American Journal of Botany 100(1): 111–125. 2013.

GRAVITROPISM AND MECHANICAL SIGNALING IN PLANTS1

MASATSUGU TOYOTA AND SIMON GILROY2
Department of Botany, University of Wisconsin, Birge Hall, 430 Lincoln Drive, Madison, Wisconsin 53706 USA

Mechanical stress is a critical signal affecting morphogenesis and growth and is caused by a large variety of environmental
stimuli such as touch, wind, and gravity in addition to endogenous forces generated by growth. On the basis of studies dating from
the early 19th century, the plant mechanical sensors and response components related to gravity can be divided into two types in
terms of their temporal character: sensors of the transient stress of reorientation (phasic signaling) and sensors capable of monitor-
ing and responding to the extended, continuous gravitropic signal for the duration of the tropic growth response (tonic signaling).
In the case of transient stress, changes in the concentrations of ions in the cytoplasm play a central role in mechanosensing and are
likely a key component of initial gravisensing. Potential candidates for mechanosensitive channels have been identified in Arabi-
dopsis thaliana and may provide clues to these rapid, ionic gravisensing mechanisms. Continuous mechanical stress, on the other
hand, may be sensed by other mechanisms in addition to the rapidly adapting mechnaosensitive channels of the phasic system.
Sustaining such long-term responses may be through a network of biochemical signaling cascades that would therefore need to be
maintained for the many hours of the growth response once they are triggered. However, classical physiological analyses and re-
cent simulation studies also suggest involvement of the cytoskeleton in sensing/responding to long-term mechanoresponse inde-
pendently of the biochemical signaling cascades triggered by initial graviperception events.

Key words: Arabidopsis thaliana; calcium; columella; endodermis; gravitropism; MCA1; mechanosensitive channels; mi-
crotubule; MscS; signal transduction.

Sessile plants, unlike motile animals, cannot move freely
from their habitat, even if the environment changes drastically.
Therefore, plants must sense stimuli precisely and adapt them-
selves to their surroundings by changing their growth and de-
velopment. Mechanical stress is one of the critical signals for
plant survival and can originate from exogenous environmental
stimuli such as touch, herbivory, wind, and rain or from endog-
enous stimuli such as the stresses and strains of turgor-pressure-
driven growth. The force of gravity also provides a constant
background of mechanical stimulation for the plant, acting as it
does upon elements ranging from organs to organelles. Indeed,
calculations of the signal generated in excess of thermal noise
by the action of gravity on cellular masses suggests that even at
the level of the organelle, gravity can generate signals for the
plant. Thus, providing that the organelle is relatively massive,
such as the nucleus or the dense, starch-filled amyloplasts, the
action of gravity upon it should generate a detectable directional
signal within the cell (Bjorkman, 1992).
The importance of such physical phenomena on plant growth
has been recognized for centuries. For example, comprehensive
descriptions of mechanical and gravity-driven responses were
presented by Knight (1803) and Darwin (1880) in the 1800s.
Yet, despite this long history of characterization and the central
role that gravity/mechanical responses play in shaping plant de-
velopment, the molecular mechanisms behind how plants trans-
late physical force to biochemical signal still remain unclear.
1 Manuscript received 7 August 2012; Manuscript accepted 9 November
2012.
The authors thank Dr. S. Swanson for critical reading of the manuscript.
This work was supported by grants from NSF and NASA to S.G. and from
TOYOBO Biotechnology Foundation, Grant-in-Aid for JSPS Fellows and
Grant-in-Aid for JSPS Fellows for Research Abroad to M.T.
2 Author for correspondence (e-mail: sgilroy@wisc.edu)
doi:10.3732/ajb.1200408
In this review, we will highlight some recent advances in under-
standing how plants perceive the physical force of gravity and
elicit appropriate growth responses. We will also discuss recent
work that has implicated ionic fluxes and the cytoskeleton as
critical players in transduction of the mechanical signal. However,
as we will see, the key, initial step(s) of gravity perception whereby
the action of gravity on a cellular mass triggers a change in a
mechanoresponsive sensor still remains to be defined.
