63
Cytoskeletal control of plant cell shape: getting the ®ne points
Laurie G Smith
The shapes of plant cells, which are de®ned by their surrounding
walls, are often important for cell function. The cytoskeleton
plays key roles in determining plant cell shape, mainly by
in¯uencing the patterns in which wall materials are deposited in
expanding cells. Studies employing cytoskeleton-disrupting
drugs, together with studies of mutants with cytoskeletal defects,
have demonstrated that both microtubules and actin ®laments
are critical for all modes of cell expansion, although their precise
roles remain poorly understood. In recent years, however,
signi®cant progress has been made in understanding the
contributions of a variety of proteins that in¯uence cell shape by
regulating the organization and polymerization of cytoskeletal
®laments in expanding cells.
Addresses
Section of Cell and Developmental Biology, Division of Biology,
University of California San Diego, 9500 Gilman Drive, La Jolla, California
92093-0116, USA
e-mail: lsmith@biomail.ucsd.edu
Current Opinion in Plant Biology 2003, 6:63±73
This review comes from a themed issue on
Growth and development
Edited by Thomas Laux and John Bowman
1369-5266/03/$ ± see front matter
ß 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S1369-5266(02)00012-2
Abbreviations
ADF
actin-depolymerizing factor
AN
ANGUSTIFOLIA
Arp2,3
Actin-related protein 2,3
AtCAP1 Arabidopsis thaliana ADENYLYL CYCLASE-ASSOCIATED
PROTEIN1
AtKTN1 Arabidopsis thaliana KATANIN1
BARS
brefeldin A adenosine diphosphate-ribosylated substrate
BOT1
BOTERO1
Brk
Brick
CA
constitutively active
CtBP
carboxy-terminal binding protein
F-actin
®lamentous actin
FRA2
FRAGILE FIBER2
GFP
green ¯uorescent protein
KCBP
KINESIN-LIKE CALMODULIN BINDING PROTEIN
MAPK
mitogen-activated protein kinase
MOR1
MICROTUBULE ORGANIZATION1
ROP
Rho of plants
SIMK
stress-induced MAPK
SPK1
SPIKE1
TON2
TONNEAU2
ZWI
ZWICHEL
Introduction
Plant cells exhibit a wide variety of shapes, which are
often functionally signi®cant. For example, the highly
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elongated shapes of root hairs, pollen tubes, trichomes
and vascular elements are vitally important to their functions. Even subtle features of cell shape can have a
signi®cant impact on function. For example, the lobed
shapes of mesophyll cells are thought to enhance their
capacity for gas exchange during photosynthesis, and the
conical shapes of petal epidermal cells affect their optical
properties so as to enhance coloration. The shapes of
plant cells are de®ned by their walls, and are acquired
during development according to the patterns in which
walls expand during organ and cell growth. Cell expansion depends on the ability of the wall to yield in a
controlled manner under the force of turgor pressure
exerted by the cell within. This is achieved through
constant breakage and re-formation of bonds between
wall components combined with controlled deposition of
new wall materials. Wall composition and structure are
critical for cell shape determination, but the cytoskeleton
also plays key roles in cell shaping, mainly because of its
in¯uence on the pattern in which wall materials are
deposited. This review highlights recent advances in
our progress toward understanding how the cytoskeleton
contributes to plant cell shape determination, and how
the polymerization and organization of cytoskeletal ®laments are regulated in expanding cells.
Diffuse growth
Most cells expand diffusely, meaning that wall extension
and the incorporation of new wall material are distributed
across the cell surface. Diffuse growth is generally anisotropic in that it is oriented preferentially along one axis.
This property is thought to be conferred mainly by the
arrangement of cellulose micro®brils, which serve as the
principle structural component of the walls of expanding
cells. Cross-linked cellulose micro®brils constrain cell
expansion, and their alignment along one axis favors
growth in the perpendicular axis.
Studies spanning four decades have demonstrated that
the orientation of diffuse growth depends critically on
microtubules. Like cellulose micro®brils, microtubules
are generally aligned perpendicular to the major axis of
cell expansion in diffusely growing cells (Figure 1a). The
treatment of diffusely expanding cells with microtubuledisrupting drugs causes them to expand more isotropically (reviewed in [1]). Moreover, recent genetic studies
have con®rmed a critical role for microtubules in orienting
diffuse cell expansion. Constitutive expression of an antisense a-tubulin gene in transgenic Arabidopsis reduced
both the mRNA levels of several a-tubulin isoforms and
the overall level of a-tubulin protein. Shortly after germination, this resulted in root growth effects that closely
resembled those produced by treatment with oryzalin, a
Current Opinion in Plant Biology 2003, 6:63±73
64 Growth and development
Figure 1
(a)
(b)
Cortical microtubule
Cytoplasmic F-actin bundle
Cortical F-actin filament
Current Opinion in Plant Biology
Schematic summary of the organization of microtubules and F-actin in
expanding cells. (a) In a diffusely growing cell, microtubules are
essentially restricted to the cell cortex and are aligned perpendicularly
to the major axis of cell expansion. In this schematic, the microtubules
are aligned transversely and cell expansion is oriented longitudinally.
