Atrazine resistance in velvetleaf (Abutilon theophrasti) due to enhanced atrazine detoxification

PESTICIDE

BIOCHEMISTRY

Atrazine

AND

PHYSIOLOGY

34, 149-163 (1989)

Resistance in Velvetleaf (Abutilon theophrasti) Enhanced Atrazine Detoxification’

Due to

JOHN W. GRONWALD,~ ROBERTN. ANDERSEN, AND CHESTERYEE United States Department of Agriculture, Agricultural Research Service and Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 Received October 24, 1988; accepted April 19, 1989 A velvetleaf (Abutilon theophrasti Medic.) biotype, originally discovered in Maryland, was lo-fold more tolerant of atrazine than a susceptible or “wild type” biotype from Minnesota. The I, values for the inhibition of shoot growth in the atrazine-resistant and susceptible biotypes were 3.0 and 0.3 @4, respectively. Electron transport in chloroplast thylakoids isolated from leaves of both biotypes was equally sensitive to atrazine. Atrazine treatment inhibited whole leafphotosynthesis in both biotypes but the extent of the initial inhibition was less and the rate of recovery was greater in the resistant biotype. Both biotypes accumulated approximately equal amounts of radiolabel during a 3-hr pretreatment with [‘4C]atrazine via hydroponic solution. Both biotypes metabolized atrazine via glutathione conjugation in stem and leaf tissue. However, the resistant biotype did so more rapidly. As measured immediately after the 3-hr pretreatment, the concentration of the glutathione conjugate of atrazine was approximately 9-fold greater in stem tissue and approximately 2-fold greater in leaf tissue of the resistant biotype. The rates of formation of the glutathione conjugate of atrazine were compared in excised leaf discs of both biotypes and the Fl hybrids obtained by reciprocal crosses. As compared to the susceptible biotype, the rate of formation of the glutathione conjugate was approximately dfold greater in leaf discs of the resistant biotype. The rate of formation of the glutathione conjugate was equivalent in Fl progeny of reciprocal crosses and intermediate between that of the parents. It is concluded that attazine resistance in the Maryland biotype is under nuclear control and is due to an enhanced capacity to detoxify atrazine via glutathione conjugation. 0 1989 Academic PW.S. I,-,c. INTRODUCTION

There are numerous reports of the appearance of biotypes of weed species that are resistant to atrazine (1, 2). In those cases where the basis for resistance has been examined, a modification at the site of action, the 32-l&a quinone-binding protein, has been identified (3, 4). This trait has been demonstrated to be maternally inherited (5, 6). Recently, an Abutilon theophrasti biotype resistant to atrazine was discovered in a field in Maryland where triazine herbicides had been applied for at least 5 con’ Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service and the Minnesota Agricultural Experiment Station. Paper No. 16,259, Scientific Journal Series, Minnesota Agricultural Experiment Station, St. Paul, MN. ’ To whom correspondence and reprint requests should be addressed.

secutive years in a continuous no-till corn production system (7). Investigations by Andersen and Gronwald (8) revealed that atrazine resistance in the Maryland biotype is not maternally inherited, but rather is controlled by a single nuclear gene exhibiting partial dominance. The objectives of this study were to quantify the degree of resistance in the biotype found in Maryland and to determine the basis for this trait. MATERIALS

AND METHODS

Chemicals. [14C]Atrazine (uniformly ring-labeled, spec act 60 pCi/mg, 98% purity) and technical grade (purity greater than 98%) atrazine [6-chloro-N-ethyl-N’(1-methylethyl)-1,3,5-triazine-2,4-diamine], hydroxyatrazine [2-hydroxy-4-ethylamino6-isopropylamino-s-triazine], N-des-isopropylatrazine [2-chloro-4-amino-6-ethyl-

149 0048-3575/89 $3.00 Copyright 8 1989 by Academic Press. Inc. AU rights of reproduction in any form reserved.

150

GRONWALD,

ANDERSEN,

amino-s-triazine], and IV-des-ethylatrazine [2-chloro-4-amino-&isopropylamino-s-triazine] were provided by Ciba-Geigy Corp. (Greensboro, NC). The GSH3 conjugate of atrazine, used as a standard, was prepared using the method of Crayford and Hutson (9). Peter’s Hydrosol was obtained from Peter’s fertilizer products, Fogelsville, Pennsylvania. All other chemicals used in this study were obtained from Sigma Chemical Co. (St. Louis, MO). Plant material. A bulk sample of velvetleaf (A. theophrusti Medic) seed from plants that had survived triazine treatments in a field near Westminster, Maryland was provided by Ritter (7). Seeds were germinated in the greenhouse and when in the early three-leaf stage were sprayed with 3.3 kg/ha of atrazine, followed 1 week later by atrazine at 6.7 kg/ha plus crop oil concentrate at 2 liter/ha. The atrazine-resistant plants used in this study were descendants of one plant that had survived the atrazine treatment. Seed of the atrazine-susceptible or “wild type” biotype were collected from a plant found in a field near Rosemount, Minnesota. Growth studies. Seeds were scarified by a 20-min treatment with concentrated H2S04 followed by rinsing with deionized water. Seeds were planted in vermiculite in a growth chamber maintained at 25”C, 55 + 5% relative humidity, with a 16-hr photoperiod. The PAR at plant height was approximately 220 uE * me2 * set-‘. After 1 week of growth, seedlings (in the two-leaf stage of growth, approximately 2.5 cm tall) were transferred to a Styrofoam template floating in a plastic tub (28 x 33 x 13 cm) containing 10 liters of nutrient solution. Holes had been cut in the Styrofoam and seedlings were positioned in the holes such that the roots extended into the nutrient solution with the shoots extending above the 3 Abbreviations used: DCPIP, 2,ddichlorophenol indophenol; GSH, ghnathione; GS-atrazine, ghrtathione conjugate of atrazine; PAR, photosynthetically active radiation; TLC, thin-layer chromatography.

