Plant Physiol. (1990) 94, 704-7090032-0889/90/94/0704/06/$01 .00/0

Received for publication April 2, 1990Accepted June 6, 1990

Site of Clomazone Action in Tolerant-Soybean andSusceptible-Cotton Photomixotrophic

Cell Suspension Cultures1

Michael A. Norman*, Rex A. Liebi, and Jack M. WidholmDepartment of Agronomy, University of Illinois, Urbana, Illinois 61801

ABSTRACT

Studies were conducted to determine the herbicidal site ofclomazone action in tolerant-soybean (Glycine max [L.] Merr. cvCorsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.]cv Stoneville 825) (COT-M) photomixotrophic cell suspensioncultures. Although a 10 micromolar clomazone treatment did notsignificantly reduce the terpene or mixed terpenoid content (mi-crogram per gram fresh weight) of the SB-M cell line, there wasover a 70% reduction in the chlorophyll (Chl), carotenoid (CAR),and plastoquinone (PQ) content of the COT-M cell line. Thetocopherol (TOC) content was reduced only 35.6%. Reductionsin the levels of Chi, CAR, TOC, and PQ indicate that the site ofclomazone action in COT-M cells is prior to geranylgeranyl pyro-phosphate (GGPP). The clomazone treatment did not significantlyreduce the flow of [14C]mevalonate ([14C]MEV) (nanocuries pergram fresh weight) into CAR and the three mixed terpenoidcompounds of SB-M cells. Conversely, [14C]MEV incorporationinto CAR and the terpene moieties of Chi, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site ofclomazone action must be after MEV. Sequestration of clomazoneaway from the chloroplast cannot account for soybean toleranceto clomazone since chloroplasts isolated from both cell linesincubated with (14C]clomazone contained a similar amount ofradioactivity (disintegrations per minute per microgram of Chi).The possible site(s) of clomazone inhibition include mevalonatekinase, phosphomevalonate kinase, pyrophosphomevalonate de-carboxylase, isopentenyl pyrophosphate isomerase, and/or aprenyl transferase.

Clomazone2 is a registered soybean herbicide that killssusceptible species by blocking pigment synthesis via inhibi-tion of the terpenoid pathway. The exact site of clomazone(dimethazone, FMC-57020, Command) action is currentlyunknown. Some of the suggested sites of action include: (a)IPP isomerase (22), (b) prenyl transferase(s) (5, 22), and (c)enzymatic phytylation of chlorophyllide (5, 6).Many reports regarding the initiation of photoautotrophic

Supported in part by funds from the Illinois Agricultural Experi-mental Station and FMC Corporation.

2Abbreviations: clomazone, [2-(2-chlorophenyl)methyl-4,4-di-methyl-3-isoxazolidinone]; IPP, isopentenyl pyrophosphate; SB-M,soybean photomixotrophic cell line; COT-M, cotton photomixo-trophic cell line; TOC, tocopherols; CAR, carotenes; PQ, plastoqui-nones; MEV, R-[2-'4C]mevalonic acid lactone; SPP, solanesyl pyro-phosphate; DAS, days after subculture.

and photomixotrophic cell suspension cultures (1 1, 12, 14,28) have indicated their potential usefulness in herbicidemechanism and site of action studies (2, 20, 23). A previousreport (17) has indicated that the response to clomazone ofSB-M (Glycine max [L.] Merr. cv Corsoy) and COT-M (Gos-sypium hirsutum [L.] cv Stoneville 825) cells used in thisstudy closely parallels that of identical cultivars of soybeanand cotton species on a whole plant level.Terpene synthesis occurs in both the chloroplast and the

cytosol with common reactions including the conversion ofacetyl-CoA to the 45 carbon containing compound SPP (Fig.1). Synthesis of the phytyl moieties of Chl, vitamin K1, andTOC from GGPP, however, occur exclusively in the chloro-plast with the exception ofvery small rates ofTOC productionwithin the cytosol (7). CAR and the terpene moiety ofPQ arealso derived from GGPP and produced solely in chloroplasts.An attempt to quantify the cellular content and the flow of['4C]MEV into CAR and the terpene components of TOC,Chl, and PQ in these cell lines has yet to be determined. Thesite of clomazone action could be determined to be eitherbefore or after GGPP, since the biosynthesis of all four of theterpenoid compounds include GGPP as their last commonprecursor (Fig. 1). Clomazone-induced reductions in the levelof MEV incorporation would clearly indicate the site ofclomazone action to be after MEV.

