The comparative cell cycle and metabolic effects of the herbicide napropamide on root tip meristems

PESTICIDE

BIOCHEMISTRY

The Comparative

AND

PHYSIOLOGY

31, 166174 (1988)

Cell Cycle and Metabolic Effects of the Herbicide Napropamide on Root Tip Meristems

JOSEPH M.DITOMASO,I Department

ofBotany,

THOMAS University

L. ROST, AND FLOYD M. ASHTON of California,

Daris,

California

95616

Received December 31, 1987, accepted March 30. 1988 Napropamide reduces the rate of entry of pea root cells into DNA synthesis and cell division by 8 and 12 hr of treatment, respectively. Protein synthesis was reduced by 22% 8 hr after treatment, and this level of inhibition remained about the same even after 48 hr. Napropamide treatment appeared to have little effect on oxygen uptake or the rate of RNA synthesis. Transfer of treated pea roots to fresh herbicide-free nutrient solution demonstrated complete recovery in both root growth and cell cycle progression. Incorporation of tritiated thymidine in recovering roots indicates that a number of dividing cells were arrested in Gz. However, the majority of total cells were in G, after 24-hr herbicide treatment. This suggests a block in both G, and G, of the cell cycle. The inhibitory effect of napropamide on the mitotic cycle may result from an inhibition in the synthesis or activity of cell cycle specific proteins. f) 1988 Academic Press, Inc. INTRODUCTION

Napropamide [2-(a-naphthoxy)NJ-diethyl propionamide] is a soil-applied herbicide reported to inhibit root elongation (l-3). The mode of action has been suggested to involve the inhibition of RNA and protein synthesis (4), as well as cell division (5). An inhibitory effect of several herbicides on various metabolic processes has been shown to influence cell cycle progression. Hess and Bayer (6) demonstrated that trifluralin disrupts mitosis by interfering with microtubule formation. Other dinitroanilines (2, 7), carbamates (8, 9>, and pronamide (10) have also been reported to interrupt spindle formation. The majority of “mitotic poisoners” (7). however, act as preprophase inhibitors, such that inhibition occurs in G, (pre-DNA synthesis phase) and G, (premitotic phase) of the cell cycle ( 11). The G, and Gz phases require the synthesis of proteins and nucleic acids to per’ Present address: Agronomy Department. Cornell University. Bradfield and Emerson Hall, Ithaca, NY 14853. ’ Abbreviations used: S. DNA synthesis phase; M, mitosis: DNP. dinitrophenol; CDAA. N,N-diallyl2-chloroacetamide: FAA, formahn:acetic acid:alcohol.

mit cells to enter into DNA synthesis phase (S)’ or mitosis (M). Ioxynil apparently prevents the entry of cells into mitosis by inhibiting RNA and protein synthesis (12, 13), dinitrophenol (DNP) acts by uncoupling respiration (9), and N,Ndiallyl-2-chloroacetamide (CDAA) acts by inhibiting protein synthesis (12). Analyses of the cell cycle with the sulfonylurea herbicide chlorsulfuron have provided the most conclusive description of the mechanism of action among preprophaseinhibiting herbicides. Rost (14) provided evidence suggesting that chlorsulfuron inhibited the progression of cells primarily from Gz to mitosis and secondarily from G, to DNA synthesis in pea roots. Ray (15) determined that inhibition in branched amino acid synthesis (Ile and Val) was responsible for the observed arrest in cell division. The site of action of chlorsulfuron was demonstrated to involve the enzyme acetolactate synthase which catalyzes the first step in the synthesis of Ile, Val, and Leu. These amino acids are essential for cell cycle progression. Napropamide is an amide herbicide primarily, but not exclusively, active on grasses. Barrett and Ashton (16) demonstrated that a IO-fold difference in sensitiv-

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ity between a susceptible species, corn, and a tolerant species, tomato, was the result of a difference in napropamide translocation. and not absorption or metabolism. The tolerance of pea to napropamide was estimated by root growth and mitotic entry studies to be intermediate between corn and tomato (5). A similar inhibitory effect on cell division suggests that the mechanism of action of napropamide is the same in corn and pea. As a result, the experiments reported in this study were conducted with peas, as they are more suitable for procedures used in analyzing the cell cycle. This investigation was designed to describe the effect of napropamide on cell cycle progression, cell metabolism, and the distribution of cells in the stages of the cell cycle. MATERIALS