THE THREE TEMPORAL PHASES OF GRAVITROPISM:
SENSING, TRANSMISSION, AND RESPONSE
Gravitropism, the oriented growth response of plants to the
direction of gravity, is traditionally divided into three major
phases: sensing (the conversion of physical force to biochemi-
cal signal), transmission (the export of biochemical signal to
cells that will exhibit gravitropic growth), and response (the
control of differential cell elongation that causes the organ to
reorient). While the molecular elements of the first phase, sens-
ing, remain largely mysterious, there has been remarkable prog-
ress in defining components of the latter two phases.
Thus, at the level of the growth response, there have been re-
cent key steps forward in our understanding of how directional
plant cell expansion can be controlled. For example, cellulose
fiber orientation plays a critical role in directing cell elongation,
and so the regulation of its production represents a critical com-
ponent of the modulation of asymmetric growth during gravitropic
response. The dynamics of the cellulose synthase complexes
(CES) responsible for laying down these fibers have now been
well characterized (Nair and DeBolt, 2011), and regulation of
CES activity by classic signaling mechanisms such as through
phosphorylation (Chen et al., 2010) have begun to be revealed.
Of particular relevance to tropic growth is the interaction of the
synthases with the cortical microtubule cytoskeleton through, for
example, the CSL1 linker protein (Li et al., 2012). The microtubule

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cytoskeleton plays an intimate role in the regulation of cell ex-
pansion through its interplay with cellulose synthesis. Cortical
microtubules beneath the plasma membrane most likely regulate
the orientation of cellulose microfibril deposition by modulating
the insertion of the CES complexes into the plasma membrane
and constraining their subsequent movement (Baskin, 2001;
Paredez et al., 2006; Gutierrez et al., 2009). Therefore, microtu-
bules must play an integral role in the regulation of asymmetri-
cal growth response during gravitropism (Taiz, 1984; Carpita
and Gibeaut, 1993; Baskin et al., 1994; Baskin, 2005). It is im-
portant to note here though, that recent evidence suggests this
regulatory interaction may be more spatially complex than first
thought; for example, it varies across different faces of the same
cell (Chan et al., 2011).
Such detailed analysis of the regulatory dynamics of cellu-
lose synthesis represents just one aspect of the many advances
being made in our understanding of the growth response com-
ponent of tropisms. For example, proteins in the wall, such as
the expansins (Cosgrove, 2005), are also known to act as modu-
lators of growth, and in the case of the expansins, they translate
wall acidification to cell expansion (i.e., they mediate “acid
growth”). As discussed later in this review, there is mounting
evidence for a role of the cytoskeleton and ionic signaling, in-
cluding proton fluxes to the apoplast, in plant mechanoperception,
providing a clear link between mechanical/gravity signaling
events and their ultimate effects on growth.
Similar to our increasing insights into the response phase of
tropic growth, there have been recent significant advances in
our understanding of the molecular interactions underlying the
transmission phase of tropic response. For example, in the
1930s, Cholodny and Went proposed that asymmetric redistri-
bution of the plant hormone auxin modulated the differential
growth that drives tropic response (Went and Thimann, 1937).
This hypothesis is now supported by a wealth of experimental
evidence. Thus, in the root, gravistimulation has been shown to
trigger asymmetrical auxin response across the cap that is
thought to reflect the lateral redistribution of the hormone that
subsequently drives differential growth (Ottenschlager et al.,
2003). In the vertical root, auxin levels appear to increase sig-
nificantly close to the start of the elongation zone (Brunoud
et al., 2012), and data from modeling auxin redistribution infers
a 2-fold increase in auxin levels on the lower side of the root
within 5 min of reorientation (Band et al., 2012). Similarly, the
auxin transporters such as the PIN, ABCB, and AUX families,
responsible for supporting gravitropism-related fluxes of this
hormone, have been identified, and for many of these proteins,
their sites of action have been localized (Petrasek and Friml,
2009). In addition, mechanisms for the regulation of these
transporters during tropic response are now being revealed,
such as the recently defined role of the GLOVEN secretory
peptides in modulating PIN2 turnover (Whitford et al., 2012).
