Influence of the herbicides hexazinone and chlorsulfuron on the metabolism of isolated soybean leaf cells

PESTICIDE

BIOCHEMISTRY

Influence

AND

PHYSIOLOGY

17,207-214

(1982)

of the Herbicides Hexazinone and Chlorsulfuron Metabolism of Isolated Soybean Leaf Cells

on the

KRITON K. HATZIOS AND CELESTIA M. HOWE Department

of Plant

Pathology

and

Physiology, Blacksburg,

Virginia Virginia

Polytechnic 24061

Institute

and

State

University.

Received November 20, 1981; accepted February 15, 1982 The effects of the herbicides hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1.3.S triazine-2,4(1H,3N)-dione] and chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2yl)aminocarbonyl]benzenesulfonamide) on the metabolism of enzymatically isolated leaf cells from soybean [Glycine max (L.) Merr., cv. ‘Essex’] were examined. Photosynthesis, protein, ribonucleic acid (RNA), and lipid syntheses were assayed by the incorporation of specific radioactive substrates into the isolated soybean leaf cells. These specific substrates were NaHYO,, [Wlleucine, [‘*C]uracil, and [Ylacetate, respectively. Time-course and concentration studies included incubation periods of 30, 60, and 120 min and concentrations of 0.1, 1, 10, and 100 @4 of both herbicides. Photosynthesis was the most sensitive and fast metabolic process inhibited by hexazinone. RNA and lipid syntheses were also inhibited significantly by hexazinone whereas the effect of this herbicide on protein synthesis was less. The most sensitive and first metabolic process inhibited by chlorsulfuron was lipid synthesis. Photosynthesis, RNA, and protein syntheses were affected significantly only by the highest concentration of this herbicide and longest exposure. Although these two herbicides may exert their herbicidal action by affecting other plant metabolic processes not examined in this study, hexazinone appears to be a strong photosynthetic inhibitor. while the herbicidal action of chlorsulfuron appeared to be related to its effects on lipid synthesis.

INTRODUCTION

Hexazinone [3-cyclohexyl-6-(dimethylamino)-l-methyl-1,3,5-triazine-2,4(1H,3H)dione] is a highly effective herbicide that is currently registered in United States and many other countries for the control of many annual and perennial broad-leaved weeds, grasses, and woody vines in noncropland areas (1, 2). In addition, hexazinone has shown promise for selective weed control in certain crops, such as alfalfa (Medicago sativa L.) and sugarcane (Saccharurn officinarum L.), and in aquatic situations (1, 2). The chemical structure of hexazinone is shown in Fig. 1. Several studies investigating the fate of hexazinone in animals, water, fish, soils, and plants are available (2-5). N-( 1,3 ,S-Triazinylaminocarbonyl)benzenesulfonamides represent a novel class of highly active he-rbicidal compounds that has been recently introduced by the agrichemical division of DuPont. The most

promising of these compounds appears to be chlorsulfuron’ (2-chloro-N-[(4-methoxy6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide) (Fig. 1). This compound has a broad spectrum of herbicidal activity and has shown excellent crop selectivity in cereals such as wheat (Ziiticum aestivum L.), barley (Hordeum vulgare L.), oats (Avena sativa L.), and rye (Secale cereafe L.) (6, 7). Studies on the mode of action of these two herbicides are limited. The mode of action of hexazinone has not been clearly established as yet (1). Because of the presence of the symmetrical triazine ring in its molecule, hexazinone is believed to act as a photosynthetic inhibitor, resembling the triazine herbicides which are well-known inhibitors of photosynthesis (8, 9). However, inhibition of photosynthesis or of any 1 Chlorsulfuron is the accepted common name of the herbicide previously referred to as DPX-4189.

