Dichloroacetamide herbicide antidotes enhance sulfate metabolism in corn roots

PESTICIDE

BIOCHEMISTRY

AND

Dichloroacetamide

PHYSIOLOGY

19, 350-360 (1983)

Herbicide Antidotes Enhance Sulfate Metabolism in Corn Roots’

CARL A. ADAMS,*ELIZABETHBLEE,~

ANDJOHN E.CASIDA~ Pesticide Chemistry and Toxicology Laboratory, Department of Entomological Sciences, University of California, Berkeley, California 94720 Received November 15, 1982; accepted February 4, 1983 The herbicide antidotes N,N-diallyl-2,2-dichloroacetamide (R25788) and 3-dichloroacetyl-2,2,5trimethyloxazolidine (R29148) at ppm levels slightly enhance the uptake of [YS]sulfate in corn roots and greatly increase its metabolism to “bound sulfide,” cysteine, and glutathione (GSH). The decrease in free sulfate content of the roots with R25788 is closely associated with an increase in GSH level. The sulfate content is decreased with an 8-hr exposure to R25788 and R29148 at 3 ppm and its decline continues through 48 hr to about 5% of the control Ievel. Effects on sulfate content are evident at 24 hr even with 0.3-l ppm of these antidotes. Several other mono- or dichloroacetamide antidotes at 30 ppm also decrease the free sulfate content of corn roots to about 34-60% of control levels within 24 hr. R25788 at 30 ppm has little or no effect at 24 hr on sulfate levels in corn leaves whether the plants are grown in the light or in the dark. R25788 and R29148 decrease sulfate levels in the leaves of milo and in whole pigweed plants, but not in barley, lambsquarters, water grass, wheat, or wild mustard. In increasing GSH biosynthesis, the antidote acts in corn prior to the reduction step to form bound sulfide; in fact, R25788 increases the specific activity of ATP sulfurylase, the first enzyme involved in sulfate assimilation. Thus, dichloroacetamides such as R25788 and R29148 provide a means to experimentally, and perhaps even practically, manipulate sulfate utilization in corn and some other plants.

Dichloroacetamide antidotes such as R125788~ and R29148 are used with the thioI Study supported in part by grants from the National Institute of Environmental Health Sciences (Grant PO1 ES00049) to JEC and the Centre National de la Recherche Scientifique and NATO to EB. * Present address: ARC0 Plant Cell Research Institute, Dublin, Calif. 94566. 3 Present address: Laboratoire de Physiologie VCgetale, Institute de Botanique, 28 rue Goethe, 67083 Strasbourg Cedex, France. 4 To whom correspondence and reprint requests should be addressed. ( Abbreviations used: butylate, S-propyl diisobutylthiocarbamate; CDAA, N,N - diallyl - 2 - chloroacetamide; DTNB, S,S’-dithiobis(2-nitrobenzoic acid); EPTC, S-ethyl dipropylthiocarbamate; GSH, glutathione; GSH S-transferase, one or more enzymes of the glutathione S-transferase type; LSC, liquid scintillation counting; mIU., specific activity of ATP sulfurylase expressed as milli-International Units, i.e., nmol/min/mg protein; NA, 1,8naphthalic anhydride; NEM, N-ethylmaleimide; PVPP, polyvinylpolypyrollidone; R25788, N,N-diallyl-2,2-dichloroacetamide; R29148, 3-dichloroacetyl-2,2,5-trimethyloxazolidine: TCA, trichloroacetic acid; Thiocarbamate sulfoxide, activated metabolite of thiocarbamate herbicide; TLC, thin-layer chromatography. 350 0048-3575183 $3.00 Copyright @ 1983 by Academic Press, Inc. All tights of reproduction in any form reserved.

carbamate herbicides EPTC and butylate to protect corn from injury without reducing the herbicidal activity (1). The antidotal action is associated with a two- to threefold increase in the GSH content (1-5) and GSH S-transferase activity of corn roots leading to more rapid detoxification of thiocarbamate sulfoxides and consequent protection of the plant (l-3). R25788 also increases the GSH content of other plant systems including corn and tobacco cell suspension and pigweed callus cultures (5, 6). The protective action of R25788 extends to several herbicides other than thiocarbamates (7) including some that readily react with GSH or undergo GSH-mediated detoxification in plants (1, 8-10). The cysteine required for synthesis of GSH in response to a dichloroacetamide antidote might be released from seed storage protein or synthesized de mvo from sulfate. We therefore examined the effects of R25788, R29148, and related compounds on sulfate content and metabolism, and on cysteine and GSH synthesis in corn and other plants.

