Acta Bot.
Neerl. 39(1),
March
1990,
19-27
p.
The effect of colchicine
microfibrils in root
and
hairs
A.M.C. Emons1, A.M.C.
J.
microtubules
on
Wolters-Arts
J.A.
Traas
2
and
Derksen
Department ofExperimental Botany, University of Nijmegen, Toernooiveld,6525
ED
Nijmegen,
The Netherlands
SUMMARY
Roots of
and
Equisetum hyemale
sativus
Raphanus
were
grown
concentrations of colchicine that slowed down root hair
initially, and
later
hairs of E.
was
untreated hairs. In
root
helicoidalafter
ordered wall
(prior
is
texture
orientation of the
deposited
with
root
in
as
orientation of
from
remained ordered. As
deposition
in orientations that differ from the
depolymerizing
microtubules and also in the absence
nascent
not
directly
microfibrils.
Key-words: colchicine, microfibrils, microtubules,
texture,
in
colchicine,
depolymerization)
of microtubules, it is concluded that microtubules do
control the orientation of
effected
texture
deposition
microtubule
to
and transverse, but
oblique
to
treatment
hairs of R. sativus the
the microfibrils changed
treatment
of the microtubules. The wall
most
hyemale
axial
The
stopped growth completely.
of
depolymerization
in
growth
root
hairs,wall
sativus.
Equisetum hyemale, Raphanus
INTRODUCTION
The mechanism that controls the ordered
deposition
et
al.
microtubules and microfibrils are
1990).
parallel
In
growing plant cells,
each
to
other;
this also
al. 1985;
of cell wall microfibrils is
controversy (Emons 1982; Traas
in the
occurs
where both microtubules and microfibrils
Emons
1989).
microfibrils
In
This has led
(for
hair
root
helicoidal
et
al.
Seagull
a
stack
Wolters-Arts
root
hairs
&
1983; Lloyd
configurations
have been
'Correspondence:
Dr
A.
Agricultural University,
address:
are
M.
axial
Wells
reported
C.
Emons,
Arboretumlaan
Station
Dijon, Cedex
de
4,
to
(Emons
of
et
found in
i.e. the
& Derksen
tip,
1986;
nascent
Physiologic Vegetale,
the
Traas
Department
BD
been found:
previous
et
Plant
Wageningen,
Institut
France.
19
lamella. In
& Heath
(Seagull
al.
(Lloyd
of
axial,
& Wells
helical,
1985).
The
lamellae. Microfibril orientation in
helical
for Allium
6703
hair,
root
polylamellate (Lloyd
steeply
1985;
have
textures
parallel-fibred
or
Derksen
1982).
of wall
crossed
of
a matter
consistently
of the
in all directions
1985;
that cortical microtubules direct the
lamellae is rotated with respect
microtubules in
expanding part
run
hypothesis
and
1981)
helicoidal wall is built of
1504-21034
the
tubes different types
(Sassen
subsequent
to
review: Heath &
& Wells
Hepler 1985; Lloyd
1985) though
& Wells
Cytology
1985;
and
general,
cortical
1980; Emons &
more
Traas
complicated
et
al.
1985).
Morphology, Wageningen
The Netherlands.
National
de la
Recherche
Agronomique,
B.V,
20
A. M.
in
Thus,
has
hairs with axial
root
often in
are
been found. In
not
microtubules
not
are
or
while in
parallel,
helical wall texture, microtubulesand
in the
addition,
in
parallel
of microfibril order after
hairs with helicoidal wall
root
with
green
nascent
such
(for
review: Itoh
of the microtubules with colchicine is
disruption
microfibrils
co-orientation
a
with helicoidal wall texture, cortical
algae
microfibrils
nascent
texture
C. EMONS ETAL.
The loss
1989).
seen as
a
strong
argument for the role of microtubules in microfibril orientation (for review: Robinson &
Such
Quader 1982).
have
experiments
not
been carried
out on
hairs and
root
on
cells with
helicoidal walls.
To
the effect of microtubule
study
root
hairs,
were
treated with the
known
rest
roots
young
In young
and axial in the
hairs of E.
root
consistently using
tip
different methods:
Emons & Wolters-Arts
1982;
(Traas
et
al.
1985)
higher plant
root
in which
plant
The present
on
hair
study reports
microtubules of
MATERIALS
Root
hairs of
Wolters-Arts
germinated
in
special
under
a
on
were
fixed
a
in
hairs
(L.)
of
(Emons
It is the
most
alignment.
studied
thoroughly
alignment.