207 0048-3575/82/030207-08$02,00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

208

HATZIOS

p\ / C-NkH,IZ : CM3

3-CYCLOHEXYL-6-(DIMETHYLAMINO)-I-METHYL1,3,5TRIAZINE2,4(lH,3Y)-DIONE HEXAZINONE

AND

HOWE

active cells can be isolated very easily from its leaves (17). The objectives of this study were to determine the metabolic site of herbicidal action of hexazinone and chlorsulfuron by examining the effects of these two herbicides on photosynthesis, protein, RNA, and lipid syntheses of enzymatically isolated leaf cells from soybean under various timecourse and concentration conditions. MATERIALS

AND

METHODS

Plant growth conditions. Individual soybean [G. max (L.) Merr., cv. ‘Essex’] plants were grown in plastic cups containFIG. 1. Chemical structures of the herbicides ing 473 ml of greenhouse soil mixture. The hexazinone and chlorsulfuron (DPX-4189). soil mixture was a 2:2:1 mixture of potting other plant biochemical process, such as medium (Weblite Corp., Blue Ridge, Va., protein, nucleic acid, or lipid synthesis, by 24064), vermiculite, and sphagnum peat hexazinone has not been documented. The moss containing a controlled-release ferpresence of an aminocarbonyl group in the tilizer (14-14-14) and limestone. The molecule of an herbicide has been de- plants were grown at 28 klux in a growth scribed as a requirement for inhibition of chamber with a 16-hr photoperiod and photosynthesis by this herbicide in studies temperatures of 30°C during the day and where quantitative structure-activity re- 20°C during the night for 3 weeks. At this lationships were analyzed (8- 10). Since time, the photoperiod of the chamber was chlorsulfuron contains in its molecule both changed to 6 hr. A short-day treatment has an aminocarbonyl group and a triazinyl been reported to be necessary for high ring, it would be reasonable to suspect that photosynthetic rates of cotton (18) and soyinhibition of photosynthesis would be in- bean (17) leaf cells, since it reduces the volved in the mode of action of this her- starch content of chloroplasts. Furtherbicide. However, preliminary studies on more, the photosynthetic rate of mature the mode of action of chlorsulfuron have soybean leaves used for cell isolation has shown that photosynthesis, protein, and been reported to increase if shading of these RNA syntheses were either unaffected at leaves proceeds their isolation (17). Thus, herbicidal rates that completely inhibited after 1 week of short-day treatment, mature growth (11) or affected significantly by high leaves, usually from the second or third herbicidal concentrations and long expoemerged trifoliate, were shaded with sure (12). aluminum foil for 1 day. On the day of cell During the past few years several studies isolation, the shaded mature leaves were on the mode of action of several herbicides illuminated for at least 1 hr and detached have made use of metabolically active iso- from the plant. lated leaf cells from selected plant species Cell isolation. All procedures were car(13-16). Soybean [Gfycine max (L.) Merr.] ried out at room temperature. The detached appears to be an appropriate plant species leaves were rinsed with distilled water, blotted, deveined, and cut into l-mm x to study the mode of action of hexazinone and chlorsulfuron, since it is sensitive to l-cm strips with a razor blade. Two to three grams of leaf tissue were then vacuum intilboth of these herbicides and metabolically 2-CHLORO-&-(4-METHOXY-6-METHYL-l,3,5TRIAZIN - 2 - YL-AMINOCAREONYLI DPX- 4169

BENZENESULFONAMIDE

MODE

OF

ACTION

OF

THE

HERBICIDES

HEXAZINONE

AND

CHLORSULFURON

209

trated with 30 ml of infiltration medium flask as the assayingmediumfor all metauntil the tissuewasfully infiltrated. The in- bolic studies.Analytical grade(100% pure) filtrationmediumfor all preparationscon- hexazinone andchlorsulfuron weredistained 20 r& MES, pH 5.8, 2% macerate (Calbiochem, LaJolla, Calif.), and 0.3% potassium dextran sulfate (Calbiochem). After vacuum infiltration, the leaf tissue was filtered through a 242~pm nylon net, the filtrate was discarded, and the leaf tissue remaining was transferred to a beaker with 30 ml of a maceration medium containing 20 mM MES, pH 5.8, 2% macerase, 0.3% potassium dextran sulfate, and 0.3 M sorbitol. The tissue was stirred slowly on a magnetic stirrer for 10 min. The suspension was filtered through the same nylon net and the filtrate discarded. The leaf tissue was transferred to another 30 ml of fresh maceration medium and was stirred for 50 to 60 min. The cells released during this period were filtered again through the nylon net and then washed three times with lo-ml aliquots of wash medium by centrifugation at 60g for 3 min. The wash medium contained 0.2 M sorbitol, 5 miI4 KN03, 2 mM Mg(NO&, and 1 m&I CaCIZ, and was buffered with 50 mM of HEPES, pH ‘7.8, for photosynthesis or with 50 mM MES, pH 5.8, for protein, RNA, and lipid synthesis assays. After each washing, the supernatant solution was removed by suction and after the final washing the cells were made up to the desired volume with incubation medium which was identical to the wash medium. For the chlorophyll determination, 1 ml of the cell suspension was added to 4 ml of 80% acetone and mixed thoroughly. The supernatant fluid was then assayed spectrophotometrically for its chlorophyll content according to the method of Amon (19). The chlorophyll content of cell preparations used in this study was 40 to 65 pg chlorophyll/ml assay medium. Time-course and concentration studies with the herbicides. Two milliliters of the cell preparation, 0.1 ml of the respective radioactive substrate containing 1 &i of radioactivity, and 0.05 ml of herbicide solution were placed in a 25-ml Erlenmyer