Recent reports (15, 17, 26, 27) have indicated that differ-ential uptake, translocation, and metabolism can only par-tially account for soybean tolerance to clomazone. Conse-quently, differences at the site of action may account forselectivity. Differences at the site ofaction could include eithera sequestration mechanism (localization of clomazone awayfrom the site of action) or differences in enzyme sensitivitybetween soybean and susceptible species. A previous report(22) has indicated that terpenoid biosynthesis in the chloro-plast of susceptible species is much more sensitive to cloma-zone than extraplastidic terpenoid formation. Also, the datacontained herein clearly demonstrate that terpene biosyn-thesis in COT-M cells is remarkably inhibited by clomazone.Isolation of intact chloroplasts from SB-M and COT-M cellstreated with ['4C]clomazone would determine if soybean tol-erance to clomazone is due, at least partially, to sequestrationofclomazone from the chloroplast, the primary site of action.The objectives of the present study were to determine: (a)

the terpene content of the cells for comparison to whole plantreports, (b) the effects of clomazone on the cellular contentand the level of ['4C]MEV incorporation into CAR, and the

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CLOMAZONE ACTION IN SOYBEAN AND COTTON CELL SUSPENSIONS

MEV

11

2

3

IPP IPP

4

DMAPP

5

GPP

Number of

Carbon Atoms

5

5

10 TERPENOID

5j COMPOUND

FPP 15 2X - Sterols

51GGPP 2 0 GA3

GGPP 20 Phytol-CHL-TOC

GGPP 20 2X - CAR

55

5X - SPP 45 PQ

Figure 1. Schematic representation of the terpenoidhigher plants. 1, MEV kinase; 2, phosphomevalonatEpyrophosphomevalonate kinase; 4, IPP isomerase; 5, prease, DMAPP, dimethylallyl pyrophosphate; GPP, geranphate; FPP, farnesyl pyrophosphate.

terpene moieties of Chl, TOC, and PQ compouncand COT-M cell lines in an effort to identifyclomazone action, and (c) if a sequestration mecdcounts for soybean tolerance to clomazone by meconcentration of radiolabeled clomazone and/ormetabolites in intact chloroplasts isolated from bot

MATERIALS AND METHODS

Cell Culture Conditions

The media and culture conditions of the soybeamax [L.] Merr cv Corsoy) and cotton (Gossypiun[L.] cv Stoneville 825) photomixotrophic cell suspitures used in this study were as described previou10 gM clomazone treatment was added to SB-M c

followed by the addition of0.98 MCi of ['4C]MEV (.Corp., Arlington Heights, IL) at 4 DAS which coirthe time of maximum Chl synthesis (Fig. 2). SB-tharvested after a 48 h pulse with ['4C]MEV or 610 AM clomazone treatment was added to COT-Minitiation of Chl synthesis (8 DAS) followed by tiof 0.88 uCi ['4C]MEV at 11 DAS. The COT-Mthen harvested after a 48 h pulse with ['4C]MEV c

The delay in addition of clomazone to COT-Mdue to the delay in the onset of CHL productionSB-M cells (Fig. 2). The Chl content (Ag/g freshboth cell lines during their growth cycles was

spectrophotometrically ( 16).

[14C]MEV Incorporation and Terpene Analysis

Terpenes were extracted from harvested cells using metha-nol and petroleum ether according to the procedures of Kri-vankova and Dadak (13). The petroleum ether fraction con-

taining CAR and the mixed terpenoid compounds from eachtreatment was then taken to dryness and resuspended in 1

mL of acetone. A 200 ,uL aliquot was then spotted on each of5 (0.25 mm) silica gel TLC plates (EM Science, Gibbstown,NJ) and developed in benzene:methanol (99:1, v/v). RF valuesfor the mixed terpenes Chl, TOC, and PQ, and CAR corre-sponded well to a previous report (3) and respectively, were

SITE OF 0.01, 0.47, 0.67, and 0.80. TOC and PQ were visualized on

SYNTHESIS the edge of the TLC plates, respectively, using the Emmene-Cytosol Engel and reduced methylene blue spray reagents (1). The

bands of CAR and the three mixed terpenoid compoundsChloroplast were then scraped into individual vials containing approxi-

mately 30 mL of 100% ethanol. Chl, CAR, TOC, and PQwere eluted from the silica gel by shaking on a reciprocal

