Plant Cell Physiol. 45(5): 573–582 (2004)
JSPP © 2004
Microtubules of Guard Cells are Light Sensitive
Maoz Lahav 1, Mohamad Abu-Abied 1, Eduard Belausov 1, Amnon Schwartz 2 and Einat Sadot 1, 3
1
The Department of Ornamental Horticulture, The Volcani Center, Bet-Dagan 50250 Israel
The Robert H. Smith Institute of Plant Science and Genetics, The Faculty of Agriculture, Food and Environmental Sciences, The Hebrew University of Jerusalem, Rehovot, Israel
2
;
Guard cells of stomata are characterized by ordered
bundles of microtubules radiating from the ventral side
toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial
arrangement of the cellulose micro-fibrils of guard cells.
However, the role of microtubules in daily cycles of opening
and closing of stomata is not clear. The organization of
microtubules in guard cells of Commelina communis leaves
was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were
organized in parallel, straight and dense bundles, in the
dark they were less straight and oriented randomly near
the stomatal pore. The effect of blue and red light on the
organization of guard cell microtubules resembled the
effects of white light and dark respectively. When stomata
were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore,
when incubated in the light, guard cell microtubules were
more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein–
tubulin α 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The
dynamics of microtubule rearrangement upon transfer of
intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light.
Our data suggest that microtubule organization in guard
cells is responsive to light signals.
stomatal aperture via signal-induced changes in their volume
and shape. Such signal-mediated movement of guard cells regulates the CO2 influx into leaves for photosynthesis and the loss
of water vapor by transpiration.
Guard cell signaling integrates hormonal stimuli, light,
CO2 levels and other environmental stimuli to regulate stomatal aperture. A network of ion channels in the plasma
membrane and the vacuolar membrane of guard cells, which
controls ion fluxes and stomatal movement, has been characterized. Overall, the current understanding of how guard cell
signalling networks operate is well advanced (Assmann and
Shimazaki 1999, Blatt 2000, Fedoroff 2002, Hetherington
2001, Ng et al. 2001, Schroeder et al. 2001a, Schroeder et al.
2001b, Zeiger 2000). However, an intriguing challenge for
future studies is to decipher how the plant cytoskeleton, which
is inherently associated with signalling (Geitmann and Emons
2000, Kost and Chua 2002, Kost et al. 1999, Lloyd and Hussey
2001, Moore et al. 1998, Nick 1998, Volkmann and Baluska
1999, Wasteneys 2002) integrates and responds to these signalling cascades.
Guard cells contain cortical microtubules that are radiating
from the ventral pore side toward the dorsal side (Marc et al.
1989a, Marc et al. 1989b, Marc and Palevitz 1990, McDonald
et al. 1993, Palevitz and Hepler 1976). This specific organization of microtubules has been implicated in guard cell development, where microtubules are thought to guide the deposition
of cellulose microfibrils in distinctive orientation and distribution, which is crucial to the ability of stomata to open and close
upon changes in guard cell volume (Palevitz and Hepler 1976).
Interestingly, unlike in other cells, such as pea epidermal
epicotyl or Arabidopsis root cells where microtubules change
orientation from transverse to longitudinal when elongation
declines (Sugimoto et al. 2000, Granger and Cyr 2001, Yuan et
al. 1994), and in leaf epidermal cells where microtubules possess different orientation at different stages of leaf development (Fu et al. 2002), in guard cells the strict organization of
radially orientated microtubules remains throughout their lifetime. This suggests that microtubules of guard cells are unique
in their function and, most likely, in their associated proteins or
effectors as well. Nevertheless, neither a mechanical role nor a
role in signaling perception is known for microtubules in
mature guard cells and their involvement in stomatal movements is controversial. Assmann and Baskin (1998) reported
that treatment with colchicine (a microtubule-disrupting agent)
Keywords: Arabidopsis thaliana — Commelina communis —
Guard cells — Light — Microtubules.
Abbreviations: ABA, abscisic acid; GFP, green fluorescent protein; TUA6, tubulin α 6; MAP, microtubule-associated protein; MBD,
microtubule-binding domain; EF-1α, elongation factor 1α; DIC, differential interference contrast.
Introduction
Guard cells are highly specialized multisensory cells that
flank stomatal pores in the leaf epidermis. Guard cells control
3
Corresponding author: E-mail, [email protected]; Fax, +972-3-9669583.