It is the hairs of this
microfibrils
nascent
was
1980).
number of concentrations of colchicine on
microfibril
tip,
roots
to
of
taken
were
stem
from
sativus
Raphanus
texture
in the
tube,
and
textures.
disturbing
exposed
to
the
cuvettes
drug
was
did
growth
the
and intact
Only
seedlings
freshly
(cj
growth,
Emons
&
.
from
seedlings
were
placed
could be examined
and in which the medium
hairs that grew in the medium
within this
or
10 ium for up
to
were
20 h.
range microtubules
stop abruptly, and cells did
adding
taken
were
concentrations of 1, 5,
chosen because
not
by
at
roots.
cuttings
stem
(L.)
in Petri dishes. Plantlets
water
cuttings
follow hair initiation and
without
these
al.
etal. 1985), immunofluorescence
different root hair wall
possess
hyemale
Root
in which
hair
el
found
was
METHODS
range of concentrations
depolymerized,
were
the effects of
well-
(Sassen
alignment
of chemically fixed material
& Heath
Seagull
moist filter paper and grown in
changed
microtubule
1989).
microfibril texture in the
Equisetum
cuvettes
used. Roots
This
AND
light microscope
could be
axial
between cortical microtubules and
species that
two
1983).
on
of the hair tube in R. sativus
net
axial microtubule
net
a
Emons & Wolters-Arts 1983) and random
show concomittant microtubuleand microfibril
hair also has
morphology,
have
they
and helicoidal in the
rest
1983), dry cleaving (Traas
not
co-alignment
tip
in
deposition
with growing hairs
sativus
from the hair
thin-sectioning
first shown (Newcomb & Bonnett 1965;
growth,
wall microfibril
chosen because
were
pm
and freeze-substitution(Emons
cell that does
The R. sativus
hyemale
on
Raphanus
species
hyemale (Emons 1982;
until 25 pm from the hair
1985).
and
random until 300
texture:
of the hair tube in E.
depolymerization
Equisetum hyemale
colchicine. These
drug
hair wall
root
of
not
prepared
were
Roots
plasmolyse.
to
up
a
concentration of 3%.
Microtubules
wall
al.
texture was
were
studied
examined
1985). Preparations
by
were
by
means
means
of immunofluorescence
of the
examined and
(Traas
dry-cleaving technique
photographed
using
a
et
al.
1985).
for cell walls
Phillips
Cell
(Sassen
EM 300
or
et
201.
RESULTS
Root
hair
growth
control hairs of
after
60-85
during
colchicine
treatment
Equisetum hyemale, growth
min
treatment
with
10
mM
was
was
followed
17-26 pm h
colchicine
1.
by
light microscopy.
Hair
growth
(three experiments,
was
In
arrested
five hairs
each).
COLCHICINE
IN ROOT
Fig.
In
sativus,
Raphanus
an
of active
application
up
to
each),
example, Fig.
colchicine. Growth
period
curve
at
in
start
stopped
a
control
curve
was
was
104 pm
reduced
growth
some cases
and
treatment
116gmh“‘ (five
with 10 him colchi-
of root hairs of R. sativus grown in 1
experiment
hairs,
in
drug; however,
varied between 48
after 60-115 min
growth
of the
in I mM colchicine.
sativus grown
to
a
-1
The
.
growth
minimum
continued in 1
mM
rate, in the
125 min after
at
colchicine for
mM
3 h.
The
morphology
of the part
grown
of E.
hyemale
during
Microtubules
time depended
Figure
were
on
was
studied after
net-axial. After 7 h in
1
original configuration;
mM
7h in
10
at
large
the
tip
to
seen
stopped.
colchicine,
most
mM
in
Their
it remains round.
Figure
depolymerization
an
untreated
root
of the microtubules
that
a
still present in the
microtubules
2c shows the
hairs,
more
net
the
be wider because the hair
2d shows
at
hair. Microtubules lie
were
microtubules deviated
(Fig. 2b). Figure
seems to
hemisphere
hairs.
and is shown for R. sativus in Table 1.
hairs of R. sativus. In these
vacuole is situated
root
colchicine many
remaining
The diameter
treatment.
twice the original and the
in R. sativus
had
hyemale
direction than that in untreated hairs
in untreated root
tips changed during
up
growth
colchicine,
after
merized and the orientation of the
where the
not
the concentration of
2a shows microtubules of E.
organization
hair
root
treatment
flattened. An increase in diameterwas
while
Raphanus
control hairs
and
1 shows
the
growth of
of the
21
of root hairs of
growth
five hairs
experiments,
cine. As
I. Growth
HAIRS
were
depoly-
from the axial
axial microtubule
area
of the hair tube
collapses
on
the slide,
hair of R. sativus treated with 10
mM
A.