Along with the advances for auxin, there is evidence for the
involvement of a host of other regulators of gravitropic response
including an asymmetrical redistribution of cytokinin (Aloni et al.,
2006), generation of reactive oxygen species (Joo et al., 2001),
and asymmetrical accumulation of inositol-1,4,5-trisphosphate
(Perera et al., 2006). However, although these elements have all
been associated with the modulation of gravitropic growth,
there is remarkably little data linking them to the initial sensory
machinery, which itself remains largely undefined. A discus-
sion of how graviperception could modulate, for example, the
distribution of auxin transporter activity is intimately linked to
the question of how physical force is translated into an initial
[Vol. 100
biochemical signaling cascade. In the following pages, we will
therefore draw together current insights from the world of me-
chanical sensing in plants and see how these might inform our
models of the networks controlling this initial perception phase
of gravitropic response.
THE TIMING OF PERCEPTION EVENTS:
PHASIC VS. TONIC MECHANOPERCEPTION SYSTEMS
To put the potential mechanisms underlying plant mecha-
nosensory transduction in context, we find it useful to divide
mechanical signals and mechanoreceptors into two types, both
of which may play a role in either initial graviperception or in
sustaining the graviresponse. Thus, animal researchers have tra-
ditionally divided mechanical stress in terms of its temporal
character: (1) short, transient, or phasic stress and (2) continu-
ous, or tonic stress (Fig. 1A). Momentary touch or wind might
be considered phasic, whereas a response to gravity or turgor
pressure requires a tonic perception/response system. Mamma-
lian cells have distinct mechanoreceptors for each kind of stress:
rapidly adapting, low threshold phasic receptors and sustained
activity, slowly adapting tonic receptors (Fig. 1B) (Schmidt
et al., 1967; Janig et al., 1968). During complex plant mechano-
stimulation such as during lodging, both receptor types would
likely interact to generate a sustained response (Fig. 2). In plants,
whether there are separate tonic and phasic mechanoreceptors or
there is a sustained downstream biochemical signal transduction
chain triggered by, e.g., an initial, transient opening of a mecha-
nosensitive ion channel, that can take the place of tonic receptors
remains unknown.
GRAVITROPISM:
MECHANORESPONSE TO THE CONTINUOUS
STIMULUS OF GRAVITY
In general, when plants are laid horizontally in darkness,
shoots (e.g., coleoptile, hypocotyl, and inflorescence stem) and
roots orient their growth directions upward and downward, re-
spectively, during a gravitropic response (Blancaflor and Masson,
2003; Morita and Tasaka, 2004; Morita, 2010). After sensing a
change in the gravity vector (gravistimulation), plants restore
their appropriate growth angle through differential growth. Dif-
ferent organs can have different maintained angles relative to
the gravity vector, their so-called gravitational set-point angles
(Digby and Firn, 1995), and these can change through develop-
ment. This observation suggests a sophisticated regulatory sys-
tem that not only triggers differential growth when an organ is
reoriented but that can also continuously monitor its angle rela-
tive to gravity. Thus, gravitropic response exhibits both rapid
phasic and extended tonic components.
The most widely accepted hypothesis for the events trigger-
ing the initial phases of this response is the starch–statolith hy-
pothesis. It is thought that high-density, starch-filled plastids
(amyloplasts) in specialized mechanosensory cells (statocytes)
of the shoot endodermis and root cap columella sediment
through the cytoplasm in response to reorientation of the cells
and initiate tropic signaling (Sack, 1991, 1997; Kiss, 2000), al-
though a second sensor shootward of the cap has been proposed
in maize roots (Wolverton et al., 2002). Signal generation may
be through interaction with cellular components such as the cy-
toskeleton or more specialized structures such as the nodal ER
January 2013] TOYOTA AND GILROY—GRAVITY AND SIGNALING 113

Fig. 1. Two kinds of mechanical stress and mechanoreceptor generating distinct signals. (A) Time course of intensity of short transient (left) and
continuous mechanical stresses (right). (B) Time course of signal amplitude induced by short transient (left) and continuous mechanical stresses (right).
Conceptual phasic and tonic mechanoreceptors generating signal 1 and signal 2, respectively, are drawn.

found in the periphery of the root cap columella (Zheng and
Staehelin, 2001). Indeed, live cell imaging shows the complex
kinetics of amyloplast sedimentation upon gravistimulation in
wild-type endodermal (Saito et al., 2005) and columella cells
(Leitz et al., 2009).