HERBICIDE MATERIALS

AND

ANTIDOTES

ENHANCE

METHODS

Chemicals

Dichloroacetamides and other candidate antidotes were obtained from Stauffer Chemical Company (Richmond and Mountain View, Calif.) as previously described (3). Na235S04 (25-40 Ci/mg) was from Amersham (Arlington Heights, Ill.). DTNB and NEM were from Aldrich Chemical Company (Milwaukee, Wise.) and agaroseME was from United States Biochemical Corporation (Cleveland, Ohio). Growth

and Treatment

of Plants

In studies on sulfate and GSH levels and on [35S]sulfate metabolism, seeds of corn (De&lb XL-25-A) (thoroughly washed with water and surface sterilized with 2.5% aqueous NaOCl) were germinated in petri dishes (9-cm diameter x l-cm depth) on filter paper wetted with distilled water or 1 mM Na,SO, (3 ml). They were transferred daily to fresh petri dishes with three plants per dish and maintained in the dark with additional treatments as appropriate until harvest of the roots or leaves at 4.5 days after germination. Alternatively, barley, milo, wheat. and corn (DeKalb XL-43-A) were grown in soil in the greenhouse for 914 days after germination prior to harvest of the leaves and stems. Wild mustard, water grass, lambsquarters, and pigweed were grown from seed on filter paper wetted with 1 mM Na2S04, 0.5 mM K2HP04, and 0.5 mM Ca(NO,),. In experiments involving ATP sulfurylase assays, corn seeds (10 g) were germinated in larger petri dishes (15-cm diameter x 7.5-cm depth) on six layers of filter paper wetted with distilled water (20 ml). [j5SlSulfate Uptake Corn Seedllngs Treatment

and

and Metabolism sample

in

preparation.

Seedlings 2.5-days old were treated with antidote added in ethanol (25 (~1) to water (2 ml). After 24 hr, they were transferred to a petri dish containing Na235S04 (10 PCi) in water (2 ml). Alternatively, the Na23S04

SULFATE

METABOLISM

351

was supplied for 24 hr followed by the antidote for 24 hr. On completion of the treatment at 4.5 days the roots were cut off, soaked in 1 M unlabeled Na,SO, (50 ml) for 10 min and then rinsed with distilled water for 5 min to remove surface or readily exchangeable Na,SO,. The roots from three seedlings were blotted with filter paper, weighed (-400 mg), and homogenized with an all-glass Potter homogenizer at 20% (w/v) in distilled water containing 1 pmol each of GSH and cysteine at &4”C. The crude homogenate and a 0.5-ml distilled water rinse of the homogenizer were combined and analyzed as follows: 50 ~1 for direct LSC, 0.8 ml for TLC separation of sulfate, cysteine and GSH, and 2 ml for bound sulfide analysis (described below). [35S]Su@te, -GSH, and -cysteine analysis by TLC. NEM (100 pmol) was added to

0.8 ml crude homogenate with shaking and after 5 min 0.2 ml 20% (w/v) TCA was added. Extraction with ether (2 ml) (to remove TCA and excess NEM) and centrifugation (27,000g x 20 min) gave a supernatant from which 50 ~1 was subjected to direct LSC and replicate 50-~1 portions to TLC. TLC used 0.25-mm cellulose MN3OOF chromatoplates (Analtech, Inc., Newark, Del.) and solvent systems as follows: (A) methanol - n - butanol - benzene -water (2: 1: 1: 1); (B) n-butanol-pyridine-wateracetic acid (30: 10: 10:3); (C) n-butanol-acetic acid-water (4: 1: 1); (D) isopropanol-water (7:3). Standard unlabeled NEM derivatives of cysteine and GSH (1 l), routinely ran on all chromatoplates, were visualized with ninhydrin spray and labeled compounds (including standard Na23SS04) with autoradiography. Gel regions containing 3sS products were scraped into scintillation vials for LSC. Two-dimensional cochromatography in each of solvent systems A-D served to identify sulfate and the NEM derivatives of cysteine and GSH. R, values were as follows (systems A-D, respectively): NEM derivative of GSH: 0.54, 0.42, 0.30, and 0.51; NEM derivative of cysteine: 0.69, 0.57, 0.50, and 0.63; sulfate: 0.38, 0.34. 0.01, and 0.20. Two additional TLC systems with

