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The mode of action of chlorsulfuron: A new herbicide for cereals
PESTICIDE
BIOCHEMISTRY
The Mode
AND
PHYSIOLOGY
of Action
17, lo-17
of Chlorsulfuron: THOMAS
Biochemicals
Department,
(1982)
A New Herbicide
for Cereals
B. RAY
E. I. du Pont de Nemours & Company, Inc., Wilmington,
Delaware 19898
Received June 4, 1981; accepted October 20, 1981 Chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide) is the active ingredient in DuPont “Glean” Weed Killer (fotmerly DPX-4189), a new herbicide for weed control in small grains as well as other uses. Continuous growth measurements of chlorsulfuron-sensitive seedlings demonstrated that the herbicide inhibits growth within 2 hr of application and by 8 hr reduces growth by 80%. This reduction in growth was closely associated with an inhibition of plant cell division. No significant effects were observed on auxin-, cytokinin-, or gibberellin-induced cell expansion. Photosynthesis, respiration, RNA synthesis, and protein synthesis were also initially unaffected under conditions where plant cell division is strongly inhibited.
INTRODUCTION
MATERIALS
Chlorsulfuron’ (2-chloro-N-[4-methoxy6-methyl1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide2 is the active ingredient in DuPont “Glean” Weed Killer (Fig. 1). This new herbicide shows promise for weed control in small grains as well as other uses. An important feature of this compound is its very high herbicidal activity at extremely low application rates. Recommended rates for weed control in wheat are between 10 and 40 g AI/ha (1, 2). Mammalian toxicity is low with an oral LD,, of 5000 mg/kg in male rats (2). Chlorsulfuron is absorbed by both roots and foliage of plants and is readily translocated. Death of treated plants is generally slow and is accompanied by chlorosis, necrosis, terminal bud death, vein discoloration, and complete inhibition of plant growth. Selectivity in crops such as wheat is primarily related to metabolism of chlorsulfuron to inactive products in tolerant crops (3). The study presented in this paper was undertaken to determine the mode of action of chlorsulfuron. 1 Proposed common name for the American tional Standards Institute. * U.S. Patent 4,167,719.
Na-
Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Plant growth measurements. Solution culture experiments were carried out with corn seedlings (two- to three-leaf stage). The seedlings were placed in small jars containing 50 ml of the herbicide solution in one-quarter strength Hoaglands. During the course of the experiment the solution in the jars was aerated. After 2 days the fresh weights of the roots and shoots were determined and the results are expressed as the net increase in fresh weights over the initial weights. The results are expressed as the mean of three replicates. Continuous growth measurements of corn seedlings were made using a linear variable differential transformer (LVDT) (Model 243-000, Trans Tech, Inc., Ellington, Conn.) essentially as described by Hsiao (4). Chlorsulfuron was sprayed onto corn seedlings with a Devilbiss Model 152 atomizer held approximately 50 cm from the plant. The herbicide was dissolved in a wetting solution containing 20% (v/v) acetone and 0.2% (v/v) Tween 20. Control plants received the wetting solution minus the chlorsulfuron. Three milliliters of solution was sprayed onto each plant. After spraying, the plants were attached to the LVDT which was connected to a strip-chart 10
0048-3575/82/010010-08$02.00/O
AND
MODE
FIG.
1. Structure
of
OF
ACTION
chlorsulfuron.
recorder. Growth rate measurements were made at 30°C in a controlled-environment room under 3500 fc of light. The LVDT was calibrated with a vertical screw-type micrometer and the growth rates were determined from the slopes of the recorder tracings using a standard curve obtained with the micrometer. The results are expressed as the average of four treated and four control plants, each of which was run on separate days. Photosynthesis and respiration. Photosynthesis was measured by following ferricyanide-catalyzed 0, evolution in chloroplasts isolated from pea leaves and ‘“CO, fixation by cells isolated from spinach leaves. Chloroplasts were prepared from pea leaves according to Lilley et al. (5). Photosynthesis was measured with a Clark-type O2 electrode (Hansatech Ltd., Norfolk, England) at 25°C and under a light intensity of 200 pElm%ec. The 1.5ml reaction volume contained the chloroplasts (55 pg chlorophyll), chlorsulfuron, isolation buffer, and 1 mM KFe(CN),. Intact leaf cells were isolated from spinach leaves according to the method of Ashton (6) with the following modifications. The maceration medium contained 1% Macerase (Calbiochem Lot 001082). After incubation in the maceration medium the isolated cells were collected by centrifugation and washed once in a maceration medium minus the Macerase and once in the incubation medium. The incubation medium contained 0.05 M Hepes, pH 7.6, 0.1 mM MgCIP, 0.1 mM CaCl,, and 0.6 M sorbitol. Photosynthesis was measured in sealed flasks containing 5 mM NaH14C0, (sp act, 0.04 $Zi/mmol, 0.05 &i/flask), chlorsulfuron, isolated cells, and incubation medium in a total volume of 2.5 ml at 25°C
OF
CHLORSULFURON
11
under a light intensity of 100 pE/m*/sec. Aliquots of 100 ~1 were removed at various times, pipetted onto filter-paper disks which were then washed with formic acid, dried, and counted in a liquid scintillation counter. Respiration was measured with an oxygen electrode using root tips isolated from pea seedlings which had been pretreated with up to 10 ppm chlorsulfuron. Seven root tips were used per assay. Plant cell expansion assays. IAA-in-. duced cell expansion was measured in subapical stem sections from 7-day-old etiolated Alaska peas. Sections, 5 mm in length, were taken from the pea stems just below the apical hook. The sections were floated on solutions containing 1 mM NaHPO,, pH 6.2, 10m7M IAA, and 10 ppm chlorsulfuron. Controls minus the chlorsulfuron, and minus chlorsulfuron and IAA were also run. All manipulations were carried out in a dark room under a green safelight. After 24 hr of dark incubation the net increase in stem section length was determined. Ten sections were used per treatment with each treatment replicated three times in an experiment. The experiment was repeated three times. Cytokinin-induced cell expansion of cucumber cotyledon was determined according to the method of Green and Muir (7). Cotyledons were excised from 5-dayold etiolated cucumber seedlings (var. Ohio MR-17) and were floated in petri dishes on solutions containing 40 mM KCl, 10 mM CaCl,, 10 ppm kinetin, and chlorsulfuron. Ten cotyledon were used per dish with three replicates per treatment. Controls contained either water, 10 ppm kinetin, or salts alone. All manipulations were carried out in a dark room under a green safelight. After 24 hr incubation in the dark, the cotyledons were weighed and the increase in fresh weight over the water controls were then determined. Gibberellic acid (GA)-induced elongation was measured using lettuce seedling hypocotyls. Lettuce (var. Grand Rapids) was germinated in petri dishes for 48 hr
12
THOMAS
B.