Consistent with such ideas, gravitropic growth responses in
roots and shoots of starch-deficient mutants (e.g., phosphoglu-
comutase [pgm]) were inhibited and their amplitudes were al-
most proportional to the amount of starch content in a genotype
(Kiss et al., 1996, 1997; Weise and Kiss, 1999). In the case of
pgm-1, this lesion in tropic response was also correlated with a
severely disrupted formation of the auxin gradient across the
root tip (Wolverton et al., 2011; Band et al., 2012). The reduced
gravitropic sensitivities in these mutants were restored by hy-
pergravity, which correlated with the resumption of sedimenta-
tion of their plastids (Fitzelle and Kiss, 2001). Similarly, an
amyloplast-less mutant (endodermal-amyloplast less 1 [eal1])
containing no intact amyloplasts in shoot endodermal cells ex-
hibited abnormal gravitropism in inflorescence stems and hypo-
cotyls, whereas the roots of the same plant contained normal
amyloplasts in columella cells and exhibited normal gravitrop-
ism (Fujihira et al., 2000; Morita et al., 2007). Furthermore,
shoot agravitropic mutants such as shoot gravitropism2 (sgr2),
sgr3, zig/sgr4, and sgr9 have nonsedimentable amyloplasts in
their endodermal cells. However, gravitropic response and sed-
imenting amyloplasts were restored by selectively expressing
the wild-type gene in the endodermis of each respective mutant
line (Kato et al., 2002; Morita et al., 2002; Yano et al., 2003;
Nakamura et al., 2011). These results all suggest that sedimen-
tation of amyloplasts plays a critical role in gravity sensing and/
or the following signal transduction. Because gravity is a force
in proportion to mass, the dense starch of the amyloplasts is
thought to increase the sensitivity of the cellular perception ma-
chinery, amplifying the signal resulting from amyloplast inter-
action with the gravi/mechanosensor (Fasano et al., 2002;
Perbal and Driss-Ecole, 2003; Leitz et al., 2009).
arg MUTANT HIGHLIGHTS A ROLE FOR THE
TOC PLASTID PROTEIN IMPORT MACHINERY IN
GRAVISENSING
Screens for Arabidopsis mutants showing altered gravitropic
responses have proven powerful tools to identify components
114 AMERICAN JOURNAL OF BOTANY
Fig. 2. Two kinds of mechanical stresses exerted simultaneously to plants. Time course of intensity of transient touch stimulation (left) and continuous
gravity stimulation (right).
of the graviresponse machinery. For example, mutant agravit-
ropic 1 (agr1), found in a screen for reduced gravitropic growth,
led to the discovery of one of the PIN auxin efflux facilitators
required to generate the hormone gradient that supports gravity-
related asymmetrical growth (Chen et al., 1998; Muller et al.,
1998). Despite providing such critical insights into the molecu-
lar components behind gravitropism, most of these mutant screens
have revealed elements of the transmission and response path-
ways rather than genes contributing to initial signaling events.
However, there are a few notable exceptions (see also the re-
view by Baldwin et al. [2013] in this issue).
Mutants in Altered Response to Gravity1 (ARG1) show le-
sions in tropic response in both roots and shoots (Sedbrook
et al., 1999). ARG1 has two paralogs in Arabidopsis, ARG-like1
and ARG-like2 (ARL1 and ARL2). Mutants in ARL1 show no grav-
itropic phenotype, whereas those in ARL2 show gravitropic de-
fects very similar to arg1, as does the arl2 arg1 double (Harrison
and Masson, 2008b; Luesse et al., 2010), implying ARG1 and
ARL2 operate in the same pathway. ARG1 and ARL2 may well
interact with each other, with membranes and also with the ac-
tin cytoskeleton (Boonsirichai et al., 2003; Harrison and Masson,
2008a). ARL2 is selectively expressed in gravisensing cells, and
both arg1 and arl2 mutants fail to relocalize the PIN3 auxin
efflux facilitator thought to contribute to generating the asym-
metrical auxin fluxes that drive tropic bending (Harrison and
Masson, 2008b). The agravitropic mutant phenotype of arg1
can be rescued by selectively expressing the wild-type gene in
only the statocytes of the plant (i.e., the columella of the root
cap or endodermal cells of the hypocotyl), further implying a
direct role in gravity sensing (Boonsirichai et al., 2003). Indeed,
analysis of amyloplast dynamics in the arg1-2 mutant suggests
it may influence tropic signaling through an effect on plastid
movement/sedimentation (Kumar et al., 2008). The structure of
the protein, however, provides few clues as to molecular func-
tion. ARG1 does contain a J-domain found in many proteins that
interact with heat shock cognate 70 to promote chaperone-like
[Vol. 100
activities (Mayer and Bukau, 2005), plus it contains a putative
protein–protein interaction domain. Together these features im-
ply ARG1 could act as an adapter/chaperone for components of
the gravity sensor complex.