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template. Seedlings were held in place by a piece of cotton. The nutrient solution consisted of 4.01 g of Ca(NO,), . 4H,O and 4.87 g of Peter’s Hydrosol. The nutrient solution pH was 5.5 and the final nutrient composition in milligrams per liter was: N, 73; P, 24; K, 105; Ca, 68; Mg, 15; S04, 58.5; Fe, 1.54; Mn, 0.51; Zn, 0.15; Cu, 0.05; B, 0.51; MO, 0.05; Cl, 0.02; and Na, 1.81. After 5 days of growth in hydroponic culture as described above, eight uniform seedlings were transferred to a Styrofoam template in a plastic tub (28 x 33 x 13 cm) containing 10 liters of aerated nutrient solution. Nutrient solution composition and environmental conditions were the same as those described above. Each treatment consisted of eight seedlings per lO-liter tub. Two days after transfer, various concentrations (0.1 to 25 ujV) of commercially formulated atrazine (AAtrex 90)4 were added to the nutrient solutions. Nutrient solutions (-+atrazine) were changed every other day during the experiment to maintain a constant level of atrazine exposure. Plants were harvested 2 weeks after initial exposure to atrazine and shoot fresh weight was determined. The I, values (atrazine concentrations required to reduce shoot fresh weight by 50%) were calculated. Chloroplast isolation and assay. Thylakoids were isolated from leaves of 9week-old atrazine-resistant and susceptible biotypes grown in soil in the greenhouse. The procedure used was that of Darr et al. (10) with modifications. All operations were conducted at 0-4”C. Five grams of leaf tissue (minus midribs) were rinsed with cold distilled water, precooled in a beaker of ice, and then homogenized in a Sorvall omnimixer (high speed, 15 set) containing 100 ml of homogenizing medium (100 nUI4 4 Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or the University of Minnesota and does not imply its approval to the exclusion of other products or vendors which may also be suitable.

ENHANCED

ATRAZINE

DETOXIFICATION

IN VELVETLEAF

151

tricine-NaOH, pH 7.8, 400 mM sorbitol, 3 ml of aerated buffer [0.2 mM KPO.,, 0.5 mM mM MgCi,, 2 mM sodium ascorbate). The Ca(NO,),, pH 7.0, -t 30 p.M technical grade homogenate was filtered through four lay- atrazine]. The shoot portion of the plant ers of cheesecloth and centrifuged at 1SOOg was suspended above the test tube with the for 5 min. The pellet was suspended in a roots extending down into the test tube and wash solution buffer (10 mM tricineexposed to the atrazine solution. The plants NaOH, pH 7.8,10 mM NaCi, 5 mM MgCi,) were incubated in a growth chamber mainand then centrifuged at 3000gfor 5 min. The tained under the environmental conditions pellet containing thyiakoid fragments was described above (growth studies). After 3 resuspended in a final suspension medium hr of atrazine exposure, the plants were re(10 mM tricine-NaOH, pH 7.8, 100 mM moved from the test tubes and the roots sorbitol, 10 mM NaCi, 5 mM MgCi,). The were rinsed with distilled water. Each plant thylakoid fraction was kept on ice in the was then transferred to another test tube dark until used. Chlorophyll concentration containing IS ml of aerated incubation mewas determined by the method of Arnon dium but without atrazine. After the appropriate time interval (0, 1, 3, 6, and 24 hr (11). Photosystem II-dependent electron after transfer), the third leaf of the plants transport was assayed in isolated thyia- was excised and four leaf discs (14 mm in koids as described by Darr et al. (10). The diameter, approx 70 to 80 mg fresh wt) were assay medium contained: 50 mM cut from the leaf with a No. 7 cork borer. Na,HPO,-NaOH, pH 6.8, 10 mM NaCi, 5 The LD-2 Hansatech oxygen electrode mM MgCi,, 100mM sorbitoi, 1 mM NH&i, (Decagon Devices, Pullman, WA) was used 30 @f DCPIP, 0.1 pM gramicidin D. As- to measure the photosynthesis rate of the says were conducted in 3 ml of assay me- excised leaf discs (12). Measurements were dium which contained 15 to 20 p.g chloro- made at 25°C under conditions of saturatphyll/ml. Technical grade atrazine prepared ing fight intensity (approx 1200 in absolute ethanol was added to the assay FE . me2 . set-’ at leaf surface) and medium. The final ethanol concentration CO, concentration (approximately 5%). did not exceed 0.1% (v/v) and had no effect Appropriate controls (minus atrazine) were on photoreduction of DCPIP. DCPIP re- run throughout the experiment to correct duction was monitored spectrophotometri- for diurnal variation in photosynthesis rate. tally at 580 nm. The suspension was crossMetabolism studies with intact plants. illuminated with actinic light which had Approximately 3-week-old plants were been filtered through a red Corning 2-58 fil- treated as described above (photosynthesis ter. The phototube was shielded from scat- determination) except that the test tube tered actinic light by a blue Coming 4-96 contained 9 ml of aerated standard incubafilter. tion medium plus 30 PM [14C]atrazine (spec Photosynthesis determination. Approxi- act 3.38 pCi/pmol). Plants were given a 3mately 3-week-old plants, grown hydropon- hr pulse of [14C]atrazine via the roots and ically as described above (growth studies), were then transferred to a test tube containwere used. Individual plants were trans- ing 9 ml of aerated incubation medium withferred to polyethylene stoppers with holes out atrazine. The plants were harvested at in the bottom to allow roots to extend be- prescribed time intervals (0, 1,6, and 24 hr) low the stopper. The shoot portion of the and divided into roots, stem (including petplant was held in an upright position in the ioies and cotyledons), and leaves. In some stopper with a piece of cotton. Plants, po- experiments, only the third leaf was samsitioned in the polyethylene stoppers, were pled. Plant parts were weighed, frozen in transferred to test tubes (16 x 125 mm) liquid nitrogen, and stored at -20°C. wrapped with aluminum foil containing 15 Plant tissues (leaves, stem, and roots)

