the effect of trifluralin on the ultrastructure … · of some root meristem areas even after...

J. Cell Sci. 15, 429-441 (1974) 429Printed in Great Britain

THE EFFECT OF TRIFLURALIN ON THE

ULTRASTRUCTURE OF DIVIDING CELLS

OF THE ROOT MERISTEM OF COTTON

(GOSSYPIUM HIRSUTUM L. 'ACALA 4-42')

D. HESS AND D.BAYERDepartment of Botany, University of California,Davis, California 95616, U.S.A.

SUMMARY

Ultrastructural studies of trifluralin-treated cells in lateral root meristems of cotton (Gossy-pium hirsutum L.) revealed that mitotic disruptions were due to the absence of microtubules.The extent of disruption varied between individual roots and correlated with the presence orabsence of microtubules. Where microtubules were absent, cells began division with a normalprophase chromosome cycle. The chromosomes did not line up along a metaphase plate, butcoalesced in the cell. If cell division had begun prior to microtubule disappearance the mitoticprocess was arrested at the stage that had been reached when the disappearance occurred.In some cell divisions randomly oriented microtubules were noted, with mitosis apparentlyarrested at those stages. Nuclear envelope reformation yielded cells that were polyploid,polymorphonucleate, binucleate, or occasionally multinucleate. If microtubules were presentand if their orientation were normal, all stages of mitosis occurred. The range of mitoticdisruption observed can be explained by the threshold concentration for microtubule dis-appearance being very near aqueous saturation of trifluralin.

INTRODUCTION

Colchicine is well known as an inhibitor of mitosis. Levan (1938) reported thatin onion roots treated with colchicine, the prophase stage of mitosis appeared normalbut the chromosomes did not arrange along an equatorial plate at metaphase. Morerecent studies have shown that a normal spindle does not form prior to division whencells are treated with this drug (Hindmarsh, 1953). If the compound is applied duringcell division, the spindle apparatus breaks down, resulting in various abnormal celldivision figures (Hindmarsh, 1953). In a detailed ultrastructural study of the effectof colchicine on mitosis of cells in wheat {Triticum vulgare L.) root tips, Pickett-Heaps(1967) stated that 'microtubules disappeared from the spindle no matter what stagein the mitotic cycle had been reached prior to colchicine application'. With an ab-sence of microtubules in these dividing cells no mitotic arrangement of chromosomesoccurred, rather the chromosomes became 'randomly scattered'. When applied attelophase no cell plate formation occurred, resulting in binucleate and 'dumbbell-shaped' nuclei.

Amato, Hoverson & Hacskaylo (1965) reported that trifluralin caused disorganizednuclear division, and prevented cytokinesis in roots of corn (Zea mays L.) and cotton

430 D. Hess and D. Bayer

(Gossypium hirsutum L.). Bayer, Foy, Mallory & Cutter (1967) found that trifluralindisrupted the mitotic process in onion (Allium cepa L., 'yellow') root tip tissue, andthat no single stage of mitosis was predominant in the treated cells. Mitotic activitywas not disrupted to the same extent in all cells, and some appeared to be undergoingnormal mitosis. Bayer et al. concluded from their light-microscopic observations thatmany of the mitotic effects of trifluralin were similar to those observed for colchicineor isopropyl-TV-phenylcarbamate (propham). In a light-microscope study Lignowski& Scott (1971, 1972) reported that trifluralin affected root tip swelling and mitosisin onion and wheat in the same manner as did colchicine. They found that thechromosome cycle proceeded through prophase in a normal manner, but thesechromosomes did not become arranged along the equatorial plate. Polyploid andmultinucleate cells were observed after tissue had been treated for 24 h. It was alsonoted that after centrifugation the chromosomes of the ' arrested metaphases' weredisplaced, indicating that spindle disruption had taken place.

Ennis (1948) reported blocked metaphase mitotic configurations in Avena rootstreated with propham. He concluded 'cytological effects induced (by propham)resemble those caused by low concentrations of colchicine'. Kiermayer (1972)described the abnormal positioning of nuclei within Micrasterias denticulata cellswhen treated with trifluralin. He suggested a microtubule disappearance or dis-organization was occurring similar to that reported for colchicine or propham.Hepler & Jackson (1969) observed disorganization of the mitotic process in endospermcells of African blood lily (Haemanthus katherinae) after treatment with propham.They reported the microtubules to be structurally similar to those in nontreatedcells, but the cells contained a 'multipolar spindle apparatus in which each poleconsisted of a radial array of microtubules'. The chromosomes were scattered aroundthe perimeter of these poles, with microtubules being between the focal area of thearray and the chromosomes. As a result of this microtubule distribution, the chromo-somes failed to align at metaphase.