352

ADAMS,

BLEE,

AND CASIDA

0.25-mm silica gel 60 F254 chromatoplates were used for confirmation in sulfate analysis, i.e., methanol - n - propanol- watercont. NH40H (10:10:2:1) giving an Rf of 0.30 and methanoldioxane-conc. N&OHwater (3:3: 1: 1) giving an Rf of 0.20 Bound [35S]sulfide analysis. The procedure of Schmidt (12) was used to differentiate bound sulfite and bound sulfide and to analyze the latter. The crude homogenate (2 ml) was placed in a Warburg flask containing in its sidearm a solution (100 ~1) of Na2S9H20 (50 kmol) in 2.5% mannitol. After sealing the flask with a septum and the sidearm with a vented glass stopper, the system was deoxygenated by alternately evacuating (-10 mm Hg) and tilling with argon (3 x). The contents of the sidearm were then mixed with those of the flask. After 30 min at 25”C, an argon flow was established through the flask into an appropriate trap of 0. I M CdC&/l% lactic acid or Carbosorb (9 ml)/Permafluor (12 ml) (Packard Instruments, Downers Grove, Ill.). The flask contents were then acidified by injection of 1 ml 20% (w/v) TCA containing 5% ascorbic acid to liberate the H235S for trapping. Radioactivity due to bound sulfite (released as 35S02) was very low so that the less-specific Carbosorb/Permatluor trap was used for routine HZ3’S determination. Sample flask and CdClz traps were heated to 50°C to insure release of SO,. Soluble protein y5S analysis. The crude homogenate was centrifuged (27,000g x 20 min), 1 ml of the supernatant was mixed with 0.25 ml 20% (w/v) TCA, and the precipitated proteins were pelleted (as above) and the pellet washed with 5% TCA until no radioactivity was found in the wash. The protein pellet was dissolved in Soluene-100 (Packard) for LSC. Protein content of a second l-ml aliquot of the original supernatant was measured by the method of Bradford (13).

Na,SO, at all stages. Weeds for sulfate analysis were also grown in 1 mM Na$O, as previously described. Roots, leaves, or whole seedlings were harvested, rinsed, weighed, frozen in liquid nitrogen, and broken into small pieces by grinding in a centrifuge tube with a glass rod. Two milliliters of 5% TCA were added to each tube and the samples were homogenized with a polytron 3 x IO-set bursts at two-thirds full power) while cooling in an ice-salt bath. The supernatant from centrifugation (27,000g x 20 min) was decanted and the pellet washed with 1 ml of 5% TCA and recentrifuged. The combined supernatants were analyzed turbidometrically for sulfate by the procedure of Jackson and McCandless (14). An agarose/BaCl, reagent was made by dissolving agarose-ME (20 mg) in distilled water (200 ml) with heat, adding BaClZ*2H20 (1 g) while stirring, and standing overnight before use. A l.O-ml aliquot of sample supernatant was mixed with 1 .O ml of agarose/BaC& reagent. Turbidity was fully developed in 30 min and was stable for 1 hr. A standard curve using known concentrations of Na,SO, in 5% (w/v) TCA was prepared in each experiment. Measurements were made against a blank (distilled water and reagent) at 500 nm with a PerkinElmer Model 576 spectrophotometer. In some cases, measurements had to be corrected for absorbance due to the sample alone. The precipitate was BaSO, and not an interfering root component since it dissolved readily and without charring in concentrated H,SO, and it coprecipitated with all the radioactivity when Na,35S0, was added to the samples before reagent was added. Neither GSH alone nor cysteine alone formed a precipitate with the reagent. This procedure determines >98% of the soluble sulfate from plant material as established by addition of standard amounts of Na,S04 to samples before homogenization.

Sulfate Content of Corn Roots and Leaves and of Other Plants Corn seedlings for analysis of sulfate content were germinated and grown in 1 mM

GSH Analysis of Corn Roots GSH was determined by a modification of the method of Lay and Casida (3). Roots of seedlings grown in 1 mM sulfate and

HERBICIDE

ANTIDOTES

ENHANCE

washed with distilled water were homogeenized at 20% (w/v) in 5% (w/v) TCA. The supernatant from centrifugation (27,000g X 20 min) of the crude homogenate was extracted with ether (2 x 2 vol). An aliquot (0.5-l ml) of the extracted supernatant was then placed in a cuvette to which was added 0.1 M phosphate buffer, pH 7.4 (2.0-2.5 ml), and then DTNB reagent (0.1 ml, 2 mg DTNB/ml buffer) with mixing. The absorbance at 412 nm was compared with a standard curve for GSH in 5% TCA treated in the same manner. Extraction

