Differential oxidative metabolism and 5-ketoclomazone accumulation are involved in Echinochloa phyllopogon resistance to clomazone.

Echinochloa phyllopogon (late watergrass) is a major weed of California rice (Oryza sativa) that has evolved cytochrome P450-mediated 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.


INTRODUCTION
Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) has been used for the last four years in California to control Echinochloa phyllopogon (Stapf) Koss., a major weed of rice, and other grass weeds in rice (Oryza sativa L.). 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 non-mevalonate isoprenoid pathway, leading to the biosynthesis of isopentenyl pyrophosphate (IPP) 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 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 fenoxaprop-ethyl and cyhalofop; the acetolactate synthase (ALS) inhibitors bispyribacsodium, bensulfuron-methyl, and penoxsulam; and the DXP synthase inhibitor clomazone (Fischer et al., 2000a;Yasuor et al., 2008;Yasuor et al., 2009). Ratios of the GR 50 (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 bio-activation of inactive clomazone to active 5ketoclomazone (Yasuor et al., 2008). Based on inhibitor data, Fischer et al. (2000b) suggested that 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 rings (ElNaggar et al., 1992, Fig. 1). Hydroxylation at the 5-methylene carbon or at the methyl group of the isoxazolidinone ring (yielding 5hydroxyclomazone 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 tolerance to clomazone may involve lower rates of bioactivation to 5-ketoclomazone than in cotton. Therefore, measuring differential accumulation of the presumed active metabolite 5ketoclomazone 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;Simiszky, 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 pre-treating E. phyllopogon plants with P450 inhibitors, such as malathion or 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 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 towards 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;Vencill et al., 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 over production of the DXP synthase was demonstrated by expressing this enzyme under the control of the cauliflower mosaic virus 35S promoter in transgenic Arabidopsis thaliana (L.) Heynh. (Carretero-Paulet 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 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 analysis (RP-LC-MS/MS) in the multi-reaction monitoring mode (MRM) to identify the biotransformation products. Thus differences in clomazone metabolism between resistant and susceptible E. phyllopogon plants were characterized and quantified.

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 to 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 susceptible plants, and that metabolites in plant extracts were different from those in the growth medium ( Fig. 2A). Three principal components (PC) 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 Echinochloa phyllopogon
According to the principal component analysis, differences in the overall clomazone metabolite profile between resistant and susceptible plants were already evident by 2 days after clomazone was added to the growth medium and became even more distinct by 4 days ( Fig. 2A). The contribution of specific compounds towards this metabolite profile differentiation between resistant and susceptible plants was characterized (Fig. 2B). As would also happen in non-sterile 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 (Supplementary 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 6 times more of this compound than susceptible plants by 48 and 96 h, respectively, after clomazone addition (Table I,  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 di-hydroxylation in the isoxazolidinone moiety of the parent compound (Fig. 1).
Resistant plants accumulated from 8 to 5 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 di-hydroxylation of the isoxazolidinone ring of clomazone. There are other di-hydroxylations 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. phyllopogont roots, which paralleled the 2-to 3-fold accumulation of this metabolite in resistant vs. 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 to susceptible plants, resistant plants accumulated greater amounts (~2-fold) of an aromatic-ring hydroxylate (VI) with a mass shift and a retention time matching that of the 3'hydroxyclomazone standard (Table I and 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 (data on growth medium metabolites is presented in the Supplementary files S2 and S3).
Assuming similar root uptake rates by these strains (Yasuor et al. 2008), 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  Fig. 3) and could correspond to the addition of either a ketone group (Holčapek 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 that was 4 to 9 times (48, 96 h) abundant in resistant vs. susceptible plant extracts (Table I). Its MRM signal fits that of a glucose conjugate of 2-chlorobenzyl alcohol (Liu et al., 1996;Holčapek 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;Ferhatoglou and Barrett 2006;Yasuor et al., 2008) have consistently suggested that clomazone toxicity is due to its oxidative conversion to the toxic 5ketoclomazone (II, Fig. 1). In our study, susceptible plants accumulated 2.5-fold more 5ketoclomazone than resistant plants by 96 h after clomazone addition (Table I, Table I) must result from an enhanced ability to detoxify this compound compared to susceptible plants. It could also be hypothesized resistant plants preferentially convert 5-hydroxyclomazone into the di-hydroxy derivative rather than into 5-ketoclomazone compared to 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 5ketoclomazone open ring derivative (Fig. 1), parallels the greater accumulation of the toxic 5ketoclomazone 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 (Siminszky, 2006;Werck-Reichhart et al., 2000). Our earlier studies demonstrated that resistance to thiocarbamate herbicides, and to ALS-and ACCase-inhibiting 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 a herbicide-resistant 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 vs. susceptible plants. The P450-mediated resistance 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 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 P450mediated, has endowed multiple-herbicide-resistant E. phyllopogon plants with cross-resistance to clomazone. Our study also provides direct evidence associating 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 where P450 herbicide metabolism in conjunction with enzymatic conjugation and photo oxidative 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).

Plant Material and Growing Conditions
Two inbred E. phyllopogon strains were used in the present study. These strains were either resistant or susceptible to the herbicides currently used for grass control in rice, 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 and from a susceptible accession. Seeds for all experiments were subjected to scarification in sulfuric acid (95-98%) for 10 min followed by rinsing with de ionized 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. solvent and the mixture 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 µm membrane Acrodisc ® 13mm Syringe Filter (Pall Corporation, MI). temperature (TEM), 300 °C; ion source gas 1 (GS1), 2.6 x 10 3 torr; ion source gas 2 (GS2), 2.6 x 10 3 torr; and interface heater (IHE), on.

Chromatographic Conditions
For chromatographic separations, a phenyl-hexyl universal guard cartridge (2.0 x 4.0 mm) and a Luna phenyl-hexyl analytical column (150 x 3 mm, particle size 3 μm, Phenomenex,Torrance, CA) were chosen because of their high affinity with the phenyl group present in the clomazone structure.
Ten-μL 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 min -1 into the 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: 61 V declustering potential (DP); 10 V entrance potential (EP); 29 V collision energy (CE); and 20 V collision cell exit potential (CXP). The first quadrupole (Q1) was set at unit resolution and Q3 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 (IDA) was used to trigger acquisition of enhanced product ion (EPI) spectra for ions exceeding 500 counts per second (cps) to confirm charge state and/or isotope pattern selection. Parameters for EPI were: scan mode, profile; scan rate 4000 atomic mass units per second (

Method Validation
The assignment of mass spectrometry 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, Waltham, MA; detailed instrument information is in the supplementary file S1) in conjunction with full scan or selected ion monitoring (SIM) with data-dependent MS/MS scans. Because clomazone contains one chlorine 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.

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