Journal of Experimental Botany, Vol. 49, No. 319, pp. 163–170, February 1998
The function of guard cells does not require an intact
array of cortical microtubules
Sarah M. Assmann1 and Tobias I. Baskin2,3
1 Biology Department, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA
2 Division of Biological Sciences, 109 Tucker Hall, University of Missouri, Columbia, MO 65211–7400, USA
Received 21 July 1997; Accepted 24 September 1997
Abstract
The development of stomatal guard cells is known to
require cortical microtubules; however, it is not known
if microtubules are also required by mature guard cells
for stomatal function. To study the role of microtubules
in guard cell function, epidermal peels of Vicia faba
were subjected to conditions known to open or close
stomata in the presence or absence of microtubule
inhibitors. To verify the action of the inhibitors, microtubules in appropriately treated epidermal peels were
localized by cryofixation followed by freeze substitution and embedding in butyl-methyl methacrylate.
Mature guard cells had a radial array of microtubules,
focused toward the thick cell wall of the pore, and the
appearance of this array was the same for stomata
remaining closed in darkness or induced to open by
light. Treatment of epidermal peels with 1 mM colchicine for 1 h depolymerized nearly all cortical microtubules. Measurements of stomatal aperture showed that
neither 1 mM colchicine nor 20 mM taxol affected any
of the responses tested: remaining closed in the dark,
opening in response to light or fusicoccin, and closing
in response to calcium and darkness. We conclude
that intact microtubule arrays are not invariably
required for guard cell function.
Key words: Colchicine, cortical microtubules, cryofixation,
guard cells, stomata, taxol, Vicia faba.
Introduction
The development of stomatal guard cells is well known
to involve cortical microtubules. In fact, developing guard
cells have been a model system for studying how microtubules determine cell shape and control the deposition of
cellulose microfibrils. These studies have found that cortical microtubules are needed to produce a pair of kidney
shaped cells surrounding an aperture, as well as to direct
the asymmetric deposition of cellulose microfibrils in the
cell wall, which enables the stoma to open and close in
response to changes in osmotic potential (Palevitz, 1981;
Sack, 1987). Once they mature, guard cells change shape
to regulate the stomatal aperture. It is reasonable to ask
whether these changes in shape require, or affect, the
cortical microtubules. But even though the role of microtubules in guard cell development was recognized as
important long ago, there has been little assessment of
the role of cortical microtubules in guard cell function.
The possible reasons why the role of microtubules in
guard cell function has been little studied are both practical and conceptual. Practically, attempts to localize
microtubules in mature stomata may have been frustrated
by the thick cell wall, which hinders chemical fixation.
Conceptually, microtubules are considered to orient microfibrils and specify cell shape; once guard cells reach
maturity, their structure is plausibly sufficient to allow
many rounds of opening and closing. However, microtubules have roles beyond shaping cells: the cytoskeleton,
including microtubules, participates in signal transduction. For example, in animal cells, actin filaments terminate in special regions of the plasma membrane enriched
in tyrosine receptor-kinases (Mochly-Rosen, 1995). For
plant cells, analogous examples probably exist, although
the evidence is fragmentary (Pickard, 1994). Of particular
relevance to stomatal regulation is a recent demonstration
that treating carrot cells with colchicine or oryzalin, which
depolymerize microtubules, significantly increased the
activity and open duration of channels permeable to
calcium ( Thion et al., 1996). Calcium is one of the prime
regulators of stomatal aperture. Treatment of epidermal
peels with calcium inhibits stomatal opening and pro-
3 To whom correspondence should be addressed. Fax: +1 573 882 0123. E-mail: [email protected]
© Oxford University Press 1998
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Assmann and Baskin
motes stomatal closure (Schwartz, 1985; Mansfield et al.,
1990), both of which effects appear to be mediated via
elevation of cytosolic calcium (Gilroy et al., 1991). The
activity of calcium channels in guard cells might, as in
carrot cells, depend on microtubules. Thus, microtubules
may be involved in the signal transduction required for
guard cell function.