RAY
after which they were transferred to test proteins extracted according to the method solutions containing 10 ppm GA with and of Smillie and Krotkov (9). Radioactivity in the final fractions was determined by liquid without 1 ppm chlorsulfuron. Hypocotyl length was measured after 24 hr. The re- scintillation counting. sults were expressed as net increase in RESULTS hypocotyl length over initial values obEffects of Chlorsulfuron on Plant Growth tained at the start of the experiment. Mitotic index. Seeds of Vicia faba were One of the most noticeable plant regerminated 48 hr in moist vermiculite at sponses of chlorsulfuron is inhibition of 28°C in the dark. Seedlings were treated by plant growth. The effect of chlorsulfuron on immersing the roots in solutions containing the net increase in fresh weight of corn chlorsulfuron. After treatment the root tips seedlings grown hydroponically in the preswere excised and fixed for 1 hr in an ence of several concentrations of the herethanol:acetic acid solution (3:l) with the bicide is shown in Fig. 2. Corn was used as fixative being changed every 20 min. After test plant because it is relatively sensitive to fixing, the root tips were heated at 60°C for chlorsulfuron as compared to wheat which 15 min in 1 N HCl and then stained with is tolerant. The net weights were obtained 2 Feulgens reagent. Squashes were prepared days after adding the herbicide. As little as by the quick freeze method of Conger and 0.001 ppm chlorsulfuron caused inhibition Fairchild (8). The mitotic index is defined of root growth while shoot growth was first as the number of dividing cells per 100 cells. inhibited at 0.01 ppm. Squashes of five treated and five control To obtain a kinetic analysis of chlorsulfuroots werre examined and an average of 700 ron inhibition of plant growth, continuous nuclei were scored per squash. The results growth measurements of corn seedlings are expressed as the means of five tips. were obtained by attaching leaves of corn For the frequency distribution, four con- seedlings to an LVDT following a foliar aptrols tips and seven treated with chlorsulfuplication of chlorsulfuron. Initial growth ron were inspected. An average of 88 figures per root were scored for the controls and 28 figures per tip for the chlorsulfurontreated roots. The results for each mitotic phase are expressed as a percentage of the total figures counted per tip. - .24 Nucleic acid and protein synthesis. Corn seedlings were germinated at 28°C in the 3 dark in moist vermiculite for 48 hr. The roots of 10 intact seedlings were then immersed in chlorsulfuron solution for 6 hr in the dark at 30°C and then placed either in [3H]thymidine (sp act, 20 Wmmol, 1 2&i/ml) to measure DNA synthesis, rH]uridine (sp act, 35.2 Wmmol, 1 &i/ml) to measure RNA synthesis, or [14C]leucine (sp act, 54.7 mCi/mmol, 0.1 &i/ml) to mea0 ,001 sure protein synthesis. After 1 hr in the CHLORSULFURON CONCENTRATION I ppml isotope solutions the roots were excised FIG. 2. Net increase in the fresh weight of roots and and soaked in ice-cold water for 15 min. shoots of corn seedlings grown for 48 hr in the presFollowing the soak, the apical 5-mm por- ence of chlorsulfuron in solution culture. Each point is tion of the roots including the tip was ex- the mean of three replicates. The error bars are the cised, weighed, and the nucleic acids and standard errors of the means.
,“v.32
‘1.1
MODE
OF
OF
ACTION
rates ranged between 30 and 40 pm/min but, within 2 hr of treatment, growth rates were significantly decreased (Fig. 3), and by 8 hr the rate was only 20% of the initial rate. The growth rate of control plants did not change over the course of the experiment. From the results of the time-course and concentration experiments, it is apparent that any metabolic process which is involved with the primary mode of action of chlorsulfuron would have to be affected at low concentrations (less than 10 ppm) and within 2 hr of treatment. Effect of Chlorsulfuron on Plant Elongation and Division
of I ppm
0
CONTROL
Y i 0’
I
2
0
Control + 1 ppm Chlorsulfuron ‘I The values
are expressed
Mitotic
index
6.4 -+ 0.42 0.9 2 0.15 as means
I
1
1 I
4
6
s
TIME lhourd
FIG. 3. Effect offoliar application of chlorsulfuron on the growth rate of corn seedlings as measured with an LVDT. Each point is the mean of four replicates. The error bars are the standard errors of the means. The average zero-time rate is 35.4 + 3.04 nmimin.
Similar results have been found in pea roots treated with chlorsulfuron (data not shown). The reduction of the number of dividing cells in the treated plant roots is consistent with the proposal that chlorsulfuron inhibits plant cell division. Analysis of the frequency distribution of the various mitotic stages in the treated root tips indicates that there is no significant change over that found in the untreated tips (Table 1). This suggests that the inhibition of cell division does not actually occur during the mitotic or M stage of the cell division cycle but rather during some other stage of the cycle. Another method for analyzing the cell division activity of plant tissue is to measure DNA synthesis. This can be conveniently
Distribution
Percentage Treatment
_
20 -
TABLE 1 on the Mitotic Index and Frequency Root Tips of Vicia faba”
Chlorsulfuron
CHLORSULFURON
40-
F b 5
Cell
Plant growth can be considered to have two components, cell expansion and cell division. To better understand how chlorsulfuron is inhibiting plant growth it was necessary to determine how chlorsulfuron may be affecting these components. Bioassays using plant hormones were employed to evaluate the effects of chlorsulfuron on cell expansion. Neither indoleacetic acid-induced elongation of subapical etiolated pea stems, cytokinininduced cell expansion of cucumber cotyledons, nor gibberellic acid-induced elongation of lettuce hypocotyls was affected by treatments of up to 10 ppm chlorsulfuron. Though chlorsulfuron had little effect on plant cell expansion, strong inhibiting effects were found on plant cell division. Table 1 shows the effect of chlorsulfuron at 1 ppm on the mitotic index of roots of V. faha. The index is reduced 87% (from 6.4 in the control to 0.9 in the treated roots).