Chloroplast shaker for 2 h. Each sample was then passed through a 0.22Mm filter (Millipore Products, Bedford, MA) to remove the

Chloroplast silica gel. Chl and CAR were pure based on Abs spectra andquantified spectrophotometrically, respectively, using the pro-

Chloroplast cedure of Holden (9) and maximum A values at 450 nm withstandard curves. TOC was quantified spectrophotometrically

pathway in using the procedure of Desai (4). The cellular levels of PQe kinase; 3, were determined using the oxidized and reduced UV A spectra

enyl transfer- according to Barr and Crane (1). Interfering UV absorbing

y'l pyrophos- materials were removed from PQ samples using a C18 Sep-Pak cartridge (Waters Associates, Milford, MA). Each car-

tridge was prewet with 5 mL of methanol followed by 5 mL

Is of SB-M ofwater. Each PQ sample in 5 mL of ethanol was then loadedthe site of onto the column. PQ were then eluted from the column withhanism ac- 5 mL of 100% hexane. This cleanup procedure was extremelyasurinz the effective in removing interfering UV absorbing compounds

clomazone:h cell lines.

in (Glycinen hirsutumFension cul-sly (17). Aells 2 DASAmershamicided withI cells wereDAS. Thecells at theie additioncells were

)r 13 DAS.I cells was

relative toweight) ofletermined

1200 P

1000 e-Cr

0)

c-

(9)

800 F

600

400

200

0

0 5 10 15 20 25TIME (Days After Subculture)

Figure 2. The Chl content (9g/g fresh weight) of SB-M and COT-Mcells throughout their representative growth cycle period. The growthcycle was initiated by subculturing cells (0.5 g) into 50 mL of freshlymade media. The durations of the entire SB-M and COT-M cell cycleswere 14 and 28 d, respectively.

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Plant Physiol. Vol. 94, 1990

from coenzyme Q standards (Sigma) exposed to identicalexperimental procedures used to isolate PQ. The PQ hexaneeluate was then taken to dryness and resuspended in 100%ethanol for quantification. Aliquots from each sample werecounted for radioactivity using liquid scintillation countingtechniques to determine the level of ['4C]MEV incorporation.

Chloroplast Extraction from Protoplast Preparations

Chloroplast isolation from protoplasts generally followedthe procedure of Robinson (19). SB-M and COT-M cells wereincubated with 2.1 ,uCi of ["4C]clomazone at 8 and 18 DAS,respectively, and harvested after a 48 h pulse. The differencein the time of clomazone addition was to allow both cell linesto attain a Chl content of 600 to 650 ,ug Chl/g fresh weight.SB-M (0.31 g) and COT-M (0.33 g) cells containing exactly200 ,ug ofChl were placed in Petri plates containing a bufferedincubation mix (B 1) of 0.15 M mannitol, 0.15 M sorbitol,0.10 M glucose, 6.0 mM CaCl2 0.7 mM K2HPO4, and 3 mMMes (pH = 5.6, KOH). Two mL of the buffer also containing0.2 g cellulase CEL (Worthington, Freehold, NJ), 0.2 g drise-lase (Kyowa Hakko Kogyo Co., Tokyo, Japan) and 0.02 gpectolyase Y23 (Karlan Chemical Corp., Torrance, CA) wasthen added to each Petri plate (final volume, 10 mL) andgently swirled on an orbital shaker in the dark for 3.5 h. Thecells were then collected by centrifugation (lOOg for 30 s) andplaced in 10 mL of fresh enzyme incubation mix and incu-bated for another 4 h. The protoplasts were then collected asbefore and resuspended in 5 mL of a buffered solution (B2)containing 0.5 M sucrose, 1 mM CaCl2, and 5 mm Mes (pH6.0, KOH) and layered on top of 5 mL of B2 solutioncontaining 15% Ficoll 400 (w/v, Pharmacia Fine Chemicals,Piscataway, NJ). A third buffered solution (B3) containing0.5 M sorbitol, 1 mm CaCl2, and 5 mM Mes (pH 6.0, KOH)was then layered on top ofthe protoplast layer and centrifugedin a swing-out rotor at lOOg for 5 min. The layer ofprotoplastson top of the Ficoll cushion was then removed and resus-pended in 8 mL of a buffered solution (B4) containing 0.33M sorbitol, 2 mm Na4EDTA, and 5 mM Hepes (pH 7.6, KOH).Protoplasts were mechanically ruptured by passing the pro-toplasts through a 15 to 20 ,m nylon mesh screen fastened atthe end of the barrel of a cutoff syringe. After two passagesthrough the nylon mesh screen the chloroplast preparationwas layered on top of 12 mL of a solution containing 40%Percoll (v/v, Sigma), 0.33 M sorbitol, 0.1% BSA and 50 mMTricine (pH 7.6, KOH). The samples were then centrifugedin a swing-out rotor at 2500g for 2 min. The chloroplast pelletwas collected by removing the Percoll layer and resuspendingin 2 mL of the B4 solution. The intactness of the chloroplastswas determined using the procedure of Heber and Santarius(8). The chloroplast preparation was then taken to drynessand resuspended in 1 mL of ethanol and centrifuged in amicrofuge for 5 min. Aliquots were then taken from eachchloroplast preparation to determine the Chl content (9) andtotal dpm using liquid scintillation spectrometry.