A straightforward way to study the role of microtubules
in guard cell function is through the use of inhibitors.
Using such an approach, evidence has recently been
obtained in support of the hypothesis that signal transduction for guard cell responses does involve the actin
cytoskeleton ( Kim et al., 1995). The analogous hypothesis
for the microtubule component of the guard cell cytoskeleton has been tested, to our knowledge, in only two
previous reports, with conflicting results. In one report,
colchicine inhibited light-dependent stomatal opening
(Couot-Gastelier and Louguet, 1992); in the other, neither
propyzamide, which depolymerizes microtubules similarly
to colchicine, nor taxol, which stabilizes microtubules,
affected the ability of stomata to close in response to
abscisic acid (Jiang et al., 1996). As stomatal opening
and stomatal closure are not simply the mechanistic
reverse of one another(Assmann, 1993), it is possible
that the above studies imply microtubules are required
for opening but not for closing. However, these investigations used different species: Tradescantia virginiana for
opening, and Vicia faba for closing, and each group
stimulated opening or closing with only a single kind of
treatment ( light or abscisic acid).
Given the general lack of information concerning
microtubule arrays of functioning guard cells, this study
set out to characterize the microtubule arrays of mature
guard cells of Vicia faba, and to test whether these arrays
are involved in transducing either stomatal opening in
response to light or fusicoccin, or stomatal closure in
response to calcium and darkness.
Materials and methods
Plant material
Plants of Vicia faba L. were grown in growth chambers under
a 10 h light (0.2 mmol m−2 s−1), 14 h dark regime, at 21/18 °C
day/night. Plants were grown in three parts Metro-Mix 360
(Scotts-Sierra Horticultural Products, Marysville, OH, USA),1
part perlite (Schundler Co., Metuchen, NJ, USA), and were
watered four times per week with quarter-strength Hoagland’s
solution. All experiments used epidermal peels from the abaxial
surface of young, fully expanded leaves of 3–4-week-old plants.
Analysis of microtubules
Epidermal peels were obtained and preincubated as described
below to maximize stomatal closure. Peels were then transferred
to incubation solution, consisting of 30 mM KCl, 0.1 mM
CaCl , 10 mM MES, pH 6.1 (note: the pH of all solutions for
2
peels was adjusted with KOH ), and subjected to 1 h of darkness
in the presence or absence of 1 mM colchicine. A third set of
peels was maintained in incubation solution for 2 h under white
light (0.17–0.19 mmol m−2 s−1; GE ‘Brite Stiks’) in order to
promote stomatal opening. Immediately following the termination of incubation, six epidermal peels from each treatment
were cryofixed. At the same time, additional peels were visually
inspected and it was confirmed that light had promoted
stomatal opening.
The protocol for preparing sections from peels for examination with light microscopy was essentially as described in Baskin
et al. (1996). For handling, the peels were supported on
tungsten wire loops coated with 1% Formvar, as described in
Lancelle et al. (1986). For cryofixation, a wire loop was
carefully brought up under a floating peel and the peel adhered
to the Formvar. Then, most of the adhering buffer was wicked
away and the sample immediately cryofixed by plunging into
liquid propane held at −180 °C. Approximately 10–15 s elapsed
between first adhering a peel to the loop and plunging. The
plunging apparatus was constructed to minimize precooling of
the samples prior to entry into the propane, accelerated samples
to c. 5 m s−1 at entry, and propelled them through the cryogen
for at least 4 cm.