Trifluralin, propham, and colchicine attack the spindle apparatus of dividing cellsand produce many similar-appearing, abnormal nuclear patterns. In studies of theultrastructural effects of propham and colchicine it was found that their methods ofspindle disruption, as discussed above, varied markedly. In the present study, themitotic process in trifluralin-treated cells of cotton root tips was studied at the ultra-structural level in order to determine the mechanism of spindle disruption, and tocompare its effects with those reported for colchicine and propham.

MATERIALS AND METHODS

Cotton seeds {Gossypium hirsutum L. 'Acala 4-42') were germinated and grown in Vermi-culite (Terra-Lite, California Zonalite Company) until they had reached the fully expandedcotyledon stage (approximately 1 week). The seedlings were then transferred to 2-5 x 20 cm,aluminum foil covered test tubes containing 1/16-strength (o-o62S-strength) nutrient solution(Hoagland & Arnon, 1950) adjusted to pH 7-2. The nutrient solutions were changed daily.The seedlings were grown under a 16-h 25 °C day, 8-h 20 °C night regime in a growth chamberfor 3 days prior to treatment with the herbicide.

Technical grade trifluralin (a)a,a-trifluoro-2,6-dinitro-A^,iV-dipropyl-/)-toluidine), 98%

Effect of trifluralin on cotton root 431

pure,* was brought to aqueous saturation (< 1 ppm, 1 ppm = 3 x IO~*M) in the nutrientsolution by shaking the mixture for 24 h. Treatments were begun by substituting the trifluralin-saturated nutrient solution for the original culture solution. The treatment period rangedfrom 24 to 96 h, and each treatment was replicated 4 times. Ten lateral root tips, each 2 mmlong, were cut from each replicate and fixed at room temperature for 1-5 h in 4 % glutaraldehydebuffered with 005 M phosphate, pH 7-2. The root tips were washed for 3 h in 6 changes ofthe phosphate buffer, and were postfixed in phosphate-buffered 2-0 % osmium tetroxide for1 "5 h. After several buffer rinses, the tissue was dehydrated in a graded acetone series andembedded in Spurr's plastic (Spurr, 1969). Longitudinal sections were cut from the corticalregion of the root tip just above the region of apical initial with a Porter-Blum ultramicrotome.The tissue was stained with a saturated solution of uranyl acetate in 50 % ethanol (Watson,1958) for 20 min and with lead citrate (Reynolds, 1963) for 4 min. Sections were viewed witha Zeiss EM-9A electron microscope.

OBSERVATIONS

The observations which follow will be limited to the occurrence of microtubulesand their orientation during the cell cycle. Other organelles in the cell appeared to bestructurally unaffected by the herbicide.

Microtubule localization in cells from untreated plants

In the interphase cell, microtubules were abundant near cell walls with theirorientation circumferential to the cell axis (Fig. 1). During nuclear division micro-tubules were present near the condensed chromatin by late prophase (Fig. 2). Atmetaphase they were attached to the chromosome kinetochores (Fig. 3), as well asextending from pole to pole without chromosome attachment. During anaphase,microtubules were present in the interzone between the 2 sets of daughter chromosomesand also between the chromosomes and the polar regions. The greatest accumulationof microtubules during cell plate formation was near the leading edge of the vesiclefusion. No evidence indicated any ultrastructural difference between spindle andcell wall microtubules, therefore they will be considered different only in locationand time of occurrence in the cell. A detailed paper on the role of microtubules incell division has been published by Newcomb (1969).

Microtubule presence and localization in trifluralin-treated cells

Trifluralin injury was manifested as an inhibition of mitosis. The degree of mitoticinhibition, although variable, was consistent within small areas of the meristem. Nocorrelation was noted between the degree of disruption and a specific area in themeristem. This gradation of injury could be divided into 2 groups. In the first,microtubules were absent, with completion of mitosis being totally blocked. In thesecond, microtubules were present, resulting in a range of mitotic disruption.