Cortical F-actin is co-aligned with microtubules, but cytoplasmic
F-actin bundles are oriented longitudinally. This drawing is based on
data for rapidly expanding cells in the elongation zone of maize roots
[23,24]. (b) In a tip-growing cell (in this case, a root tip), both F-actin
bundles and microtubules are longitudinally oriented. A meshwork
of fine actin filaments is found in the sub-apical region. This drawing is
based on data that are summarized in [28,29].
microtubule-disrupting drug [2]. An interesting insight
into the effects of microtubules on the polarity of diffuse
growth has recently come from analysis of two lefty
mutations of Arabidopsis, which cause left-handed helical
twisting of cell growth in roots and other organs [3]. Two
different lefty mutant a-tubulin proteins have identical
missense mutations. These proteins are incorporated into
microtubules but destabilize them, causing the microtubules to adopt a helical arrangement that predicts the
twisted growth pattern.
Recently, genetic studies have led to the identi®cation of
a few of the undoubtedly large number of proteins that
play critical roles in determining the orientation of diffuse
cell expansion by regulating microtubule dynamics or
arrangement in diffusely expanding cells. The Arabidopsis
TONNEAU2 (TON2) gene is required for normal spatial
organization of cortical microtubules in expanding and
dividing cells, and encodes a protein predicted to function
as a regulatory subunit for a type-2A protein phosphatase
[4]. Thus, understanding the function of TON2 in
microtubule organization will probably depend on de®ning the substrates of the associated phosphatase. Arabidopsis thaliana KATANIN1 (AtKTN1) is a katanin-like
protein that is encoded by the mutationally de®ned
Current Opinion in Plant Biology 2003, 6:63±73
FRAGILE FIBER2 (FRA2), BOTERO1 (BOT1) and
ECTOPIC ROOT HAIR3 (ERH3) genes [5±7]. Reduced
growth anisotropy in the cells of fra2 and bot1 mutants is
associated with abnormal microtubule organization [5,6].
As katanins have been shown to function as microtubulesevering proteins [8], work on AtKTN1 implicates microtubule severing as an important mechanism for achieving
the proper organization of microtubules in expanding
cells. The Arabidopsis MICROTUBULE ORGANIZATION1
(MOR1) gene encodes a protein that is homologous to
structural microtubule-associated proteins (MAPs) of the
highly conserved MAP215 class [9]. Shifting temperaturesensitive mor1-1 and mor1-2 mutants to restrictive temperatures causes the rapid fragmentation and disorganization of cortical microtubules in expanding cells of several
tissues, which is associated with loss of growth anisotropy.
Other mutant alleles of MOR1 reveal that this gene also
plays an essential role in cytokinesis [10]. MOR1 protein
binds to microtubules in vitro and co-localizes with microtubules at all stages of the cell cycle in vivo [10], con®rming its role in stabilizing/organizing microtubule
arrays via direct association with microtubules.
A question that remains to be answered fully is that of
how microtubules in¯uence the orientation of diffuse cell
growth. In a wide variety of plant species and cell types,
cellulose micro®brils are deposited into the wall in a
pattern mirroring that of cortical microtubules, and the
pharmacological disruption of microtubules is often associated with changes in the pattern of cellulose deposition.
These observations have led to the hypothesis that cortical microtubules determine the cellulose deposition
pattern by guiding the movement of cellulose synthase
complexes through the plasma membrane (e.g. [11]).