The Effect
13
CHLORSULFURON
+ SEM.
Prophase
Metaphase
46 +- 2.2 51 k 2.9
21 t 3.1 20 + 4.1
of Mitotic
Stages
distribution Anaphase 19 r 0.9 15 k 2.5
Telophase 15 t 1.0 14 + 2.5
in
14
THOMAS
determined by measuring [3H]thymidine incorporation into DNA. The effects of chlorsulfuron on plant cell division as measured by [3H]thymidine into DNA of roots of corn seedlings is shown in Fig. 4. The roots of the intact seedlings were treated for up to 6 hr in 1 ppm chlorsulfuron followed by a 1-hr treatment with [3H]thymidine. Analysis of the amount of radioactivity in the DNA of the roots indicated that 1 hr of treatment was sufficient to inhibit subsequent thymidine incorporation. By 6 hr the amount of [3H]thymidine incorporated was only 20% of the initial value. Measurement of total uptake of [3H]thymidine by the root tips showed no decrease during the first 4 hr of treatment when cell division was inhibited by 70% (Fig. 4 top curve) indicating that the inhibition of thymidine incorporation into DNA by chlorsulfuron is not due to an inhibition of thymidine uptake.
e u40-
B.
RAY
Figure 5 shows a dose-response curve for chlorsulfuron inhibition of plant cell division as measured by thymidine incorporation into DNA. With 6 hr of treatment, inhibition could be detected with as little as 0.01 ppm chlorsulfuron. Thus as with plant growth, cell division is rapidly inhibited by low concentrations of the herbicide. Effect of Chlorsulfuron on Photosynthesis and Respiration The data in Table 2 indicate that chlorsulfuron has no direct effect on photosynthesis. High levels of chlorsulfuron (100 ppm) caused no inhibition of ferricyanidecatalyzed photosynthetic O2 evolution in isolated pea chloroplasts. Preincubation of the chloroplasts for up to 2 hr with 100 ppm chlorsulfuron prior to measuring photosynthesis also had no inhibitory effect. The slightly higher rates after the 2-hr preincubation may have been due to loss of chloroplast integrity allowing better penetration of ferricyanide into the chloroplasts. As with photosynthetic 0, evolution in isolated chloroplasts, photosynthetic 14C0, fixation in isolated spinach leaf cells was also unaffected by chlorsulfuron. Concen-
I-ATION
e f Jo2 w z20s g
lo-
TIME,
hrr.
course for the effects of 1 ppm chlorsulfuron on the uptake and incorporation of [3H]thymidine into DNA as measured in roots of corn seedlings. Seedlings were incubated in the herbicide for up to 6 hr followed by a l-hr incubation in [3H]thymidine. The zero-time values are 4.60 X IO’ dpmlg fresh wt for uptake and 2.23 x lo6 dpmlg fresh wt for incorporation into DNA. FIG.
4. Time
CHLORSULFURON,
ppm
5. Dose response of the inhibition by chlorsulfuron of [3H]thymidine incorporation into DNA in roots of corn seedlings. Ten seedlings were used per concentration and were treated with the herbicide for 6 hrfollowed by 1 hr in [3H]thymidine. The control value is 3.67 x lo6 dpmlg fresh wt. FIG.
MODE
OF
ACTION
OF
CHLORSULFURON
TABLE Effect
2
of Chlorsulfuron on Photosynthetic 0, Evolution and Photosynthetic ‘4c0, Fixation in Isolated
Reaction
1s
in Isolated Pea Chloroplasts Spinach Leaf Cells
Treatment
Rate prnol O,/hr/mg Chl
0, evolution (ferricyanide reduction)
Control + 100 ppm Chlorsulfuron” 2-hr Preincubation + 100 ppm chlorsulfuron
29 33 39
Fmol CO,lhr/mg Chl WO, fixation
Control + 100 ppm Chlorsulfuron 2-hr Control 2-hr Incubation + 100 ppm chlorsulfuron
’ 100 ppm chlorsulfuron
= 2.8
x
Synthesis
Since both protein synthesis and RNA synthesis are necessary for cell division to occur, inhibition of either of these two processes would result in an inhibition of DNA synthesis and cell division. The effects of chlorsulfuron on protein and RNA synthe-
41
DISCUSSION
Consistent with field studies showing high herbicidal activity (l), laboratory experiments described here have shown that
of Chlorsulfuron
on RNA
Control 6.06
x 106
1.02 x 10’
3
and Protein
Precursor incorporation
RNA ([3H]uridine) Protein ([Ylleucine)
38
sis in corn root tips are shown in Table 3. After a 6-hr treatment with 1 ppm chlorsulfuron, there was no inhibition of protein synthesis as measured by [14C]leucine incorporation into protein. There was a slight (28%) reduction in RNA synthesis as measured by [ 3H]uridine incorporation into RNA. Under the same conditions, [3H]thymidine incorporation into DNA is inhibited 80-90%. Thus, under conditions where cell division as measured by DNA synthesis is strongly inhibited, little or no effect is seen on RNA and protein synthesis.