573
574
Guard cell microtubules
Fig. 1 Differences in microtubule organization in guard cells incubated in the light or in the dark. Leaves of Commelina communis were incubated in the light (A–C) or in the dark (D–F). Epidermal peels were fixed and microtubules were labeled with anti-tubulin antibody. Images were
acquired by a confocal microscope and are three-dimensional reconstructions of a series of optical sections. (A–C) Rotation around the axis of the
pore of stomata incubated in the light: (A) –35°; (B) 0°; (C) +35°. (D–F) Rotation around the axis of the pore of stomata incubated in the dark:
(D) –35°; (E) 0°; (F) +35°. Scale bar = 10 µm.
failed to interfere with stomatal function, suggesting that microtubules are not required for stomatal movements. In contrast,
Fukuda et al. (1998) reported that propyzamide (a microtubuledisrupting herbicide) and taxol (a microtubule stabilizing
agent) interfered with stomatal function, suggesting that microtubules are required for stomatal movements. Marcus et al.
(2001) used several microtubule-disrupting herbicides as well
as over-expression of microtubule-binding domain of microtubule associated protein 4 – green fluorescent protein (GFP–
MBD) and showed that microtubules are necessary for stomatal
opening in a specific manner prior to the ionic fluxes. Similarly,
over-expression of the microtubule-associated protein elongation-factor-1-α (EF1-α) inhibited stomatal opening upon white
light illumination (Moore and Cyr 2000). In addition, whereas
abscisic-acid (ABA) induction of stomatal closure was accompanied by microtubule fragmentation in guard cells of Vicia faba
(Jiang et al. 1996), guard cell microtubules of Commelina communis (Eun and Lee 1997) and Arabidopsis thaliana (Lemichez
et al. 2001) remained intact after ABA treatment. On the other
hand, guard cells of V. faba showed dynamic changes in microtubule organization during stomatal opening and closing
(Fukuda et al. 1998, Fukuda et al. 2000, Yu et al. 2001).
In the present research we have compared the organization and integrity of microtubules in guard cells when incubated in the light or in the dark. We report here that, in the
light, microtubules are radiating straight from the ventral side
of guard cells and are arranged in parallel arrays. In contrast, in
the dark, microtubules range from less parallel to randomly oriented at the region closer to the ventral side of the cylindrical
guard cell. This “light-condition configuration” is cell volumeindependent and was reproduced by blue light illumination, but
not by red light. In addition, we show that microtubules of
guard cells incubated in the light are more stable. A different
organization of microtubules was also observed in live guard
cells of A. thaliana expressing GFP–tubulin. The kinetics of the
changes of microtubules in stomata transferred from dark to
light is demonstrated.
Results
The organization of guard cell microtubules differs between
leaves that are incubated in the light or in the dark
It has been previously shown that in guard cells of V. faba
microtubules change during cycles of opening and closing of
stomata (Fukuda et al. 1998, Fukuda et al. 2000, Yu et al.
2001).
In the present study we followed the organization of
microtubules in guard cells of C. communis after exposing
detached leaves to different light treatments. Following the
treatment, abaxial epidermes were peeled, fixed, and microtu-
Guard cell microtubules
575
Fig. 2 Blue and red light confers the light and dark
effects on guard cell microtubules respectively. Abaxial
leaf surfaces of C. communis were illuminated either by
red or blue light. Epidermal peels were then fixed and
stained with an anti-tubulin antibody (2a upper panel) or
examined under differential interference contrast microscopy (2a lower panel). Guard cells were scored for the
straight and parallel “light-condition configuration” of
microtubules (light-like), non straight and non parallel
“dark-condition configuration” organization (dark like),
or “intermediate” (2b). Experiments were reproduced several times. Scale bar of fluorescent images = 10 µm, and
of differential interference contrast (DIC) images =
20 µm.
bules were immuno-stained with anti-tubulin antibody. Images
were acquired by a confocal microscope as follows; series of
optic sections were acquired, projected and three-dimensional
reconstruction of the whole cell was performed and rotated right
or left. For the whole movie of rotating stomata see supplemental data where movies 1 and 2 correspond to stomata in the light
and in the dark respectively. This approach enabled us to follow
individual microtubules along and surrounding the whole cell.