22
T able 1
.
Effectof colchicine on microtubules in root
hairs of Raphanus
sativus.
tubules
was
scored
cence.
Quantitative
by
The
means
of micro-
presence
of immunofluores-
measurements
not
were
possible
Duration of treatment (h)
Concentrationof
1
colchicine (mM)
5
10
1
5
+
10
( —)
+/-
Most microtubules
microtubules
tubules
present,
no
present, partial effect, (
present, strong
effect.
Fig.
2.
+/-
/+
+
effect, ( + /—)
+
)
no
or
some
few micro-
M. C.
EMONS ET AL
COLCHICINE
Fig.
2.
IN ROOT
Microtubules
in
(b) Equisetum hyemale
sativus treated with
HAIRS
root
treated
10 mM
hairs
with
colchicine
23
visualized
10 mM
immunofluorescence,
by
colchicine
during
7 h.
(a)
(c) Raphanus
Equisetum hyemale,
sativas control,
control,
(d) Raphanus
during l-5h.(x 1250.)
colchicine for 1-5 h: only short microtubules in the original longitudinal orientation
observed.
ground
Immunofluorescence
staining
microtubule
(Fig.
2d),
probably
depolymerization.
microtubules after colchicine
shown).
longer
After 5 h in
10
mM
pictures
The
of colchicine-treated hairs
derived
from
occurrence
treatment was
tubulin
of
confirmed
colchicine, approximately
contained microtubules
(Table 1).
the
by
show much
monomers
short
as
a
longitudinally
electron
are
back-
result
of
oriented
microscopy (data
80% of the hairs of R. sativus
not
no
A.
24
Fig.
3. Surface
during
6 h.
according
(a)
views of inner cell wall
Random
to the
long
The cell wall
was
as
at
axis of the hair.
texture
of
of root
the hair
root
(x
transverse
deposited
typical
(Figs
3b
18
hair of
a
Equisetum hyemale
tip. (b-d)
144,
bar:
hairs of E.
in the control hairs with
the hair tube the
and
texture
random
The
500
hyemale,
Arrow:
EMONS ETAL
after treatment with
last-deposited
nm.)
M. C.
long
lamellae
with
the hair
mM
colchicine
axis of the hair.
observed after 6 h in 10
texture at
10
different orientations
tip (Fig. 3a),
him
colchicine,
and further into
lamellae of the helicoidal wall contained longitudinal,
d, respectively)
nascent
lamella showed different orientations
oblique
microfibrils. The microfibrils in the lastat
different locations
along
the
hair,
as
IN ROOT
COLCHICINE
found in the control
25
HAIRS
preparations (Emons
counter-clockwise and the
1989).
The rotation mode of the helicoid
between microfibrils in
angle
was
lamellae (30°) did
adjacent
not
differ significantly from the control.
The microfibril orientation in control
hairs
grown
treatment
with 1
is
as
to
much
the
the
hairs
narrower
grown in
colchicine the orientation of the last
In
4a).
some
of the hairs the
majority
oblique
than in
more
him
from the control (Fig.
the
of R. sativus
preparations
axial, but in
was
long axis of
moist air.
deposited
hairs random nascent wall
main orientation of the
the hair (Fig.
observed,
texture was
differ. Active microfibril
almost reached the
hair
drug
in
treatment
deposition
because the helical
place
characteristic that does
a
fibril orientations
oriented
the last
taken after
micrographs
microfibrils,
deposited
resembles
a
be observed. The
can
for 7 h with 10
treatment
not
in
occur
is
helicoidal
transverse
the
to
subsequent
long
arc
not
texture
growing
Figures
4c
longitudinally
lamellais
axis of the hair. This
and would be halfa helicoidal
texture
were
colchicine. Three micro-
mM
oldest lamella contains
deepest,
the microfibril orientation of the
one
was
again deviating by
control hairs in which the 25 pm from the hair tip exhibits random microfibrils.
are
but in
concentration of 1 him, the results did
have taken
must
tip (Fig. 4b);
a
1-h
a
microfibrils
newly deposited
with individual microfibrils
4b)
After
microfibrils differed
45° from the main orientation. Areas with axial microfibril orientation
as
still observed. After 2 h of
and d
root
the deviation of individualmicro fibrilsfrom the axial orientation was
water
which
45°,
to
up
in
oblique
and
configuration
new
in thin section.