solved in methanol and made up to volume with distilled water so that the final methanol concentration was less than 1%. Herbicide concentrations of 0.1. 1, 10, and 100 $V were used in all assays. The respective radioactive substrates used in each metabolic study were NaH14C0, (sp act, 44.4 mCi/mmol) for photosynthesis, L-[U14C]leucine (sp act, 290 mCilmmo1) for protein synthesis, [2-14C]uracil (sp act, 55 mCi/mmol) for RNA synthesis, and [1,214C]acetic acid (sp act, 56.2 mCi/mmol) for lipid synthesis. The Erlenmeyer flasks with the assay mixtures were sealed and placed in a shaking water bath at 25°C. The flasks were illuminated from above with a combination of fluorescent and incandescent lamps with an intensity of 5.2 klux at the level of the flasks. The assay mixtures were incubated for 30,60, and 120 min. At the end of each incubation period, samples were collected and treated accordingly for each metabolic process studied. A detailed description of the specific procedures followed for the treatment of the collected samples has been given elsewhere (15). The radioactivity of the treated samples was determined by radioassay with a liquid scintillation spectrometer (Beckman LS250 series) having a counting efficiency of more than 90%. Photosynthesis, protein, RNA, and lipid syntheses were calculated as counts per minute (cpm) of the 14C from the respective radioactive substrate incorporated into the cells per 100 pg of chlorophyll. The results were also calculated as percentage inhibition caused by each concentration of hexazinone or chlorsulfuron as compared to the controls (zero inhibition). All assays were replicated three times. The data were analyzed for variance in a completely randomized design with a factorial arrangement of the treatments. Duncan’s multiple range test was used to separate the means.

HATZIOSANDHOWE RESULTS

AND

DISCUSSION

The effects of hexazinone and chlorsulfuron on photosynthesis, protein, RNA, and lipid syntheses of isolated soybean leaf cells are presented in Tables 1 through 4. Inhibition of photosynthesis reached a maximum within 30 min of incubation with the high concentrations (10 and 100 PM) of hexazinone. However, inhibition of photosynthesis by the low concentrations of hexazinone (0.1 and 1 @4) appeared to be a function of time and it increased from 34 and 63% at 30 min to 60 and 90%, respectively, after 120 min (Table 1). These results are in agreement with those of a recent study (21) which documented the strong effects of hexazinone on the photosynthetic electron transport of isolated spinach chloroplasts. Thus, hexazinone appeared to be a very strong inhibitor of photosynthesis with inhibition rates comparable to those of the triazine or substituted urea herbicides (9, 13, 21). Chlorsulfuron, on the other hand, did not appear to act as an inhibitor of photosynthesis. Inhibition of photosynthesis by chlorsulfuron was insignificant at any incubation period and at

any concentration examined (Table 1). These results are in agreement with the findings of Ray (11) who has also reported that chlorsulfuron is not a photosynthetic inhibitor. DeVilliers e? al. (12), however, reported that chlorsulfuron at concentrations of 100 and 500 $l4 inhibited significantly photosynthesis and photosynthetic electron transport of isolated navy bean leaf cells or chloroplasts, respectively. These concentrations of chlorsulfuron, however, are far in excess of those that completely inhibit plant growth (11). The absence of any inhibition of photosynthesis by chlorsulfuron at herbicidal concentrations, despite the presence of an aminocarbonyl moiety in its molecule (Fig. l), may be due to the benzenesulfonamide moiety in the molecule of chlorsulfuron which could probably reduce the activity or binding efficiency of this herbicide to the appropriate chloroplast site for inhibition of photosynthesis to occur. Thus, the mode of action of chlorsulfuron appears to resemble that of the herbicide siduron [I-(2-methylcyclohexyl)-3-phenylurea] which is the only substituted phenylurea that does not inhibit