Statistical Analysis

All treatments were replicated three to four times, dataaveraged, and standard error of the means calculated. Three

to four subsamples were taken from one flask for each pointon the Chl synthesis curves and means and standard error ofthe means calculated.

RESULTS AND DISCUSSION

Pigment Synthesis Curves

The Chl content of SB-M cells at 0 DAS (1,1 11 g/g freshweight) was approximately twice that of COT-M cells (583,ug/g fresh weight, Fig. 2). The CHL synthesis curves weredetermined prior to the ["4C]MEV incorporation and ['4C]clomazone sequestration experiments. The initial time of Chlsynthesis in the ['4C]MEV incorporation experiments wasdetermined visually by comparison to flasks of SB-M andCOT-M cells treated at 0 DAS with a 100 and 10 Mm cloma-zone treatment, respectively. These treatments which inhibitCHL synthesis in both cell lines permitted the detection byvisual observation the initial time of Chl synthesis in SB-Mand COT-M cells in the untreated flasks of the ['4C]MEVincorporation experiments.The Chl content of both cell lines decreases early in the

growth cycle, rises steadily, and then oscillates near the initialChl concentration. The early decline in the Chl content isprobably due to a dilution of the chloroplast pool duringlogarithmic growth 1 to 3 DAS (17), while the upward trendis probably due to Chl synthesis in newly developed plastids.The time of ['4C]MEV addition (4 DAS) to the SB-M cellscorresponded to the initial time of Chl synthesis. Since theinitiation of Chl synthesis occurs later in COT-M cells, [14C]MEV was added (11 DAS) at the time of maximum Chlsynthesis in the COT-M cell line. The Chl synthesis curverepresents only one cell cycle. Although the cellular Chlcontent and the initiation of CHL synthesis changes slightlyfrom cycle to cycle, the overall shape of the curve remainsthe same. The reason for the random fluctuations in thecellular CHL content is unknown. As of this date, the Chlcontent of the SB-M and COT-M cells at the time of subcul-ture are, respectively, 820 and 938 ug/g fresh weight.

Terpene Synthesis and [14C]MEV Incorporation

Over 90% ofthe ['4C]MEV was recovered for all treatments(data not shown). The 10 gM clomazone treatment did notsignificantly reduce the level (,ug/g fresh weight) of Chl, TOC,CAR, and PQ in SB-M cells (Table I). Conversely, the 10 Mmtreatment caused at least a 70% reduction in the level of Chl,CAR, and PQ levels in COT-M cells (Table I). The TOClevels in COT-M cells were also reduced but to a lesser extent(35.6%). The small decrease in the percentage of TOC maybe due to a low level of TOC production in COT-M cellsbetween the time of clomazone addition (8 DAS) and theharvest time (13 DAS). The clomazone-induced inhibition ofChl, CAR, TOC, and PQ synthesis in susceptible COT-Mcells indicate that the site of clomazone action must be priorto GGPP production. These data support the hypotheses ofSandmann and Boger (21) and Duke et al. (5) that the site ofclomazone action is prior to GGPP production and that thelack of phytylation of chlorophyllide is due to inhibition ofGGPP production.