It is worth noting here that during fixation the turgor pressure
of guard cells is lost but cytoskeletal structures are preserved
(Assmann and Baskin 1998, and our own data). It is shown that
in guard cells incubated in the light most of the microtubules
are straight, and that they radiate from the ventral side of the
cylindrical guard cell toward the dorsal side, encircle the dorsal
side and return back toward the ventral side. Most of the microtubules are arranged in parallel arrays. This three-dimensional
view conforms to previous observations and models describing
in detail microtubule arrangement in guard cells (Marc et al.
1989a, Marc et al. 1989b, Marc and Palevitz 1990). In contrast, in guard cells incubated in the dark there are microtubules with random orientation that form crossovers mainly near
the ventral pole. Interestingly, the differences between the light
and dark configurations of microtubules were less pronounced
when experiments were carried out with epidermal peels.
Blue and red light effects on microtubules of guard cells resemble the white light and dark effects respectively
Blue light is one of the environmental stimulators affecting opening of stomata (Assmann and Shimazaki 1999,
Assmann et al. 1985, Gorton et al. 1993, Schroeder et al.
2001a, Shimazaki et al. 1986, Zeiger 2000). Blue light perception by guard cells is mediated by the receptors phototropins 1
and 2 (Kinoshita et al. 2001), and leads to phosphorylation of
the C-terminus of a plasma membrane ATPase and its activation (Kinoshita and Shimazaki 1999). Since changes in microtubule organization were observed upon illumination by white
light (Fig. 1), we analyzed the relative efficiency of the blue
and the red spectral regimes in affecting microtubule organization. The abaxial side of C. communis leaves was illuminated
by blue or red light for 2.5–3 h, removed, fixed and immunostained for microtubules. Fig. 2aA and 2b show that guard cells
of leaves illuminated by blue light possessed microtubules
arranged in the white-light-condition configuration, that is,
in straight and parallel bundles. Nevertheless, the blue-lightcondition configuration of microtubules differs from that of the
white light configuration with more crossing over of microtubules observed near the ventral zone (Fig. 2aA). Guard cells
illuminated with red light possessed microtubules that resembled the dark-condition configuration (Fig. 2aB, 2b). This suggests that blue light can initiate a signaling pathway affecting
microtubule organization in guard cells.
The microtubule organization is dependent on light but not on
stomatal opening or on the activation of plasma membrane
ATPase
Next we addressed the question of whether microtubule
organization was dependent on stomatal aperture. Leaves were
treated in the dark with fusicoccin, a drug that activates plasma
576
Guard cell microtubules
Fig. 3 Microtubule organization is dependent on light but
not on stomatal opening. Leaves of C. communis were either
incubated in the light (3aA), or dark (3aB), or in the dark in
the presence of fusicoccin (Fc) (3aC). Following these treatments epidermal peels were fixed, stained for microtubules
with anti tubulin antibody (3a upper panel), or examined
under differential interference contrast microscopy (3a lower
panel). Guard cells were scored for the straight and parallel
“light-like” microtubule organization, non-straight and
non-parallel “dark like” organization, or “intermediate”.
Experiments were reproduced several times. Scale bar of
fluorescent images = 10 µm, and of DIC images = 20 µm.
membrane ATPases, and, eventually, leads to stomatal opening
(Assmann and Schwartz 1992, Johansson et al. 1993). It is
demonstrated in Fig. 3aC that fusicoccin, which was applied to
the intact leaf via the incubation medium, promoted partial
opening of the stomata in the dark. Nevertheless the microtubule configuration in these guard cells resembled the darkcondition configuration (Fig. 3aB, C, 3b). Taken together, the
data suggest that blue light affected microtubules independent
of stomatal aperture and upstream or irrespective to the activation of plasma membrane ATPase.