Microtubules absent after trifluralin treatment. At any one time, cells in the meri-stem were at many different stages of the cell cycle, therefore initial microtubuledisappearance influenced all stages of mitosis where microtubules were involved.Mitosis was arrested at that stage of division where the microtubule disappearance

• L. E. Peterson of Eli Lilly and Co., has stated (Lignowski & Scott, 1972) that the 2%impurities in technical grade trifluralin do not have biological activity.

28 c EL 15


occurred. With the mitotic sequence unable to proceed, nuclear envelope reformationoccurred yielding various interphase nuclear patterns characteristic of the stage atwhich mitosis was arrested. Nuclear divisions arrested at late prophase or metaphasewere characterized by chromosomes being arranged in an abnormally small group inthe clear zone (Fig. 4). Nuclear envelope reformation resulted in polyploid nuclei.Chromosomes in cells arrested at anaphase remained in 2 distinct groups (Fig. 5).Nuclear envelope reformation around chromosomes in this distribution resulted inpolyploid polymorphonucleate or binucleate cells. If microtubular inhibition occurredduring early telophase the vesicles did not become organized in the interzone betweenthe 2 sets of chromosomes. Without vesicle alignment, vesicle fusion and thus cellplate formation did not occur, resulting in binucleate cells. If cell plate formationhad begun prior to microtubule disappearance, fusion of vesicles ceased when micro-tubule disappearance occurred, yielding cells with partially formed cell walls (Fig. 6).

Microtubule absence in interphase cells did not stop the occurrence of mitoticdivision attempts. The mitotic cycle appeared normal until microtubule involvementshould have occurred. The chromosomes condensed and divided in a normal mannerand the nuclear envelope began to disperse as prophase progressed. By late prophasemicrotubules were not present near the condensed chromatin (Fig. 7) although theyhad been observed at this stage in untreated tissue (Fig. 2). The chromosomes didnot align along the equatorial plate at metaphase but instead coalesced in the clearzone (Fig. 8), causing the division sequence to be arrested. Occasionally one or morechromosomes became separated from the main group. Nuclear envelope reformationaround arrested division figures resulted in polyploid, polymorphic nuclei (Fig. 9).Thus in meristem areas where microtubules were absent, the cell cycle sequence was:interphase, prophase (Fig. 7), blocked metaphase (Fig. 8), interphase.

Microtubules present after trifluralin treatment. Microtubules were present in cellsof some root meristem areas even after treatment for 96 h. In many instances thenumber of microtubules observed in the cell was reduced when compared to thoseobserved in untreated cells at the same stage in the cell cycle. If microtubules wereobserved along the wall in interphase cells, they also occurred in the nuclear divisionsof adjacent cells. Regardless of the duration of treatment, where microtubules werepresent and their orientation was normal, all stages of cell division were observed.In some instances, microtubules present during cell division were abnormallyoriented (arrow, Fig. 10). During metaphase, the disoriented microtubules werealways found near the chromosome, with some exhibiting kinetochore attachment(Fig. 10).

DISCUSSION

Trifluralin treatment often induces a range of effects from near normal mitosis tosevere colchicine-like disruption. Trifluralin is relatively insoluble in water ( < 1 ppm,Eli Lilly Co. Tech. Bull.), and thus treatment concentrations must be low unlessalcohol or some other substance is added to the nutrient solution to increase solubility.Using Haemanthus katherinae liquid endosperm cells, Jackson & Stetler (1973)


found that trifluralin caused a reduction in the number of microtubules in dividingcells. They used concentrations of o-i—100 ppb (parts per billion) as well as 'water-saturated' solutions of trifluralin. We propose that the threshold concentrations forcomplete microtubule disappearance and thus complete blockage of mitotis is near theaqueous saturated concentration. Therefore, the degree of saturation achieved willdetermine the completeness of microtubule disappearance. Levan (1938), in a studyusing colchicine, reported a threshold time and concentration for significant dis-ruption of onion root tip cells of 4 h at 50-100 ppm. Using rat liver, Brues & Cohen(1936) observed that 0-02-0-05 mg colchicine/100 g body weight resulted in onlypartial disruption of mitosis. More recently Jokelainen (1968) reported that with atreatment of 0-012 mg/ioog body weight some microtubules were present in thedividing cells of rat liver.

Borisy & Taylor (1967) reported that colchicine can bind to protein subunits ofmicrotubules in such a way as to prevent microtubule assembly. They found thisbinding was reversible, and that it did not involve a chemical modification of thecolchicine molecule. Therefore, at high concentrations the colchicine-binding siteof the protein subunits would be occupied and no construction of microtubuleswould occur. If uptake of a microtubule disrupting compound is highly localized, itsmobility poor or its concentration low, one would expect to find some microtubulespresent and thus a gradation of mitotic disruption would result.