TABLE The Effect
54
10m4M.
trations of chlorsulfuron up to 100 ppm had no effect on CO, fixation even after incubating the cells for 2 hr (Table 2). This concentration of chlorsulfuron is equivalent to a 0.28 n&I solution of the herbicide. Like photosynthesis, plant respiration is also initially unaffected by chlorsulfuron. Rates of 0, uptake by pea roots treated with 10 ppm of the herbicide for as long as 48 hr were the same as control rates (data not shown). Effects on Protein
49
Synthesis
in Corn
Root
Tips
(dpmig fresh wt) Chlorsulfuron 4.41 x 106 1.14 x
10’
Percentages of control 72 112
Note. Seedlings were treated for 6 hr with 1 ppm chlorsulfuron followed by a I-hr treatment with [“HIuridine to measure RNA synthesis or [Wlleucine to measure protein synthesis.
16
THOMAS
chlorsulfuron is active as a plant growth inhibitor at 0.001 ppm (2.8 x 10mgM) in sensitive plants such as corn. At levels where chlorsulfuron inhibits plant growth, major physiological processes such as respiration, photosynthesis, and protein synthesis are not initially affected. However, in sensitive species, plant cell division is inhibited within l-2 hr of treatment with 0.01 ppm (2.8 x lop8 M) of the herbicide. The possible effects of chlorsulfuron on the metabolic and physiological processes described in this paper were measured after relatively short treatment times. It can be assumed that with longer treatment times secondary effects would become apparent. Concentrations of chlorsulfuron used in this study were at the relatively low levels of 0.001 to 10 ppm. This is between 2.8 x 10Pg and 2.8 x low5 M. These low concentrations were used not only because adequate responses could be obtained at these levels, but because use of high concentrations could cause secondary effects which are not related to the primary mode of action. DeVilliers et al. have reported that at very high concentrations (greater than 10e4 M) chlorsulfuron inhibits photosynthesis (10). The results presented in this paper and elsewhere (11) have shown that the concentration of chlorsulfuron required to inhibit photosynthesis is 10,000 times greater than that needed to inhibit plant growth and cell division. Thus the inhibition of photosynthesis by chlorsulfuron at high concentrations is a secondary effect as concluded by DeVilliers (10). Consistent with the results presented previously, no inhibitory effect on photosynthesis, respiration, protein synthesis, or RNA synthesis has been found with concentrations of chlorsulfuron below 10e4 M (10,ll). At present, the exact primary site of action of chlorsulfuron is not known. Several herbicides are known to inhibit cell division (12). Both dinitroanilines and N-phenylcarbamates block plant cell division, and, although the mechanism and site of action for these herbicides are not clear, some type of interaction with plant microtubules
B. RAY
has been suggested (13-18). Unlike the dinitroaniline and N-phenylcarbamate herbicides, chlorsulfuron does not alter the frequency distribution of the various mitotic stages in treated plant tissue. This suggests that chlorsulfuron is blocking some required process which occurs prior to the actual cell division step of the cell division cycle. ACKNOWLEDGMENTS Appreciation is extended to Dr. James Hutchison for the measurements of photosynthesis in spinach leaf cells and to Robert Jankowski for his technical assistance. REFERENCES 1. H. L. Palm, J. D. Riggleman, and D. A. Allison, Worldwide review of the new cereal herbicideDPX4189, Proc. Brit. Crop Prot. Co& Weeds 1, 1 (1980).
2. G. Levitt, H. L. Ploeg, R. C. Weigel, Jr., and D. J. Fitzgerald, 2-Chloro-N-[4-methoxy-6methyl-1,3,5-triazin-2-yl)amino-carbonyl]benzenesulfonamide, a new herbicide, J. Agric. Food Chew. 29, 416 (1981). 3. P. B. Sweetser and J. M. Hutchison, Metabolism of chlorsulfuron by plants, Pestic. Biochem.
Physiol. 17,
18 (1982).
4. T. C. Hsiao, E. Acevedo, and D. W. Henderson, Maize leaf elongation: Continuous measurements and close dependence on plant water stress, Science 163, 590 (1970). 5. R. C. Lilley, M. P. Fitzgerald, K. G. Rienits, and D. A. Walker, Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations, New Phytol. 75, 1 (1975). 6. F. F. Ashton, 0. T. DeVilhers, A. M. Glenn, and W. B. Duke, Localization of metabolic sites of action of herbicides, Pestic. Biochem. Physiol. 7, 122 (1977).
J. Green and R. M. Muir, The effect of potassium on cotyledon expansion induced by cytokinins, Physiol. Plant. 43, 213 (1978). 8. A. D. Conger and L. M. Fairchild, A quick freeze method for making smear slides permanent, 7.
Stain
Technol.
28, 281 (1953).
R. M. Smilhe and G. Krotkov, The estimation of nucleic acids in some algae and higher plants, Cannd. J. Bot. 38, 31 (1960). IO. 0. T. DeVilliers, M. L. Vandenplas, and H. M. Koch, The effect of DPX4189 on biochemical processes in isolated leaf cells and chloroplasts, 9.
Proc.
Brit.
Crop
Prof.
Conf.
Weeds
1, 237
(1980). 11. T. B. Ray, Studies on the mode of action of
MODE OF ACTION DPX4189, Proc. Brit. Crop Prot. Conf. Weeds 1, 7 (1980). 12. D. E. Moreland, Mechanisms of action of herbicides, Annu. Rev. Plant Physiol. 31, 597 (1980). 13. L. W. Young and N. D. Camper, Trifluralin effects on tobacco callus tissue: Mitosis and selected metabolic effects, Pestic. Biochem. Physiol. 12, 117 (1979). 14. S. J. Parka and 0. F. Soper, The physiology and mode of action of the dinitroaniline herbicides, Weed Sri. 25, 79 (1977).
OF CHLORSULFURON
17
15. E. M. Lignowski and E. G. Scott, Effect of trifluralin on mitosis, Weed Sci. 20, 267 (1972). 16. J. Hacskaylo and V. A. Amato, Effect of trifluralin on roots of corn and cotton, Weed. Sci. 16. 513 (1968). 17. T. L. Rost and D. E. Bayer, Cell cycle population kinetics of pea root tip meristems treated with propham, Weed Sci. 24, 81 (1976). 18. P. K. Hepler and W. T. Jackson, Isopropyl iVphenylcarbamate affects spindle microtubule orientation in dividing endosperm cells of Haemanthus katherinae Baker. J. Cell Sci. 5. 727 (1969).