DISCUSSION
Treatment with colchicine
root
hair
Equisetum hyemale,
300 pm from the
which does
tip,
the
not
treatment
treatment
contain
effected
with colchicine the
normal arrangement of
showed
root
entered the cell, and that
This is
certainly
shifts after
with
area
not
drug
the
whether this
area
following
treatment
that
suggested
not
in E.
find any
hyemale
Since
the
(Ledbetter
available
as
Seagull
a
and
two
the
tip
it was
latter,
tip
wall microfibrils up
is
not
so
long
possible
drug
in E.
of E.
hyemale
a
hairs had
root
deposited before
to
the hair
seen
root
that
argued
the
a
hairs
drug
had
deposited.
tip
becomes covered
in the control. Because the
hyemale
and the transition from
determineby
means
of
generation
dry-cleaving
liquid suspension
of
microfibril
bundles
cultures of Vicia faba.
result of characteristics inherent in
triggered by
to
treatment.
the
reported
In
microfibril orientation in the tube
not
to
on
the hairs derived
addition,
microfibrils had been
close
phenomena
colchicine in
the
cytoplasm.
The hair tube of these
no new
texture
result of the
are
In
effect
significant
bulbous appearance.
a
formed hair
treatment
(1988)
mM
bundling
disruption of normal
They
the cellulose
microtubule function. We
of microfibrils in treated cells than in the
controls,
neither
in R. sativus.
hypothesis
& Porter
(for
at
gradual,
these bundles
nor
had
tip.
a
of the
when grown in colchicine. It could be
and the random
microfibrils,
with 3
more
treatment
in R. sativus. In the
case
so
synthesizing machinery
wider hair
helicoidal wall had been
during drug
decreased
&
Weerdenburg
a
newly
texture
a
with random microfibrils
random to helicoidal
did
hairs
treatment
oriented
helically
reinforcing secondary
randomly oriented microfibrils.
helicoidalwall
perfect
a
in the tube of these
and had
microtubules,
disrupting polar organization
from the trichoblasts during colchicine
After
the
depolymerized
growth rate, possibly by
reviews:
that
1963),
microtubules orientate
more
Bershadsky
data
on
the
& Vasiliev
microfibrils
was
first
postulated
function of microtubules have
1988; Derksen
et
al.
1990).
Their
become
possible
A.
26
Fig.
4. Surface views
of inner cell wall
(a) Longitudinal microfibrils
colchicine,
in hairs
in
4c)
after 7 h in
exocytosis
affects the
also
amount
probably
enzymes,
role
of root
the control,
hairs of
Raphanus
the
inserted into the
by maintaining
cell
10 mM
colchicine.
amount
of
before
(x
18
5
from the hair
treatment),oblique
216, bar:
rosettes,
the
membrane (Emons
morphology during growth
EMONS ETAL
deposited during colchicine
pm
500
nm.)
of wall matrix material
particle
plasma
sativus
(b) Oblique microfibrils
(c, d) Subsequent longitudinal(original layer
(long arrows
role in
in
M. C.
after
Short
arrows:
in
long axes
1
mu
of hair.
into the wall and
microfibril
1985). They
treatment,
1 h
and transverse orientations
brought
putative
tip
serve a
synthesizing
morphogenetic
when the wall is still
plastic.
These
COLCHICINE
IN
ROOT HAIRS
functions taken into account,
27
one
would expect that depolymerization of microtubules
affects the direction of microfibrils without
fibril orientation
The present
do
which
polarity, by
orientate the microfibrils
tion of the microtubules
(Table
connected
membrane
the
plasma
and
ordered
is
to exert
colchicine. It
disrupted by
control micro-
also
It is
mean
a
function in cell
possible,
naturally
that
means
the
does
in
not occur
note
tightly
be
to
of cellulose
movement
microfibrils
to
depolymeriza-
that microtubules need
on
but that
important
before the
place
their influence
and that this connection
synthesizing complexes
cells
1). This could
is
tip growth
during deposition.
that the shift in microfibril orientation in R. sativus takes
to
they actually
offer further evidence that microtubules have
and maintenanceof cell
directly
not
that
postulating
direction.
own
experiments
morphogenesis
they
their
by
number of
a
committed
are
to
without the intervention of cortical microtubules.
deposition
ACKNOWLEDGEMENT
We
are
grateful
Professor Dr M. M. A. Sassen for his support and encouragement.
to
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