., 2000a). They represent strains derived after three successive selfing cycles from a multiple-herbicide-resistant accession and a susceptible accession. Seeds for all experiments were subjected to scarification in sulfuric acid (95%–98%) for 10 min, followed by rinsing with deionized water for 2 min. After scarification, seeds were surface sterilized in 3% (v/v) sodium hypochlorite for 3 min followed by thorough washing with sterile distilled water. Seeds were pregerminated for 5 d on wet Whatman No. 1 paper within closed petri dishes set in a growth chamber set at 26°C/10°C day/night temperatures and 16-h daylight under 500 mmol m22 s21 photosynthetic photon flux density delivered by a mixture of incandescent and fluorescent lights. Assay plants were cultured hydroponically by placing one pregerminated seed of each accession in a 6-mL glass tube containing 2 mL of


Oxidative Metabolism, 5-Ketoclomazone, and Clomazone Resistance

sterile half-strength Hoagland solution, which had been prepared using sterile distilled water and then filtered through a 0.22-mm membrane before use.

Chemicals Commercial clomazone (Command 3ME; 311 g L 21 ), analyticalgrade reference samples of clomazone (greater than 99% chemical purity), and reference standards (97%–99% pure) for the transformation products 5-hydroxyclomazone, 5-ketoclomazone, open ring clomazone, 3#-hydroxyclomazone, 4#,5-dihydroxyclomazone, 2-chlorobenzoic acid, and 2-chlorobenzyl alcohol were provided by FMC Corporation. HPLC-grade acetonitrile was purchased from Burdick and Jackson (VWR International). The purity of organic solvents was verified by LC-MS each time before using. Extra-pure formic acid was purchased from EMD. The ultrapure water was supplied by an in-house Millipore system. Fresh aqueous buffers for LC-MS were prepared on the working day.

Selection of Clomazone Metabolites for MS Differential clomazone metabolism between resistant and susceptible E. phyllopogon strains was assessed by subjecting plant extract and growth medium samples to LC-MS/MS using the LightSight 2.0 software (Applied Biosystems/MDS SCIEX) for data acquisition. The specific transition products screened for were selected from the literature or by the LightSight predictive algorithm, which was optimized using clomazone (m/z 240) as the reference parent compound. The identity of the clomazone metabolites was verified by comparison with known standards and/or by obtaining accurate masses and isotope ratios.

started at 3% B, increased linearly to 60% B within 6 min, and then ramped to 95% B for 9 min; lastly, the column was washed, equilibrated for 9 min, and kept at 50°C before the next injection.

LightSight-Generated Acquisition Methods Clomazone phase I and II metabolites in plant and growth medium were screened with the LC-MS/MS instrument in positive mode and using the LightSight metabolite identification software with predictive MRM. This software package enabled a direct comparison of control (no clomazone) and clomazone-incubated samples to identify new peaks potentially arising from clomazone metabolism in plants and the growth medium. The parameters for the MRM scan were as follows: 61-V declustering potential; 10-V entrance potential; 29-V collision energy; and 20-V collision cell exit potential. The first quadrupole (Q1) was set at unit resolution and Q3 was set at low resolution. Dwell and pause times for each MRM channel and between mass ranges were 5 and 2.5 ms, respectively. Information-dependent acquisition was used to trigger the acquisition of enhanced product ion spectra for ions exceeding 500 counts per second to confirm charge state and/or isotope pattern selection. Parameters for enhanced product ion were as follows: scan mode, profile; scan rate, 4,000 atomic mass units per second; linear ion trap fill time, 5 ms; dynamic fill time, on; declustering potential, 40 V; collision energy spread, 25 V; collision energy, 60 V; collision cell exit potential, 20 V. Thus, MRM allowed the identification and monitoring of the following transitions: m/z 240/125, m/z 204/125, m/z 242/125, m/z 254/125, m/z 256/125, m/z 256/141, m/z 272/125, m/z 272/141, m/z 272/157, m/z 268/125, and m/z 320/125, where the first value corresponds to the target ion identified in the first MS/MS quadrupole (Q1) and the second value is for the fragmentation product (corresponding to either the aromatic or the isoxazolidinone moiety of the original clomazone molecule) identified with the third quadrupole (Q3).