Microtubules of guard cells in the light are more resistant to
oryzalin treatment
We next asked whether the different configuration of
microtubules in guard cells incubated in the light vs. dark is
accompanied by a difference in their stability. Leaves were pre-
incubated in white light or in the dark for 2.5 h and then transferred to buffer containing 5 µM oryzalin, a microtubule disrupting herbicide, for different periods of time, from 5 min to
60 min. Oryzalin binds dimers of α and β plant tubulin with
high affinity to form an oryzalin/tubulin complex that eventually leads to microtubule depolymerization. It was found that in
the light, mild disruption of microtubules was observed in
~10% of guard cells after 5, 15 or 30 min of oryzalin treatment
(Fig. 4aG, H, I, 4b). After 60 min of treatment with oryzalin in
the light, 70% of guard cells contained severely disrupted
microtubules (Fig. 4aJ, 4b). In contrast, when leaves were incubated in the dark prior to and during the oryzalin treatment,
severe disruption of microtubules was observed already 5 min
following the application of the drug in 40% of guard cells
(Fig. 4aB). More than 50% of guard cells possessed severely
disrupted microtubules after 15 and 30 min of oryzalin treat-
Guard cell microtubules
577
Fig. 4 Microtubules of guard cells incubated in the light are more
resistant to oryzalin treatment than microtubules of guard cells in the
dark. Leaves of C. communis were incubated either in the light or in
the dark and then transferred to the same buffer containing oryzalin in
the light or in the dark for additional time periods as indicated. (a) Epidermal peels were fixed and stained with anti-tubulin antibody. (A, F)
Control without oryzalin; (B, G) 5 min; (C, H) 15 min; (D, I) 30 min;
(E, J) 60 min. (A–E) white light; (F–J) dark. (b) Guard cells were
scored for disrupted or intact microtubules; the chart represents the
average of three experiments. Scale bar = 10 µm.
ment (Fig. 4a C, D, 4b) while after 60 min, 80% of guard cells
in the dark contained no microtubules (Fig. 4aE, 4b). DMSO
alone at 0.05% did not affect microtubule organization (not
shown). Interestingly, similar results were obtained when
leaves were incubated with 1 mM colchicine (another microtubule disrupting agent). Microtubules of guard cells that were
incubated in the light were hardly affected by colchicine treatment of 1 h while after this time, microtubules of guard cells
that were incubated in the dark were severely disrupted (not
shown). The mild treatment of intact leaves with microtubule
disrupting agents revealed the difference in microtubule stability.
Light sensitivity of microtubules in live GFP–tubulin expressing
cells
In order to further confirm the differences in the configuration of guard cell microtubules observed in the light or dark,
we followed GFP-labeled microtubules in A. thaliana expressing GFP–tubulin α 6 (TUA6) (Ueda et al. 1999). Leaves were
incubated in the dark or in the light and guard cells of the abaxial side of the leaf were inspected by the confocal microscope.
It is demonstrated in Fig. 5 that when leaves were incubated in
the light, guard cell microtubules were straight and parallel
while in dark treated leaves more than 50% of guard cells pos-
sess a diffuse pattern of GFP, suggestive of unpolymerized
tubulin. In order to follow the dynamic changes of microtubules
in guard cells, leaves were incubated in the dark, transferred to
light while on the microscope stage, and the organization of
microtubules followed in single stomata. The results of two different experiments are demonstrated in Fig. 6. It is shown that
after 2 h in the dark, the GFP signal was diffuse, suggesting
that the microtubules were mostly depolymerized. However,
following 0.5 h of white light illumination, organized microtubules appeared, although some microtubules with different orientations remained (Fig. 6B, E, arrows). Following 1 h of light
illumination, microtubules became organized in parallel arrays
(Fig. 6C, F, arrows). Interestingly, during this time in the light,
microtubules of the subsidiary cells changed their highly variable organization to become perpendicular to the guard cell
microtubules (Fig. 6C, F, arrows). Similarly, microtubules of
guard cells and of subsidiary cells were perpendicular in stomata complexes of C. communis (Fig. 1B, 2aA). Moreover,
Fig. 6 demonstrates that the changes in microtubule organization in guard cells preceded the increase in stomatal aperture.
These results confirm and reinforce our data obtained from
fixed cells.
578
Guard cell microtubules
Fig. 5 Differences in Guard cell microtubules of GFP–TUA6 Arabidopsis plants incubated in the dark or light. Leaves of
Arabidopsis plants expressing GFP-TUA6
were incubated in the dark or in the light and
then inspected by the confocal microscope.
(A) Open stomata; (B) closed stomata. Each
image is a projection of six optical sections,
1 µm apart, covering the whole stomata top
to bottom. Scale bar 10 µm.