Although often stated as an established fact, proof for
this longstanding hypothesis has been elusive. The literature is sprinkled with observations that are not readily
accommodated by this hypothesis; for example, cellulose
micro®brils can sometimes align locally in the absence of
microtubules (reviewed in [12]). Alternative models have
been proposed to explain the alignment of cellulose
micro®brils, but these models do not account for the
in¯uence that microtubules appear to have on this process
(e.g. [13]). A recent, comprehensive review of this topic
culminated in a model for cellulose micro®bril alignment
through `templated incorporation' that can account for a
wide variety of relevant observations [12]. Illustrated in
Figure 2, this model proposes that nascent cellulose
micro®brils can be aligned by their binding interactions
with existing micro®brils, and can also be oriented through
binding interactions with plasma membrane-associated
proteins that are linked directly or indirectly to microtubules on the opposite site of the membrane. To account
for changes in cellulose deposition pattern that are often
observed to accompany the re-orientation of microtubules,
the model proposes that the microtubule-based system
generally takes precedence when guidance provided by
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Cell shape Smith 65
Figure 2
(a)
Nascent microfibril
PM
(b)
PM
MT
Membrane proteins
Scaffold
Wall matrix
Current Opinion in Plant Biology
Model for the orientation of cellulose microfibrils in the cell wall by `templated incorporation' [12]. (a) The orientation of a nascent cellulose microfibril is
specified by a scaffold that binds to an extant microfibril. (b) The orientation of the nascent cellulose microfibril is specified by a scaffold that
binds to plasma membrane (PM) proteins, which are oriented with respect to a cortical microtubule (MT). For simplicity, plasma membrane proteins
are shown binding the microtubule directly but there may be intermediaries. Although wall-dependent and microtubule-dependent scaffolds are
drawn separately here, the model proposes that both scaffolds are usually present simultaneously and cooperate to orient nascent microfibrils.
Redrawn from [12] with permission.
existing micro®brils con¯icts with that provided by microtubules. This could be explained if the binding interactions between nascent micro®brils and microtubulelinked guidance molecules were tighter than those
between nascent micro®brils and existing micro®brils.
Finally, it must be recognized that microtubules may have
more than one role in diffuse cell expansion. Indeed,
growth alterations have occasionally been associated with
the loss or altered organization of microtubules apparently
without corresponding alterations in the pattern of cellulose deposition (e.g. [14±16]). Given that wall components
besides cellulose and callose are introduced via secretion,
another possible role for cortical microtubules in diffuse
growth is the local guidance of secretory vesicles to appropriate sites in the plasma membrane. Evidence supporting
this notion comes from analysis of Arabidopsis fra2 mutants,
which lack the putative microtubule-severing katanin,
AtKTN1. The walls of these mutants are de®cient in both
cellulose and hemicellulose, a secreted wall component [5].
Although the role of microtubules in regulating diffuse
cell expansion has received more attention than those of
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other cytoskeletal components, it has become increasingly clear that actin ®laments also play an important role.
Unlike microtubules, however, ®lamentous actin (Factin) seems to act mainly by promoting cell expansion
per se rather than by controlling the pattern of expansion.
This conclusion is based in part on the effects of actindisrupting drugs, which slow diffuse cell expansion in a
variety of plant organs without altering the polarity of
growth [17,18]. Moreover, altering the levels of proteins
that regulate actin polymerization also causes changes in
growth rates. Consistent with its known biochemical
function, overexpression of actin-depolymerizing factor
(ADF) in transgenic Arabidopsis caused a partial loss of Factin and reduced growth, whereas reduction of ADF
levels through expression of an antisense version of the
gene promoted the formation of excess F-actin and excess
growth [19]. Overexpression of AtCAP1 (Arabidopsis thaliana ADENYLYL CYCLASE-ASSOCIATED PROTEIN1), which is related to animal and yeast CAPs
that inhibit actin polymerization in vitro and in vivo,
reduced cell expansion in transgenic tobacco plants
[20]. Furthermore, overexpression of AtCAP1 essentially
halted the growth of cultured tobacco Bright Yellow-2
Current Opinion in Plant Biology 2003, 6:63±73
66 Growth and development
(BY-2) cells while causing a dramatic loss of F-actin in
these cells [20]. Alterations in levels of pro®lin, an actinmonomer-binding protein that can either promote or
inhibit actin polymerization depending on the experimental conditions, have had variable effects on cell
growth. Nevertheless, these studies also broadly support
the conclusion that F-actin has an impact on the rate of
diffuse cell expansion [21,22].
Little is known about how the actin cytoskeleton promotes diffuse cell expansion. Unlike microtubules, which
are essentially restricted to the cell cortex except during
cell division, F-actin bundles permeate the cytoplasm
(e.g. [23]; see Figure 1a). F-actin is also present in the
cortex of expanding cells, where it tends to be co-aligned
with microtubules (e.g. [24]; see Figure 1a). Actin-disrupting drugs can cause alterations in the arrangement of
cortical microtubules (e.g. [24,25]), suggesting a role for
cortical F-actin in organizing cortical microtubules. Given
the different impacts of disrupting F-actin and microtubules, however, the effects of F-actin on microtubule
organization are unlikely to completely explain the Factin's role in diffuse cell expansion. By analogy to a wellestablished role for F-actin in tip-growing cells (discussed
later), a likely role for F-actin in diffuse growth is to
transport secretory vesicles to the cell surface [19,26].