and Assay of ATP Sulfurylase

Corn seedlings 2.5 days old were treated by adding R25788 to the culture medium. After up to 72 hr exposure in the light, the shoots (1 g) were excised, chopped, and ground in a Potter homogenizer in 0.1 M pH 7.4 sodium phosphate buffer (5 ml) containing 10 mM cysteine, 1 mM MgC&, and 500 mg PVPP. The homogenate was filtered through four layers of cheesecloth, centrifuged 15 min at lO,OOOg, and 1 ml of the supematant was layered on a Sephadex G25 column (1 x 20 cm) which was then eluted with the same buffer, collecting l-ml fractions. ATP sulfurylase activity was normally confined to fractions 3 to 5 so these fractions were used for assay. Enzyme isolation was carried out below 10°C at all stages and the protein content was measured according to Bradford (13). ATP sulfurylase activity was determined spectrophotometrically as the APS-dependent generation of ATP in the presence of PPi according to Schwenn et al. (1.5) except that NaF (1 mM) and p-chloromercuribenzoate (20 t&f) were added to the reaction mixture as phosphatase and ATPase inhibitors, respectively. The reaction rate was determined from the linear portion of the curve, after the initial lag phase with a duration depending on the enzyme activity and due to the nature of coupled assays (16). The required hexokinase and glucose 6 - phosphatase were “salt-free” preparations (Sigma, St. Louis, MO.). In these condi-

SULFATE

METABOLISM

353

tions, in the presence of saturating amounts of APS (0.8 mM), PPi (0.8 mM), and Mg’* (2 n&f) the rate was directly proportional to the amount of ATP sulfurylase. Specific activity is expressed as mIU milli-International Units (mIU). RESULTS

Effects of Antidotes on [3SS]SuIfate Uptake and Metabolism in Corn Roots [35S]Surfate uptake. Exposure of corn roots for 24 hr to 3 or 30 ppm R25788, R29148, or CDAA appears to slightly increase their [3SS]sulfate uptake whereas NA is without significant effect (Table 1). [“S]Sulfate metabolism. Of the 3sS detected in the roots, -5% was a mixture of cysteine and GSH, identified by conversion to their NEM derivatives and two-dimensional TLC cochromatography in four different solvent systems. R25788 and R29148 decrease the proportion of total radioactivity due to [35S]sulfate and increase the 35S content of protein-bound sulfide, [35S]cysteine, and [3sS]GSH (Table 2). Antidote levels of 0.3 ppm have a detectable effect on sulfate metabolism and levels of 3 ppm have a pronounced influence on the content of sulfur compounds measured. Although bound [3SS]sulfite proved difficult to measure by the method used, it appeared to be affected little or not at all by the antidotes. [3S]-Labeled metabolites tabulated as “unTABLE 1 Effects of Four Antidotes on [35S]Sulfate Uptake in Corn Roots

Antidote R25788 R29148 CDAA NA

35S Content relative to control (%) at indicated ppm antidote” -3 30 I____ 1222 8 112 _t 32 138 j- 34 158 k 26 130 j: 26 120 r 12 102% 6 92 ‘- 13

a Roots exposed to antidote for 24 hr and then to [Yj]sulfate for 24 hr. Means and SE values for six replicates from two experiments. SE values for the control were 9- 12% of the mean in each case.

354

ADAMS,

BLEE,

AND CASIDA

TABLE 2 Effects of Two Antidotes on [34S&Yulfate Metabolism in Corn Roots Y!i Content (%) at indicated ppm antidote Relative to total W

YS Compound R25788 treatment Sulfate Bound sulfide Cysteine GSH Unknownb R29148 treatment Sulfate Bound sulfide Cysteine GSH Unknown*

0

0.3

Relative to control

3

30

0.3

3

30

62 0.10 1.9 5 31

*3 2 0.02 20.1 21 26

56 0.11 2.8 8 33

-r-7 2 0.01 2 0.5 +-1 * 9

45 0.23 2.5 10 43

*5 rt 0.03 f 0.3 52 25

31 0.17 4.1 15 50

-+5 f 0.04 f 1.4 23 25

90 110 147 160 77

73 230 132 200 100

50 170 216 300 116

58 0.30 6.7 5 30

26 f 0.04 + 2.0 ? 0.3 26

53 0.42 5.0 5 37

+6 2 0.10 ” 0.3 ” 0.3 *7

31 2.0 9.1 8 50

24 aO.l -c 4.2 k 0.5 k7

27 0.60 6.0 9 57

55 2 0.06 2 1.8 -r- 0.5 k-8

91 140 134 loo 123

53 667 136 160 167

47 200 90 180 190

a Roots exposed to [Yilsulfate for 24 hr and then to antidote for 24 hr. Means and SE values for three replicates in each of three experiments. b Compounds in TLC regions other than those for sulfate and the NEM derivatives of cysteine and GSH.

known” (Table 2) are likely to include small peptides and a minor amount of APS. The 35Scontent of corn root soluble protein (510% of total root 3sS at 24 hr) was marginally increased by 30 ppm R25788 while R29148 at 30 ppm had no significant effect on the 3sS content (Table 3).

GSH content are essentially identical (Table 4). Values for the level of sulfate in untreated roots are the same as values for GSH in those treated with 30 ppm R25788.