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METHODS

Preparation of plant material. Pea (Pisum sativum L. cv Alaska) seeds were sterilized in 5.25% sodium hypochlorite for 5 min. Sterilized seeds were aseptically placed on autoclaved gauze and rinsed with sterile deionized water. Approximately 200 seeds were planted in 190 by IOO-mm crystallization dishes containing sterile vermiculite saturated with one-quarter-strength sterile Hoagland’s solution (17). Crystallization dishes were covered with autoclaved aluminum foil and placed in an incubation chamber at 26°C in the dark. After 4 days, the seedlings were removed and 1 to 2 cm of the root tips were excised and transferred to 125-ml Erlenmeyer flasks containing 48 ml sterile White’s medium (18) at pH 6.5. The flasks were covered with aluminum foil and immediately placed on an orbital shaker at 80 rpm in a growth chamber at 26°C in the dark. Root excisions and transfers were conducted under aseptic conditions. Mitotic index analysis. Six to eight l-cm pea root tips incubated in White’s medium for 24 hr and transferred to treatment solution (0, 0.31, 0.63, 1.25, 2.5, 5.0, 10.0, or

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20.0 FM napropamide) were examined at each sample time. Roots were grown at room temperature, fixed in absolute ethanol:glacial acetic acid (3:1, v/v) for 2 to 24 hr, hydrated, hydrolyzed for 30 min in 5 N HCl at room temperature, rinsed in distilled water, and stained for 3 to 6 hr in Feulgen reagent in the dark. Two 2-mm tips were squashed on each microscope slide, and slides were made permanent by placing them on dry ice and later mounting the coverslip with Euparal. One thousand cells per slide were scored for percentage of mitotic figures (mitotic index). At least three slides were scored per data point. Experiments involving peas were repeated twice, and only standard errors greater than 5% of the mean are indicated on each graph. Metabolic studies. Uptake and incorporation of tritiated thymidine, uridine, and amino acid mixture were measured by scintillation counting to determine the rates of napropamide inhibition on DNA, RNA, and protein synthesis, respectively. Ten roots per flask were pulse labeled for 1 hr prior to completion of herbicide treatment. Isotopes (ICN and Schwarz/Mann) were used at the following concentrations: [3H]thymidine (0.5 uCi/ml, sp act 6 mCi/ mmol), [3H]uridine (0.9 $i/ml, sp act 25 Ci/mmol), and [3H]amino acid mixture (0.6 &i/ml, sp act 234 mCi/mg). Labeled roots were removed and washed in 2 mM cold thymidine, uridine, or distilled water. The terminal 2 mm of the root tips (10 roots/ treatment) were cut, placed in cryogenic vials, and stored in a liquid nitrogen freezer at - 60°C. DNA, RNA, and proteins were isolated by macerating root tips in 2 ml cold 80% ethanol (v/v) in a hand tissue grinder. The ground tissue was poured over a GF/A Whatman filter on a vacuum filter apparatus. The filter was washed four times with approximately 8 ml cold 80% ethanol, dried at 60°C for 2 hr, placed into scintillation vials with 10 ml Amersham PCS scintillation fluid, allowed to sit for 12 hr, and counted for 10 min in a Beckman LS 8000