Consistent with this model of plastid function being a critical
component of the initial gravisensing machinery, analysis of the
arg1 mutant has revealed a role for the plastid proteins of the Trans-
locon of the Outer envelope of Chloroplasts (TOC) in gravisensing
(Stanga et al., 2009). Thus, two TOC genes were identified as ge-
netic enhancers of the reduced gravitropism phenotype seen in
arg1. The TOC complex acts to translocate preplastid proteins
across the outer envelope in a GTP-dependent manner and Arabi-
dopsis has three major classes of elements: TOC159, TOC75, and
TOC34 (Soll and Schleiff, 2004). A hypermorphic allele of
TOC75-III, which encodes one of the TOC75 isoforms that
forms the central pore of the translocation complex (Baldwin et al.,
2005), enhances the agravitropic phenotype seen in arg1 (Stanga
et al., 2009), although interpretation of this phenotype is complex
because this TOC75 allele has pleiotropic effects on growth and
development. However, a second modifier of the arg1 phenotype
had a much more limited effect on the plant but also modified the
arg1 gravitropism phenotype. The responsible mutation was found
to be in TOC132, a paralog of TOC159 (Stanga et al., 2009), which
encodes a component thought to play a role in plastid preprotein
recognition during translocation (Perry and Keegstra, 1994), i.e.,
TOC132 may act as a receptor on the plastid surface. This observa-
tion would fit well with the starch–statolith model for gravipercep-
tion where TOC proteins, especially TOC132 would be well
situated to play a role in mediating plastid–gravisensor interac-
tions. However, defining precisely how the TOC complex on the
amyloplast might play a role in gravisensing has proven difficult.
On their own, these mutants in TOC show no gravitropic defects,
i.e., they must be in the arg1 background to impact upon gravitrop-
ism (Stanga et al., 2009). Thus, at present, it seems less likely that
they play a direct role as components of a TOC/ARG1 plastid-
localized gravity/mechanoreceptor.
January 2013] TOYOTA AND GILROY—GRAVITY AND SIGNALING
The potential complexity of an ARG1-related signaling com-
plex is hinted at from analysis of the arg1 pgm-1 and arl2 pgm-1
double mutants. The pgm-1 gene is expected to affect the gravi-
tropic response through its role in plastid function, but the arg1
pgm-1 and arl2 pgm-1 mutants show an enhanced inhibition in
tropic response relative to either arg1 or arl2 alone (Guan et al.,
2003), implying that pgm-1 does not operate in the same genetic
pathway to gravitropic response as these other two genes. Cell
fractionation and localization of ARG1 fused to GFP suggest
that this protein associates with a variety of endomembranes in
puncta and may be related to vesicle trafficking but not with
plastids. ARL2 is associated with the plasma and internal mem-
branes (Harrison and Masson, 2008b). Thus, the genetic inter-
actions between ARG1 and TOC75-III and TOC132 do not
seem to reflect a stable physical interaction at the plastid sur-
face. Considering its protein–protein interaction and J-domains,
ARG1 may be required for the correct targeting/folding of a
component of the gravitropic sensing/transduction pathway.
This function could occur in a compartment distinct from the
sensing element supported by TOC action at the plastid. The
TOC complex might then act as a nonessential enhancer of the
action of the gravisensor complex.