TABLE 1 The Effect of Hexazinone and Chlorsulfuron on Photosynthesis of Isolated Soybean Cells” Incubation time (min) 30

60

120

Hexazinone t&W 0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100

14C02 fixation (cpm/lOO pg Chl) 50,373 33,255 18,638 4,576 3,989 91,432 39,926 24,686 6,786 5,429 188,681 75,472 19,041 15,983 16,008

d e,f g h h b e f,g h h a c f,g g g

Inhibition m 0 34 63 91 92 0 56 73 93 94 0 60 90 92 92

Chlorsulfuron 6-W

14C02 fixation (cpm/lOO pg Chl)

0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 i 10 100

50,129 49,459 50,765 52,369 48,632 95,829 95,915 86,550 95,095 93,313 191,630 195,935 184,998 194,249 169,773

d d d d d c c c c c a a a,b a a,b

Inhibition” m 0 1 -1 -4 3 0 0 10 1 3 0 -2 4 -1 12

u Means within columns with similar letters are not signiticantly different at the 5% level by Duncan’s multiple range test. b A minus (-) sign in front of a percentage value indicates stimulation instead of inhibition.

MODE

OF ACTION

OF THE HERBICIDES

HEXAZINONE

211

AND CHLORSULFURON

photosynthesis (20). Siduron has been char- concentration of 500 pM and after 120 min acterized as an inhibitor of cell division and of incubation, inhibited strongly the protein plant growth (20) and a similar mode of synthesis of isolated navy bean leaf cells. action has been proposed for the herbicide From the results of all these studies it can chlorsulfuron by Ray (11). be concluded that chlorsulfuron at normally Protein synthesis of isolated soybean leaf applied herbicidal concentrations does not cells was not inhibited significantly by any appear to act as an inhibitor of protein concentration of hexazinone after 30 min of synthesis. incubation time (Table 2). After 60 and 120 RNA synthesis of isolated soybean leaf min of incubation time, hexazinone at high cells was inhibited significantly by all but concentrations caused significant inhibition 0.1 pM concentrations of hexazinone at 30 of protein synthesis (Table 2). However, and 60 min of incubation time (Table 3). even in this instance, the magnitude of the After 120 min of incubation, RNA synthesis observed inhibition of protein synthesis by of soybean cells was inhibited significantly hexazinone was small indicating that pro- even by the lowest hexazinone concentratein synthesis is not a target site involved in tion of 0.1 pM (Table 3). This strong effect the primary mode of action of this her- of hexazinone on RNA synthesis of isolated bicide. Chlorsulfuron at concentrations of soybean leaf cells may be an indirect effect 10 and 100 PM caused significant inhibition of this herbicide caused by its primary acof protein synthesis of isolated soybean leaf tion on photosynthesis which can result in cells but only after 120 min of incubation the reduction of metabolic energy (ATP), (Table 2). However, the percentage values available for driving other metabolic proof these inhibitions were small. Ray (11) has cesses within the cell. Since nucleic acid reported that chlorsulfuron at 2.8 pM was metabolism in plants is driven by metabolic not inhibitory to protein synthesis of corn energy derived from ATP, the effects of root tips. DeVilliers et al. (12), however, many herbicides that act as strong photohave reported that chlorsulfuron at the high synthetic inhibitors on RNA or DNA synTABLE 2 The Effect of Hexazinone and Chlorsulfuron on Protein Synthesis of Isolated Soybean Cells” Incubation time (min) 30