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scintillation counter. Each experiment was repeated at least three times and graphs were plotted as dpm incorporated per root vs time. Standard errors less than 5% of the mean are not presented. The exchangeable fraction was measured by placing 1 ml of the wash solution in scintillation fluid, counting it, multiplying by total volume of wash solution, and dividing by the number of roots sampled. Oxygen uptake. Two-centimeter pea root tips were cultured as previously described. Herbicide solution at 3 or 20 piV was added to flasks for appropriate times. Ten 2-mm tip sections of pea roots were excised and placed in 2 ml O,-saturated White’s medium in a Rank Brothers oxygen electrode chamber. The chamber temperature was held constant at 25°C by a Laudai Brinkmann K-2/RD circulator. After oxygen consumption was measured for 10 to 15 min, the roots were removed, blotted dry, weighed, oven dried, and reweighed. The rate of O2 uptake was plotted as nanomoles O2 per minute per milligram fresh weight against time of napropamide exposure. A similar curve was obtained by plotting nanomoles O2 per minute per milligram dry weight and nanomoles O? per minute per 10 roots against time of herbicide exposure (not shown). Standard error bars less than 5% of mean are not presented. Microautoradiography experiments. Pea roots were cultured in treatment solution and [3H]thymidine, fixed in absolute alcohol:glacial acetic acid (3: I, v/v), and stained in Schiff’s reagent. The 2-mm root tips were squashed on dry microscope slides coated with a solution consisting of 5 g/liter gelatin and 0.5 g/liter chromium potassium sulfate in water. Slides with root squashes were frozen on dry ice for 2 to 5 min and stored in absolute ethanol. Slides were hydrated, dipped in Kodak NTB2 liquid photographic emulsion, dried, and exposed in plastic slide boxes for 7 to 10 days at 4°C in the dark. Exposed slides were developed, dehydrated through an alcohol series, and permanently mounted with Euparal. One

AND

ASHTON

thousand cells in each slide were scored for labeled mitotic figures, total labeled cells, and total mitotic figures. Experiments were represented by three replicates with only standard errors greater than 5% displayed. Pea root cytological studies. Pea roots in White’s solution were fixed in forma1in:acetic acid:alcohol (FAA) (19) after 24 hr in treatment solution. Fixed root tips were dehydrated, embedded in Paraplast, sectioned longitudinally at 10 pm, and stained with safranin 0 and fast green (19). Slides were permanently mounted with Euparal. Mitotic figures were scored on a Zeiss RA-38 light microscope at x400 magnification. Data were recorded as mitotic figures per square millimeter in the 0.5 to 1.5-mm region proximal to the root apex. Several median and close to median sections of four to six roots per treatment were scored. Recovery experiments. Pea roots treated with herbicide solution for 24 or 48 hr were transferred to fresh herbicide-free White’s medium for an additional 24 to 48 hr. The mitotic index was determined. Root growth recovery was monitored by measuring six roots per flask, two flasks per treatment, at 24-hr intervals. The means of two repetitions are presented. In an additional experiment, the mitotic index was measured at 24-hr intervals in pea roots exposed to treatment solution plus sterile 0.1% casein hydrolysate in water. No recovery in the mitotic index occurred after 48 hr of herbicide treatment (data not presented). Microspectrophotometry. Permanently mounted slides of untreated and 24-hr napropamide-treated pea root squashes were analyzed for their relative amounts of nuclear DNA. The cell cycle positions of the nuclei were determined on a Zeiss photomicroscope filtered to a Zonax 3651 cytospectrophotometer. Sixty-two cells in a total of four treatment slides and I26 cells in a total of six control slides were measured at 560 nm. The relative DNA values were determined using version 824701 of

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METABOLIC

EFFECTS

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NAPROPAMIDE

the Zeiss Bioscan program. Measurements were adjusted by determining relative DNA content in one-half telophase (2C = G,) and late prophase (4C = GJ figures of dividing pea root cells. RESULTS

Mitotic entry response. The effect of 24hr napropamide treatment on cell division in roots of peas is presented in Fig. 1. The Z,, value (herbicide concentration resulting in 50% of maximum inhibition) was approximately 3.0 piVZ. At 10 FM napropamide, nearly 80% reduction in the mitotic index was observed. No further reduction in the number of dividing cells was observed even when the concentration was increased to 20 or 50 PM (not shown). In Fig. 2, the percentage of mitotic tigures in pea roots is plotted against time of herbicide exposure. In the 20qM treatment, the mitotic index began to drop 12 hr after herbicide exposure. No reduction in the mitotic index was detected at 3 FM until 16 hr of exposure. By 48 hr, however, both 3 and 20 PM napropamide resulted in an 87% drop in the percentage of mitotic figures. It is noteworthy that even after 48 hr of treatment the mitotic index never dropped below 0.6%.

$W

Napropamide

FIG. 1. The effect of a 24-hr napropamide treatment ut various concentrations on the percentuge of mitotic figures in peas. Stundurd errors less than 57~ sf the mean ure not shown.