RAPID IONIC CHANGES DURING GRAVIPERCEPTION
If mutant screens have yet to clearly define the perception
machinery, could correlating potential signals to the timing of
gravitropic sensing help to identify candidates? The gravitropic
growth response plays out over many minutes to hours. Thus, it
takes more than 1 h for horizontally laid inflorescence stems of
Arabidopsis to bend perpendicularly (Fukaki et al., 1996), and
primary roots of Arabidopsis roots can take >6 h to reorient to
the vertical from a 90° turn (Blancaflor et al., 1998). However,
the induction of differential growth occurs much more quickly,
e.g., it was evident in a high resolution growth analysis of roots
within 10 min (Selker and Sievers, 1987; Mullen et al., 2000),
setting a timeframe for the transmission and response phases of
gravitropic bending. Presentation time, the minimum time of
gravistimulation to elicit an obvious gravitropic growth response,
is estimated at ~3 min in Arabidopsis inflorescence stems (Fukaki
et al., 1996), less than 10 s in cress roots (Hejnowicz et al., 1998),
or 1 s in a variety of species (Perbal et al., 2002), indicating that
gravity sensing is carried out in this short period.
Behrens et al. (1985) attempted to detect rapid changes in
membrane potential induced by gravistimulation in a columella
cell of cress roots to ask if ion channel activities might be part
of the initial rapid signaling system. Membranes in the lower-
side columella cells start to depolarize at 8 s after 45°-gravis-
timulation and reach a maximum depolarization at 170 s. This
short latent period of 8 s (Behrens et al., 1985) corresponds to
the presentation time in cress roots (Larsen, 1969; Hejnowicz
et al., 1998). On the other hand, membranes in the upper-side
columella cells start to hyperpolarize at 58 s after gravistimula-
tion and continue to hyperpolarize even without gravistimula-
tion. Similar results were obtained from outer cortical cells
within the elongation zone of mung bean roots (Ishikawa and
Evans, 1990). These results suggest that gravistimulation in-
duces a rapid and characteristically asymmetric electrical re-
sponse with respect to the gravity vector. Such electrical
changes likely reflect ionic fluxes across the plasma membrane,
suggesting that gravistimulation leads to the asymmetrical acti-
vation/inhibition of plasma membrane channels and pumps.
115
IONIC SIGNALS IN PLANT GRAVITROPIC RESPONSES:
CA2+ CHANGES IN RESPONSE TO GRAVISTIMULATION
Ca2+ is a ubiquitous cellular regulator in a great variety of
plant responses including growth, development, and morpho-
genesis (Bush, 1995; White and Broadley, 2003; Hepler, 2005;
Dodd et al., 2010), and a possible involvement of changes in
cytoplasmic [Ca2+] in gravitropic responses has been repeatedly
suggested (Trewavas and Knight, 1994; Sinclair and Trewavas,
1997; Fasano et al., 2002; Pickard, 2007). Indeed, recent ad-
vances in Ca2+ imaging technology have enabled the detection
of cytoplasmic [Ca2+] changes in response to gravistimulation
in both roots and shoots. Thus, Gehring et al. (1990b) reported
increases in [Ca2+] induced by gravistimulation in maize co-
leoptiles using a cytoplasmic [Ca2+] fluorescent indicator. [Ca2+]
increased in the lower side of the horizontally oriented coleop-
tiles several minutes after gravistimulation, suggesting that
gravistimulation is transduced into [Ca2+] increase. However,
neither detailed kinetics of the [Ca2+] increase, nor the underly-
ing molecular mechanisms have been examined. The lower side
of the coleoptile is the expected site of growth stimulation trig-
gered by accumulation of auxin, potentially placing the Ca2+
increases in the auxin response pathway.
In contrast, cytoplasmic [Ca2+] changes induced by gravis-
timulation were not initially detected in roots of Arabidopsis
thaliana using a similar [Ca2+] fluorescent indicator (Legue
et al., 1997), although this lack most likely reflects the limited
sensitivity of the imaging system available at this time. More
recent imaging using the genetically encoded Yellow Cameleon
(YC) 3.6 Ca2+ sensor did reveal a gravistimulation-induced
wave of Ca2+ on the lower side of the root that moves from root
tip toward the elongation zone with the same kinetics as pre-
dicted for auxin (Monshausen et al., 2011), again potentially
placing the Ca2+ increases in the auxin response pathway. To-
gether with the finding that auxin itself can induce Ca2+ signals
(Gehring et al., 1990a; Tretyn et al., 1991; Irving et al., 1992;
Ayling et al., 1994; Plieth and Trewavas, 2002; Shishova and
Lindberg, 2010; Monshausen et al., 2011), these observations
suggest that the spreading gravitropic Ca2+ increase is likely
reflecting auxin-triggered signaling during the transmission
phase of the tropic response. The Ca2+ increase could regulate
further auxin transport through Ca2+-dependent regulation of
elements such as the pinoid kinase (Robert and Offringa, 2008).