60

120

Hexazinone CPM) 0 0.1 10 100 0 0.1 10 100 0 0. I 10 100

[W]Leucine incorporated (cpm/lOO wg Chl) 8,479 9,749 8,451 8,215 7.170 21,047 23,715 17,612 15,483 14,257 44,326 46,690 30,183 31,636 23,861

g f,g g g g c,d c d,e d e,f a a b b c

Inhibition” (%I 0 -15 0 16 0 -12 17 27 32 0 -5 29 32 46

Chlorsulfuron CPM) 0 0.1 10 100 0 0.1 10 100 0 0.1 10 100

[*‘C]Leucine incorporated (cpm/lOO pg Chl) 11,536 12,994 11,336 12.360 12,232 23,207 26,600 25,080 24,370 17,862 44,075 46,605 39,604 34,599 27,434

e e e e e cd C cd cd d,e a a ah b

C

Inhibition” (%) 0 -12 2 -7 -6 0 -14 -8 -5 23 0 -6 10 22 38

” Means within columns with similar letters are not significantly different at the 5% level by Duncan’s multiple range test. ’ A minus (-) sign in front of a percentage value indicates stimulation instead of inhibition.

212

HATZIOS

,AND HOWE TABLE

The Effect

Incubation time (min) 30

of Hexazinone

Hexazinone OLM) 0

0.1 1 10 100 60

0

120

0.1 1 10 100 0 0.1 1 10 100

and Chlorsulfuron

[14C]Uracil incorporated (cpm/lOO pg Chl) d,e d,e f 1,235 f 1,404 f

3,876 3,996 2,040

6,554 5,363 2,829

1,786 1,674 12,007 8,553

4,518 2,800

2,815

c

c,d e,f f f a b d e,f e,f

3

on RNA

Inhibition* (%I 0 -3

48 68 63 0

18 57 73 75

0 29 62

77 77

Synthesis

Chlorsulfuron 6-M

of Isolated

Soybean

[‘4c]uracil incorporated (cpm/lOO pg Chl)

0 0.1 1 10 100

3,860 4,586 4,239 3,589

0

6,636 7,339 7,608

0.1 1 10 100 0 0.1 1 10 100

Cells”

d,e d,e

d,e

Inhibitionb (%I 0

- 18 - 10

e

7

3,441 e

11

c c c

7,161 c 5,200 d 12,302 a 13,747 a 12,453 a 12,943 a 9,448 b

0

- 10 -14 -7

22 0 - 12 -1 -5 23

n Means within columns with similar letters are not significantly different at the 5% level by Duncan’s multiple range test. * A minus (-) sign in front of a percentage value indicates stimulation instead of inhibition.

thesis have been considered as indirect effects caused by their action on photosynthesis (9, 16). However, further experimental work is needed to rule out a direct effect of hexazinone on RNA synthesis of isolated soybean leaf cells. RNA synthesis of soybean cells was inhibited significantly by chlorsulfuron only at the highest concentration of 100 pM and after 60 or 120 min of incubation (Table 3). However, even in this instance, the magnitude of the observed inhibition was small, indicating that inhibition of RNA synthesis is not the mode of action through which chlorsulfuron exhibits its phytotoxicity. Evidence for the absence of strong inhibition of RNA synthesis by chlorsulfuron has been also presented by other investigators (11). Extremely high concentrations (500 pM) of chlorsulfuron, however, have been reported to inhibit very strongly the RNA synthesis of isolated navy bean cells after 60 or 120 min of incubation time. Lipid synthesis of isolated soybean leaf cells was inhibited significantly by all concentrations of hexazinone except the lowest concentration of 0.1 pM and at all incuba-

tion periods examined (Table 4). The inhibition percentage values of the incorporation of [14C]acetate into soybean leaf cells by hexazinone at 1, 10, and 100 pM reached maximum values as early as 30 min of incubation and then remained more or less constant with subsequent increases in exposure time (Table 4). Chlorsulfuron at concentrations as low as 1 pM inhibited significantly lipid synthesis of isolated soybean leaf cells as early as 30 min of exposure time (Table 4). After 120 min of incubation, lipid synthesis was inhibited significantly even by the lowest concentration of chlorsulfuron examined (Table 4). Inhibition of lipid synthesis of soybean leaf cells by 1 and 10 pM of chlorsulfuron appeared to be independent of the incubation time, while the inhibition of lipid synthesis by the highest concentration of chlorsulfuron examined (100 pM) increased slightly with concomitant increases of the incubation time (Table 4). DeVilhers er al. (12) have reported that the incorporation of [14C]acetate into isolated leaf cells of navy beans (Phaseolus vulgaris L.) was inhibited significantly by chlorsulfuron only when it was used at 100