Tip growth
Tip growth is a mode of polarized cell expansion in which
wall extension and the incorporation of new wall material
are focused at a single site on the cell surface. Thus, a tipgrowing cell elongates unidirectionally. Pollen tubes and
root hairs are the only well-characterized tip-growing cell
types in angiosperms. However, there may be other cell
types that also employ this mode of elongation, at least
some of the time, as suggested by the observation that
cells in xylogenic suspension culture appear to expand via
tip growth [27].
Actin-disrupting drugs either slow or stop tip growth
(reviewed in [28]), not unlike their effects on diffusely
growing cells. Actin ®laments are arranged in longitudinal
bundles that run along the length of pollen tubes and root
hairs (see Figure 1b). The myosin-driven movement of
secretory vesicles along these actin cables transports them
to the vicinity of the growth site, where their fusion adds
new cell wall material and membrane [29]. However, Factin has long been suspected to play one or more additional roles in tip growth. This notion has recently been
strengthened by the observation that tip growth is considerably more sensitive to a variety of actin-disrupting
treatments than is the cytoplasmic streaming that drives
long-range vesicle transport to the tip region [30].
Consideration of other possible roles for F-actin in tip
growth has focused mainly on the population of ®laments
near the growth site, where the majority of recent studies
reveal a sub-apical network of ®ne actin ®laments (see
Current Opinion in Plant Biology 2003, 6:63±73
Figure 1b). This network sometimes appears as a collar
around the base of the growth site [31±33,34]. However,
time-lapse analysis of living tobacco pollen tubes has
demonstrated that this F-actin population is highly
dynamic, and individual ®lament bundles transiently
penetrate into the extreme tip [35]. A wide variety of
models have been proposed to explain what F-actin might
do to promote tip growth besides driving long-range
vesicle transport via cytoplasmic streaming (reviewed
[28,29]). Among these, the most widely favored ideas
seem to be that actin ®laments near the tip are somehow
involved in the local regulation of vesicle docking or
fusion, or in facilitating endocytosis, which is needed
to remove excess membrane. A variety of actin-binding
proteins with de®ned biochemical functions are present
in tip-growing cells and contribute to the regulation of
actin dynamics (pro®lin, ADF) or actin bundling (villin;
reviewed in [29]).
A growing body of work demonstrates that Rac/Rho/
Cdc42-related small GTPases, which form a distinct
family in plants (called ROPs [Rho of plants], with 11
members in Arabidopsis), also play critical roles in tip
growth, including the regulation of actin dynamics. Arabidopsis ROP1 and the closely related ROP5 are enriched
at pollen-tube tips and are required for the properly
polarized growth of pollen tubes [36±38]. Functional
studies of ROP1 have demonstrated that it plays roles
both in the formation of the tip-high Ca2‡ gradient that is
characteristic of tip-growing cells [37] and in the regulation of actin dynamics at the tip [35]. More recently, three
other closely related members of the Arabidopsis ROP
family, ROP2, ROP4 and ROP6, have been implicated in
the polarization of root hair growth. Antibody localization
showed that ROP proteins are enriched at root hair tips
[39]. In particular, ROP2 is expressed in elongating root
hairs and green ¯uorescent protein (GFP)::ROP2 is
enriched in root tips [34]. The expression of constitutively active (CA) forms of ROP2, ROP4 and ROP6
caused the depolarization of root hair growth; in the case
of CA-ROP4 and CA-ROP6, this was associated with delocalization of the calcium gradient [34,39]. The expression of dominant negative ROP2 caused the loss of the
®ne F-actin meshwork from the tip region of root hairs
and greatly reduced root hair elongation [34]. Thus,
ROP2, ROP4, and ROP6 are likely to promote properly
polarized root hair growth by playing roles that are analogous to those of ROP1 and ROP5 in pollen tubes.
Another recently identi®ed regulator of root hair growth
with an actin-related function is the mitogen-activated
protein kinase (MAPK), stress-induced MAPK (SIMK)
[40]. This protein is enriched in root hair tips. The
expression of a hyperactivated form of SIMK either
accelerated or prolonged root hair growth, and treatment
with the MAPK inhibitor UO 126 caused abnormal root
hair growth. Studies using drugs have indicated that
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Cell shape Smith 67
F-actin is necessary both for tip localization and for the
activation of SIMK. Although the substrates of SIMK
have not yet been identi®ed, a possible role for SIMK in
polarized vesicle traf®cking at the tip was suggested by
observations of aberrant vesicle behavior in living UO
126-treated hairs. Thus, SIMK could provide a link
between F-actin and the regulation of exocytosis and/
or endocytosis at the tip.