Ca2+ may also trigger downstream changes, for example, altera-
tions in wall pH related to the modulation of cell elongation via
acid growth (Monshausen et al., 2011).
Gravistimulation-induced cytoplasmic [Ca2+] increases were
also seen in whole Arabidopsis seedlings expressing apoae-
quorin, a Ca2+ sensitive bioluminescent protein (Fig. 3) (Plieth
and Trewavas, 2002; Toyota et al., 2008b). Hundreds of Arabi-
dopsis seedlings were mounted on a rotatable wheel in front of
a photomultiplier tube and subjected to 135°-gravistimulation
by rotation. The seedlings showed a biphasic [Ca2+] increase
consisting of a Ca2+ spike lasting ~20 s and second Ca2+ in-
crease that peaked at around 90 s and declined within approxi-
mately 15 min (Plieth and Trewavas, 2002). Toyota and colleagues
(2008b) were able to extend these results to measurements
on single seedlings where the gravistimulated [Ca2+] increase
was localized to the leaf petiole and hypocotyl. Furthermore,
the second [Ca2+] increase was shown to be dependent on
rotational angle but not angular velocity, whereas the first
[Ca2+] spike showed the opposite properties. These results sug-
gest that the first cytoplasmic [Ca2+] increase is induced by
116 AMERICAN JOURNAL OF BOTANY [Vol. 100

Fig. 3. Ca2+ concentration increases in response to two kinds of mechanical stresses: a short, transient wind and continuous gravity. Arabidopsis seed-
lings expressing apoaequorin were repeatedly stimulated by a wind with different forces (lower arrows), turned 180° (upper black arrow) and turned back
to 0° (upper gray arrow). (M. Toyota, T. Furuichi, H. Tatsumi, and M. Sokabe, unpublished data).

rotation/mechanical stimulation whereas the second is respon-
sive to gravistimulation, a conclusion strongly supported by
experiments under microgravity (M. Toyota et al., unpublished
data) and hypergravity (Toyota et al., 2007).
Pharmacological analyses suggest that the second [Ca2+] in-
crease is induced by influx through mechanosensitive channels
at the plasma membrane and efflux from intracellular Ca2+-
stores. The Ca2+ changes were also partially disrupted by ap-
plication of latrunculin B, suggesting a role for actin filaments.
Intriguingly, actin has been shown elsewhere to play a role in
the activation of mechanosensitive channels by mechanical
stress (Guharay and Sachs, 1984; Zhang et al., 2007; Hayakawa
et al., 2008; Kiyoshima et al., 2011) and to participate as a likely
inhibitor of the graviresponse (Hou et al., 2003, 2004; Palmieri
and Kiss, 2005) (see also the review by Blancaflor [2013] in
this issue). Although the duration of this second [Ca2+] increase
is short when compared to the extended time-course of full
gravitropic response (Fig. 3), Ca2+ is well characterized as trig-
gering a host of Ca2+-dependent proteins such as calmodulin
and the Ca2+-dependent protein kinases (Poovaiah et al., 1987;
Toyota et al., 2008a). These proteins could convert the transient
ionic change to a long-lived biochemical cascade and so con-
tribute to the extended phase of graviresponse.
Although Ca2+ is usually discussed as a cytoplasmic regulator,
apoplastic fluxes of this ion may also play a key role in gravitro-
pism. For example, gravitropic bending was nearly completely
inhibited by extracellularly applied Ca2+ chelators such as EGTA
in maize roots (Lee et al., 1983b; Bjorkman and Cleland, 1991)
and oat coleoptiles (Daye et al., 1984). Gravistimulation also
caused a downward redistribution of apoplastic Ca2+ across
the horizontally oriented root tip of maize within approximately
10 min (Lee et al., 1983a; Bjorkman and Cleland, 1991) that was
dependent on an intact root cap (Björkman and Cleland, 1991).