The dynamics of microtubules of guard cells transferred from
dark to light
In order to study the dynamics of microtubule organization in guard cells that were transferred from dark to light, we
followed the changes in microtubule arrangement in single stomata every 5 min, upon transferring the leaves from dark to
light. As shown in Fig. 7a, microtubular organization changed
from a diffuse fluorescent signal to clear filaments within the
first 5 min of white light illumination. Furthermore, while
transverse microtubules retain their orientation through out the
experiment, “disoriented” oblique microtubules appeared and
disappeared from image to image (Fig. 7a arrows). This is further demonstrated in Fig. 7b, in which the images after 20, 25
and 30 min (from Fig. 7a) were colored red, blue and green
respectively and projected together. It is shown that while several transverse microtubules contain white pixels, indicating
co-localization of the three colors throughout the 10-min
period, and therefore delineating static microtubules (Fig. 7b
arrow), dis-oriented microtubules are indicated in blue (Fig. 7b
arrow heads). A movie of this experiment (supplementary
movie 3) shows clearly that oblique microtubules are transient.
In addition, reorientation of microtubules at the subsidiary cells
can be observed (supplementary movie 3). At this stage it is not
known whether the oblique microtubules in guard cells, shrink
as a result of catastrophe, or reorient and co-align with transverse microtubules during this time. Nevertheless, at the end of
the process, when stomata are wide open, oblique microtubules were no longer observed Fig. 5A.
Discussion
A number of reports have described changes in microtubule organization in mature guard cells during cycles of openFig. 6 Changes in microtubule organization of guard cells transferred from dark to light. Leaves of Arabidopsis plants expressing
GFP–TUA6 were incubated in the dark for 2 h prior to the experiments. Shown here are two different experiments (A–C and D–F) of
single stomata inspected by the confocal microscope. The first image
acquisition (A, D) showing microtubule organization in the dark. The
leaf was then illuminated by white light while positioned on the microscope stage. Two more image acquisitions were performed after 0.5 h
(B, E) and 1 h (C, F) of white light illumination. It is shown that after
dark incubation the GFP signal in guard cells is diffuse (AD). After
0.5 h of light illumination, organized microtubules appear but they are
not uniformly oriented (arrows in B and E). Microtubules of guard
cells are parallel to each other and perpendicular to those of subsidiary
cells after 1 h of light (C and F arrows). Scale bar = 10 µm.
Guard cell microtubules
579
Fig. 7 The dynamics of guard cell microtubules during the transition of leaves from
dark to light. Leaves of Arabidopsis plants
expressing GFP–TUA6 were incubated in
the dark for 2 h. (a) Time-lapse image
acquisition of single stomata was performed every 5 min for 35 min as indicated. During this time the microscope
stage was illuminated by white light in
between image acquisitions. Arrows mark
temporary oblique microtubules. (b) The
images of 20, 25 and 30 min were colored
red, blue, and green respectively, and projected together. White pixels show static
microtubules (arrows), blue/red/green pixels show dynamic microtubules (arrow
heads). Scale bar = 5 µm.
ing and closing of stomata. Fukuda and colleagues followed the
organization of microtubules in guard cells of V. faba every 2 h
for 24 h (Fukuda et al. 1998, Fukuda et al. 2000). They found
that in the morning hours, microtubules were organized in par-
allel arrays perpendicular to the axis of the pore, the same as
was previously described (Marc et al. 1989a, Marc et al. 1989b,
Marc and Palevitz 1990, McDonald et al. 1993, Palevitz and
Hepler 1976). However, in the night microtubules were dis-
580
Guard cell microtubules
rupted, shorter and more randomly oriented (Fukuda et al.
1998, Fukuda et al. 2000). While Fukuda and colleagues fixed
the cells and immunostained them, Yu and colleagues followed
microtubules in live guard cells of V. faba after microinjection
of fluorescent tubulin. Similarly, they observed fine parallel
arrays of microtubules when stomata were open in the light but
in the dark, microtubules were crooked in appearance (Yu et al.
