J. Cell Sci. 15, 429-441 (1974)
Printed in Great Britain
429
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.BAYER
Department of Botany, University of California,
Davis, California 95616, U.S.A.
SUMMARY
Ultrastructural studies of trifluralin-treated cells in lateral root meristems of cotton (Gossypium 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 or
absence of microtubules. Where microtubules were absent, cells began division with a normal
prophase chromosome cycle. The chromosomes did not line up along a metaphase plate, but
coalesced in the cell. If cell division had begun prior to microtubule disappearance the mitotic
process was arrested at the stage that had been reached when the disappearance occurred.
In some cell divisions randomly oriented microtubules were noted, with mitosis apparently
arrested at those stages. Nuclear envelope reformation yielded cells that were polyploid,
polymorphonucleate, binucleate, or occasionally multinucleate. If microtubules were present
and if their orientation were normal, all stages of mitosis occurred. The range of mitotic
disruption observed can be explained by the threshold concentration for microtubule disappearance being very near aqueous saturation of trifluralin.
INTRODUCTION
Colchicine is well known as an inhibitor of mitosis. Levan (1938) reported that
in onion roots treated with colchicine, the prophase stage of mitosis appeared normal
but the chromosomes did not arrange along an equatorial plate at metaphase. More
recent studies have shown that a normal spindle does not form prior to division when
cells are treated with this drug (Hindmarsh, 1953). If the compound is applied during
cell division, the spindle apparatus breaks down, resulting in various abnormal cell
division figures (Hindmarsh, 1953). In a detailed ultrastructural study of the effect
of 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 stage
in the mitotic cycle had been reached prior to colchicine application'. With an absence of microtubules in these dividing cells no mitotic arrangement of chromosomes
occurred, rather the chromosomes became 'randomly scattered'. When applied at
telophase no cell plate formation occurred, resulting in binucleate and 'dumbbellshaped' nuclei.
Amato, Hoverson & Hacskaylo (1965) reported that trifluralin caused disorganized
nuclear 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 trifluralin
disrupted the mitotic process in onion (Allium cepa L., 'yellow') root tip tissue, and
that no single stage of mitosis was predominant in the treated cells. Mitotic activity
was not disrupted to the same extent in all cells, and some appeared to be undergoing
normal mitosis. Bayer et al. concluded from their light-microscopic observations that
many of the mitotic effects of trifluralin were similar to those observed for colchicine
or isopropyl-TV-phenylcarbamate (propham). In a light-microscope study Lignowski
& Scott (1971, 1972) reported that trifluralin affected root tip swelling and mitosis
in onion and wheat in the same manner as did colchicine. They found that the
chromosome cycle proceeded through prophase in a normal manner, but these
chromosomes did not become arranged along the equatorial plate. Polyploid and
multinucleate cells were observed after tissue had been treated for 24 h. It was also
noted that after centrifugation the chromosomes of the ' arrested metaphases' were
displaced, indicating that spindle disruption had taken place.
Ennis (1948) reported blocked metaphase mitotic configurations in Avena roots
treated 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 cells
when treated with trifluralin. He suggested a microtubule disappearance or disorganization was occurring similar to that reported for colchicine or propham.
Hepler & Jackson (1969) observed disorganization of the mitotic process in endosperm
cells of African blood lily (Haemanthus katherinae) after treatment with propham.
They reported the microtubules to be structurally similar to those in nontreated
cells, but the cells contained a 'multipolar spindle apparatus in which each pole
consisted of a radial array of microtubules'. The chromosomes were scattered around
the perimeter of these poles, with microtubules being between the focal area of the
array and the chromosomes. As a result of this microtubule distribution, the chromosomes failed to align at metaphase.