Comparative Effects of R25788 on Sulfate Utilization and GSH Synthesis in Corn Roots

R29148 at 3 and 30 ppm detectably

The sulfate and GSH contents were not significantly affected in this experiment by 0.3 ppm R25788 but at 3 and 30 ppm R25788 the decrease in sulfate level and increase in TABLE 3 Effects of Two Antidotes at 30 ppm on % Content of Corn Root-Soluble Protein Protein W contenP (dpm/mg x lo+) Antidote

Control

Antidote

R25788 R29148

82 5 26 382 -c 58

134 * 52 444 + 56

n Roots exposed to antidote for 24 hr. Means and SE values for three replicates in each of three experiments with R25788 and five replicates in each of two experiments with R29148.

Effects of Antidotes on Sulfate Utilization in Corn Roots Time-response relationships. R25788 and

decrease

TABLE 4 Effects of R25788 on Sulfate and GSH Contents of Corn Roots Micromoles R25788 W-f0 Total content 0 0.3 3 30 Change in content due to antidote 0.3 3 30

Sulfate 1.68 1.55 1.27 0.94

2 0.02 2 0.14 -c 0.14 k 0.07

0.13c 0.41 0.74

per gramb Thiol 0.76 0.82 1.25 1.64

? 0.09 2 0.10 2 0.10 +- 0.20

O.OB 0.49 0.88

a Roots exposed to antidote for 24 hr. * Means and SE values for nine replicates three experiments. c Not significantly different from control.

from

HERBICIDE

ANTIDOTES

ENHANCE

the sulfate content of the roots by 8 hr and the content steadily declines for 48 hr to less than 5% of the control value (Fig. 1). The two antidotes give similar results at 3 and 30 ppm. In confirmation, after 48 hr exposure to 3 or 30 ppm R25788 and R29148, roots of older seedlings (5.5 days old) also contain about 5% of the sulfate content of control roots (i.e., 0.10 + 0.04 pmol/g). Dose - response relationships. R25788 lowers the sulfate content in a dose-dependent manner from 0.3 to 30 ppm and appears to be more effective than R29148 (Fig. 2). These dichloroacetamides are much more potent than compounds 3 and 4 which have a significant effect at 30 ppm (Table 5) but not at 3 or 10 ppm. Structure-activity relationships. The most effective compounds at 30 ppm in lowering the root sulfate content are 1 or R25788, 2 or R29148, and the monoallyl and monochloro analogs of R25788 (i.e., 3 and 4 or CDAA, respectively) (Table 5). Less effective dichloroacetamides, although still ac-

T I

SULFATE

355

METABOLISM

tive, are 5 (substituting an isopropyl group for an ally1 group of R25788), 6 (R28725, the 5-desmethyl analog of R29148), and 7 (the saturated analog of R25788). The other compounds at 30 ppm, including NA, have little or no effect on root sulfate content (Table 5). Effects of R25788 and R29148 on Sulfate Content of Leaves or Whole Plants The sulfate content of corn shoots is not affected by R25788 or R29148 at 30 ppm for plants held either in the light or in the dark (Table 6). The sulfate level is only slightly higher for control plants held in the light after germination than for etiolated plants held in the dark. R25788 and R29148 lower the sulfate content of milo leaves but not of barley or corn leaves (Table 7). The extent of sulfate depletion in milo is the same for both compounds. Wheat showed a significant increase in sulfate content with both R25788 and R29148. Neither antidote had a signif-

T

2.c R25788

R29 I48

i I .5

I .c

0.5

Exposure

Time,

hr

FIG. 1, Effect of time of exposure to two antidotes on sulfate content of corn roots. Antidotes at 3 (0) and 30 (A) ppm. SE shown by bars above or below the mean (0, LI) for three replicates in each of three experiments. The control value (0 antidote) of 1.61 k 0.22 pmol sulfatelg is applicable throughout the experimental period.

356

ADAMS,

BLEE,

AND

CASIDA

I .4

7i

0.6

0.4 -0.3

1

’

I

I

I

I

3

IO

Antidote,

30

ppm

Effect of concentration of two antidotes with 24 hr exposure on sulfate content of corn roots. SE shown by bars above or below the mean (0, A) for three replicates in each of three experiments. FIG. 2.

icant effect on wild mustard, water grass, or lambsquarters, but R25788 caused a 46% drop in sulfate content of pigweed, which had a high control sulfate content.