Sample Preparation In California rice culture, clomazone is applied as a granular formulation, which is dropped onto the surface of flooded paddy soils. Clomazone can be absorbed by roots and shoots and is transported apoplastically (Vencill et al., 1990b). Fresh leaf samples (100 mg) of E. phyllopogon plants hydroponically treated at the two- to three-leaf stage with 50 mM clomazone for 48 and 96 h were mixed with 0.5 mL of water-acetonitrile-isopropanol (4:3:1, v/v/v), and the mixture was placed into a 2-mL microcentrifuge tube. Tissues were homogenized in a stainless-steel ball mill (MM 301; Retsch) with a prechilled (280°C) tube holder for 0.5 min at 30 cycles s21; the homogenate was then sonicated in an ultrasonic bath for 1 min and agitated in an orbital shaker for 5 min at 4°C in the dark. The mixture was centrifuged at 16,000g for 2 min, and the supernatant was transferred to a clean tube. The pellets were resuspended in 0.5 mL of extraction solvent, and the mixture was centrifuged at 16,000g for 2 min; the supernatant was combined with the initial extracted volume. Growth medium samples were prepared by collecting 2 mL from the hydroponic solution at 0, 48, and 96 h after clomazone had been added to the solution; samples were taken from media with and without plants. In all cases, tubes were incubated in a growth chamber as described earlier. The samples were cleaned through a 0.45-mm membrane Acrodisc 13-mm Syringe Filter (Pall).

Instrumentation LC-MS/MS was conducted using an API 4000 Q-Trap hybrid triple quadrupole linear ion trap mass spectrometer (Applied Biosystems/MDS SCIEX) interfaced with an Acquity (Waters) HPLC system. All the LC-MS/MS experimental events were controlled using the LightSight 2.0 software. The TurboIonSpray ion source conditions were optimized and set at the following values: curtain gas, 1.0 3 103 torr (1 torr = 133.3 Pa); collision gas, high; ion spray voltage, 5.2 kV; temperature, 300°C; ion source gas 1, 2.6 3 103 torr; ion source gas 2, 2.6 3 103 torr; and interface heater, on.

Chromatographic Conditions For chromatographic separations, a phenyl-hexyl universal guard cartridge (2.0 3 4.0 mm) and a Luna phenyl-hexyl analytical column (150 3 3 mm, particle size 3 mm; Phenomenex) were chosen because of their high affinity with the phenyl group present in the clomazone structure. Tenmicroliter samples were injected, and degassed solutions of formic acid/ ultrapure water 0.1% (v/v; eluent A) and of formic acid/acetonitrile 0.1% (v/v; eluent B) were pumped at 0.3 mL min21 into the HPLC system. The gradient


Method Validation The assignment of MS peaks to specific clomazone metabolites was validated by comparison with analytical standards and by obtaining accurate masses and isotope ratios using a high-resolution HPLC-LTQ-Orbitrap instrument (Thermo Scientific; for detailed instrument information, see Supplemental File S1) in conjunction with full-scan or selected ion monitoring with data-dependent MS/ MS scans. Because clomazone contains one chlorine atom, the unique chlorine isotopic signature was also used for validation. Mass spectral data were corrected using the MassWorks 2 software (Cerno Bioscience).

Statistical Procedure Peak areas for each detected clomazone metabolite were automatically integrated using Analyst software (version 1.4.1; Applied Biosystems/MDS SCIEX). Preliminary data exploration was accomplished using principal component analysis. In our experiments, treated plants were continuously exposed to clomazone throughout the assays; thus, metabolic profiles included large amounts of parent compound. We removed the high-abundance clomazone peak from the principal component analysis to facilitate group clustering among clomazone metabolites. A scree plot of successive eigenvalues was used to select the number of principal components to be retained. A score plot allowed visual detection of data clustering with respect to the main principal components, and a loading plot was used to illustrate the strength of positive and negative correlations of the original variables (clomazone biotransformation products) with each principal component. Experimental treatments were factorial combinations of clomazone levels (0 and 50 mM), incubation times (0, 48, and 96 h), and E. phyllopogon strains (herbicide resistant and susceptible), which were arranged within a completely randomized design with three replications and conducted twice in time. Each plant extract or growth medium sample (n = 6 for each treatment) was analyzed twice in the LC-MS/MS system. Data from both experiments were pooled, and peak area data from each run were log transformed to homogenize variances prior to ANOVA. Means were separated using Tukey’s honestly significant difference test with a = 0.05. Analyses were conducted using the JMP software (version 8, 2008; SAS Institute).

Supplemental Data The following materials are available in the online version of this article. Supplemental File S1. HPLC-LTQ-Orbitrap setup.

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Supplemental File S2. Clomazone metabolic profiling in plants and growth medium. Supplemental File S3. RP-LC-MS/MS analysis of clomazone metabolites in growth medium.

ACKNOWLEDGMENT We thank FMC Corporation for providing clomazone analytical standards. Received January 12, 2010; accepted March 2, 2010; published March 5, 2010.

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