Trifluralin, propham, and colchicine attack the spindle apparatus of dividing cells
and produce many similar-appearing, abnormal nuclear patterns. In studies of the
ultrastructural effects of propham and colchicine it was found that their methods of
spindle disruption, as discussed above, varied markedly. In the present study, the
mitotic process in trifluralin-treated cells of cotton root tips was studied at the ultrastructural level in order to determine the mechanism of spindle disruption, and to
compare 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 Vermiculite (Terra-Lite, California Zonalite Company) until they had reached the fully expanded
cotyledon 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 chamber
for 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 nutrient
solution by shaking the mixture for 24 h. Treatments were begun by substituting the trifluralinsaturated nutrient solution for the original culture solution. The treatment period ranged
from 24 to 96 h, and each treatment was replicated 4 times. Ten lateral root tips, each 2 mm
long, were cut from each replicate and fixed at room temperature for 1-5 h in 4 % glutaraldehyde
buffered with 005 M phosphate, pH 7-2. The root tips were washed for 3 h in 6 changes of
the phosphate buffer, and were postfixed in phosphate-buffered 2-0 % osmium tetroxide for
1 "5 h. After several buffer rinses, the tissue was dehydrated in a graded acetone series and
embedded in Spurr's plastic (Spurr, 1969). Longitudinal sections were cut from the cortical
region 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 with
a Zeiss EM-9A electron microscope.
OBSERVATIONS
The observations which follow will be limited to the occurrence of microtubules
and their orientation during the cell cycle. Other organelles in the cell appeared to be
structurally unaffected by the herbicide.
Microtubule localization in cells from untreated plants
In the interphase cell, microtubules were abundant near cell walls with their
orientation circumferential to the cell axis (Fig. 1). During nuclear division microtubules were present near the condensed chromatin by late prophase (Fig. 2). At
metaphase they were attached to the chromosome kinetochores (Fig. 3), as well as
extending from pole to pole without chromosome attachment. During anaphase,
microtubules were present in the interzone between the 2 sets of daughter chromosomes
and also between the chromosomes and the polar regions. The greatest accumulation
of microtubules during cell plate formation was near the leading edge of the vesicle
fusion. No evidence indicated any ultrastructural difference between spindle and
cell wall microtubules, therefore they will be considered different only in location
and time of occurrence in the cell. A detailed paper on the role of microtubules in
cell 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 mitotic
inhibition, although variable, was consistent within small areas of the meristem. No
correlation was noted between the degree of disruption and a specific area in the
meristem. This gradation of injury could be divided into 2 groups. In the first,
microtubules were absent, with completion of mitosis being totally blocked. In the
second, microtubules were present, resulting in a range of mitotic disruption.
Microtubules absent after trifluralin treatment. At any one time, cells in the meristem were at many different stages of the cell cycle, therefore initial microtubule
disappearance 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
432
D. Hess and D. Bayer
occurred. With the mitotic sequence unable to proceed, nuclear envelope reformation
occurred yielding various interphase nuclear patterns characteristic of the stage at
which mitosis was arrested. Nuclear divisions arrested at late prophase or metaphase
were characterized by chromosomes being arranged in an abnormally small group in
the 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 in
polyploid polymorphonucleate or binucleate cells. If microtubular inhibition occurred
during early telophase the vesicles did not become organized in the interzone between
the 2 sets of chromosomes. Without vesicle alignment, vesicle fusion and thus cell
plate formation did not occur, resulting in binucleate cells. If cell plate formation
had begun prior to microtubule disappearance, fusion of vesicles ceased when microtubule disappearance occurred, yielding cells with partially formed cell walls (Fig. 6).
Microtubule absence in interphase cells did not stop the occurrence of mitotic
division attempts. The mitotic cycle appeared normal until microtubule involvement
should have occurred. The chromosomes condensed and divided in a normal manner
and the nuclear envelope began to disperse as prophase progressed. By late prophase
microtubules were not present near the condensed chromatin (Fig. 7) although they
had been observed at this stage in untreated tissue (Fig. 2). The chromosomes did
not align along the equatorial plate at metaphase but instead coalesced in the clear
zone (Fig. 8), causing the division sequence to be arrested. Occasionally one or more
chromosomes became separated from the main group. Nuclear envelope reformation
around 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 cells
of some root meristem areas even after treatment for 96 h. In many instances the
number of microtubules observed in the cell was reduced when compared to those
observed in untreated cells at the same stage in the cell cycle. If microtubules were
observed along the wall in interphase cells, they also occurred in the nuclear divisions
of adjacent cells. Regardless of the duration of treatment, where microtubules were
present and their orientation was normal, all stages of cell division were observed.