Effect of R2.5788 on ATP Sulfurylase Activity in Corn Roots and Shoots

Preliminary studies indicated that R25788 induces an increase in ATP sulfurylase activity in extracts of both roots and shoots. Subsequent experiments focused on the Effect of R25788 on Soluble Protein Content of Corn Roots more active shoot extracts, The shoot enzyme activity reaches a maximum of 167% R25788 at 30 ppm has little or no effect relative to the control after exposure to 3 on the corn root soluble protein level (mil- ppm R25788 and decreases in activity at 30 ligrams per gram fresh weight; two sets of ppm (Fig. 3); this decrease is not reflected experiments with three or four samples in the sulfate level (Table 6) although the each) either for the 27,000g supematant 24 growth and treatment conditions were hr after treatment (7.5 -+ 0.9 vs 7.3 f 0.9 somewhat different. At 3 ppm R25788 the and 8.2 -e 0.7 vs 8.9 k 1.3 for control vs specific activity of ATP sulfurylase remains antidote) or for the 10,OOOg supernatant 48 higher than that of the control for at least hr after treatment (5.5 f 1.2 vs 7.3 f 1.2 72 hr (Fig. 4). The activation probably does and 2.7 + 0.4 vs 3.5 k 0.9 for control vs not involve direct action on the enzyme antidote). since under the standard in vitro assay con-

HERBICIDE

ANTIDOTES

ENHANCE

SULFATE

351

METABOLISM

TABLE 5 Effects of 12 Antidotes and Related Compounds on Sulfate Utilization

in Corn Roots

Sulfate content

Antidote or analo@

-

Relative to control (%Y

No.

Structure

1

Cl,CHC(O)N(CH,CH=CH&

0.42 k 0.06

34

Cl,CHC(O)N~(CH& CI,CHC(O)NHCH,CH=CH, CICH,C(O)N(CH,CH=CHJ,

0.69 0.62 0.67 0.84

iz 0.12 2 0.20 2 0.07 _f 0.14

46 51 55 60

C12CHC(0)NCH&H,0C(CH& Cl,CHC(O)N(CH,CH,CH,), CH,C(O)N(CH,CH=CH& Cl,CHC(O)NHC(CH& HOCH,C(O)N(CH,CH=CH& HOC(O)C(O)N(CH,CH=CH,), 1,I-Naphthalic anhydride

0.85 0.92 1.19 1.05 1.33 1.33 1.63

+ 0.08 4 0.10 -c 0.21 + 0.27 -t 0.22 ” 0.40 2 0.21

70 76 85 86 95 95 120

6 7 8 9 10 11 12

~LMoW

a Names or designations are as follows: 1 R25788, 2 R29148, 4 CDAA, 6 R28725, and 12 NA. Antidotal activity is rated as follows: superior for 1,2, and 6, good for 5 and 9, moderate for 3,4, and 7 and little or none for 8 and 10 (3). b Roots exposed to 30 ppm antidote or analog for 24 hr. Means and SE values for three replicates in each of three experiments. r Control values (pm01 sulfate/g) for different test series were as follows: 1, 1.24 t 0.22; 2, 1.49 + 0.25: 3, 4, 6, 7, and 9, 1.22 + 0.15; 5, 8, and 10-12, 1.40 t 0.22.

ditions R25788 at 1 mM does not alter ATP sulfurylase activity. The R25788-enhanced shoot enzyme activity noted with purified enzyme preparations is also evident with crude enzyme prepared without the steps of centrifugation and filtration on Sephadex .

TABLE 6 Effects of Two Antidotes and Growth in Light or Dark on Sulfate Content of Corn Shoots Sulfate conteneb (PmoUg) Antidote” R25788 treatment Control Antidote R29148 treatment Control Antidote

Light

Dark

2.5 2 0.3 2.4 t 0.3

2.0 _f 0.2 1.8 2 0.2

2.3 f 0.2 2.0 r+ 0.2

2.0 f. 0.2 2.1 2 0.3

u Roots exposed to 30 ppm antidote for 24 hr. * Means and SE values for three replicates in each of two experiments.

DISCUSSION

Dichloroacetamide herbicide antidotes affect several aspects of plant sulfur metabolism. They increase the nonprotein thiol content of corn and other plant systems determined by the relatively nonspecific DTNB method but interpreted as an increase in GSH content (2, 3, 5) or analyzed by a more specific method (4). R25788 also increases the content of NJ-dipropylcarbamoyl derivatives of GSH and cysteine in corn roots treated with EPTC sulfoxide, based on TLC cochromatography for identification (17). The present study confirms that R25788 enhances GSH synthesis in that it increases the [35S]sulfate-treated root levels of both [35S]GSH and P5S]cysteine, identified by converting them to NEM derivatives for characterization. R25788 and R29148 increase the labeling not only of GSH and cysteine but also of protein and bound sulfide. The increased 35Sin protein probably originates in the most