152

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were homogenized with a Polytron tissue homogenizer (setting No. 6 for 1 min) in 20 ml of 95% methanol. Leaf tissue was homogenized twice, whereas stem and root tissue were homogenized once. The extraction efficiency for all tissues was greater than 98%. The homogenate was centrifuged (83Og, 5 min) to remove debris. Methanol was removed from the extract in vacua at 30°C. The volume of the remaining aqueous phase was adjusted to 2 ml, centrifuged as described above, and an aliquot (100 ~1) was counted by liquid scintillation spectroscopy. An additional lOO-p,l aliquot of the extract was applied to a TLC plate (Anasil H7, 250 km), which had been converted to reverse phase by spraying with 10% mineral oil in hexane (13). Hexane was removed by heating the plates for several minutes at 75°C. Atrazine and metabolites in the extract were separated by developing the TLC plates with methanol:H,O (20:80, v/v). The radiolabel in the extracts was identified as atrazine and GS-atrazine on the basis of cochromatography with authentic standards. In addition to the TLC solvent system described above, the identity of the GSH conjugate was confirmed by cochromatography with an authentic standard of GS-atrazine on silica (Anasil H7) TLC plates in the following solvent systems: (I) benzene:acetic acid (50:4, v/v) (14); (II) benzene:ethanol:acetic acid (10: 10:1, v/v/v) (15); (III) n-butanol:acetic acid:H,O (12:3:5, v/v/v) (15), (IV) solvent B (benzene:ethyl acetate:acetic acid:H,O; 25:50:20:3, v/v/v/v), and solvent C (nbutanol:acetic acid:H,O; 60: 15:25, v/v/v). Plates were developed three times in solvent B and once in solvent C, with all developments in the same direction (16). After separating atrazine and GSatrazine in the plant extracts on the reverse-phase Anasil H7 plates with methanol:H,O (20:80, v/v), a TLC linear analyzer (Berthold Model 2B 282) was used to quantify the relative amounts of radiolabe1in the two forms. The absolute concentrations of atrazine and GS-atrazine separated by TLC were determined by multiply-

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YEE

ing the relative percentages of atrazine and GS-atrazine measured by the TLC linear analyzer by the total dpms found in the tissue extract. Metabolism studies with leaf discs. Two types of metabolism studies were conducted with excised leaf discs: (I) atrazine metabolism in leaf discs of susceptible and resistance biotypes during a 3-hr continuous exposure to [14C]atrazine, and (II) atrazine metabolism in leaf discs of the susceptible and resistant biotypes and the Fl hybrids measured 24 hr after a 2-hr pulse with [14C]atrazine. For (I), 15leaf discs (9 mm in diameter, approx 120mg fresh wt) were excised from the second and third leaves of approximately 3-week-old plants. The leaf discs, from either the resistant or susceptible plants, were placed in a 50-ml beaker containing 5 ml of a medium containing 0.5 m&f Ca(NO&, 0.2 mM KPO,, pH 7.0, and 30 pM [‘4C]atrazine (spec act 4.1 l.&i/tJ,rnol). The final concentration of ethanol was 1% (v/v). Leaf discs were infiltrated for 3 min in a vacuum desiccator. The beakers containing the leaf discs were placed on a gyrating shaker in a growth chamber, 25°C 220 p,E . me2 * set-‘. After 0, 1, and 3 hr, the external labeled medium was removed by filtering the 15 leaf discs on a Buchner funnel. The leaf discs were rinsed three times with 100 ml of distilled water, frozen in liquid nitrogen, and stored at -20°C until atrazine and GSatrazine were extracted and quantified as described above. For (II), 20 leaf discs (8 mm in diameter, approx 100 mg fresh wt) were excised from mature leaves of approximately 3-month-old greenhouse-grown plants. The leaf discs were vacuum infiltrated in the 14C-labeledmedium described above. The beakers containing the leaf discs were placed on a gyrating shaker at room temperature (23°C). After 2 hr, the leaf discs were removed from the labeled medium by filtering with a Buchner funnel, rinsed three times with 100 ml of distilled water, and placed in a petri dish (15 x 100 mm) which contained filter paper (Whatman No. 1) saturated with the incubation

ENHANCED

ATRAZINE

DETOXIFICATION

IN VELVETLEAF

153

medium minus atrazine. The discs were in- in the time course of development and the cubated in a growth chamber under contin- expression of injury symptoms in leaf tissue uous illumination (220 FE * mW2* set- ‘) of the two biotypes. In the case of the refor 24 hr at 25°C. Leaf discs were then fro- sistant biotype, a 48-hr exposure to 5 +V zen in liquid nitrogen and stored at -20°C atrazine via hydroponic solution inhibited until atrazine and GS-atrazine were ex- growth (shoot fresh weight) by 17% (data tracted and quantified as described above. not shown). During this period, veinal chlorosis developed on the second leaf (plants RESULTS in the three-leaf stage of growth). During Growth response. Differences in growth the next 5 days, veinal chlorosis intensified response between the atrazine-resistant in the second leaf and was also expressed in and susceptible biotypes were quantified by the third leaf. After 7 days of exposure to 5 exposing young seedlings to a range of atra- $l4 atrazine, shoot fresh weight of the rezine concentrations (0.1 to 25 p&) via hy- sistant biotype was 40% of the control (undroponic solution. The resistant biotype treated) value. In the case of the susceptiwas lo-fold more tolerant of atrazine than ble biotype, a 48-hr exposure to 5 PM atrathe susceptible biotype (Fig. 1). Iso values zine inhibited shoot growth by 41%. In for the susceptible and resistant biotypes contrast to the resistant biotype, veinal were 0.3 and 3.0 ~JM,respectively. chlorosis did not develop in leaf tissue durAs was reported earlier (8), the resistant ing 48 hr of exposure. Rather, the entire biotype grew somewhat less vigorously surface of the second leaf exhibited a very than the susceptible biotype. At the termi- slight chlorosis. After 72 hr, necrotic lenation of the growth response studies sions developed on the second leaf and af(when plants were 4 weeks old), the shoot ter 5 days of exposure to 5 p&f atrazine, all fresh weight of the untreated resistant bio- leaf tissue of the susceptible biotype was type was 34% less than that of the untreated completely necrotic. susceptible biotype. Chloroplast assays. Atrazine was equally Injury symptoms. There was a difference inhibitory to electron transport, as mea-

.

RESISTANT

0 SUSCEPTIBLE

0

10-7

ATRAZINE

10-S

CONCENTRATION

10-S

(Ml

FIG. 1. Growth response of shoot tissue of susceptible and resistant A. theophrasti biotypes grown in the presence of atrazine. I,, values (in parentheses) represent the atrazine concentration required to reduce shoot fresh weight by 50%. Values represent the mean + SE (n = 16). SE provided when larger than data point. Shoot fresh weights of the untreated resistant and susceptible biotypes at the termination of the experiment where 13.2 f 0.6 (SE) and 20.0 +- 1.3 g, respectively.