Om

4 Hours

After

Trestment

FIGS. 2 and 3. The effect qf napropamide on the mitotic index (Fig. 2) and the incorporation of [‘Hjthymidine into DNA (Fig. 3) in 2-mm excised pea root tips; control (0). 3 +M (O), 20 PM(~). Five-day etiolatedpea seedlings were excised and incubated for 24-hr in White’s medium prior to application of treatment solution. Standard errors less than 5% of the mean are not shown.

DNA, protein, and RNA synthesis. The rate of DNA synthesis was measured after 1 hr [3H]thymidine exposure at each sample time. The rate of progression from G, through S was estimated as the amount of labeled isotope incorporated. The exchangeable fraction of 13H]thymidine in control and treated pea roots varied only slightly within 24 hr, indicating that the differences in thymidine incorporation were not the result of differences in the rate of thymidine uptake. Similar results were also observed with the exchangeable fraction of [3H]amino acid mixture and [3H]uridine (data not shown). The effect of napropamide on DNA syn-

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thesis is presented in Fig. 3. The rate of [3H]thymidine incorporation declined 8 hr after napropamide treatment. By 24 hr, the rate of DNA synthesis was reduced by 80 and 89% in 3 and 20 PM treatments, respectively. The decline in DNA synthesis occurred prior to the drop in the percentage of mitotic figures (Figs. 2 and 3). This suggests that inhibition of DNA synthesis may lead to the subsequent reduction in cell division. The effect of napropamide on protein synthesis was determined by estimating the rate of [3H]amino acid incorporation. Results are plotted as dpm incorporated against time of herbicide exposure (Fig. 4). A 22% reduction in the rate of protein synthesis was observed after 8 hr of treatment.

4 moo-

.;” 3000;; 2 . *oooI

E I

Protein

RNA

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synthesis

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ASHTON

Within 48 hr of treatment, at either rate, protein synthesis was not inhibited by more than 35%. The rate of RNA synthesis following napropamide treatment is shown in Fig. 5. No inhibitory effect was observed in the rate of [3H]uridine incorporation within 48 hr of treatment. It should be noted that [3H]amino acid mixture and [3H]uridine incorporation measure the overall rate of protein and RNA synthesis and do not distinguish the inhibition of specific proteins or RNAs. Oxygen uptuke. The rate of 0, uptake in treated roots varied little from those of control within 24 hr exposure to 3 and 20 p~I4 napropamide (Fig. 6). Only at 48 hr of treatment was a significant reduction (44%) observed in herbicide-treated roots. These results indicate that treated roots respire at near normal rates, even during a concurrent decline in cell division. Cell population studies. In Fig. 7, [3H]thymidine was added to untreated roots and to roots already exposed to 24 hr treatment solution. The average duration of G, plus one-half the time of mitosis can be estimated as the time between [3H]thymidine addition and the point at which 50% of the maximum number of mitotic figures are labeled. The average value was estimated to be 11 hr for treated roots and 8 hr in control

synthesis

Treatment

FIGS. 4 and 5. The effect of naprupamide on the incorporation of [3H]amino acid mixture into proteins (Fig. 4) and [‘Hjuridine into RNA (Fig. 5) in 2-mm excised pea root tips; control (O), 3 FM (0, 20 (LM (W). Standard errors less than 5% of the mean are not shown.

8 12 16 20 24 28 32 36 40 44 48 Hours

After

Treatment

FIG. 6. The effect of napropamide on oxygen uptake in 2-mm excised pea root tips; control (0). 3 (IM (0). 20 WM (W). Standard errors are indicated.