358

ADAMS,

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AND CASIDA

TABLE 7 Effects of Two Antidotes on Sulfate Content of Crops and Weeds Sulfate content Relative to control (%)

kMol/ga Species

Control

Crops-leaves and stems only Barley (Hordeum vulgare) Milo (Sorghum bicolor) Wheat (Triticum aestivum Var. Anza) Corn (Zea mays Var. XL-43-A) Weeds-whole plantsc Wild mustard (Brassica kaber) Water grass (Echinochloa crus-galli) Lambsquarters (Chenopodium album) Pigweed (Amaranthus retroflexus)

3.5 2.6 2.5 1.5

R25788

Z!T0.8 2 0.2 * 0.6 _’ 0.3

3.2 f 2.6 f 1.3 k 4.1 5

0.2 0.3 0.4 0.6

R29148

R25788

R29148

4.6 t 1.6 ? 4.5 I 1.6 k

1.0 0.4 0.1 0.4

4.0 _’ 1.3 k 3.9 I 1.3 2

0.7 0.1 0.2 0.3

131 62 180 106

114 SO 156 87

3.3 2.3 1.4 2.2

0.2 0.2 0.4 0.4

3.9 2 1.0 2.2 k 0.3 1.1 ” 0.4 -

103 88 108 54

122 85 85 -

f 2 f f

u Means and SE values for three replicates in each of two experiments. b Crops grown in soil treated with 5 lb antidote/acre by preplant incorporation and harvested 9 days (or 14 days for wheat) after germination. e Weeds grown in sand for 2 weeks and then watered with 30 ppm antidote and harvested after 24 hr.

part via [YSJcysteine since protein labeling is very high relative to protein-bound sulfide. However, the antidote-induced increase in protein-bound sulfide content indicates an effect at a step early in the sulfate to cysteine metabolic pathway. These findings are confirmed by establishing a close association between GSH and cysteine content determined by the DTNB method and sulfate level determined turbidometrically, i.e. the lowered sulfate level is due mostly to GSH (and cysteine) synthesis.

R25788,

The sulfate level of corn roots is very sensitive to R25788 and R29148 as established with both 35S introduced in tracer levels of sulfate and unlabeled compound used at 1 miV as a nutrient solution. The turbidometric analyses were greatly facilitated by increasing the root sulfate content in control plants; this was accomplished with sulfate at concentrations which are not unusual for plant nutrient solutions or soils. The site of action of the dichloroacetamides in enhancing sulfate assimilation ap-

I 1

ppm

FIG. 3. Effect of concentration of R25788 with 24-hr exposure on the specific activity of corn shoot ATP sulfurylase. Means and SE values for three to1four replicates.

24

I

I

48

72

Exposure

Time,

hr

4. Effect of time of exposure to R25788 (0 or 3 ppm) and light on the specific activiry of corn shoot ATP sulfurylase. Means of three replicates. FIG.

HERBICIDE

ANTIDOTES

ENHANCE

pears to be in formation or utilization of “active sulfate” or in the subsequent reduction steps. The first enzyme in the pathway, ATP sulfurylase, is enhanced by R25788 in corn roots and shoots. This appears to be due to an increased level of extractable enzyme per se. Thus, Sephadex purification studies indicate that the enhanced activity does not result from formation of an activator or removal of an inhibitor in antidote-treated plants. The decrease in ATP sulfurylase specific activity a few days after treatment may be associated with increased protein content on growth of the leaves. R25788 enhances sulfate utilization and GSH levels in corn roots but only the GSH level is affected in shoots or leaves (3). The absence of an effect on sulfate in leaf tissue may be due to the dose used (30 ppm) or to differences between green and nongreen tissue linked to chloroplastic functions. In the case of ATP sulfurylase, an activity increase in shoots was evident at 3 ppm but a decrease in activity was observed at 30 ppm R25788. While this enzyme activity is enhanced in corn leaf tissue, the overall utilization of sulfate is unchanged. Part of the sulfate metabolic pathway in tobacco cells responds similarly in that net GSH synthesis is stimulated by R25788 in heterotrophically grown cells but not in photoheterotrophically grown cells (6), thus paralleling the present study. The overall rate of sulfate assimilation is controlled only in part by ATP sulfurylase levels (18) so the activity of this enzyme in green tissue may not be the limiting factor in sulfate utilization. Dichloroacetamides affecting sulfate utilization are generally active as antidotes for thiocarbamates in corn, e.g., R25788 and R29148. Two R25788 metabolites (compounds 10 and 11) (19) are inactive as antidotes (3; personal communication in 1981 from M.-M. Lay) and in enhancing sulfate utilization. However, some very effective antidotes (compounds 6 and 9) also have relatively little effect on the sulfate level. In contrast, R25788 and R29148 enhance