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CLOMAZONE ACTION IN SOYBEAN AND COTTON CELL SUSPENSIONS

Table I. Effects of Clomazone on the Content of CAR and Three Mixed Terpenoid Compounds in SB-M and COT-M Cells

Cell culture and terpenoid compound extraction and quantification procedures were as described in"Materials and Methods."

Terpenoid Cell Line Clomazone Terpenoid InhibitionCompound Conc. Content

JIM Ag/g fresh wt %

Chl SB-M 0 150 ± 8.010 145±6.1 3.3

COT-M 0 163±14.510 48±6.1 70.6

CAR SB-M 0 20.9 ± 2.61 0 18.7 ± 3.5 10.5

COT-M 0 63.8 ± 3.410 9.1 ±2.7 85.7

TOC SB-M 0 18.8 ± 0.710 18.5± 0.3 1.6

COT-M 0 14.6 ± 1.610 9.4 ± 0.8 35.6

Po SB-M 0 12.8 ± 0.610 10.9±1.5 14.8

COT-M 0 25.0 ± 1.910 7.4 ± 1.3 70.4

Since Chl, CAR, PQ, and most TOC are produced inplastids, then the primary site of clomazone action must bewithin the chloroplast. This observation is supported by aprevious report (22) that indicated that terpene biosynthesisin the chloroplast is four times more sensitive to clomazoneinhibition than extraplastidic terpene formation. Other phys-iologically significant compounds derived solely or partiallyfrom the terpenoid pathway that are produced outside ofplastids include sterols (endoplasmic reticulum) and ubiqui-none (mitochondria). It is an extremely remote possibility,however, that the immediate and complete lack of pigmentsin leaf tissue of susceptible species treated with clomazone isa secondary effect caused by a lack of either sterol or ubiqui-none production or any other terpenoid compound producedsolely in the cytosol. Furthermore, leaves of clomazone-sus-ceptible species that are devoid of pigments grow otherwisenormally until food reserves from endosperm or cotyledonsare exhausted (15). Also, the growth and development of roottissue which is devoid of chloroplasts is not significantlyinhibited by clomazone in susceptible species (15). The clom-azone-induced decline in the PQ pool observed in COT-Mcells also supports a previously conjectured hypothesis thatone or more component of the photosynthetic electron trans-port chain is either missing or nonfunctional in clomazonetreated tissues of susceptible species (6).The relative quantities (,tg/g fresh weight) of the terpenoid

compounds isolated from SB-M cells (Chl > CAR > TOC -

PQ) is consistent with a previous report (25). Although theCHL content was greater than CAR in COT-M cells therewas a greater level of PQ relative to the TOC content. Thesedata indicate that there is a greater cellular PQ pool relativeto the TOC pool within the first 13 DAS in COT-M cells.

The relatively low level ofTOC in COT-M cells may accountfor the small inhibitory (35.6%) effect of clomazone.There was significant ['4C]MEV incorporation (nCi/g fresh

weight) into all four terpenoid compounds within the timeframes used in this study based on control treatments fromboth cell lines (Table II). The amount of ['4C]MEV incorpo-rated in the phytyl moiety of Chl in COT-M cells was signif-icantly higher than that of SB-M cells. The 10 gM clomazonetreatment caused a slight but nonsignificant increase in thelevel of ['4C]MEV incorporation into the phytyl moieties ofCHL and TOC in SB-M cells (Table II). There was a signifi-cant clomazone-induced increase in the level of ['4C]MEVincorporated into CAR of SB-M cells. The reason for theapparent trend of clomazone-induced increases in the levelsof ['4C]MEV incorporation into these terpenoid compoundsis unknown. There was a small nonsignificant decrease, how-ever, in the level of ['4C]MEV incorporation into PQ. Con-versely, the 10 lM clomazone treatment caused a minimumof a 72% reduction in the level of ['4C]MEV incorporationinto CAR and the three mixed terpenes of the COT-M cells(Table II). These data indicate that the site of clomazoneaction is after MEV synthesis in susceptible COT-M cells.Specific activities of CHL, CAR, TOC, and PQ were notdetermined since the total cellular content or pool size of['4C]MEV and unlabeled MEV was not determined. It is notpossible to interpret the effects of clomazone on terpenesynthesis based on specific activities without knowing theeffects of clomazone on the MEV content of both cell lines.