BIOCHEMISTRY
The Mode
AND
PHYSIOLOGY
of Action
17, lo-17
of Chlorsulfuron: THOMAS
Biochemicals
Department,
(1982)
A New Herbicide
for Cereals
B. RAY
E. I. du Pont de Nemours & Company, Inc., Wilmington,
Delaware 19898
Received June 4, 1981; accepted October 20, 1981 Chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide) is the active ingredient in DuPont “Glean” Weed Killer (fotmerly DPX-4189), a new herbicide for weed control in small grains as well as other uses. Continuous growth measurements of chlorsulfuron-sensitive seedlings demonstrated that the herbicide inhibits growth within 2 hr of application and by 8 hr reduces growth by 80%. This reduction in growth was closely associated with an inhibition of plant cell division. No significant effects were observed on auxin-, cytokinin-, or gibberellin-induced cell expansion. Photosynthesis, respiration, RNA synthesis, and protein synthesis were also initially unaffected under conditions where plant cell division is strongly inhibited.
INTRODUCTION
MATERIALS
Chlorsulfuron’ (2-chloro-N-[4-methoxy6-methyl1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide2 is the active ingredient in DuPont “Glean” Weed Killer (Fig. 1). This new herbicide shows promise for weed control in small grains as well as other uses. An important feature of this compound is its very high herbicidal activity at extremely low application rates. Recommended rates for weed control in wheat are between 10 and 40 g AI/ha (1, 2). Mammalian toxicity is low with an oral LD,, of 5000 mg/kg in male rats (2). Chlorsulfuron is absorbed by both roots and foliage of plants and is readily translocated. Death of treated plants is generally slow and is accompanied by chlorosis, necrosis, terminal bud death, vein discoloration, and complete inhibition of plant growth. Selectivity in crops such as wheat is primarily related to metabolism of chlorsulfuron to inactive products in tolerant crops (3). The study presented in this paper was undertaken to determine the mode of action of chlorsulfuron. 1 Proposed common name for the American tional Standards Institute. * U.S. Patent 4,167,719.
Na-
Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Plant growth measurements. Solution culture experiments were carried out with corn seedlings (two- to three-leaf stage). The seedlings were placed in small jars containing 50 ml of the herbicide solution in one-quarter strength Hoaglands. During the course of the experiment the solution in the jars was aerated. After 2 days the fresh weights of the roots and shoots were determined and the results are expressed as the net increase in fresh weights over the initial weights. The results are expressed as the mean of three replicates. Continuous growth measurements of corn seedlings were made using a linear variable differential transformer (LVDT) (Model 243-000, Trans Tech, Inc., Ellington, Conn.) essentially as described by Hsiao (4). Chlorsulfuron was sprayed onto corn seedlings with a Devilbiss Model 152 atomizer held approximately 50 cm from the plant. The herbicide was dissolved in a wetting solution containing 20% (v/v) acetone and 0.2% (v/v) Tween 20. Control plants received the wetting solution minus the chlorsulfuron. Three milliliters of solution was sprayed onto each plant. After spraying, the plants were attached to the LVDT which was connected to a strip-chart 10
0048-3575/82/010010-08$02.00/O
AND
MODE
FIG.
1. Structure
of
OF
ACTION
chlorsulfuron.
recorder. Growth rate measurements were made at 30°C in a controlled-environment room under 3500 fc of light. The LVDT was calibrated with a vertical screw-type micrometer and the growth rates were determined from the slopes of the recorder tracings using a standard curve obtained with the micrometer. The results are expressed as the average of four treated and four control plants, each of which was run on separate days. Photosynthesis and respiration. Photosynthesis was measured by following ferricyanide-catalyzed 0, evolution in chloroplasts isolated from pea leaves and ‘“CO, fixation by cells isolated from spinach leaves. Chloroplasts were prepared from pea leaves according to Lilley et al. (5). Photosynthesis was measured with a Clark-type O2 electrode (Hansatech Ltd., Norfolk, England) at 25°C and under a light intensity of 200 pElm%ec. The 1.5ml reaction volume contained the chloroplasts (55 pg chlorophyll), chlorsulfuron, isolation buffer, and 1 mM KFe(CN),. Intact leaf cells were isolated from spinach leaves according to the method of Ashton (6) with the following modifications. The maceration medium contained 1% Macerase (Calbiochem Lot 001082). After incubation in the maceration medium the isolated cells were collected by centrifugation and washed once in a maceration medium minus the Macerase and once in the incubation medium. The incubation medium contained 0.05 M Hepes, pH 7.6, 0.1 mM MgCIP, 0.1 mM CaCl,, and 0.6 M sorbitol. Photosynthesis was measured in sealed flasks containing 5 mM NaH14C0, (sp act, 0.04 $Zi/mmol, 0.05 &i/flask), chlorsulfuron, isolated cells, and incubation medium in a total volume of 2.5 ml at 25°C
OF
CHLORSULFURON
11
under a light intensity of 100 pE/m*/sec. Aliquots of 100 ~1 were removed at various times, pipetted onto filter-paper disks which were then washed with formic acid, dried, and counted in a liquid scintillation counter. Respiration was measured with an oxygen electrode using root tips isolated from pea seedlings which had been pretreated with up to 10 ppm chlorsulfuron. Seven root tips were used per assay. Plant cell expansion assays. IAA-in-. duced cell expansion was measured in subapical stem sections from 7-day-old etiolated Alaska peas. Sections, 5 mm in length, were taken from the pea stems just below the apical hook. The sections were floated on solutions containing 1 mM NaHPO,, pH 6.2, 10m7M IAA, and 10 ppm chlorsulfuron. Controls minus the chlorsulfuron, and minus chlorsulfuron and IAA were also run. All manipulations were carried out in a dark room under a green safelight. After 24 hr of dark incubation the net increase in stem section length was determined. Ten sections were used per treatment with each treatment replicated three times in an experiment. The experiment was repeated three times. Cytokinin-induced cell expansion of cucumber cotyledon was determined according to the method of Green and Muir (7). Cotyledons were excised from 5-dayold etiolated cucumber seedlings (var. Ohio MR-17) and were floated in petri dishes on solutions containing 40 mM KCl, 10 mM CaCl,, 10 ppm kinetin, and chlorsulfuron. Ten cotyledon were used per dish with three replicates per treatment. Controls contained either water, 10 ppm kinetin, or salts alone. All manipulations were carried out in a dark room under a green safelight. After 24 hr incubation in the dark, the cotyledons were weighed and the increase in fresh weight over the water controls were then determined. Gibberellic acid (GA)-induced elongation was measured using lettuce seedling hypocotyls. Lettuce (var. Grand Rapids) was germinated in petri dishes for 48 hr
12
THOMAS
B.