SULFATE

359

METABOLISM

sulfate assimilation in milo and redroot pigweed yet do not protect these plants from thiocarbamate herbicides even though R25788 increases the GSH content of pigweed cell cultures (5). These apparent discrepancies may be due to the multiple requirements for antidotal activity, i.e., effective in elevating GSH levels and GSH Stransferase activity (3) and sufficient persistence to provide protection through the critical growth stages. Dichloroacetamide herbicide antidotes such as R25788 have a pronounced effect on sulfate metabolism in some plants, thereby providing useful probes into the process and control of sulfate assimilation. They offer a means to experimentally, and perhaps even practically, enhance sulfur metabolism in plants where this nutrient may be limiting. REFERENCES 1. F. M. Pallos and J. E. Casida, Eds., “Chemistry and Action of Herbicide Antidotes.” Academic Press, New York, 1978. 2. M. -M. Lay, J. P. Hubbell, and J. E. Casida. Dichloroacetamide antidotes for thiocarbamate herbicides: Mode of action, Science 189, 287 (1975). 3. M. -M. Lay and J. E. Casida, Dichloroacetamide antidotes enhance thiocarbamate sulfoxide detoxification by elevating corn root glutathione content and glutathione S-transferase activty, Pestic.

Biochem.

Physiol.

6, 442 (1976).

4. R. D. Carringer, C. E. Rieck. and L. P. Bush. Effect of R-25788 on EPTC metabolism in corn (Zea mays), Weed Sci. 26, 167 (1978). 5. G. Ezra and J. Gressel, Rapid effects of a thiocarbamate herbicide and its dichloroacetamide protectant on macromolecular syntheses and glutathione levels in maize cell cultures, Prstic. Biochem. Physiol. 17, 48 (1982). 6. H. Rennenberg, C. Birk. and B. Schaer, Effect of N,N-diallyl-2.2-dichloroacetamide (R-25788) on efflux and synthesis of glutathione in tobacco suspension cultures, Phytochemisrry 21, 5 (1982). 7. G. R. Stephenson and F. Y. Chang, Comparative activity and selectivity of herbicide antidotes. in “Chemistry and Action of Herbicide Antidotes,” (F. M. Pallos and J. E. Casida. Eds.) p. 35, Academic Press, New York, 1978. 8. G. L. Lamoureux and D. S. Frear. Pesticide me-

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10.

11.

12.

13.

14.

ADAMS,

BLEE,

tabolism in higher plants: In vitro enzyme studies, Amer. Chem. Sot. Symp. Ser. 97,77 (1977). G. L. Lamoureux, L. E. Stafford, and F. S. Tanaka, Metabolism of 2-chloro-N-isopropylacetanilide (propachlor) in the leaves of corn, sorghum, sugarcane, and barley, .Z.Agric. Food Chem. 19, 346 (1971). J. R. C. Leavitt and D. Penner, In vitro conjugation of glutathione and other thiols with acetanilide herbicides and EPTC sulfoxide and the action of the herbicide antidote R-25788, J. A&c. Food Chem. 27, 533 (1979). B. States and S. Segal, Thin-layer chromatographic separation of cystine and the N-ethylmaleimide adducts of cysteine and glutathione, Anal. Biochem. 27, 323 (1969). A. Schmidt, “Untersuchungen zum Mechanismus der Photosynthetischen Sulfatreduktion IsoIierter Chloroplasten,” Ph.D. thesis, Giittingen, Federal Republic of Germany (1968). M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248 (1976). S. G. Jackson and E. L. McCandless, Simple,

AND CASIDA

15.

16. 17.

18.

19.

rapid, turbidometric determination of inorganic sulfate and/or protein, Anal. Biochem. 90, 802 (1978). J. D. Schwenn, H. El-Shagi, A. Kemena, and E. Petrak, On the role of S: Sulfotransferases in assimilatory sulfate reduction by plant cell suspension cultures, Pfanta 144, 419 (1979). I. H. Segel, “Enzyme Kinetics,” p. 85, Wiley, New York, 1975. J. P. Hubbell and J. E. Casida, Metabolic fate of the N,N-dialkylcarbamoyl moiety of thiocarbamate herbicides in rats and corn, J. A&c. Food Chem. 25, 404 (1977). L. Bergmann, J. -D. Schwenn, and H. Urlaub, Adenosine triphosphate sulfurylase and O-acetylserin sullhydrylase in photoheterotropically and heterotrophically cultured tobacco cells, 2. Natutforsch. 35C, 952 (1980). J. B. Miaullis, V. M. Thomas, R. A. Gray, J. J. Murphy, and R. M. Hollingworth, Metabolism of R-25788 (N,N-diallyl-2,2-dichloroacetamide) in corn plants, rats and soil, in “Chemistry and Action of Herbicide Antidotes” (F. M. Pallos and J. E. Casida, Eds.), p. 109, Academic Press, New York, 1978.