154

GRONWALD,

ANDERSEN,

sured by DCPIP reduction, in chloroplasts isolated from resistant and susceptible biotypes (Fig, 2). The atrazine concentration required to reduce the rate of DCPIP reduction by 50% (Is,,) in chloroplasts isolated from either biotype was 0.18 p&. These results indicate that tolerance is not due to a modification at the site of herbicide action; the 32-kDa quinone-binding protein on the reducing side of photosystem II (3, 4). In many respects, this result was expected as resistance in this biotype is not cytoplasmitally inherited (8) as is resistance associated with a modification at the 32-kDa quinone-binding protein (5, 6). E&cts on photosynthesis. It is well established that atrazine is a photosynthesis inhibitor (3). The effect of this herbicide on photosynthesis was determined in both susceptible and resistant biotypes (Fig. 3A). A 3-hr exposure to 30 pM atrazine via hydroponic solution inhibited photosynthesis in the third leaf of both resistant and susceptible biotypes. However, the degree of inhibition was not as great and the recovery was faster in the resistant biotype. In the susceptible biotype, photosynthesis was inhibited by 90% after atrazine treatment. There was little or no recovery during the

ATRAZINE

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YEE

initial 3 hr after treatment. Between 3 and 6 hr, photosynthesis recovered to 55% of the control rate. Approximately 24 hr was required for complete recovery of photosynthesis. In contrast, photosynthesis in the third leaf of the resistant biotype was inhibited by about 35% after atrazine treatment and complete recovery occurred after 3 hr. Metabolism in the third leaf. The differential recovery of photosynthesis in susceptible and resistant biotypes after atrazine treatment (Fig. 3A) suggested there may be differences in the ability of the two biotypes to metabolize atrazine. In order to determine whether this was the case, the kinetics of atrazine metabolism were evaluated in the same tissue (third leaf) and under the same environmental conditions as were used to determine the effects of atrazine on photosynthesis (Fig. 3B and 3C). Atrazine was metabolized to a GSH conjugate in the third leaf of both resistant and susceptible biotypes. Other metabolites (hydroxyatrazine, des-ethylatrazine, and des-isopropylatrazine) , if formed during the 24-hr study, were beyond the detection limits of the instrumentation used. The identity of the GS-atrazine conjugate was based on cochromatography with an authentic stan-

CONCENTRATION

(M)

2. Effect of atrazine on DCPIP reduction in chloroplast thylakoids isolated from atrazineresistant and susceptible biotypes. The atrazine concentration required to reduce the rate of DCPIP reduction by 50% (Z& was 0.18 @for chloroplasts isolated from both resistant and susceptible biotypes. FlG.

ENHANCED

RECOVERY

ATRAZINE

PERIOD

DETOXIFICATION

(h)

(A) Recovery ofphotosynthesis in the third leaf of susceptible (0) and resistant (0) A. theophrasti biotypes following a 3-hr treatment with [14Cjatrazine via hydroponic solution. At zero time, seedling roots were transferred from a medium containing [‘*CIatrazine to the same medium minus atrazine. See Materials and Methods for details. Rates of 0, evolutionfor untreated controls at zero time were 531 2 91 (SE) and 513 ‘- 80 ~01 0,/g fresh wtihr in susceptible and resistant biotypes, respectively. (B) Atrazine concentrations in the third leaf of susceptible (0) and resistant (e) biotypes following a 3-hr pretreatment with l’4Clatrazine via hydroponic solution. (C) GS-atrazine concentrations in the third leaf of susceptible (0) and resistant (0) biotypes following a 3-hr pretreatmenr with [“C]atrazine via hydroponic solution. FIG.

3.

155

IN VELVETLEAF

sult, GS-atrazine accounted for 57% of the total radiolabel in the third leaf of the resistant biotype as compared to 22% in the third leaf of the susceptible biotype. The concentration of atrazine in the third leaf of the susceptible biotype at 0 hr (immediately after the 3-hr pretreatment) was 2.4-fold greater than that in the resistant biotype. There was a rapid decline in atrazine levels in the third leaf of the susceptible biotype during the first hour after atrazine treatment. Concomitant with this decline was an increase in the levels of GS-atrazine. There was little change in the ievels of atrazine or GS-atrazine in the third leaf of the resistant biotype during the 24-hr recovery period. In both the resistant and susceptible biotypes, there appeared to be a background level of atrazine that was not available for metabolism or binding at the site of action. During the 24-hr recovery period, photosynthesis completely recovered in both biotypes, yet the estimated concentration of atrazine in the third leaf of both biotypes was about 18 nmol/g fresh wt (Fig. 3B). Differences

in metabolism

among

tis-

sues. The fact that less radiolabel moved to the third leaf of the resistant biotype suggested that the translocation of [‘4C]atrazine was restricted in the root and/ or stem. To evaluate this, the levels of atrazine and GS-atrazine were measured in root, stem, and leaf tissue of susceptible dard using the solvent systems described (Table I) and resistant (Table 2) biotypes 0, under Materials and Methods. 1, 6, and 24 hr after a 3-hr pulse with After the 3-hr pretreatment with [14C]atrazine via hydroponic solution. On [“Clatrazine (at zero time), the total con- both a gram fresh weight basis (zero time, centration of radiolabel (atrazine plus GS- Tables 1 and 2) and on a whole plant basis atrazine) was 30 and 42 nmol/g fresh wt in (data not shown), resistant and susceptible the third leaf of the resistant and suscepti- biotypes accumulated approximately equal ble biotype, respectively (Fig. 3B and 3C). amounts of radiolabel during the 3-hr preThe concentration of GS-atrazine was ap- treatment. However, the distribution of raproximately twofold greater in the leaf of diolabel within the plant and its form (parthe resistant biotype. As compared to the ent herbicide or metabolite) differed bethird leaf of the susceptible biotype, less tween the two biotypes. atrazine moved to the third leaf of the reImmediately after the pulse with atrazine sistant biotype and that which entered the (at zero time), root tissue of both biotypes Ieaf was metabohzed more rapidly. As a re- contained approximately 25% of the total

55.7

9.6

Cl

5.9

3.8 f 1.1 1.6 + 0.3 0.5 * 0.2

GS-Atrazine

and GS-Atrazine

15.6 2 3.4 22.5 t 1.4 17.6 k 0.6

Atrazine

of Atrazine

TABLE

1

Atrazine

28.0

_____ GS-Atrazine

25.9

9.8

nmoVg fresh wtb 6.3 2 1.8 2.8 +- 0.4 0.7 2 0.1

1

Incubation

20.1

Biotype

11.5

8.6 + 1.9 2.3 f 0.2 0.6 + 0.1

GS-Atrazine

36.4

6

theophrasti

12.9 2 2.1 5.0 2 0.4 2.2 f 0.1

____ Atrazine

period (hr)”