When microtubules were absent in trifluralin-treated root cells, there was no move-ment of chromosomes to form a metaphase plate after completion of prophase;rather the chromosomes coalesced in the clear zone in an unorganized array (Fig. 8).Using colchicine, Pickett-Heaps (1967) reported that when microtubules were absentduring prophase and early metaphase, the chromosomes did not line up along ametaphase plate. He also reported that nuclear envelope reformation around chromo-somes of arrested cell divisions sometimes resulted in polymorphic nuclei. In tri-fluralin-arrested cell divisions, similar polymorphic nuclei (Fig. 9) were observedwhere nuclear envelope reformation had occurred around abnormal chromosomegroupings.

Abnormal microtubule orientation was observed in some nuclear divisions wheremicrotubules were present (Fig. 10). This disorientation is not the sole cause ofspindle disruption. Whaley, Dauwalder & Kephart (1966) reported that after treatingthe roots of several plant species with 80 ppm colchicine ' spindle fibers were ob-served but they lacked a definite orientation'. Loss of birefringence near the polesand an overall reduction in spindle length were noted by Inoue (1952) when col-chicine was applied at low concentrations (io~5 M) to Chaetopterus oocytes.

Esau & Gill (1965) and Pickett-Heaps & Northcote (1966) have suggested thatmicrotubules that are present in the zone between the 2 daughter nuclei during lateanaphase are concerned with the movement of vesicles to the cell plate region.Pickett-Heaps (1967) reported that with the destruction of microtubules by colchicineduring telophase, no cell plate initiation occurred. Vesicles were present in trifluralin-treated cells lacking microtubules, yet they did not move into the cell plate regionduring division. Where cell plate formation had begun prior to microtubule dis-

28-2


appearance, partially formed cell walls were found (Fig. 6) which were similar tothose observed in colchicine-treated root tips (Pickett-Heaps, 1967).

A similarity exists between the ultrastructural effects of trifluralin and colchicinein that both cause the disappearance of microtubules if present in sufficient con-centration. Increasing evidence indicates that a variety of agents that are known todisrupt microtubules do so by several different mechanisms. The mechanism ofaction of trifluralin may not be identical to that of colchicine (i.e. reacting with thesame protein-binding site as colchicine) yet the effect on the microtubules and thuson mitosis is the same.

The authors would like to thank Drs R. H. Falk and T. L. Rost for their critical review ofthis manuscript. This research was supported by Cooperative Regional Research Project W-108.

REFERENCES

AMATO, V. A., HOVERSON, R. R. & HACSKAYLO, J. (1965). Microanatomical and morphologicalresponses of corn and cotton to trifluralin. Proc. Ass. sth. agric. Wkrs. p. 234 (abstr.).

BAYER, D. E., FOY, C. L., MALLORY, T . E. & CUTTER, E. G. (1967). Morphological and histo-logical effects of trifluralin on root development. Am. J. Bot. 54, 945—952.

BORISY, G. G. & TAYLOR, E. W. (1967). The mechanism of action of colchicine. Binding ofcolchicine-3H to cellular protein. J. Cell Biol. 34, 525-533.

BRUES, A. M. & COHEN, A. (1936). Effects of colchicine and related substances on cell division.Biochem. J. 30, 1363-1368.

ENNIS, E. R., JR. (1948). Some cytological effects of O-isopropyl N-phenyl carbamate uponAvena. Am. J. Bot. 35, 15-21.

ESAU, K. & GILL, R. H. (1965). Observations on cytokinesis. Planta 67, 168-181.HEPLER, P. K. & JACKSON, W. T. (1969). Isopropyl iV-phenylcarbamate affects spindle micro-

tubule orientation in dividing endosperm cells of Haemanthus katherinae Baker. J. CellSci. 5, 727-743.

HDTOMARSH, M. M. (1953). The effect of colchicine on the spindle of root tip cells. Proc. Litm.Soc. N.S.W. 77, 300-306.

HOAGLAND, D. R. & ARNON, D. I. (1950). The water culture method for growing plants withoutsoil. Calif, agric. exp. Sta. Cir. 547.