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s 10 12 14 16 1s 20 22 2.4 3H-Thymidine

Exposure

FIG. I. The effects of napropamide on the duration of GZ and the progression of lubeled and unlabeled cells from G? + M in cells of excised pea roots: control, total mitotic figures (O), labeled mitotic figures (m); 20 PM, total mitoticfigures (0). labeled mitoticfigures (Cl). Continuous [3Hjthymidine exposure began 48 hr after roots were excised and 24 hr after the applrcatzon of treatment solution. Standurd errors less than 5% of the mean are not shown.

roots. This suggests a prolongation in the time required for treated cells to progress through Gz and into mitosis. The rate at which cells progress from G, * S can be estimated by the slope of the curve represented by the percentage labeled interphase cells (Fig. 8). In untreated roots, the rate of cells advancing from G, -+

Hours

After

3H-Thymidine

Exposure

FIG. 8. Progression of cells from G, -+ S in 2-mm excisedpea root tips. Continuous c3HJthymidine exposure followed a 24-hr incubation in control (0) or 20 p.M napropamide treatment solution (0). Standard error bars are presented.

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S is 2.56%/hr, whereas treated roots progressed at 0.78%/hr, a 70% reduction. Mitofic figure distribution. Longitudinal sections of pea roots were examined to determine whether the inhibitory action of napropamide on dividing cells occurs preferentially in cortical or vascular regions of the meristem. Mitotic figures were counted within a distance of 1.5 mm basipetally from the junction with the root cap. The number of mitotic figures in median longitudinal sections of treated and untreated roots were counted for the occurrence of mitotic figures in the cortical and vascular tissues. The ratio of mitotic figures in vascular tissue to cortical tissue per square millimeter in untreated roots was 2.22 ? SE 0.3 1, whereas the ratio in treated roots was 1.99 _+ SE 0.29. The treatments did not differ significantly suggesting that the activity of napropamide is not tissue specific. Root gronjth and cell cycle recovery kinetics. Twenty-four-hour napropamidetreated pea roots were transferred to fresh herbicide-free White’s medium to determine whether the activity of napropamide could be reversed. The growth of pea roots transferred to 20 pM napropamide medium was severely reduced by 24 hr, and was completely inhibited within 48 hr. A subsequent transfer to fresh herbicide-free medium resulted in full recovery of root growth from 24 to 48 hr after transfer (Fig. 9). The mitotic index completely recovered following transfer from 24-hr treatment in 20 p.M napropamide to herbicide-free medium (Fig. 10). Within 24 hr of transfer the percentage of mitotic figures increased from 1.82 to 4.62% Microautoradiography following 13H]thymidine treatment was used to compare the number of labeled to nonlabeled mitotic figures in roots after transfer to herbicide-free medium (Fig. 11). After 4 hr slightly less than 1% of the total cells observed were in mitosis. This represents the cells dividing even after napropamide treat-

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Hours

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ROST,

I

Hours

After

Treatment

9 and 10. Recovery

qf root growth (Fig. 9) and cell division (Fig. IO) fi>llowing war&r of24hr napropamide-treated excised pea roots to fresh herhitide-free medium; antreated (O), 20 FM (W. treated roots transferred to fresh herbicide-free mediam (0). Each data point represents the mean of two experiments. Arrows point to treatment which u’as transferred at either 24- or 48-hr herbicide e.rposure. FIGS.

ASHTON

Hours

Excision

“I

AND

ment. The percentage of nonlabeled mitotic figures rapidly increased within 10 hr and subsequently dropped to 0.18% at 24 hr. This indicates that a large number of dividing cells were in G, prior to recovery, suggesting a napropamide-induced block in G,. Labeled mitotic figures denote the progression of cells from S -+ G2 --, M. The first labeled mitotic figures were observed 6 hr after exposure to [3H]thymidine, following transfer to herbicide-free medium. A more rapid increase in the percentage of labeled cells entering mitosis coincided with a drop in the percentage of nonlabeled mi-

After 3H-Thymidine in Herbicide-Free

Exposure Medium

FIG. 11. Progression of cells from G, ---, S -+ Gz and,from S + G2 + M in 24-hr napropamide-treated roots transferred to herbicideyfree medium. Progress of cells through the cell cycle hjas monitored by adding [3Hjthymidine to recovery medium. The curve representing percentage nonlabeled mitotic figures (0) MWS obtained by subtructing labeled (0) from total mitotic figures (m). The total number of labeled cells (a) is represented by the right L’ertical axis. Standard errors less than 5% of the mean are not shown.