MODE

OF

ACTION

OF

THE

HERBICIDES

HEXAZINONE

TABLE The Effect

Incubation time (min) 30

60

120

of Hexazinone

Hexazinone (~‘4 0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100

and Chlorsulfuron

[W]Acetate incorporated (cpm/lOO pg Chl) 26,361 21,727 15,407 2,544 1,251 56,026 39,932 25,844 6,109 4,459 97,262 97,885 63,388 13,509 11,185

d,e d,e,f f g g b,c c,d d,e f,g g a a b f f

0 18 42 91 95 0 29 54 89 92 0 0 35 86 89

213

CHLORSULFURON

4

on Lipid

Inhibition (%I

AND

Synthesis

Chlorsulfuron Wf) 0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100

of Isolated

Soybean

Cells”

rQ4cetate incorporated (cpm/lOO /~,g Chl) 25,075 17,954 13,483 11,106 5,883 49,939 42,115 30,677 18,884

e,f f,g,h g,h g,h h b,c c,d d,e e,f,g

8,140 g,h 89,086 55,070 58,936 40,897 8,965

a b b c,d g,h

Inhibition m’o) 0 28 46 56 77 0 16 39 62 84 0 38 34 54 90

’ Means within columns with similar letters are not significantly different at the 5% level by Duncan’s multiple range test.

@4 or higher concentrations. The results of this study indicate that chlorsulfuron caused significant inhibition of lipid synthesis of isolated soybean leaf cells at concentrations as low as 0.1 @VI. Thus, it appears that lipid synthesis of soybean leaf cells is more sensitive to chlorsulfuron than the lipid synthesis of isolated navy bean leaf cells. The inhibitory effects of some herbicides such as the s-triazines, substituted phenylureas, acylanilides, and pyridazinones on plant lipid synthesis have been considered as indirect effects of their primary inhibition of photosynthesis, photophosphorylation, or oxidative phosphorylation (9, 16). Thus, the observed inhibition of lipid synthesis by hexazinone may be an indirect effect of this herbicide caused by its primary effect on photosynthesis. However, further work is needed to document whether this effect of hexazinone on lipid synthesis is a direct or an indirect one. The strong inhibition of lipid synthesis that was caused by chlorsulfuron appears to be a direct effect of this herbicide on lipid synthesis, since its effects on photosynthesis of isolated soybean leaf cells were insignificant.

The lowest concentration of hexazinone that inhibited significantly any of the four metabolic processes examined was 0.1 +I4 which at 30 min inhibited significantly photosynthesis of isolated soybean leaf cells (Table 1). Protein, RNA, and lipid syntheses were essentially unaffected by this concentration of hexazinone at the same incubation period of 30 min (Tables 2-4). At the highest concentration of 100 PM and maximum exposure time of 120 min, hexazinone inhibited photsynthesis by 92%, protein synthesis by 46%, RNA synthesis by 77%, and lipid synthesis by 89%. Therefore, it appears that photosynthesis as measured by CO, fixation of isolated soybean leaf cells was the most sensitive and first metabolic process inhibited by hexazinone. The effects of high concentrations of hexazinone on RNA and lipid syntheses of isolated soybean cells indicate that these processes may be also involved as target sites in the ultimate action of this compound as herbicide. Protein synthesis was less sensitive to hexazinone than any other metabolic process examined. The lowest concentration of chlorsulfuron that was inhibitory to any of the four

214

HATZIOS

AND

metabolic processes of the isolated soybean leaf cells was 0.1 ,uM which at 120 min of incubation inhibited significantly lipid synthesis (Table 4). Photosynthesis, protein, and RNA syntheses were not affected by this concentration of chlorsulfuron at any exposure time (Tables l-3). At the highest concentration of 100 pM and maximum exposure time of 120 min, chlorsulfuron inhibited photosynthesis by 12%, protein synthesis by 38%, RNA synthesis by 23%, and lipid synthesis by 90%. Although chlorsulfuron could exert its herbicidal action by affecting other metabolic processes not examined in this study (ll), the obtained results appeared to indicate that lipid synthesis is involved as a target site in the action of this herbicide. Photosynthesis, protein, and RNA syntheses were not inhibited strongly by this herbicide and, therefore, they do not appear to be target sites in its herbicidal action. ACKNOWLEDGMENTS