Roles for microtubules in tip growth have received relatively little attention, mainly because treatment of tipgrowing cells with microtubule-inhibiting drugs often
shows little or no effect (reviewed in [28]). Recent work
has shown, however, that treatment of growing Arabidopsis root hairs with microtubule-interacting drugs causes
them to display a wavy growth pattern and to undergo
branching [41]. Root hair branching was also observed in
transgenic Arabidopsis plants that expressed an antisense
a-tubulin gene [2]. These results suggest that microtubules play a role in stabilizing the growth site in Arabidopsis root hairs, but how they do this remains unclear. In
tip-growing cells of angiosperms, microtubules are generally found in longitudinal, helical, or net-axial arrays.
These arrays extend further into the tip in root hairs than
in pollen tubes (reviewed in [28,29]; see Figure 1b).
Given the proposed role of microtubules in diffusely
growing cells, an interesting possibility is that microtubules may function to direct cellulose deposition at the
growth site of root hairs. In contrast to pollen tubes, in
which there is very little cellulose at the tip [29], a
randomly oriented network of cellulose is found at the
growth site of root hairs. However, Bibikova et al. [41]
propose that the role of microtubules is to somehow
restrict the location of the tip-high Ca2‡ gradient to a
single site.
The initiation of tip growth involves the selection of a
growth site and the initial bulging of the cell wall at that
site. It is distinct from tip growth proper, involving
different mechanisms and different genes (reviewed in
[42]). The initial bulging of root hairs is predicted by
local changes in the pH of the cell wall [43], by the local
accumulation of the cell wall-loosening enzymes expansin [44] and xyloglucan endotransglycosylase [45], and by
the local enrichment of ROPs [34,39]. Surprisingly, the
localization of most of these early indicators of bulge
formation is insensitive to both actin- and microtubuledisrupting drugs, suggesting a cytoskeleton-independent
mechanism for bulge site selection [39,44,45]. Nevertheless, actin ®laments become reoriented with respect to
the bulge site during a very early stage of its formation
[44]. Moreover, recent analysis of the phenotype resulting
from a mutation in the Arabidopsis ACTIN2 gene clearly
indicates a role for actin in selecting and focusing the
bulge at the appropriate site on the surface of root hairforming cells [46]. A possible role for ROP2 in regulating
the proper organization of F-actin at the bulge site is
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indicated by the formation of multiple bulges on individual root hair-forming cells when wildtype ROP2 is
overexpressed [34]. Earlier work had suggested an
analogous role for ROP1 in establishing the site of tip
growth in pollen grains [37].
Generation of complex cell shapes:
trichomes and pavement cells
Many plant cells have relatively simple shapes that are
acquired through either diffuse or tip growth (e.g. rectangular or tubular shapes, respectively), whereas others
have more complex shapes involving growth patterns that
are less well characterized. Among these are epidermal
trichomes that have branched shapes, such as those in
Arabidopsis, and epidermal pavement cells that have
marginal lobes, which are found almost universally among
angiosperms (see Figure 3). The involvement of the
cytoskeleton in the multidirectional cell expansion patterns that are involved in generating these complex cell
shapes has received increasing attention over the past few
years, and some interesting advances have been made
recently.
Super®cially, the outgrowth of unbranched trichomes
resembles that of root hairs. However, studies employing
microtubule-depolymerizing drugs, combined with
examination of the arrangement of microtubules and wall
polymers, have indicated that cotton ®bers (which are
unbranched trichomes that emerge from the seed surface)
grow via a highly polarized form of diffuse growth rather
than by tip growth [47,48]. In line with these early
®ndings, more recent work has shown that the initial,
polarized outgrowth of Arabidopsis trichomes and trichome branches is sensitive to microtubule-interacting
drugs, which cause a more isotropic growth pattern
[49,50]. Moreover, mutations in genes that encode putative microtubule/tubulin-interacting proteins interfere
with the formation of trichome branches. These mutations include those affecting the AtKTN1 gene (which
encodes a putative microtubule-severing katanin) [5], and
weak mutations in the KIESEL and PORCINO genes
(which encode tubulin-folding co-factors) [51,52]. As
expected for a microtubule-dependent and polarized
diffuse growth process, the emergence of trichomes
and trichome branches involves the organization of microtubules into arrays with a net alignment transverse to the
axis of elongation [53]. Although actin-disrupting drugs
do not affect the initial, polarized outgrowth of trichomes
and trichome branches, they severely inhibit the subsequent, rapid elongation of trichome branches [49,50].