Tissues of the Susceptible Abutilon Pretreatment with [‘4CjAtrazine

13.8 + 2.3 11.1 2 0.5 3.1 k 0.1

-

in Various after

17.0

11.8 + 1.3 3.6 2 0.3 1.6 rc_0.1

Atrazine

24

11.1

8.4 + 1.2 2.3~0.1 0.4 iI 0.1

GS-Atrazine

39.5

0, I, 6, and 24 hr

o After 3-hr pretreatment with 30 PM [‘4C]atrazine. See Materials and Methods for details. b Values represent the mean 2 SE for three experiments (n = 7). The mean fresh weight of leaf, stem (including petiole and cotyledons), and root tissue was 0.58, 0.29, and 0.32 g, respectively.

Total Percentage of total radiolabel as GS-atrazine

Leaf Stem Root

Tissue

Levels

%

F

5 g g

z E F p

;

40.8

5.9 + 1.2 16.9 2 1.4 18.0 4 1.1

Atrazine

24.6

8.2 k 2.3 15.0 2 1.9 1.4 r 0.2

GS-Atrazine

37.6

0

and GS-Atrazine

17.6

26.4

fresh wt” 9.2 t 1.4 15.9 2 1.2 1.3 k 0.2

GS-Atrazine

nmoVg 5.1 k 0.6 8.6 +- 1.1 3.3 t- 0.5

Atrazine

1

18.0

6.6 f 0.9 9.6 f 1.3 1.8 5 0.2

Atrazine

6

26.4

9.1 2 1.0 15.9 + 1.4 1.4 4 0.1

GS-Atrazine

17.7

7.1 f 1.3 9.3 2 1.3 1.3 f 0.1

Atrazine

theophrasti Biotype 0, 1, 6, and 24 hr

Incubation period (hr)”

TABLE 2 in Various Tissues of the Resistant Abutilon after Pretreatment with [“CjAtrazine

24

22.8

10.3 2 1.3 11.3 2 1.2 1.2 _’ 0.1

GS-Atrazine

60.0 59.5 56.3 __a After 3-hr pretreatment with 30 )*&I [‘4C]atrazine. See Materials and Methods for details. b Values represent the mean 2 SE for three experiments (n = 7). The mean fresh weight of leaf, stem (including petioles and cotyledons), and root tissue was 0.54, 0.28, and 0.27 g, respectively.

Total Percentage of total radiolabel as GS-atrazine

Leaf Stem Root

Tissue

Levels of Atratine

2 ,$J * rl %

2 5:

4$ 8 5

g

2

E

5

? 3 g

kz

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GRONWALD,

ANDERSEN,

radiolabel accumulated in the plant (data not shown) and almost all was in the form of atrazine (Tables 1 and 2). There was a rapid loss of radiolabel from the roots of both biotypes during the first hour after transfer from the solution containing [14C]atrazine. This radiolabel was identified as [14C]atrazine. Price and Balke (17) also reported a rapid efflux of atrazine from velvetleaf roots when rinsed or transferred to an atrazine-free solution. We, as well as Price and Balke (17), attribute this loss to the rapid, passive eftlux of the lipophilic atrazine molecule across membranes. There was little metabolism of atrazine by roots of either biotype. The levels of GSatrazine in root extracts from the resistant biotype were slightly higher than those found in the susceptible biotype. However, the GS-atrazine found in the root extracts may actually represent metabolism occurring in stem tissue. Root tissue was excised at the level of the hydroponic solution and may have included some stem tissue. There were significant differences in the amount of radiolabel and its identity in stem tissue of the two biotypes. More radiolabel accumulated in the stem of the resistant biotype and a much greater proportion was in the form of the GSH conjugate (Tables 1 and 2). The levels of GS-atrazine (at zero time) were approximately ninefold greater in the stem of the resistant biotype. The amount of radiolabel in stem tissue decreased during the 24-hr period following atrazine treatment in both biotypes. This change, which was more apparent in the susceptible biotype, cannot be attributed to movement of radiolabel from stem to leaf tissue as there was little, if any, increase in radiolabel in leaf tissue during this period. This loss of radiolabel could be partly due to the basipetal movement of atrazine in the phloem. Such movement has been reported to occur in A. theophrasti (18). However, for the most part, this loss probably represents the efflux of atrazine from the basal portion of the stem. The basal 1 to 1%” portion of the stem was periodically ex-

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posed to atrazine during the atrazine treatment due to the aeration of the medium bathing the roots. Therefore, after transfer from the labeled medium to an unlabeled medium, the rinsing of this portion of the stem by the aerated medium would promote the passive efflux of atrazine as occurred in root tissue. Efflux from the stem of the resistant biotype was less, probably due to the fact that a much larger fraction of the radiolabel entering the stem was metabolized to GS-atrazine which, being more polar, is less able to passively efflux from the tissue. Immediately after atrazine treatment (at zero time), 35 and 47% of the total radiolabe1absorbed by the plant was found in leaf tissue of the resistant and susceptible biotypes, respectively (data not shown). The concentration of GS-atrazine was approximately twofold greater in leaf tissue of the resistant biotype. Of the total radiolabel in leaf tissue at zero time, 58 and 20% were in the form of the GSH conjugate in resistant and susceptible biotypes, respectively. Hence, as was observed in studies that focused on only the third leaf (Fig. 3), less atrazine was translocated to total leaf tissue in the resistant biotype during the 3-hr pulse and that which was translocated to leaf tissue was conjugated with GSH at a faster rate. Metabolism in leaf discs. As measured in leaves of intact plants exposed hydroponically to atrazine, the formation of the GSatrazine conjugate was more rapid in leaf tissue of the resistant biotype (Fig. 3, Tables 1 and 2). However, because the enhanced metabolism in the stem reduced the amount of atrazine moving to leaf tissue of the resistant biotype, the inherent difference in capacity of leaf tissue of the two biotypes to metabolize atrazine could not be properly evaluated. In order to evaluate the difference in atrazine conjugating capacity in leaf tissue of the resistant and susceptible biotypes, atrazine metabolism was measured in leaf discs exposed exogenously to [‘4C]atrazine. The levels of GS-