RAY
after which they were transferred to test proteins extracted according to the method solutions containing 10 ppm GA with and of Smillie and Krotkov (9). Radioactivity in the final fractions was determined by liquid without 1 ppm chlorsulfuron. Hypocotyl length was measured after 24 hr. The re- scintillation counting. sults were expressed as net increase in RESULTS hypocotyl length over initial values obEffects of Chlorsulfuron on Plant Growth tained at the start of the experiment. Mitotic index. Seeds of Vicia faba were One of the most noticeable plant regerminated 48 hr in moist vermiculite at sponses of chlorsulfuron is inhibition of 28°C in the dark. Seedlings were treated by plant growth. The effect of chlorsulfuron on immersing the roots in solutions containing the net increase in fresh weight of corn chlorsulfuron. After treatment the root tips seedlings grown hydroponically in the preswere excised and fixed for 1 hr in an ence of several concentrations of the herethanol:acetic acid solution (3:l) with the bicide is shown in Fig. 2. Corn was used as fixative being changed every 20 min. After test plant because it is relatively sensitive to fixing, the root tips were heated at 60°C for chlorsulfuron as compared to wheat which 15 min in 1 N HCl and then stained with is tolerant. The net weights were obtained 2 Feulgens reagent. Squashes were prepared days after adding the herbicide. As little as by the quick freeze method of Conger and 0.001 ppm chlorsulfuron caused inhibition Fairchild (8). The mitotic index is defined of root growth while shoot growth was first as the number of dividing cells per 100 cells. inhibited at 0.01 ppm. Squashes of five treated and five control To obtain a kinetic analysis of chlorsulfuroots werre examined and an average of 700 ron inhibition of plant growth, continuous nuclei were scored per squash. The results growth measurements of corn seedlings are expressed as the means of five tips. were obtained by attaching leaves of corn For the frequency distribution, four con- seedlings to an LVDT following a foliar aptrols tips and seven treated with chlorsulfuplication of chlorsulfuron. Initial growth ron were inspected. An average of 88 figures per root were scored for the controls and 28 figures per tip for the chlorsulfurontreated roots. The results for each mitotic phase are expressed as a percentage of the total figures counted per tip. - .24 Nucleic acid and protein synthesis. Corn seedlings were germinated at 28°C in the 3 dark in moist vermiculite for 48 hr. The roots of 10 intact seedlings were then immersed in chlorsulfuron solution for 6 hr in the dark at 30°C and then placed either in [3H]thymidine (sp act, 20 Wmmol, 1 2&i/ml) to measure DNA synthesis, rH]uridine (sp act, 35.2 Wmmol, 1 &i/ml) to measure RNA synthesis, or [14C]leucine (sp act, 54.7 mCi/mmol, 0.1 &i/ml) to mea0 ,001 sure protein synthesis. After 1 hr in the CHLORSULFURON CONCENTRATION I ppml isotope solutions the roots were excised FIG. 2. Net increase in the fresh weight of roots and and soaked in ice-cold water for 15 min. shoots of corn seedlings grown for 48 hr in the presFollowing the soak, the apical 5-mm por- ence of chlorsulfuron in solution culture. Each point is tion of the roots including the tip was ex- the mean of three replicates. The error bars are the cised, weighed, and the nucleic acids and standard errors of the means.
,“v.32
‘1.1
MODE
OF
OF
ACTION
rates ranged between 30 and 40 pm/min but, within 2 hr of treatment, growth rates were significantly decreased (Fig. 3), and by 8 hr the rate was only 20% of the initial rate. The growth rate of control plants did not change over the course of the experiment. From the results of the time-course and concentration experiments, it is apparent that any metabolic process which is involved with the primary mode of action of chlorsulfuron would have to be affected at low concentrations (less than 10 ppm) and within 2 hr of treatment. Effect of Chlorsulfuron on Plant Elongation and Division
of I ppm
0
CONTROL
Y i 0’
I
2
0
Control + 1 ppm Chlorsulfuron ‘I The values
are expressed
Mitotic
index
6.4 -+ 0.42 0.9 2 0.15 as means
I
1
1 I
4
6
s
TIME lhourd
FIG. 3. Effect offoliar application of chlorsulfuron on the growth rate of corn seedlings as measured with an LVDT. Each point is the mean of four replicates. The error bars are the standard errors of the means. The average zero-time rate is 35.4 + 3.04 nmimin.
Similar results have been found in pea roots treated with chlorsulfuron (data not shown). The reduction of the number of dividing cells in the treated plant roots is consistent with the proposal that chlorsulfuron inhibits plant cell division. Analysis of the frequency distribution of the various mitotic stages in the treated root tips indicates that there is no significant change over that found in the untreated tips (Table 1). This suggests that the inhibition of cell division does not actually occur during the mitotic or M stage of the cell division cycle but rather during some other stage of the cycle. Another method for analyzing the cell division activity of plant tissue is to measure DNA synthesis. This can be conveniently
Distribution
Percentage Treatment
_
20 -
TABLE 1 on the Mitotic Index and Frequency Root Tips of Vicia faba”
Chlorsulfuron
CHLORSULFURON
40-
F b 5
Cell
Plant growth can be considered to have two components, cell expansion and cell division. To better understand how chlorsulfuron is inhibiting plant growth it was necessary to determine how chlorsulfuron may be affecting these components. Bioassays using plant hormones were employed to evaluate the effects of chlorsulfuron on cell expansion. Neither indoleacetic acid-induced elongation of subapical etiolated pea stems, cytokinininduced cell expansion of cucumber cotyledons, nor gibberellic acid-induced elongation of lettuce hypocotyls was affected by treatments of up to 10 ppm chlorsulfuron. Though chlorsulfuron had little effect on plant cell expansion, strong inhibiting effects were found on plant cell division. Table 1 shows the effect of chlorsulfuron at 1 ppm on the mitotic index of roots of V. faha. The index is reduced 87% (from 6.4 in the control to 0.9 in the treated roots).