2001). In the present study, we have followed the microtubule
configuration in guard cells of C. communis by immunostaining fixed tissues and in A. thaliana live cells expressing GFP–
tubulin. We characterized the changes of microtubule organization in the light and dark and separated the light effect from the
putative stomatal aperture effect. Similar to Fukuda and Yu we
show that microtubules in guard cells of C. communis, although
not crooked in appearance in the dark, are organized differently from microtubules in the light. Three-dimensional
confocal images presented here revealed that in the dark,
microtubules were disordered and especially near the ventral
zone possess random orientation. In contrast, in the light guard
cells contain straight parallel arrays of microtubules that radiate from the tip of the ventral side to the dorsal side, and surround the cell perpendicular to its long axis. In addition, it is
demonstrated here that these well-organized microtubule arrays
in the light are more resistant to mild treatment with microtubule-disrupting agents such as oryzalin and colchicine. Oryzalin, a dinitroaniline herbicide, binds plant tubulin dimers very
efficiently (Hugdahl and Morejohn 1993), and leads to inhibition of microtubule polymerization (Morejohn et al. 1987a).
Interestingly, the affinity of oryzalin to polymerized microtubules is lower than to unpolymerized tubulin (Hugdahl and
Morejohn 1993). Thus, the higher sensitivity of microtubules of
guard cells in the dark to oryzalin may suggest a shift in the
equilibrium of polymerized/unpolymerized tubulin toward the
unpolymerized state in the dark. Furthermore, oryzalin binds
plant tubulin dimers much more efficiently than colchicine
(Morejohn et al. 1987b, Hugdahl and Morejohn 1993) and the
mechanism of oryzalin binding to plant tubulin differs from
that of colchicine. While the affinity of oryzalin to plant tubulin is high, the oryzalin/tubulin complex can dissociate completely. In contrast, the affinity of colchicine to plant tubulin is
much lower but the complex colchicine/tubulin does not readily dissociate (Hugdahl and Morejohn 1993). Nevertheless, we
obtained similar results with both drugs, showing higher stability of guard cell microtubules in the light. Resistance of microtubules to disrupting agents can reflect stabilization of the
polymer by microtubule-associated proteins (MAPs). These
can affect the dynamic instability of microtubules by reducing
the rate of tubulin dissociation. Therefore, light-induced reorganization and stabilization of transverse microtubules can be
mediated by specific MAPs that might possess a role in stomatal function. Indeed, it was shown that over-expression of
microtubule-associated proteins GFP–MBD (Marcus et al.
2001) or GFP–EF-1α (Moore and Cyr 2000) inhibited lightinduced stomatal opening. This suggests a competition on
microtubule-binding sites between the exogenous GFP chimeras and endogenous microtubule-associated proteins playing a
role in light-induced stomatal opening. In addition, it was
shown that light and fusicoccin effects on stomatal aperture
were synergistic (Assmann and Schwartz 1992), suggesting
that light may initiate both ATPase-dependent and -independent
signaling pathways affecting stomatal opening. Taken together,
these data and our results demonstrating that fusicoccin in the
dark did not affect microtubule organization, suggest that a
fusicoccin-independent pathway induced by light might involve
microtubules. The isolation of guard cell-specific microtubuleassociated proteins is a fascinating challenge still to be
explored.
We show here that blue and white light had a similar effect
on guard cell microtubules. Our observation that blue light can
promote the “light-specific configuration” of microtubules suggests the existence of a blue light signaling pathway targeting a
specific microtubule-associated protein or effector. The blue
light effect on stomatal aperture is mediated by phot1 and
phot2 receptors (Kinoshita et al. 2001). Phot1 is a serine/threonine protein kinase, the substrates of which are still unknown
(reviewed in Lin 2002). It is a fascinating assumption that the
blue light receptors might initiate a signaling pathway activating microtubule-associated proteins that may lead to microtubule reorganization. It is worth noting, that a blue light effect
on microtubule reorganization and reorientation was previously reported in maize coleoptiles, where a shift from transverse to longitudinal orientation of microtubules was observed
on the side facing the blue light irradiation (Nick 1998, Nick et
al. 1990).
Further, we demonstrate dynamic changes in microtubules of live guard cells of Arabidopsis plants expressing GFP–
TUA6. Guard cells of these plants exerted microtubule reorganization shortly after the application of light to a leaf previously
incubated in the dark. Close examination revealed that oblique
microtubules had a short lifetime while transverse microtubules formed and eventually conferred an array of parallel
microtubules surrounding the cell. Dynamic instability of discordant microtubule and/or their co-alignment with neighboring microtubules characterize the transition of microtubule
orientation from transverse to longitudinal, or vice versa, when
elongation declines or in response to various plant hormones
(Himmelspach et al. 1999, Shibaoka 1994, Yuan et al. 1994).