We express our sincere appreciation to Dr. J. D. Riggleman of DuPont, Wilmington, Delaware, for providing the analytical grade samples of hexazinone and chlorsulfuron used in this study. REFERENCES

1. W. R. Mullison, “Herbicide Handbook,” p. 479, 4th ed., Weed Science Society of America, Urbana, Ill., 1977. 2. R. C. Rhodes and R. A. Jewell, Metabolism of Y-labeled hexazinone in the rat, J. Agric. Food Chem. 28, 303 (1980). 3. R. C. Rhodes, Studies with “C-labeled hexazinone in water and bluegill sunfish, .I. Agric. Food Chem. 28, 306 (1980). 4. R. C. Rhodes, Soil studies with 14C-labeled hexazinone, J. Agric. Food Chem. 28, 311 (1980). 5. R. F. Holt, Determination of hexazinone and metabolite residues using nitrogen-selective gas chromatography, .I. Agric. Food Chem. 29, 165 (1981). 6. H. L. Palm, J. D. Riggleman, and D. A. Allison, Worldwide review of the new cereal herbicide DPX-4189, Proc. Brit. Crop Prot. Conf. Weeds 1, 1 (1980). 7. G. Levitt, H. L. Ploeg, R. C. Weigel, Jr., and

HOWE

D. J. Fitzgerald, 2-Chloro-N-[(4-methoxy-6methyl-1,3,5-triazin-2-y)amino-carbonyl]benzenesulfonamide, a new herbicide, J. Agric. Food Chem. 29, 416 (1981). 8. K. H. Biichel, Mechanisms of action and structure-activity relations of herbicides that inhibit photosynthesis, Pestic. Sci. 3, 89 (1972). 9. D. E. Moreland, Mechanism of action of herbicides, Annu. Rev. Plant Physiol. 31, 597 (1980).

10. A. Trebst and W. Draber, Structure-activity correlations of recent herbicides in photosynthetic reactions, in “Advances in Pesticide Science” (H. Geissbuhler, Ed.), Part 3, p. 836, Pergamon, Oxford, 1979. 11. T. B. Ray, Studies on the mode of action of DPX4189, Proc. Brit. Crop Prot. Conf. Weeds 1, 7 (1980). 12. 0. T. DeVilhers, M. L. Vandenplas, and H. M. Koch, The effect of DPX-4189 on biochemical processes in isolated leafcells and chloroplasts, Proc.

Brit.

Crop

Prot.

Conf.

Weeds

1, 237

(1980). 13. F. M. Ashton, 0. T. DeVilliers, R. K. Glenn, and W. B. Duke, Localization of metabolic sites of action of herbicides, Pestic. Biochem. Physiol. 7, 122 (1977). 14. F. M. Ashton and R. K. Glenn, Influence of chloro-, methoxy-, and methylthio-substitutions of bis(isopropylamino)-s-triazine on selected metabolic processes, Pestic. Biochem. Physiol.

11, 201 (1979).

15. K. K. Hatzios and D. Penner, Localizing the action of two thiadiazolyl herbicidal derivatives, Pestic.

Biochem.

Physiol.

13, 237 (1980).

16. E. M. Porter and P. G. Bar&, Use of single leaf cells to study mode of action of SAN 6706 on soybean and cotton, Weed Sci. 25,60 (1977). 17. J. C. Servaites and W. L. Ogren, Rapid isolation of mesophyll cells from leaves of soybean for photosynthetic studies, Plant Physiol. 59, 587 (1977). 18. D. W. Rehfeld and R. G. Jensen, Metabolism of separated leaf cells. III. Effects of calcium and ammonium on product distribution during photosynthesis in cotton cells, Plant Physiol. 52, 17 (1973). 19. Arnon, D. I., Copper enzymes in isolated chloroplasts: Polyphenoloxidase in Beta vulgaris L., Plant Physiol. 24, 1 (1949). 20. W. E. Splittstoesser and H. J. Hopen, Responses of bentgrass to siduron, Weeds 15, 52 (1967). 21. W. K. McNeil, J. F. Stritzke, and E. Basler, Mode of action of hexazinone and tebuthiuron on several woody species, Proc. South. Weed Sci. Sot. 34 (1982), in press.