In some instances, microtubules present during cell division were abnormally
oriented (arrow, Fig. 10). During metaphase, the disoriented microtubules were
always found near the chromosome, with some exhibiting kinetochore attachment
(Fig. 10).
DISCUSSION
Trifluralin treatment often induces a range of effects from near normal mitosis to
severe colchicine-like disruption. Trifluralin is relatively insoluble in water ( < 1 ppm,
Eli Lilly Co. Tech. Bull.), and thus treatment concentrations must be low unless
alcohol or some other substance is added to the nutrient solution to increase solubility.
Using Haemanthus katherinae liquid endosperm cells, Jackson & Stetler (1973)
Effect of trifluralin on cotton root
433
found that trifluralin caused a reduction in the number of microtubules in dividing
cells. They used concentrations of o-i—100 ppb (parts per billion) as well as 'watersaturated' solutions of trifluralin. We propose that the threshold concentrations for
complete microtubule disappearance and thus complete blockage of mitotis is near the
aqueous saturated concentration. Therefore, the degree of saturation achieved will
determine the completeness of microtubule disappearance. Levan (1938), in a study
using colchicine, reported a threshold time and concentration for significant disruption 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 only
partial disruption of mitosis. More recently Jokelainen (1968) reported that with a
treatment of 0-012 mg/ioog body weight some microtubules were present in the
dividing cells of rat liver.
Borisy & Taylor (1967) reported that colchicine can bind to protein subunits of
microtubules in such a way as to prevent microtubule assembly. They found this
binding was reversible, and that it did not involve a chemical modification of the
colchicine molecule. Therefore, at high concentrations the colchicine-binding site
of the protein subunits would be occupied and no construction of microtubules
would occur. If uptake of a microtubule disrupting compound is highly localized, its
mobility poor or its concentration low, one would expect to find some microtubules
present and thus a gradation of mitotic disruption would result.
When microtubules were absent in trifluralin-treated root cells, there was no movement 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 absent
during prophase and early metaphase, the chromosomes did not line up along a
metaphase plate. He also reported that nuclear envelope reformation around chromosomes of arrested cell divisions sometimes resulted in polymorphic nuclei. In trifluralin-arrested cell divisions, similar polymorphic nuclei (Fig. 9) were observed
where nuclear envelope reformation had occurred around abnormal chromosome
groupings.
Abnormal microtubule orientation was observed in some nuclear divisions where
microtubules were present (Fig. 10). This disorientation is not the sole cause of
spindle disruption. Whaley, Dauwalder & Kephart (1966) reported that after treating
the roots of several plant species with 80 ppm colchicine ' spindle fibers were observed but they lacked a definite orientation'. Loss of birefringence near the poles
and an overall reduction in spindle length were noted by Inoue (1952) when colchicine was applied at low concentrations (io~5 M) to Chaetopterus oocytes.
Esau & Gill (1965) and Pickett-Heaps & Northcote (1966) have suggested that
microtubules that are present in the zone between the 2 daughter nuclei during late
anaphase are concerned with the movement of vesicles to the cell plate region.
Pickett-Heaps (1967) reported that with the destruction of microtubules by colchicine
during telophase, no cell plate initiation occurred. Vesicles were present in trifluralintreated cells lacking microtubules, yet they did not move into the cell plate region
during division. Where cell plate formation had begun prior to microtubule dis28-2
434
D. Hess and D. Bayer
appearance, partially formed cell walls were found (Fig. 6) which were similar to
those observed in colchicine-treated root tips (Pickett-Heaps, 1967).