The Effect
13
CHLORSULFURON
+ SEM.
Prophase
Metaphase
46 +- 2.2 51 k 2.9
21 t 3.1 20 + 4.1
of Mitotic
Stages
distribution Anaphase 19 r 0.9 15 k 2.5
Telophase 15 t 1.0 14 + 2.5
in
14
THOMAS
determined by measuring [3H]thymidine incorporation into DNA. The effects of chlorsulfuron on plant cell division as measured by [3H]thymidine into DNA of roots of corn seedlings is shown in Fig. 4. The roots of the intact seedlings were treated for up to 6 hr in 1 ppm chlorsulfuron followed by a 1-hr treatment with [3H]thymidine. Analysis of the amount of radioactivity in the DNA of the roots indicated that 1 hr of treatment was sufficient to inhibit subsequent thymidine incorporation. By 6 hr the amount of [3H]thymidine incorporated was only 20% of the initial value. Measurement of total uptake of [3H]thymidine by the root tips showed no decrease during the first 4 hr of treatment when cell division was inhibited by 70% (Fig. 4 top curve) indicating that the inhibition of thymidine incorporation into DNA by chlorsulfuron is not due to an inhibition of thymidine uptake.
e u40-
B.
RAY
Figure 5 shows a dose-response curve for chlorsulfuron inhibition of plant cell division as measured by thymidine incorporation into DNA. With 6 hr of treatment, inhibition could be detected with as little as 0.01 ppm chlorsulfuron. Thus as with plant growth, cell division is rapidly inhibited by low concentrations of the herbicide. Effect of Chlorsulfuron on Photosynthesis and Respiration The data in Table 2 indicate that chlorsulfuron has no direct effect on photosynthesis. High levels of chlorsulfuron (100 ppm) caused no inhibition of ferricyanidecatalyzed photosynthetic O2 evolution in isolated pea chloroplasts. Preincubation of the chloroplasts for up to 2 hr with 100 ppm chlorsulfuron prior to measuring photosynthesis also had no inhibitory effect. The slightly higher rates after the 2-hr preincubation may have been due to loss of chloroplast integrity allowing better penetration of ferricyanide into the chloroplasts. As with photosynthetic 0, evolution in isolated chloroplasts, photosynthetic 14C0, fixation in isolated spinach leaf cells was also unaffected by chlorsulfuron. Concen-
I-ATION
e f Jo2 w z20s g
lo-
TIME,
hrr.
course for the effects of 1 ppm chlorsulfuron on the uptake and incorporation of [3H]thymidine into DNA as measured in roots of corn seedlings. Seedlings were incubated in the herbicide for up to 6 hr followed by a l-hr incubation in [3H]thymidine. The zero-time values are 4.60 X IO’ dpmlg fresh wt for uptake and 2.23 x lo6 dpmlg fresh wt for incorporation into DNA. FIG.
4. Time
CHLORSULFURON,
ppm
5. Dose response of the inhibition by chlorsulfuron of [3H]thymidine incorporation into DNA in roots of corn seedlings. Ten seedlings were used per concentration and were treated with the herbicide for 6 hrfollowed by 1 hr in [3H]thymidine. The control value is 3.67 x lo6 dpmlg fresh wt. FIG.
MODE
OF
ACTION
OF
CHLORSULFURON
TABLE Effect
2
of Chlorsulfuron on Photosynthetic 0, Evolution and Photosynthetic ‘4c0, Fixation in Isolated
Reaction
1s
in Isolated Pea Chloroplasts Spinach Leaf Cells
Treatment
Rate prnol O,/hr/mg Chl
0, evolution (ferricyanide reduction)
Control + 100 ppm Chlorsulfuron” 2-hr Preincubation + 100 ppm chlorsulfuron
29 33 39
Fmol CO,lhr/mg Chl WO, fixation
Control + 100 ppm Chlorsulfuron 2-hr Control 2-hr Incubation + 100 ppm chlorsulfuron
’ 100 ppm chlorsulfuron
= 2.8
x
Synthesis
Since both protein synthesis and RNA synthesis are necessary for cell division to occur, inhibition of either of these two processes would result in an inhibition of DNA synthesis and cell division. The effects of chlorsulfuron on protein and RNA synthe-
41
DISCUSSION
Consistent with field studies showing high herbicidal activity (l), laboratory experiments described here have shown that
of Chlorsulfuron
on RNA
Control 6.06
x 106
1.02 x 10’
3
and Protein
Precursor incorporation
RNA ([3H]uridine) Protein ([Ylleucine)
38
sis in corn root tips are shown in Table 3. After a 6-hr treatment with 1 ppm chlorsulfuron, there was no inhibition of protein synthesis as measured by [14C]leucine incorporation into protein. There was a slight (28%) reduction in RNA synthesis as measured by [ 3H]uridine incorporation into RNA. Under the same conditions, [3H]thymidine incorporation into DNA is inhibited 80-90%. Thus, under conditions where cell division as measured by DNA synthesis is strongly inhibited, little or no effect is seen on RNA and protein synthesis.
TABLE The Effect
54
10m4M.
trations of chlorsulfuron up to 100 ppm had no effect on CO, fixation even after incubating the cells for 2 hr (Table 2). This concentration of chlorsulfuron is equivalent to a 0.28 n&I solution of the herbicide. Like photosynthesis, plant respiration is also initially unaffected by chlorsulfuron. Rates of 0, uptake by pea roots treated with 10 ppm of the herbicide for as long as 48 hr were the same as control rates (data not shown). Effects on Protein
49
Synthesis
in Corn
Root
Tips
(dpmig fresh wt) Chlorsulfuron 4.41 x 106 1.14 x
10’
Percentages of control 72 112
Note. Seedlings were treated for 6 hr with 1 ppm chlorsulfuron followed by a I-hr treatment with [“HIuridine to measure RNA synthesis or [Wlleucine to measure protein synthesis.