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atrazine were approximately fivefold greater in the resistant biotype after a 3-hr exposure to 30 a atrazine (Fig. 4). The untake of 1’4Clatrazine was greater in the leaf discs of the resistant biotype but this reflects enhanced metabolism. Atrazine metabolism in the Fl hybrids. The inheritance of atrazine resistance in the Maryland biotype is controlled by a single nuclear gene exhibiting partial dominance (8). As measured in terms of a growth response, the Fl hybrids obtained by reciprocal crosses of resistant and susceptible parents exhibited an intermediate level of resistance. We examined atrazine metabolism in leaf discs of the resistant and susceptible parents and the Fl hybrids (Table 3). Similar to what was found for growth response (8), the reciprocal Fl hybrids exhibited the same rate of formation of the GS-atrazine conjugate, which was intermediate between the susceptible and resistant parents (Table 3). Collectively, the results of this investigation and our previous study (8) indicate that the enhanced rate of formation of GS-atrazine in stem and leaf tissue of the resistant biotype is not maternally inherited but rather is the expression of a nuclear gene exhibiting partial dominance ,

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TABLE 3 Levels of Atrazine and GS-Atrazine in Leaf Discs of Resistant and Susceptible Biotypes and the Fl Generation Obtained by Reciprocal Crosses Parent or Fl

Atrazine

GS-Atrazine

nmol/g fresh wt 2.3 f 0.2 14.5 k 1.3 16.1 + 0.6 16.9 f 0.8

Susceptible

Resistant Fl (resistant 0 x susceptible c?) Fl (susceptible P x resistant c?)

16.4 r 1.2

10.2 2 0.4

12.7 ‘- 1.1

10.1 * 0.6

Note. Levels of atrazine and GS-atrazine measured 24 hr after a Zhr exposure to [14C]atrazine. See Materials and Methods for details. Values are means t SE (n = 4). DISCUSSION

The results of this study indicate that the basis for the lo-fold greater atrazine tolerance in the A. theophrasti biotype found in Maryland is enhanced atrazine detoxification via GSH conjugation. Resistance is not due to a modification at the site of action or differences in atrazine uptake. There was less translocation of atrazine from roots to leaves in the resistant biotype but this reflects enhanced metabolism in the stem. The evidence that enhanced detoxification via GSH conjugation is the basis for

150 0 RESISTANT 0 SUSCEPTIBLE GS-ATRAZINE _____ _.. ATRAZINE

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PERIOD

(h)

FIG. 4. Levels of atrazine and GS-atrazine in leaf discs of the resistant and susceptibie biotypes during a 3-hr exposure to atrazine. Values represent means i SE (n = 3). SE bars shown when larger than data point.

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resistance is as follows. First, after a 3-hr exposure to atrazine the concentration of GS-atrazine is approximately ninefold greater in stem tissue and twofold greater in leaf tissue of the resistant biotype (zero time, Tables 1 and 2). Second, the lesser reduction in photosynthesis and the rapid recovery after atrazine treatment in the resistant biotype are correlated with the enhanced rate of GSH conjugation (Fig. 3). Third, the capacity for GSH conjugation of atrazine in the reciprocal Fl hybrids of the resistant and susceptible biotypes is intermediate between that of the resistant and susceptible parents (Table 3). This pattern of inheritance for GSH conjugation in the Fl hybrids is the same as that observed for the inheritance of atrazine resistance evaluated in terms of a growth response (8). Although the rate of atrazine detoxification via GSH conjugation was more rapid in the resistant biotype, leaf tissue of the susceptible biotype exhibited a significant capacity to detoxify atrazine via GSH conjugation. It is widely known that atrazine tolerance in certain grasses [corn, sorghum, Johnsongrass (Sorghum halepense (L.) Pers), and Sudan grass [S. sudanense (Piper) Stapfl is related to their ability to rapidly detoxify atrazine via GSH conjugation (19). It is of interest that A. theophrasti, a dicot, is also able to detoxify atrazine via GSH conjugation. This may account for the fact that the susceptible or wild type A. theophrasti biotype is only moderately susceptible to atrazine (8). Additional evidence supporting a role for GSH conjugation in regulating atrazine tolerance in velvetleaf is the finding that tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl) oxirane] synergizes atrazine in A. theophrasti (20). Tridiphane synergizes atrazine in certain weed species by forming a GSH conjugate that inhibits the glutathione S-transferase catalyzing GSH conjugation of atrazine (21). The results of this study show enhanced GSH conjugation with atrazine in both leaf and stem tissue of the atrazine-resistant

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biotype. However, the greatest difference in GS-atrazine levels between the two biotypes is in the stem where the GS-atrazine concentration found after a 3-hr pulse with [‘4C]atrazine was approximately ninefold greater in the resistant biotype (Tables 1 and 2). These results suggest that metabolism in the stem plays an important role in conferring atrazine resistance when the herbicide is taken up through the roots as occurs during exposure via hydroponic solution or when atrazine is applied preemergence in the field. The differential rate of atrazine metabolism in the stem of the two biotypes is a major factor regulating the kinetics of inhibition and the recovery of photosynthesis in leaf tissue after hydroponic exposure to atrazine (Fig. 3). In the resistant biotype, the enhanced capacity for conjugation in the stem reduces the amount of atrazine moving to leaf tissue and the atrazine which is translocated to leaf tissue is rapidly detoxified because of the enhanced GSH conjugation capacity in this tissue. Once taken up by the roots, atrazine is carried to leaf tissue in the xylem with transpiration being the driving force for upward movement. The xylem elements and tracheids are devoid of cytoplasm and hence are not capable of enzymatic detoxification of atrazine via GSH conjugation. How is it then that atrazine is being metabolized during the course of apoplastic movement to leaf tissue? Although movement is primarily in the apoplast, it is known that lipophilic herbicides such as atrazine can diffuse laterally into surrounding cells in the vascular bundle (22) and probably into parenchyma cells of the pith. Apparently, one or more of these cell types has the capacity to conjugate atrazine with GSH in A. theophrusti and this capacity is greater in the cells of the resistant biotype. Conjugation of atrazine with GSH yields a water-soluble metabolite which is considered to be less mobile in the plant (19). Hence, atrazine may readily diffuse from the xylem to surrounding cells during apoplastic movement