A similarity exists between the ultrastructural effects of trifluralin and colchicine
in that both cause the disappearance of microtubules if present in sufficient concentration. Increasing evidence indicates that a variety of agents that are known to
disrupt microtubules do so by several different mechanisms. The mechanism of
action of trifluralin may not be identical to that of colchicine (i.e. reacting with the
same protein-binding site as colchicine) yet the effect on the microtubules and thus
on mitosis is the same.
The authors would like to thank Drs R. H. Falk and T . L. Rost for their critical review of
this manuscript. This research was supported by Cooperative Regional Research Project W-108.
REFERENCES
AMATO, V. A., HOVERSON, R. R. & HACSKAYLO, J. (1965). Microanatomical and morphological
responses 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 of
colchicine- 3 H 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 upon
Avena. 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 microtubule orientation in dividing endosperm cells of Haemanthus katherinae Baker. J. Cell
Sci. 5, 727-743.
HDTOMARSH, M. M. (1953). T h e 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 without
soil. Calif, agric. exp. Sta. Cir. 547.
INOUE, S. (1952). The effect of colchicine on the microscopic and submicroscopic structure of
the mitotic spindle. Expl Cell Res. Suppl. 2, 305-318.
JACKSON, W. T. & STETLER, D. A. (1973). Regulation of mitosis. IV. An in vitro and ultrastructural study of effects of trifluralin. Can. J. Bot. 51, 1513-1518.
JOKELAINEN, P. T . (1968). The effect of colchicine on die kinetochores and mitotic apparatus
in the rat. J. Cell Biol. 39, 68 a-69 a.
KIERMAYER, O. (1972). Beeinflussung der postmitotischen Kernmigration von Micrasterias
denticulata Br6b. durch das Herbizid Trifluralin. Protoplasma 75, 421-426.
LEVAN, A. (1938). T h e 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 endoplasmic reticulum during mitosis and cytokinesis in wheat meristems. J. Cell Sci. 1, 100—120.
REYNOLDS, E. S. (1963). T h e use of lead citrate at high p H as an electron-opaque stain in
electron microscopy. J. Cell Biol. 17, 208-212.
Effect of trifluralin on cotton root
435
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 early
stage in cell plate formation. J. Ultrastruct. Res. 15, 169-180.
SPURR,
{Received 15 August 1973)
ABBREVIATIONS ON PLATES
ch
er
m
mt
chromosomes
endoplasmic reticulum
mitochondria
micro tubules
n
V
to
nucleus
vacuole
cell wall
436
D. Hess and D. Bayer
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 to
appear near the chromosomes, x 25200.
Fig. 3. Metaphase chromosomes arranged along the equatorial plate with microtubules attached at the kinetochores. The microtubules radiate toward the poles of
the spindle apparatus, x 21000.
Effect of trifluralin on cotton root
437
438
D. Hess and D. Bayer
Fig. 4. Arrested metaphase division figures as a result of microtubule disappearance at
late prophase or metaphase. x 7000.
Fig. 5. Arrested anaphase division figure as a result of microtubule disappearance
occurring 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 the
wall, x 15400.
Effect of trifluralin on cotton root
c
439
f
•
* • * ,
*
• ;v''
^
w
#
I .
i
* .
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, with
the division sequence terminated in an aggregated chromosome configuration,
x 9500.
Effect of trifluralin on cotton root
Fig. 9. Nucleus after nuclear reformation of a blocked metaphase or disrupted anaphase. Note the thin connexions (arrows) between each of the nuclear segments
indicating that this is a uninucleate and not a trinucleate cell, x 8200.
Fig. 10. Trjfluralin treatment has not caused complete disappearance of microtubules
in this metaphase division figure. The chromosomes are aligned along a metaphase
plate but the microtubules are disoriented and seem to be radiating in all directions
from the chromosomes. The short microtubules (arrow) may be seen as a result of
their orientation being oblique with respect to the plane of sectioning, or they may be
genuinely shortened as a result of contraction or breakdown, x 19600.
441