Samples were freeze substituted in freshly-opened acetone
containing 1% acidified dimethoxypropane (Aldrich Chemical
Corp. Milwaukee, WI. USA) to remove water ( Kaeser, 1989)
for 48 h at −80 °C in a commercial freezer. Vials were then
removed from the freezer and allowed to gradually warm to
−4 °C over an 18 h period, and then to room temperature over
4–6 h. Temperatures were monitored with a thermocouple
thermometer immersed in acetone. All steps after substitution
were done at room temperature. Tissue, still maintained on the
Formvar loops, was infiltrated with the following solutions
(each with an incubation period of 1 h): 100% acetone; 25%
resin: 75% acetone, 50% resin: 50% acetone; 75% resin: 25%
acetone, 100% resin. A final infiltration with 100% resin was
performed overnight. All infiltrations were performed on a
rotator. Resin comprised 80% butyl-methacrylate, 20% methylmethacrylate (Aldrich), 0.5% benzoinethylether (Aldrich). The
resin mixture was degassed with nitrogen for 20 min and stored
at −20 °C.
For embedding, the stem of the wire loop was severed and
the loop portions were placed individually in capsules manufactured with flat bottoms ( TAAB Laboratories Ltd. Aldermaston,
Berks, UK ). Resin was polymerized under long-wavelength UV
light at 4 °C. Capsules were placed on a glass plate about 4 cm
above an 8 W source for 20 h. Embedded wire was carefully
cut out of the blocks prior to sectioning. Semi-thin sections
(1.75 mm thick) were cut dry on an ultramicrotome, placed in
droplets of water, and affixed to slides coated with
3-aminopropyltriethoxy silane (Angerer and Angerer, 1991) by
heating briefly on a slide warmer for 2–5 min at 60 °C.
Sections were extracted in acetone for 10 min and then
immediately rehydrated in PBS containing 0.05% Tween-20
(PBS-Tween). Sections were rinsed (0.1% Tween-20 in PBS),
incubated in primary antibody (2 h at 37 °C ), rinsed in PBSTween (3×10 min), incubated in secondary antibody (2 h at
37 °C ), rinsed in PBS-Tween (3×10 min), and mounted in
an antifading reagent ( Vectashield; Vector Laboratories,
Burlingame, CA). Antibodies were diluted and applied in 1%
BSA, 0.1% azide and 0.05% Tween-20 in PBS.
Antibodies used were as follows. Monoclonal anti-a-tubulin,
raised against sea urchin axonemes (B-5-1-2, Sigma) was diluted
151000, and the secondary, goat anti-mouse Fab fragments
conjugated with Cy3 (Jackson Immuno-Research Laboratories,
West Grove, PA, USA), was diluted 15200. Fluorescence
microscopy was performed with conventional epifluorescence
Microtubules in stomatal function 165
(Zeiss Axioplan), with fluorescence from Cy-3 observed through
a standard rhodamine filter cube.
8–24 sections per peel, 4–6 peels per treatment were examined;
each section contained numerous stomata. There were no
detectable differences between peels from the same treatment.
Stomatal movement assays
For experiments on stomatal opening, bifoliate leaves were
excised from plants and placed in distilled water under darkness
for approximately 30 min before obtaining epidermal peels.
Peels were floated cuticle side up on pre-incubation solution
(1 mM CaCl , 10 mM MES, pH 6.1) for a minimum of 30 min.
2
This pre-incubation serves to randomize the peels and to
promote stomatal closure (Schwartz et al., 1995). An assessment
of baseline stomatal aperture was taken by measuring with an
optical micrometer 15 stomatal apertures on each of three
epidermal peels. Peels were then transferred to 4.5 cm diameter
plastic Petri dishes containing an incubation solution consisting
of 30 mM KCl, 0.1 mM CaCl , 10 mM MES, pH 6.1, either
2
alone, or with 1 mM colchicine or 20 mM taxol. For experiments
with taxol, controls were incubated in the same concentration
of DMSO (0.2%, v/v) as was added with the taxol. Dishes were
placed in either white light (as described above) or darkness.
Stomatal aperture measurements (15 apertures on each of three
epidermal peels) were made for each treatment after 1, 2 and
3 h of incubation. For experiments testing the effect of 1 mM
colchicine on stomatal opening induced by fusicoccin, fusicoccin
was added to the incubation solution to a final concentration
of 10 mM. All fusicoccin incubations were performed under
darkness for 3.5 h, after which time stomatal apertures were
quantified as described above. All experiments were replicated
at least three times, and values shown represent means±SE.