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in A. theophrasti but once conjugated with GSH, it does not readily diffuse back into the xylem. The net result is greater accumulation of radiolabel in the stem of the resistant biotype due to enhanced metabolism. Besides this study, there is another report which suggests that metabolism of photosynthesis inhibitors in the stem may contribute to tolerance when the herbicide enters the plant via the roots. Fedtke and Schmidt (23) reported that stem metabolism was important in the tolerance of soybean cultivars to metribuzin (4-amino6-l-( l ,l-dimethylethyl)-3-(methylthio)1,2,4-triazin-5(4H)-one) when herbicide exposure was via hydroponic solution. Soybean cultivars that were tolerant to metribuzin metabolized a greater percentage of the herbicide in the stem resulting in less translocation to the leaves. It should be noted, however, that the mechanism of metribuzin detoxification in soybean differs from that of atrazine detoxification in A. theophrasti. Atrazine is metabolized via GSH conjugation in A. theophrasti, whereas metribuzin is metabolized via Ndealkylation in soybean. Although the results suggest that the stem metabolism of atrazine plays a role in the tolerance of the atrazine-resistant biotype when entry of the herbicide is primarily via the roots, atrazine metabolism in the stem is not necessary for the expression of atrazine resistance. The differential tolerance of the susceptible and resistant A. theophrasti biotypes is also expressed when atrazine is applied primarily to leaf tissue as occurs during postemergence applications. The biotype from Maryland will tolerate postemergence applications of 10 kg/ha atrazine which kills the susceptible biotype (8). In stem and leaf tissue, atrazine levels did not change substantially during the period 6 to 24 hr after the atrazine pulse (Tables 1 and 2). It appears that there is a background level of atrazine which is unavailable for metabolism via GSH conjugation

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or for binding at the site of action. This is clearly shown in Fig. 3 where approximately 50% of the radiolabel in the third leaf of the resistant biotype remained as atrazine 24 hr after atrazine treatment, yet photosynthesis had completely recovered within 3 hr of atrazine treatment. Based on the assumption that 1 g fresh weight of tissue is equivalent to 1 ml (24), the estimated atrazine concentration in the third leaf of the resistant biotype 24 hr after atrazine treatment is approximately 18 p,M. In view of the I,, for inhibition of photosystem IIdependent electron transport (Fig. 2), one would expect that if this atrazine was available at the site of action, photosynthesis would be completely inhibited. The results of Shimabukuro et al. (25) also indicate that a background level of atrazine exists in corn leaf tissue after atrazine treatment. Photosynthesis in corn leaf discs pretreated for 1.5 hr with 37 ~.LM [14C]atrazine recovered to 90% of the control rate within 4 hr after treatment, yet 32% of the radiolabel extracted from the leaf discs was in the form of atrazine. It is likely that this background level of atrazine in A. theophrasti and corn leaf tissue is unavailable for metabolism or binding at the site of action because it is either (a) bound to matrices such as the cell wall or (b) sequestered into lipophilic matrices such as cellular membranes or the wax and cutin layers on the leaf surface. While there is little evidence to suggest that atrazine binds strongly to hydrophilic matrices such as cell walls (18), there is evidence that this herbicide will partition into hydrophobic matrices such as cellular membranes. Darmstadt et al. (26) estimated that the concentration of atrazine in isolated corn root protoplasts was 36% greater than that in the ambient medium and suggested that it was the result of atrazine partitioning into membranes. McCloskey and Bayer (27) reported that wheat leaf protoplasts had an internal atrazine concentration of 62 pJ4 when incubated in a medium containing 6.2 $l4 atrazine. The authors suggested that this was due to non-

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specific adsorption or partitioning of atrazine into cellular constituents such as lipids. There are approximately 50 weed species with biotypes which have developed resistance to triazine herbicides (28). In those cases where the mechanism of resistance has been examined, it has been shown to be due to a modification at the site of action, i.e., the 32-kDa quinone-binding protein. As far as the authors are aware, this is the first report of the appearance of “developed” atrazine resistance in a weed species where the primary basis for resistance is not a modification at the site of action. Rather, enhanced herbicide detoxification accounts for the lo-fold greater tolerance in the A. theophrasti biotype found in Maryland. There is one other report indicating that selection pressure applied as a result of repeated herbicide applications can select for weed biotypes with an enhanced detoxification capacity. Gressel et al. (29) reported that a simazine-resistant biotype of Brachypodium distachyon was found in Israel along roadsides where triazine herbicides (simazine, atrazine) had been applied repeatedly. Although the resistant biotype of B. distachyon had an enhanced capacity to partially detoxify atrazinc via N-dealkylation, this modification was reported to be of secondary importance in the resistance observed. The primary basis for resistance in this biotype was a modification at the site of action. Pesticide resistance among insects (30, 31) and fungi (32) has developed because of (a) modifications at the site of action, (b) enhanced detoxification, or (c) decreased uptake and/or translocation to the target site. Therefore, it should not be surprising that enhanced detoxification has also appeared as a mechanism of herbicide resistance in weeds. It is likely that in the future, under conditions where appropriate selection pressure is applied, other weed biotypes will appear which exhibit resistance to a herbicide or class of herbicides due to enhanced detoxification.