Clomazone Sequestration Experiments

Over 95% of the ['4C]clomazone was recovered from eachtreatment (data not shown). Although the recovery of the

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Plant Physiol. Vol. 94, 1990

Table II. Effects of Clomazone on [14C]MEV Incorporation into CAR and Three Mixed TerpenoidCompounds of SB-M and COT-M CellsSB-M and COT-M cells were harvested after a 48 h pulse with 0.98 ,Ci (6 DAS) and 0.88 MACi (13

DAS) [14C]MEV, respectively. Cell culture and terpene extraction and MEV incorporation quantificationprocedures were as described in "Materials and Methods."

Terpenoid Cell Line Clomazone [4C]MEV Inhibition

Compound Conc. IncorporationMM nCi/g fresh wt %

Chi SB-M 0 9.0 ± 0.310 10.2±0.3 0

COT-M 0 15.8±1.210 2.6±0.4 83.5

CAR SB-M 0 3.8 ± 0.810 6.1±0.5 0

COT-M 0 3.9 ± 0.410 0.8 ± 0.1 79.5

TOC SB-M 0 3.8 ± 0.610 5.3±0.1 0

COT-M 0 3.3 ± 0.110 0.9 ± 0.2 72.7

PQ SB-M 0 0.83 ± 0.2010 0.64±0.19 22.9

COT-M 0 0.75 ± 0.0410 0.11 ± 0.02 85.3

original 200 ,ug of Chl from SB-M (8.3 ,ug) and COT-M (7.3,ug) cells was only 4.2 and 3.7%, respectively, chloroplastsfrom all treatments were greater than 90% intact (Table III).There was no- significant difference in the total level of radio-activity (dpm/,ug Chl) in chloroplasts isolated from SB-M andCOT-M cells. These data clearly indicate that soybean toler-ance to clomazone is not due to sequestration of clomazonefrom the chloroplast, the primary site of clomazone action.Since differential uptake, translocation, and metabolism (con-version to inactive form) does not account for clomazoneselectivity (15, 17, 26, 27), differences in enzyme sensitivityappears to be the mechanism of selectivity assuming thatparental clomazone is the active form of the herbicide. Dif-ferences in enzyme sensitivity accounts for selectivity of her-bicides such as aryloxyphenoxypropionates (10, 24) and cy-

clohexadiones (18).

CONCLUSIONS

The relative contents of Chl, CAR, TOC, and PQ of bothcell lines compared well to those observed in whole plants.Terpene synthesis in SB-M cells was not affected by a 10 Mm

clomazone treatment but caused a significant decline in boththe quantity (,ug/g fresh weight) and level of ['4C]MEV incor-poration into CAR and the terpene moieties of Chl, TOC,and PQ ofCOT-M cells. These observations indicate that thesite of clomazone action is before GGPP and after MEV. Thepotential site of clomazone action corresponds to the fiveenzymes that catalyze the conversion of MEV to GGPP.These enzymes include mevalonate kinase, phosphomevalon-ate kinase, pyrophosphate mevalonate decarboxylase, IPPisomerase, and/or a prenyl transferase.Soybean tolerance to clomazone is not due to sequestration

Table Ill. Recovery of [14C]Clomazone and/or '4C-Metabolites from SB-M and COT-M CellChloroplastsSB-M and COT-M cells were incubated with 2.1 IACi [14C]clomazone at 8 and 18 d after subculture,

respectively. The total level of radioactivity and Chi content of isolated chloroplasts were determinedafter a 48 h exposure to [14C]clomazone as described in "Materials and Methods."

Chi TotalCell Line Intactness Activity Total dpm/ug Chi

Total Recovery

Mg % % dpm

SB-M 8.3 ± 1.8 4.2 >90 407 ± 45 49 ± 17COT-M 7.3± 0.9 3.7 >90 264 ±47 36±5

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CLOMAZONE ACTION IN SOYBEAN AND COTTON CELL SUSPENSIONS

of the herbicide away from the chloroplast (the primary siteof clomazone action), implicating differences in enzyme sen-

sitivity as the primary mechanism of clomazone selectivity.Future studies will be conducted to more precisely deter-

mine the site of clomazone action.

ACKNOWLEDGMENTS

We thank Harold Butler for his many helpful discussions and BenGardner for his excellent technical assistance in preparation of thefigures.

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