To induce open stomata whose closing responses could be
assessed, biofoliate leaves were excised from plants and
submerged abaxial side up in distilled water under white light
(as described above) for approximately 2.5 h. Epidermal peels
were then made and, for randomization, were floated cuticle
side up in the incubation solution used for opening experiments.
The entire peeling process required no more than 10 min. Peels
were then transferred to a low-salt, high-calcium incubation
solution (15 mM KCl, 0.5 mM CaCl , 10 mM MES, pH 6.1),
2
either alone, or with 1 mM colchicine, 20 mM taxol, or with the
equivalent amount of DMSO. Stomatal apertures were measured
as described above initially and following a 1 h incubation in
darkness. The experiment was repeated three times.
Chemicals
Fusicoccin (Sigma) was prepared as a 1 mM aqueous stock
solution, colchicine (Sigma) was prepared as a 100 mM aqueous
stock, and taxol (Sigma) was prepared as a 10 mM stock in
DMSO; all stocks were stored at −20 °C in darkness. The
efficacy of the taxol stock was confirmed by its ability to coldstabilize bovine brain microtubules assembled in vitro.
Results
Analysis of cortical microtubules
To localize cortical microtubules in guard cells, epidermal
peels of Vicia faba were cryofixed and, following freeze
substitution in acetone, embedded in butyl-methylmethacrylate and microtubules were examined with
indirect immunofluorescence in semi-thin sections.
Cryofixation was used because the thick-walled guard
cells may be particularly difficult to fix with conventional,
chemical fixatives. Although cryofixation at ambient pressure, as used here, may result in ice crystallizing in the
interior of specimens even as thin as epidermal peels,
which comprise only a single cell layer, it has been shown
that the damage from these crystals is unresolvable
through the light microscope, and preservation in cryofixed cells even with ice crystal damage is superior to
that of aldehyde-fixed cells by several criteria (Baskin
et al., 1996).
The appearance of microtubule arrays was first compared in open and closed guard cells ( Fig. 1A–H ). The
cortical microtubules were abundant and generally well
organized, radiating outwards from the region of the cell
closest to the pore. Despite the clear overall organization
of the microtubules, divergent microtubules sometimes
occurred. Comparing the structure of the cortical array
between guard cells in dark and light treated peels, no
consistent difference was seen (Fig. 1, compare A–D
to E–H ).
For the dark-treated peels, the images in Fig. 1(A–D)
appear to show open stomata, but at the time of cryofixation the relatively closed status of dark-treated stomata
was verified on companion peels to those frozen. The
images in Fig. 1 do not provide a reliable indication of
the size of the stomatal aperture in vivo. For example, the
apertures shown in Fig. 1 are nearly double the maximal
aperture for light treatment measured in peels (see below).
The pore may have been distorted physically because of
the hydrodynamics of freezing; but, on the other hand,
the paradermal sections used are unlikely to have
sampled the pore at its narrowest position. In a species
like V. faba, the pore is narrowest at medial depths from
the surface, but towards the mesophyll and the outside,
the pore widens out (Pallas and Mollenhauer, 1972;
Raschke, 1979). Therefore, at the focal plane used to
measure stomatal apertures in vivo, a paradermal section
will pass through the narrow part of the pore, cutting the
guard cells more-or-less at mid-plane, and thus transecting
cortical microtubules ( Fig. 1F, left-hand guard cell ).
Whereas, to localize a portion of the cortical array in
face view, as seen in Fig. 1, a paradermal section is
required that passes near the top or bottom faces of the
stoma, and which will therefore pass through a wider
region of the pore.