During trichome branch elongation, actin ®laments are
arranged in longitudinal bundles similar to those seen in
tip-growing cells [49,50]. However, analysis of the growth
pattern in elongating trichome branches reveals wall
extension along the length of the branch, so branch
elongation is not a tip growth process (M HuÈlskamp,
personal communication). Thus, F-actin probably plays
Current Opinion in Plant Biology 2003, 6:63±73
68 Growth and development
similar roles in elongating trichome branches to those that
it plays in other diffusely growing cell types undergoing
rapid elongation.
Figure 3
(a)
More than a dozen genes are required for proper trichome
morphogenesis in Arabidopsis (reviewed in [54]). Phenotypic analyses of mutants that are affected in some of
these genes, combined with molecular analyses of the
corresponding gene products, are beginning to shed light
on the cytoskeleton-dependent mechanisms that govern
trichome shape. The Arabidopsis ZWICHEL (ZWI) gene,
which is required for the normal elongation of trichome
stalks and the formation of the full complement of trichome branches, encodes a kinesin [55]. This kinesin has
also been named KCBP (KINESIN-LIKE CALMODULIN-BINDING PROTEIN) and shown to have Ca2‡/
calmodulin-regulated, minus end-directed microtubule
motor activity [56]. ZWI/KCBP functions in cell division,
pollen tube growth, and pollen germination, as well as in
trichome morphogenesis [57±59]. Although it remains
possible that ZWI/KCBP functions in trichomes to transport an as yet unidenti®ed cargo to speci®c cellular sites,
current information suggests that its function is probably
to promote the formation of microtubule arrays that are
critical for branch formation and stalk elongation. This
conclusion is supported, albeit indirectly, by data implicating a role for KCBP in the formation or maintenance of
microtubule arrays during cell division [59], and by the
observation that treatment of developing zwi trichomes
with taxol (a microtubule-stabilizing drug) can stimulate
the formation of a normal number of trichome branches
[53]. Localization of ZWI in expanding trichomes, and
detailed comparison of microtubule organization in wildtype and zwi trichomes, might help to further elucidate
the role of ZWI in trichome morphogenesis.
(b)
Recently, an intriguing connection has been made
between ZWI and another gene that promotes trichome
branch formation, ANGUSTIFOLIA (AN ). In addition to
its function in trichomes, AN is also required for properly
oriented growth of other leaf cell types [60]. Subtle
defects in microtubule organization have been observed
in the expanding leaf cells of an mutants, both trichomes
and other cell types [61,62], indicating that AN could
act directly at the level of microtubule organization to
promote normally polarized cell expansion. The cloning
of AN by two research groups showed that it encodes a
protein that is related to CtBP (carboxy-terminal binding
protein)/BARS (brefeldin A adenosine diphosphateribosylated substrate) proteins. These proteins have see-
(c)
Current Opinion in Plant Biology
Scanning electron micrographs of complex cell shapes. (a) A typical,
three-branched Arabidopsis trichome. (b) Arabidopsis epidermal
pavement cells with large, irregular, lobes that interdigitate to form a
jigsaw-puzzle-like arrangement. Stomata are interspersed among the
Current Opinion in Plant Biology 2003, 6:63±73
pavement cells. (c) Maize epidermal pavement cells with small,
regularly arranged, finger-like lobes that interdigitate to form a
zipper-like arrangement. A cell file with three stomata is shown at the
top of the figure.
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Cell shape Smith 69
mingly divergent functions as transcriptional co-repressors and as proteins that are directly involved in the
regulation of Golgi dynamics in animal cells [61,62].
AN::GFP fusion proteins are found in the nucleus and
cytoplasm of onion epidermal cells [61] and predominantly in the nucleus of Arabidopsis cells [62], but the
localization of AN::GFP in expanding trichomes has not
been reported. Thus, the connection between AN and
microtubules is not obvious from the sequence of this
protein or from its localization. Nevertheless, there is
some evidence that AN interacts directly with ZWI,
and thus also with microtubules. Plants that are doubly
heterozygous for certain alleles of an and zwi have abnormal trichome morphogenesis, and ZWI and AN have
been shown to interact in the yeast two-hybrid system
[61]. Further work will be required to determine how
AN and ZWI may work together to organize microtubules
for the proper formation of trichome branches.
The morphogenesis of lobed epidermal pavement cells,
like that of trichomes, involves multi-directional cell
expansion that depends on both microtubules and Factin. Numerous studies have examined the organization
of microtubules in lobe-forming cells in both the epidermis and mesophyll of various species. The emergence of
lobes is consistently preceded by the reorganization of
cortical microtubules into a series of bands that are
associated with the formation of local, cellulosic wall
thickenings (e.g. [63±66]). Lobes subsequently emerge
between these microtubule bands/wall thickenings (see
Figure 4a). These observations suggest that the thinner
regions of the wall are more extensible than the thicker
regions, and therefore that the thinner regions bulge out
to form lobes as the cell expands under the force of turgor
pressure. Further evidence of an essential role for microtubules in the formation of pavement cell lobes is provided by the failure to form lobes of the epidermal cells of
the leaves of Arabidopsis fra2 mutants, which lack the
putative microtubule-severing katanin AtKTN1 [5].