totic figures. By 24 hr nearly all observed mitotic figures were labeled. The summation of the labeled and nonlabeled mitotic figure curves, represented as the percentage of total mitotic figures, changed only slightly after about 10 hr. The entry of cells from G, --, S is represented by the total percentage of labeled cells (Fig. 11). After an initial 4-hr delay, the progression of cells from G, -+ S steadily increased to a level similar to that observed at 24 hr in untreated roots (Fig. 8). This, again. would indicate a complete recovery of cell cycle progression within 24 hr of transfer to herbicide-free medium. Cell cycle distribution. The total number of cells in G, and G2 was estimated by determining the relative nuclear DNA content per nucleus. If only a G2 block occurred, a shift in the percentage of cells from a 2C amount of DNA to a 4C amount should result. However, the cell cycle distribution after 24 hr of napropamide treatment demonstrated a slight accumulation of cells in the G, (2C) phase (Fig. 12), suggesting an additional block in G,.

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Relative

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Content

12. Histograms of the cell cycle distribution in 2-mm pea root tip meristems. Relative DNA content was determined on Fuelgen stained nuclei after 24-hr incubation in treatment solution. Cells in G, = 2C amount of DNA: cells in G? = 4C amount of DNA. FIG.

DISCUSSION

Results of this investigation indicate that napropamide severely inhibited DNA synthesis and mitotic entry. However, cell division was never completely inhibited regardless of the herbicide concentration (to 50 l~,l)/n or length of exposure (to 72 hr). The point of cell cycle arrest was determined through microautoradiographic and spectophotometric analyses. Results demonstrate a block in both G, and G?, and an increase in the time required for cells to progress through Gz. The G, block may lead to the observed inhibition in DNA synthesis, whereas the drop in the mitotic index would result from both DNA synthesis inhibition and the block in G,. It is important to note that the reduced mitotic index following napropamide treatment is probably the result of an incomplete inhibition in all cells. The distribution of mitotic figures in treated root tips appeared to be random throughout the meristem. In addition, the labeling index after 96 hr of treatment was greater than 50% (data not shown), indicating that most cells eventually progressed through the S phase of the cell cycle. The arrest of cells in G, and G, following napropamide treatment supports the princi-

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pal control point hypothesis proposed by Van’t Hof and Kovacs (20). The theory is characterized by two major independent sites of control in the mitotic cycle, one in G, and another in G,. Carbohydrate starvation (21) and other chemical inhibitors (14. 22) have also been shown to result in a block in cycle progression and an accumulation of cells in G, and G, (23). Casein hydrolysate recovery experiments demonstrate that the arrest of division at various points in the cell cycle is not the result of an inhibition in amino acid synthesis, as was evidenced with chlorsulfuron (24). However, cell division in treated roots recovered when transferred to fresh herbicide-free medium. The initiation of this recovery was preceded by a 4-hr delay. A similar delay was reported when sucrose was added to carbohydrate-starved pea roots (25). It was suggested by Van’t Hof that this lag period represents the time in which proteins are synthesized in sufficient quantity to facilitate the continued progression of cells previously inhibited in G, and Gz. The results of these experiments have provided clear evidence implicating the inhibition of cell division and DNA synthesis in the mode of action of napropamide. However, the primary site of napropamide activity remains in doubt. The S-hr delay preceding the drop in DNA synthesis suggests that napropamide is acting on one or a series of biochemical events which eventually evoke an inhibitory effect on DNA synthesis and cell division. Perhaps the site of napropamide inhibition is a cell cycle specific RNA or protein ordinarily found in abundance within the cell. Cell cycle progression would appear normal until the substance was significantly depleted. REFERENCES

I. Y. Eshel, J. Katan, and D. Palevitch, Selective action of diphenamid and napropamide in pepper (Capsicum annuum L.) and weeds, Weed Res. 13, 379 (1973). 2. F. M. Ashton and A. S. Crafts, “Mode of Action