The absence of any apparent effects of colchicine or
taxol on stomatal responses (see below) could indicate
that intact microtubules are not required for stomatal
responses; alternatively, negative results could indicate
that these drugs failed to enter the guard cell cytosol. For
guard cells in epidermal peels of V. faba, as used here,
Jiang et al. (1996) have already obtained evidence that
taxol entered the cells and stabilized microtubules, as
expected. To see if colchicine depolymerized guard cell
166
Assmann and Baskin
Fig. 1. Micrographs of cortical microtubules in guard cells of Vicia faba, localized with an anti-a-tubulin antibody in sections (1.75 mm thick) from
epidermal peels that were cryofixed, freeze-substituted, and embedded in butyl-methyl-methacrylate. (A–D) Dark-treated peels; ( E–H ) light-treated
peels; (I–L) dark-treated peels plus 1 mM colchicine. Note the well organized radial arrays in both dark and light treatments, and the presence of
high background and few microtubules in the colchicine treatment. Magnification is the same for all panels: 1172×, scale bar=15 mm.
microtubules, microtubules were localized in epidermal
peels treated with 1 mM colchicine for 1 h. Guard cells
had brightly staining cytosol ( Fig. 1I–L), and in most
images there were no microtubules detectable ( Fig. 1I,
J ). A few guard cells had some microtubules remaining
( Fig. 1K, L). Bright cytosolic staining as well as remnant
microtubules are typical of plant cells treated with inhibitors that depolymerize microtubules (Cleary and
Microtubules in stomatal function 167
Fig. 2. Effect of colchicine or taxol on stomatal aperture versus time
for epidermal peels of Vicia faba. (A) Peels were incubated ±1 mM
colchicine. (B) Peels were incubated ±20 mM taxol (or 0.2% DMSO
for controls). Data are means of three replicate experiments ±SE.
There was no significant effect of taxol or colchicine at any time.
Fig. 3. Effect of colchicine or taxol on stomatal aperture of epidermal
peels of Vicia faba induced to open with 10 mM fusicoccin (A) or
induced to close to with 0.5 mM CaCl in the dark (B). Baseline
2
represents the apertures measured immediately prior to treatment. The
inductive stimulus was given alone (Control ), or with 1 mM colchicine,
0.2% DMSO, or 20 mM taxol, and apertures were measured after 3.5 h
(A) or 1 h (B). Data are means of three replicate experiments ±SE.
There was no significant effect of taxol or colchicine.
Hardham, 1988; Seagull, 1990; Baskin et al., 1994). This
result shows that 1 h of incubation in colchicine was
sufficient to depolymerize the great majority of cortical
microtubules.
Thus, under all conditions assayed, in the presence of
taxol or colchicine guard cells responded normally.
Epidermal peel experiments
The requirement for microtubules in guard cell function
was analysed by treating peels with 1 mM colchicine or
20 mM taxol and measuring stomatal apertures at various
times thereafter. These doses typically are saturating for
affecting microtubules in plant cells. Neither the rate nor
the extent of stomatal opening in the light was significantly affected by colchicine (Fig. 2A) or by taxol
( Fig. 2B). In addition, neither drug promoted stomatal
opening in the dark. Although there was modest stomatal
opening in darkness, such opening has been reported
previously and is attributed to the release of ‘backpressure’ on the guard cells when epidermal cells lose
turgor as a result of injury from the peeling process
( Edwards et al., 1976; Klein et al., 1996). As an alternative
to light, fusicoccin was used to drive stomatal opening
and it was found that colchicine had no effect on this
response with 5 mM (not shown) or 10 mM fusicoccin
( Fig. 3A). Finally, open stomata were closed by treatment
with 0.5 mM CaCl and darkness, and again, significant
2
effects of colchicine or taxol were not found (Fig. 3B).