16
THOMAS
chlorsulfuron is active as a plant growth inhibitor at 0.001 ppm (2.8 x 10mgM) in sensitive plants such as corn. At levels where chlorsulfuron inhibits plant growth, major physiological processes such as respiration, photosynthesis, and protein synthesis are not initially affected. However, in sensitive species, plant cell division is inhibited within l-2 hr of treatment with 0.01 ppm (2.8 x lop8 M) of the herbicide. The possible effects of chlorsulfuron on the metabolic and physiological processes described in this paper were measured after relatively short treatment times. It can be assumed that with longer treatment times secondary effects would become apparent. Concentrations of chlorsulfuron used in this study were at the relatively low levels of 0.001 to 10 ppm. This is between 2.8 x 10Pg and 2.8 x low5 M. These low concentrations were used not only because adequate responses could be obtained at these levels, but because use of high concentrations could cause secondary effects which are not related to the primary mode of action. DeVilliers et al. have reported that at very high concentrations (greater than 10e4 M) chlorsulfuron inhibits photosynthesis (10). The results presented in this paper and elsewhere (11) have shown that the concentration of chlorsulfuron required to inhibit photosynthesis is 10,000 times greater than that needed to inhibit plant growth and cell division. Thus the inhibition of photosynthesis by chlorsulfuron at high concentrations is a secondary effect as concluded by DeVilliers (10). Consistent with the results presented previously, no inhibitory effect on photosynthesis, respiration, protein synthesis, or RNA synthesis has been found with concentrations of chlorsulfuron below 10e4 M (10,ll). At present, the exact primary site of action of chlorsulfuron is not known. Several herbicides are known to inhibit cell division (12). Both dinitroanilines and N-phenylcarbamates block plant cell division, and, although the mechanism and site of action for these herbicides are not clear, some type of interaction with plant microtubules
B. RAY
has been suggested (13-18). Unlike the dinitroaniline and N-phenylcarbamate herbicides, chlorsulfuron does not alter the frequency distribution of the various mitotic stages in treated plant tissue. This suggests that chlorsulfuron is blocking some required process which occurs prior to the actual cell division step of the cell division cycle. ACKNOWLEDGMENTS Appreciation is extended to Dr. James Hutchison for the measurements of photosynthesis in spinach leaf cells and to Robert Jankowski for his technical assistance. REFERENCES 1. H. L. Palm, J. D. Riggleman, and D. A. Allison, Worldwide review of the new cereal herbicideDPX4189, Proc. Brit. Crop Prot. Co& Weeds 1, 1 (1980).
2. G. Levitt, H. L. Ploeg, R. C. Weigel, Jr., and D. J. Fitzgerald, 2-Chloro-N-[4-methoxy-6methyl-1,3,5-triazin-2-yl)amino-carbonyl]benzenesulfonamide, a new herbicide, J. Agric. Food Chew. 29, 416 (1981). 3. P. B. Sweetser and J. M. Hutchison, Metabolism of chlorsulfuron by plants, Pestic. Biochem.
Physiol. 17,
18 (1982).
4. T. C. Hsiao, E. Acevedo, and D. W. Henderson, Maize leaf elongation: Continuous measurements and close dependence on plant water stress, Science 163, 590 (1970). 5. R. C. Lilley, M. P. Fitzgerald, K. G. Rienits, and D. A. Walker, Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations, New Phytol. 75, 1 (1975). 6. F. F. Ashton, 0. T. DeVilhers, A. M. Glenn, and W. B. Duke, Localization of metabolic sites of action of herbicides, Pestic. Biochem. Physiol. 7, 122 (1977).
J. Green and R. M. Muir, The effect of potassium on cotyledon expansion induced by cytokinins, Physiol. Plant. 43, 213 (1978). 8. A. D. Conger and L. M. Fairchild, A quick freeze method for making smear slides permanent, 7.
Stain
Technol.
28, 281 (1953).
R. M. Smilhe and G. Krotkov, The estimation of nucleic acids in some algae and higher plants, Cannd. J. Bot. 38, 31 (1960). IO. 0. T. DeVilliers, M. L. Vandenplas, and H. M. Koch, The effect of DPX4189 on biochemical processes in isolated leaf cells and chloroplasts, 9.
Proc.
Brit.
Crop
Prof.
Conf.
Weeds
1, 237
(1980). 11. T. B. Ray, Studies on the mode of action of
MODE OF ACTION DPX4189, Proc. Brit. Crop Prot. Conf. Weeds 1, 7 (1980). 12. D. E. Moreland, Mechanisms of action of herbicides, Annu. Rev. Plant Physiol. 31, 597 (1980). 13. L. W. Young and N. D. Camper, Trifluralin effects on tobacco callus tissue: Mitosis and selected metabolic effects, Pestic. Biochem. Physiol. 12, 117 (1979). 14. S. J. Parka and 0. F. Soper, The physiology and mode of action of the dinitroaniline herbicides, Weed Sri. 25, 79 (1977).
OF CHLORSULFURON
17
15. E. M. Lignowski and E. G. Scott, Effect of trifluralin on mitosis, Weed Sci. 20, 267 (1972). 16. J. Hacskaylo and V. A. Amato, Effect of trifluralin on roots of corn and cotton, Weed. Sci. 16. 513 (1968). 17. T. L. Rost and D. E. Bayer, Cell cycle population kinetics of pea root tip meristems treated with propham, Weed Sci. 24, 81 (1976). 18. P. K. Hepler and W. T. Jackson, Isopropyl iVphenylcarbamate affects spindle microtubule orientation in dividing endosperm cells of Haemanthus katherinae Baker. J. Cell Sci. 5. 727 (1969).