INOUE, S. (1952). The effect of colchicine on the microscopic and submicroscopic structure ofthe mitotic spindle. Expl Cell Res. Suppl. 2, 305-318.

JACKSON, W. T. & STETLER, D. A. (1973). Regulation of mitosis. IV. An in vitro and ultra-structural study of effects of trifluralin. Can. J. Bot. 51, 1513-1518.

JOKELAINEN, P. T . (1968). The effect of colchicine on die kinetochores and mitotic apparatusin the rat. J. Cell Biol. 39, 68 a-69 a.

KIERMAYER, O. (1972). Beeinflussung der postmitotischen Kernmigration von Micrasteriasdenticulata Br6b. durch das Herbizid Trifluralin. Protoplasma 75, 421-426.

LEVAN, A. (1938). The effect of colchicine on root mitosis in Allium. Hereditas 24, 471-486.LIGNOWSKI, E. M. & SCOTT, E. G. (1971). Trifluralin and root growth. PL Cell Physiol, Tokyo

12, 701-708.LIGNOWSKI, E. M. & SCOTT, E. G. (1972). Effects of trifluralin on mitosis. Weed Sci. 20,

267-270.NEWCOMB, E. H. (1969). Plant microtubules. A. Rev. PL Physiol. 20, 253-288.PICKETT-HEAPS, J. D. (1967). The effect of colchicine on the ultrastructure of dividing plant

cells, xylem wall differentiation and distribution of cytoplasmic microtubules. Devi Biol. 15,206-236.

PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (1966). Organization of microtubules and endo-plasmic reticulum during mitosis and cytokinesis in wheat meristems. J. Cell Sci. 1, 100—120.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-212.


SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy.J. Ultrastruct. Res. 26, 31-43.

WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals.II. Application of solutions containing lead and barium. J. biophys. biochem. Cytol. 4,475-478.

WHALEY, W. G., DAUWALDER, M. & KEPHART, J. E. (1966). The Golgi apparatus and an earlystage in cell plate formation. J. Ultrastruct. Res. 15, 169-180.

{Received 15 August 1973)

ABBREVIATIONS ON PLATES

chermmt

chromosomesendoplasmic reticulummitochondriamicro tubules

nV

to

nucleusvacuolecell wall


All electron micrographs are longitudinal sections of cotton root tip meristematic cells.Unless noted, scale markers are equivalent to i#o fim.

Figs. 1-3. Microtubule distribution in untreated tissue. Scale markers 0-5 fim.Fig. 1. Microtubules adjacent to a longitudinal cell wall in an interphase cell,

x 52300.

Fig. 2. Nuclear division at prophase. At this stage microtubules have begun toappear near the chromosomes, x 25200.

Fig. 3. Metaphase chromosomes arranged along the equatorial plate with micro-tubules attached at the kinetochores. The microtubules radiate toward the poles ofthe spindle apparatus, x 21000.


Fig. 4. Arrested metaphase division figures as a result of microtubule disappearance atlate prophase or metaphase. x 7000.Fig. 5. Arrested anaphase division figure as a result of microtubule disappearanceoccurring at anaphase. Note the abundance of endoplasmic reticulum. x 10 000.Fig. 6. Disruption of cell division at telophase due to disappearance of microtubules.The cell wall has a distorted appearance and extension has ceased at both ends of thewall, x 15400.


c f

• * • * , i

* * . I• ;v''

^w

# I .

44° D. Hess and D. Bayer

Fig. 7. Prophase division figure from tissue area where microtubules were absent.Prophase was not affected by absence of microtubules. x 13300.Fig. 8. Arrested metaphase division figures in cells containing no microtubules, withthe division sequence terminated in an aggregated chromosome configuration,x 9500.


Fig. 9. Nucleus after nuclear reformation of a blocked metaphase or disrupted ana-phase. Note the thin connexions (arrows) between each of the nuclear segmentsindicating that this is a uninucleate and not a trinucleate cell, x 8200.Fig. 10. Trjfluralin treatment has not caused complete disappearance of microtubulesin this metaphase division figure. The chromosomes are aligned along a metaphaseplate but the microtubules are disoriented and seem to be radiating in all directionsfrom the chromosomes. The short microtubules (arrow) may be seen as a result oftheir orientation being oblique with respect to the plane of sectioning, or they may begenuinely shortened as a result of contraction or breakdown, x 19600.

the effect of trifluralin on the ultrastructure … · of some root meristem areas even after...

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