Discussion
Microtubule organization in mature stomata
In the mature guard cells of Vicia faba, as shown in the
cryofixed sections, cortical microtubules beneath the paradermal walls are arranged in a radial array ( Fig. 1). The
organization of microtubules in the mature guard cells of
species other than grasses has not been extensively studied,
but the radial arrangement seen here is consistent with
electron micrographs of guard cells at late stages of
development in the monocotyledon, onion (Palevitz and
Hepler, 1976) and at maturity in the dicotyledons, pea
(Singh and Srivastava, 1973) and bean (Galatis and
Mitrakos, 1980). In these studies, guard cell apertures
were not measured, but they were presumably open (or
opening) because the methods describe harvesting greenhouse-grown plants. The radial arrangement of microtubules is also consistent with the radial arrangement of
cellulose microfibrils known to be present in the guard
cell walls (Sack, 1987). Interestingly, in V. faba, a radial
arrangement has also been recently reported for actin
filaments ( Kim et al., 1995), which indicates that guard
168
Assmann and Baskin
cells share with several other plant cell types the
co-alignment of microtubules and actin filaments
( Fukuda and Kobayashi, 1989; Lancelle and Hepler,
1991).
When the plane of the section occasionally transected
the epidermis, microtubule arrays were imaged interior
to the dorsal or ventral guard cell walls. These arrays
contained many more divergent microtubules than the
radial arrays beneath paradermal walls, and sometimes
appeared to be completely random (not shown). In dicotyledonous guard cells, others have noted divergent microtubules in similar planes (Singh and Srivastava, 1973;
Galatis and Mitrakos, 1980). The dorsal and ventral
arrays were not viewed frequently enough to determine
if there was any difference between light and dark
treatments.
Influence of turgor pressure on microtubule organization
It is of interest to compare microtubule organization
between open and closed stomata because these states
differ in turgor pressure. Although the absolute difference
is not known with certainty, a 4 mm difference in aperture,
as occurred here between light and dark treatments, is
likely to reflect a turgor pressure change of around
0.8 MPa (Franks et al., 1995).Turgor pressure is predicted
by a prominent model to affect microtubule organization
significantly ( Williamson, 1990), but despite this there
have been few studies that have examined the effects of
different turgor pressures on the organization of the
cortical array. Although treatment of mesophyll cells with
one of several osmolytes, at greater than plasmolysing
levels, completely depolymerized cortical microtubules
(Bartolo and Carter, 1991), it is not clear what such an
extreme treatment means physiologically. For less extreme
changes, the results are conflicting: the orientation of
microtubules was changed by treatment of pea epicotyls
with levels of sucrose that were not quite plasmolysing
( Roberts et al., 1985), whereas the organization of microtubules was not changed by growing maize roots in a low
water potential treatment that reduces turgor by 0.4 MPa
(Liang et al., 1994). Similarly, for guard cells, we report
here no conspicuous difference in the organization of
microtubules over a physiological range of turgor change,
i.e. between light and dark-treated guard cells (Fig. 1).
Therefore, changes in turgor pressure (and osmolarity)
within the range that prevails in vivo need not interfere
with the organization of cortical microtubules.
Cortical microtubules are known to be dynamic structures (Hush et al., 1994; Wymer and Lloyd, 1996). Even
though microtubule organization was not affected by
changing turgor pressure or osmolarity, either parameter
might have affected microtubule dynamics. In T. virginiana, microtubules were present in opening stomata
but were absent in closed stomata(Couot-Gastelier and
Louguet, 1992), and in stomata closed by exogenous
abscisic acid, microtubule arrays were short and fragmented (Jiang et al., 1996). Although exogenous abscisic
acid may have directly fragmented microtubules, this
seems unlikely given that treatment of pea stems with this
hormone enhances the stability of microtubules
(Sakiyama and Shibaoka, 1990). Alternatively, the absent
or fragmented microtubules reported previously in closed
stomata could be explained if microtubules in closed
stomata were more dynamic and hence more difficult to
fix chemically than those of open stomata. Chemical
fixation, as used by the above two groups, takes several
minutes (see discussion in Baskin et al., 1996), a time
that exceeds the half-life of cortical microtubules, as
measured in several plant cell types (Hush et al., 1994;
Wymer and Lloyd, 1996). Therefore, it is possible that
cortical microtubules in closed stomata are destabilized
compared to those of open stomata, perhaps as a consequence of the lowered turgor pressure. Images of
dynamic cytoskeletal structures localized in chemically
fixed preparations need to be interpreted with caution.