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The basis for enhanced GSH conjugation of atrazine in the resistant A. theophrasti biotype is not known. The GSH conjugation of atrazine is catalyzed by a glutathione S-transferase (19). Hence, an increase in GSH levels and/or glutathione Stransferase activity toward atrazine as substrate could be involved. These possibilities are currently being investigated. ACKNOWLEDGMENTS We thank Ciba-Geigy Corp. for providing [t4C]atrazine, technical grade atrazine, and its metabolites (hydroxyatrazine, des-ethyl and des-isopropyl derivatives). We also express our appreciation to Ron Ritter of the University of Maryland for providing the seed of the atrazine-resistant A. rheophrusri biotype. REFERENCES 1. J. D. Bandeen, G. R. Stephenson, and E. R. Cowett, Discovery and distribution of herbicide-resistant weeds in North America, in “Herbicide Resistance in Plants” (H. M. LeBaron and J. Gressel, Eds.), pp. P-30, Wiley, New York, 1982. 2. J. Gressel, Modes and genetics of herbicide resistance in plants, in “Pesticide Resistance: Strategies and Tactics for Management”, pp. 54-73, Nat. Acad. Press, Washington, DC, 1986. 3. C. J. Amtzen, K. Pfister, and K. E. Steinback, The mechanism of chloroplast triazine resistance: Alterations in the site of herbicide action, in “Herbicide Resistance in Plants” (H. M. LeBaron and J. Gressel, Eds.), pp. 185214, Wiley, New York, 1982. 4. J. Hirschberg and L. McIntosh, Molecular basis of herbicide resistance in Amarunthus hybridus, Science 222, 1346 (1983). 5. K. R. Scott and P. D. Putwain, Maternal inheritance of simazine resistance in a population of Senecio vulgaris, Weed Res. 21, 137 (1981). 6. V. Souza-Machado and J. D. Bandeen, Genetic analysis of chloroplast atrazine resistance in Bras&a campestris-cytoplasmic inheritance, Weed Sci. 30, 281 (1982). 7. R. L. Ritter, Triazine resistant velvetleaf and giant foxtail control in no-tillage corn, Proc. Northeast. Weed Sci. Sot. 40, 50 (1986). 8. R. N. Andersen and J. W. Gronwald, Noncytoplasmic inheritance of atrazine tolerance in velvetleaf (Abutilon theophrasti), Weed Sci. 35, 4% (1987). 9. J. V. Crayford and D. H. Hutson, The metabolism of the herbicide, 2-chJoro4(ethylamino)6-(1-cyano-1-methylethylamino)-s-triazine in the rat, Pestic. Biochem. Physiol. 2, 295 (1972).

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10. S. Darr, V. Souza-Machado, and C. J. Amtzen, Uniparental inheritance of a chloroplast photosystem II polypeptide controlling herbicide binding, Biochim. Biophys. Acta. 634, 219 (1981). 11. D. 1. Amon, Copper enzymes in isolated chloroplasts: Polyphenoloxidase in Beta vulgaris, Plant Physiol. 24, 1 (1949). 12. T. J. Delieu and D. A. Walker, Simultaneous measurement of oxygen evolution and chlorophyll fluorescence from leaf pieces, Plan? Physiol. 73, 534 (1983). 13. E. Stahl, Special techniques, in “Thin-layer Chromatography: A Laboratory Manual” (E. Stahl, Ed.), p. 37, Academic Press, New York, 1%5. 14. R. H. Shimabukuro, R. E. Kadunce, and D. S. Frear, DeaikyIation of atrazine in mature pea plants, J. Agric. Food Chem. 14, 392 (1966). 15. R. H. Shimabukuro, W. C. Walsh, G. L. Lamoureux, and L. E. Stafford, Atrazine metabolism in sorghum: Chloroform-soluble intermediates in the N-dealkylation and glutathione conjugation pathways, J. Agric. Food Gem. 21, 1031 (1973). 16. R. H. Shimabukuro, Atrazine metabolism and herbicidal selectivity, Plant Physiol. 42, 1269 (1%7). 17. T. P. Price and N. E. Balke, Characterization of rapid atrazine absorption by excised velvetleaf (Abutilon theophrasti) roots, Weed Sci. 30, 633 (1982). 18. T. P. Price and N. E. Balke, Comparison of atrazine absorption by underground tissues of several plant species, Weed Sci. 31, 482 (1983). 19. R. H. Shimabukuro, Detoxication of herbicides, in “Weed Physiology“ (S. 0. Duke, Ed.), Vol. II, pp. 215-240, CRC Press, Boca Raton, FL, 1985. 20. R. J. Ehr and F. G. Burroughs, A study of the interaction between tridiphane and atrazine on broadleaf weeds, NCWCC Proc. 41, 78 (1986). 21. G. L. Lamoureux and D. G. Rusness, Tridiphane [2-(3,5-dichlorophenyl)-2(2,2, 2-trichloroethyl) oxirane] an atrazine synergist: Enzymatic conversion to a potent glutathione S-transferase inhibitor, Pestic. Biochem. Physiol. 26, 323 (1986).

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22. F. D. Hess, Herbicide absorption and translocation and their relationship to plant tolerances and susceptibility, in “Weed Physiology” (S. 0. Duke, Ed.), Vol. II, pp. 191-214, CRC Press, Boca Raton, FL, 1985. 23. C. Fedtke and R. R. Schmidt, Behavior of metribuzin in tolerant and susceptible soybean varieties, in “Pesticide Chemistry: Human Welfare and the Environment” (J. Miyamoto and P. C. Keamey, Eds.), Vol. 3, pp. 177-182, Pergamon, Elmsford, NY, 1983. 24. T. P. Price and N. E. Balke, Characterization of atrazine accumulation by excised velvetleaf (Abutilon theophrastt) roots, Weed Sci. 31, 14 (1983). 25. R. H. Shimabukuro, G. L. Lamoureux, and D. S. Frear, Glutathione conjugation: A mechanism for herbicide detoxication and selectivity in plants, in “Chemistry and Action of Herbicide Antidotes” (F. M. Pallos and J. E. Casida, Eds.), pp. 133-149, Academic Press, New York, 1978. 26. G. L. Darmstadt, N. E. Balke, and L. E. Schrader, Use of corn root protoplasts in herbicide absorption studies, Pesric. Biochem. Physiol. 19, 172 (1983). 27. W. B. McCloskey and D. E. Bayer, Atrazine and glyphosate absorption by wheat leaf proto. plasts, WSSA Abst. 28, 61 (1988). 28. H. M. LeBaron, Genetic engineering for herbicide resistance, Weed Sci. 35, 2 (Suppl. 1) (1987). 29. J. Gressel, Y. Regev, S. Malkin, and Y. Kleifeld, Characterization of an s-triazine-resistant biotype of Brachypodium distachyon, Weed Sci. 31, 450 (1983). 30. F. J. Oppenoorth, Biochemistry and genetics of insecticide resistance, in Comprehensive Insect Physiology, Biochemistry and Pharmacology (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 12, pp. 731-773, Pergamon, Elmsford, NY, 1985. 31. F. W. Plapp Jr., Genetics and biochemistry of in* secticide resistance in arthropods: Prospects for the future, in “Pesticide Resistance: Strategies and Tactics for Management,” pp. 74-86, Nat. Acad. Press, Washington, DC, 1986. 32. S. G. Georgopoulos, Plant pathogens, in “Pesticide resistance: Strategies and Tactics for Management”, pp. 100-110, Nat. Acad. Press, Washington, DC, 1986.