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of Herbicides,” pp. 91-117, Wiley-lnterscience, New York, 1981. A. R. Putnam and J. F. Hancock, Influence of napropamide on strawberry growth and fruit yield, HortScience 17, 650 (1982). F. M. Ashton and R. K. Glenn, Influence of Dand L-isomers of napropamide on selected metabolic processes and plant growth, J. Plant Growth Regul. 1, 271 (1982). J. M. DiTomaso, “The Action of the Herbicide Napropamide on the Structure, Cell Cycle, Metabolism and Polyamine Levels in Roots,” PhD Dissertation, Univ. California, Davis, 1987. F. D. Hess and D. Bayer. The effect of trifluralin on the ultrastructure of dividing cells of the root meristem of cotton (Gossypium hirsutum L. ‘acala 4-42’). J. Cell Sci. 15, 429 (1974). F. D. Hess, Determining causes and categorizing types of growth inhibition induced by herbicides, in “Biochemical Responses Induced by Herbicides” (D. E. Moreland, J. B. St. John. and F. D. Hess, Eds.), pp. 207-230, Amer. Chem. Sot. Symp. Ser. No. 181. Washington, DC, 1981. T. L. Rost and D. E. Bayer, Cell cycle population kinetics of pea root tip meristems treated with propham, Weed Sci. 24, 81 (1976). T. L. Rost and S. L. Morrison, The comparative cell cycle and metabolic effects of chemical treatments on root tip meristems. II. Propham, chlorpropham, and 2,4-dinitrophenol, Cytologia 49, 61 (1984). P. G. Bartels and J. L. Hilton, Comparison of trifluralin, oryzalin, pronamide, propham, and colchicine treatments on microtubules. Pestic. Biochem. Physiol. 3, 462 (1973). F. D’Amato. Cyto-histological investigations of antimitotic substances and their effects on patterns of differentiation, Caryologiu 13, 339 (1960). J. D. Mann, L. S. Jordan, and B. E. Day, A survey of herbicides for their effect upon protein synthesis, Plant Physiol. 40, 840 (1965). D. E. Moreland, S. S. Malhotra. R. D. Gruenhagen. and E. H. Shokrah. Effects of herbi-

tides on RNA and protein syntheses, Weed Sci. 17, 556 (1969). 14. T. L. Rost, The comparative cell cycle and metabolic effects of chemical treatments on root tip meristems. III. Chlorsulfuron, J. Plant Growth Rep/. 3, 51 (1984). 15. T. B. Ray, Site of action of chlorsulfuron. Inhibition of valine and isoleucine biosynthesis in plants, Planf Physiol. 75, 827 (1984). 16. M. Barrett and F. M. Ashton, Napropamide uptake, transport, and metabolism in corn (Zea mays) and tomato (Lycopersicon esculentum), Weed Sci. 29, 697 (1981). 17. F. H. Wilt and N. K. Wessells, “Methods in Developmental Biology.” p. 604, T. Y. Crowell, New York, 1967. 18. P. R. White, “A Handbook of Plant Tissue Culture,” p. 103, Cattel, Lancaster, PA, 1943. 19. D. A. Johansen, “Plant Microtechnique,” pp. 8082, McGraw-Hill, New York, 1940. 20. J. Van’t Hof and C. J. Kovacs, Mitotic cycle regulation in the meristem of cultured roots: The principal control point hypothesis, in “Advances in Experimental Medicine and Biology: The Dynamics of Meristem Cell Populations” (M. W. Miller and C. C. Kuehnert, Eds.). Vol. 18. pp. 15-32. Plenum, New York, 1972. 21. J. Van? Hof. D. P. Hoppin. and S. Yagi, Cell arrest in G, and Gz of the mitotic cycle of Vicia fahu root meristems. Amer. J. But. 60, 889 t 1973). 22. A. B. Rickinson. The effect of low concentration actinomycin D on the progress of cells through the cell cycle. Cell Tissue Kinet. 3, 335 (1970). 23. T. L. Rost, Responses of the plant cell cycle to stress, in “Mechanisms and Control of Cell Division” (T. L. Rost and E. M. Jr. Gifford, Eds.), pp. 1 I I-143, Dowden. Hutchinson and Ross, Stroudsburg, PA, 1977. 24. T. L. Rost and T. Reynolds, Reversal of chlorsulfuron-induced inhibition of mitotic entry by isoleucine and valine, Plant Physiol. 77, 481 (1985). 25. J. Van? Hof. The regulation of cell division in higher plants. in “Basic Mechanisms in Plant Morphogenesis,” No. 25, pp. 152-165, Brookhaven Symposia in Biology, 1973