Until recently, the role of F-actin in lobe formation has
received relatively little attention. One study showed that
cortical F-actin was organized into bands that coincided
with microtubule bands in expanding wheat mesophyll
cells; drug treatments suggested a role for F-actin in the
organization of microtubule bands [25]. However, recent
studies have suggested a different role for F-actin in the
formation of lobes in epidermal pavement cells. In both
maize and Arabidopsis, local enrichments of cortical F-actin
are found at the sites of lobe emergence, and these enrichments persist at lobe tips as they elongate ([67,68,69];
see Figure 4a). Thus, the organization of F-actin at lobe
tips is reminiscent of that seen in tip-growing cells, except
that the F-actin enrichment appears to extend further into
Figure 4
(a)
Wild type
Microtubule
F-actin
(b)
Brick mutant
Current Opinion in Plant Biology
A schematic summary of cytoskeletal organization in expanding epidermal pavement cells of a maize leaf. Microtubules are shown in black and actin
in red. (a) In a wildtype cell, lobe formation is associated with the organization of microtubules into bands that are focused at lobe sinuses. In addition,
localized enrichments of cortical F-actin are found at the sites of lobe emergence and at the tips of elongating lobes. (b) Pavement cells of brk1,
brk2 and brk3 mutants expand to the same extent as wildtype pavement cells but without forming lobes. During cell expansion, the cortical
microtubule bands that are associated with lobe formation in the wildtype are present (although somewhat less distinct than those in the wildtype), but
localized enrichments of F-actin in the cell cortex are not observed. The drawing summarizes data from [67,68].
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Current Opinion in Plant Biology 2003, 6:63±73
70 Growth and development
the extreme apex of emerging lobes than of root tips or
pollen tubes. These observations raise the possibility that
lobe formation involves a tip growth-like process that
occurs at multiple sites along the cell margin, in addition
to a microtubule-dependent process that helps to localize
growth to speci®c sites. Whatever the role of actin may be,
a variety of genetic studies have recently converged to
support the conclusion that local F-actin polymerization is
indeed critical for lobe formation and have shed new light
on the molecular mechanisms involved.
Mutations in the Arabidopsis SPIKE1 (SPK1) gene have a
variety of effects on cell and organ growth, including loss
of lobes from epidermal pavement cells and lack of
trichome branching [70]. The spatial segregation of
microtubule- and actin-enriched areas of the cell cortex
found in expanding wildtype pavement cells is not found
in the corresponding spk1 mutant cells, which instead
have a uniform distribution of both classes of ®laments.
SPK1 encodes a protein whose carboxy-terminal domain
is related to the signature domain of CDM-family (CED5, DOCK180, MYOBLAST CITY family) proteins,
which are implicated in reorganization of the cytoskeleton in animal cells in response to diverse extracellular
cues [70]. Thus, the SPK1 protein may mediate the
reorganization of the cytoskeleton in response to extracellular signals that govern the pattern of lobe formation.
The CDM domain itself interacts with Rac GTPase,
suggesting that SPK1 might interact with a ROP GTPase.
Although various aspects of the spk1 mutant phenotype
suggest that SPK1 acts primarily at the level of microtubule organization, Rac and ROP GTPases are better
known for their involvement in regulating the actin
cytoskeleton. An important question for future work is
thus whether SPK1 directly regulates the organization of
microtubules, F-actin, or both. If SPK1 does interact with
a ROP GTPase, then a good candidate is Arabidopsis
ROP2, discussed earlier in connection with the tip growth
of root hairs. Interestingly, ROP2 also plays a crucial role
in epidermal lobe formation [69]. Similar to its distribution in root hairs, GFP::ROP2 is enriched at the tips of
emerging lobes. The expression of constitutively active
ROP2 caused de-localization of cortical F-actin and a
relatively uniform pattern of expansion, resulting in the
formation of larger pavement cells that lacked lobes. The
expression of dominant negative ROP2 inhibited the
formation of localized enrichments of cortical F-actin
and reduced the outgrowth of lobes [69]. Thus, these
results suggest that ROP2 regulates lobe formation, at
least in part, by activating the assembly of cortical F-actin
in discrete regions of the cell cortex, a role similar to that
played by ROP2 in root hairs. The possibility of a direct
interaction between SPK1 and ROP2 will be an interesting one to explore in the future.