However, the machinery underling microtubule reorganization
in guard cells transferred from dark to light is still to be determined.
We believe that the results presented here support the
assumption that microtubules of guard cells do have a role in
daily cycles of opening and closing, a role that is still to be
determined.
Guard cell microtubules
581
Material and Methods
Acknowledgments
Plants
C. communis plants were grown from seeds in pots containing
peat in a growth chamber at a constant temperature of 25°C and 16 h
light/8 h dark cycles. The youngest fully expanded leaves of 5- to 7week-old plants were used for the experiments.
A. thaliana seeds of plants expressing GFP–TUA6, kindly provided by Dr. Takashi Hashimoto (Nara Institute of Science and Technology, Nara, Japan), were seeded in MS agar plates (Murashige and
Skoog 1962) and after 2.5 weeks transferred to pots with peat. GFP
tubulin was detected in 2- to 3-week-old seedlings.
We thank Prof. Benjamin Geiger from the Department of Molecular Cell Biology, the Weizmann Institute of Science, Rehovot, Israel,
for fruitful discussions and help.
Light and drug treatments
The experiments were performed in a temperature-controlled
room at 25±1°C. Light was provided from above by a bank of fluorescent tubes at photosynthetic photon flux density of 200 µmol m–2 s–1 at
the leaf surface. Blue or red light was obtained by filtering white light
through blue or red Plexiglas (Rohm and Haas, Hayward, CA, U.S.A.).
The photosynthetic photon flux density of red and blue light was
adjusted to 20 µmol m–2 s–1 at the leaf level. All the treatments were
performed using detached leaves, while at the end of the treatments the
abaxial epidermis was removed by forceps and was fixed immediately.
Uniform stomatal opening to a range of 10–14 µm was achieved in C.
communis by incubating the leaves under water surface in Petri dishes
in the light for about 2–3 h. Stomata in leaves that were treated similarly but kept in the dark attained an aperture of no more than 4 µm
within 2–3 h. Stomatal opening in the dark was achieved by holding
the leaves under the surface of incubation medium that contained
10 mM MES buffer adjusted to pH 6.1 with KOH and 2 µM fusicoccin (Sigma F0537). The fusicoccin treatment caused stomatal opening
(mainly near the central vein) in the dark to 9±2 µm within 3 h. Oryzalin (PS-410 Sigma) was prepared as 10 mM stock solution in DMSO
and used at a final concentration of 5 µM and colchicine (Sigma
C3915) was prepared at 100 mM in H2O and used at a final concentration of 1 mM.
Immunofluorescence staining, confocal microscopy and image analysis
The following antibodies were used: a monoclonal anti-tubulin
antibody (Sigma, DM 1A T9026) and goat anti-mouse conjugated to
Cy3 (Jackson 115-165-072).
Epidermal peels were fixed for 1 h in PME buffer (50 mM PIPES
pH 6.9, 5 mM EGTA, 2 mM MgSO4) containing 3% paraformaldehyde. The peels were then washed in PBS and membrane permeability
was obtained by the freeze shattering method (Wasteneys et al. 1997).
Blocking was performed in PBS with 1% BSA and 0.05% Triton X100 at 4°C overnight. Incubations with the antibodies were for 1 h at
room temperature, followed by 3×30 min washing with PBS. The
peels were then mounted in Elvanol under a cover slip. Live cell imaging was carried out by mounting intact Arabidopsis leaves under a
cover glass, and performing time lapse image acquisition of XYZ
scanning. Images were acquired by an Olympus IX81/FV500 confocal
microscope using an argon laser (488 nm), a green helium/neon laser
(543 nm) and the following objectives; PLAPO60Xoil /NA 1.4 WD
0.15 mm, or PLAPO60XW/LSM /NA1.00 WD 0.15 mm. Image analysis was carried out using the MICA software (Cytoview, Israel).
Supplementary material
Supplementary material mentioned in the article is available to
online subscribers at the journal website: www.pcp.oupjournals.org.
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(Received September 5, 2003; Accepted February 23, 2004)