Microtubules and signal transduction in guard cells
Guard cells mediated stomatal opening in response to
light or fusicoccin, and mediated stomatal closure in
response to darkness and calcium, regardless of the presence of 1 mM colchicine. Colchicine at this concentration
depolymerized most microtubules, as expected. It is a
formal possibility that the remnant microtubules in the
colchicine-treated cells were sufficient for the required
signal transduction; however, this would be highly
unusual because levels of depolymerization comparable
to that of Fig. 1 inhibit if not abolish microtubulemediated responses, to our knowledge without exception.
In addition, stomatal responses were not changed by
taxol, which affects microtubules in the opposite manner,
that is, by stabilizing them. That taxol stabilizes microtubules in epidermal peels of V. faba, as used here, has
previously been shown (Jiang et al., 1996). From these
results, we concluded that microtubule arrays are not
involved in transducing these diverse opening and closing signals.
These results are partially consistent with those of
previous researchers. Jiang et al. (1996) stimulated closure
of V. faba stomata in the light by application of 10 mM
abscisic acid and, consistent with the results of the experiments here, the stomata closed regardless of whether
microtubules had been depolymerized by propyzamide or
stabilized by taxol. By contrast, light-dependent stomatal
opening in T. virginiana was inhibited by colchicine
(Couot-Gastelier and Louguet, 1992). However, the
inhibition of opening by 1 mM colchicine required a 4 h
pretreatment to become manifest, and when colchicine
was removed, the guard cells recovered the ability to open
Microtubules in stomatal function 169
despite the fact that microtubules were still not observed.
Therefore, the reported inhibition by colchicine could
have resulted from a non-specific effect of this compound.
Taken together, it appears that microtubules are required
neither for stomatal opening in response to physical
( light) or chemical (fusicoccin) stimuli, nor for stomatal
closure in response to physical (darkness) or chemical
(abscisic acid, calcium) stimuli.
These results are consistent with a recent report in
which colchicine had no short-term effect on the activity
of the potassium channels involved in uptake for stomatal
opening: When colchicine was applied to Xenopus laevis
oocytes expressing the KAT1 gene, which encodes these
channels, no immediate effects on potassium currents
were observed (Marten and Hoshi, 1997). Interestingly,
the absence of a role for microtubule arrays is opposite
to that reached for microfilament arrays by Kim et al.
(1995). They observed that poisoning the microfilament
system by either cytochalasin or phalloidin diminished
stomatal responses. Thus, microfilaments may be the
predominant cytoskeletal element involved in the signal
transduction pathways of guard cells.
Our finding no requirement for microtubules in guard
cell function prompts us to ask: Why do guard cells
maintain these costly structures? Only a subset of
responses were examined and microtubules could be
required for others. Additionally, guard cell function was
only studied over the course of several hours, which is
brief compared with the lifespan of a functioning stoma.
It may be that continued deposition of oriented cellulose
microfibrils, governed by oriented microtubules, is
required to maintain appropriate wall structure in the
face of repeated rounds of opening and closing. Therefore,
a requirement for microtubules would only have been
detected had peel responses been examined over a period
of days, which experimentally is difficult if not impossible.
Assessing any requirement for microtubules in guard cell
physiology over the long term will require alternative
approaches to epidermal peels; perhaps one such would
be the identification and study of mutants with defective
microtubule arrays.
Acknowledgements
We thank Dr Jim Frazier, Department of Entomology, Penn
State University for use of his plunge freeze apparatus
(blueprints for which are available from him upon request), Dr
Richard Cyr and Mr Rich Moore (PSU ) for performing the
taxol experiments on bovine microtubules, and Ms Jan Wilson
(MU ) for superb technical assistance. This research was
supported by a tri-agency (NSF/DOE/USDA) ‘‘Cytonet’’ grant
to SMA and TIB, and by NSF grant MCB-9316319 to SMA,
and by a grant to TIB from the US Department of Energy
(award No. 94ER20146), which does not constitute endorsement
by that Department of views expressed herein.
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