In maize, three Brick genes (Brk1, Brk2 and Brk3) are
required for the formation of lobes in epidermal paveCurrent Opinion in Plant Biology 2003, 6:63±73
ment cells [67,68]. The microtubule bands that are
associated with lobe formation in wildtype expanding
pavement cells are also present in the corresponding cells
of all three brk mutants. However, local enrichments of
cortical F-actin that are found in wildtype cells at the sites
of lobe initiation and at the tips of emerging lobes fail to
form in brk cells ([67,68]; see Figure 4b). These results
support the conclusion that local enrichments of cortical
F-actin play a crucial role in lobe formation in epidermal
pavement cells, a role that extends beyond simply promoting the formation of microtubule bands. In addition,
the results point to a role for the Brk gene products in
stimulating the formation of the F-actin enrichments.
Mosaic analysis and double-mutant analysis of brk mutations indicated that all three Brk genes act in a common
pathway in which each gene has a distinct function [68].
The Brk1 gene encodes a novel 8-kDa protein that is
highly conserved in both plants and animals [67].
Recent biochemical studies have implicated the mammalian homolog of BRK1 in the regulation of actin polymerization via its activation of the Arp2,3 (Actin-related
protein 2,3) complex. Putative components of the Arp2,3
complex are encoded by predicted genes in various plant
genomes. When activated by proteins that respond to
localized intracellular or extracellular cues, this complex
nucleates the polymerization of new actin ®laments at
speci®c sites in the cell [71]. The putative mammalian
ortholog of BRK1, HSPC300, is found in a multi-protein
complex with the Arp2,3 activator WAVE, which is regulated by Rac [72]. Although the intact WAVE/
HSPC300-containing complex is inactive, WAVE and
HSPC300 dissociate from the rest of the complex in
the presence of GTP::Rac to form a two-protein subcomplex that activates Arp2,3-dependent actin polymerization. Thus, BRK1 most likely functions by associating
with an activator of the Arp2,3 complex in plant cells.
Interestingly, however, no proteins that are obviously
related to WAVE can be identi®ed in the Arabidopsis
genome. The hypothetical BRK1-associated activator
cannot, therefore, be predicted on the basis of sequence
but may be identi®able as a BRK1-interacting protein or
may correspond to the product of the Brk2 or Brk3 gene.
Another interesting possibility for future investigation is
that the Arabidopsis BRK1 is part of a complex whose
activity in stimulating local actin polymerization is regulated by ROP2 (or perhaps by another ROP).
Conclusions
Studies on the cytoskeletal regulation of plant cell morphogenesis have mainly focused on the role of microtubules in diffuse growth and F-actin in tip growth. It has
become increasingly clear, however, that both diffuse and
tip growth, along with the multidirectional growth patterns that are involved in the morphogenesis of trichomes
and epidermal pavement cells, involve a collaboration
between F-actin and microtubules. In recent years, a wide
www.current-opinion.com
Cell shape Smith 71
variety of proteins have been identi®ed that contribute to
cell shape determination by regulating the organization
and polymerization of cytoskeletal ®laments. Approaches
that use a combination of genetic, cell biological, and
biochemical tools are making signi®cant progress in
understanding the functions of these proteins and their
interactions with the cytoskeleton. However, the precise
roles of cytoskeletal ®laments themselves in promoting
appropriate patterns of cell expansion are still poorly
understood. Genetic approaches have not yet helped
much in this area, but may provide progress if mutants
can be identi®ed that have cell morphogenesis defects
that are not due to abnormal cytoskeletal organization or
to loss of particular wall components. Subsequent analysis of the corresponding gene products might reveal
functions in mediating interactions of the cytoskeleton
with the cellulose-deposition machinery, the secretory
system, Ca2‡ channels, and so on. Likewise, biochemical
approaches that are aimed at identifying cytoskeletoninteracting proteins that are associated with the plasma
membrane or the vesicles of expanding cells might be
productive. In the years ahead, the availability of fully
sequenced genomes for Arabidopsis and rice together with
an ever-growing set of tools for reverse genetics, the
analysis of protein±protein interactions, and advanced
imaging methods will facilitate progress in all of the
approaches used to improve our understanding of the
cytoskeletal regulation of plant cell shape.
Acknowledgements
Thanks to Mary Frank, Heather Cartwright and many other colleagues for
stimulating discussions over the years on the subject material of this review,
to Martin HuÈlskamp for contributing information before publication, and to
Tobias Baskin for Figure 2. Work on plant cell shape in the author's
laboratory is supported by National Science Foundation grants IBN-9817084
and IBN-0212724.
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Current Opinion in Plant Biology 2003, 6:63±73