These observations suggest that the Ca2+ gradient across the
roots is closely related to gravity sensing and/or gravitropic re-
sponse. In addition, when an agar block containing Ca2+ was
asymmetrically applied to one side of the root surface, the roots
bent toward the Ca2+ source (Lee et al., 1983b).
These apoplastic Ca2+ fluxes appear to be intimately involved
with auxin because auxin transport inhibitors such as triiodo-
benzoic acid (TIBA) or naphthylphthalamic acid (NPA) blocked
the gravity-induced Ca2+ redistribution in maize and pea roots
(Lee et al., 1984). Furthermore, polar auxin transport across
gravistimulated maize roots was enhanced by Ca2+ locally ap-
plied to the root tip in an agar block (Lee and Evans, 1985). The
extended timing and interrelationship to auxin suggests such
apoplastic Ca2+ fluxes may well represent part of the transmis-
sion/response systems of gravitropic response. Consistent with
such ideas, the physical properties of the cell wall are intimately
linked to Ca2+ levels through, e.g., Ca2+-dependent cross-link-
ing of the acidic groups in pectin. Many enzymes are also sub-
ject to Ca2+-dependent regulation. Indeed, classic Ca2+-dependent
regulatory proteins such as calmodulin have been reported in
the extracellular matrix (Cui et al., 2005). Thus, alterations in
January 2013] TOYOTA AND GILROY—GRAVITY AND SIGNALING
extracellular Ca2+ could play a very direct role in modulating
wall properties and so help drive gravitropic growth. However,
the molecular elements such as Ca2+ pumps and channels that
drive these apoplastic fluxes and how an amyloplast-linked
perception system regulates these components have yet to be
defined.
In addition to Ca2+, pH is known to play a critical role modu-
lating both cytoplasmic and wall activities. Using fluorescent
pH indicators, several groups have reported gravistimulated
changes in pH in the columella cells of the root cap, coleoptiles,
and maize pulvini (Gehring et al., 1990b; Scott and Allen, 1999;
Fasano et al., 2001; Johannes et al., 2001). However, only in the
maize pulvinus were the pH changes shown to be asymmetric
and so capable of carrying directional information as to the
gravity vector (Johannes et al., 2001). Changes in pH are likely
to affect many cellular activities ranging from enzyme function
to the partitioning of hormones such as auxin. Thus, rather than
directly carrying gravitropic information, changes in pH may
be acting as a more global regulator by altering the efficiency
with which gravitropic signals are exported from the perceiving
cells. Consistent with these ideas, when pH changes in colu-
mella cells were blocked by chemical agents such as bafilomycin
A1 (a vacuolar H+-ATPase inhibitor), benzoic acid, or photo-
release of H+ from chemically caged protons, root gravitropism
was reduced but not abolished (Scott and Allen, 1999; Fasano
et al., 2001).
MECHANORESPONSIVE CHANNELS AND THE
GRAVIRESPONSE
Although gravity signaling and response appear closely re-
lated to ionic fluxes, especially Ca2+, no mechanically respon-
sive channel has been directly linked to the molecular machinery
of gravity perception. Indeed, up until a few years ago, there
were few molecular candidates for such channel activities in
plants. However, an examination of ion fluxes and channels re-
lated to the touch response in plants does provide some useful
insights into how the gravisensor may be operating.
Mechanically induced cytoplasmic [Ca2+] spikes have been
observed in tobacco cotyledons (Knight et al., 1991, 1993),
mesophyll protoplasts, leaf epidermal strips, and guard cells
(Haley et al., 1995; Wood et al., 2000), in the moss Physcomi-
trella patens (Haley et al., 1995; Russell et al., 1996) and in
Arabidopsis roots (Legue et al., 1997; Monshausen et al., 2009;
Richter et al., 2009). The amplitude of the [Ca2+] spike was de-
pendent on stimulus intensity (Haley et al., 1995) and frequency
(Knight et al., 1991; Legue et al., 1997). Similarly, wind also
induces a transient [Ca2+] spike in a stimulus-intensity depen-
dent manner in Nicotiana (Knight et al., 1992; Haley et al.,
1995) and Arabidopsis seedlings (Fig. 3) (Plieth, 2001; Plieth
and Trewavas, 2002). These results suggest that physical force
is transduced into ion fluxes as a likely initial perception event,
but unlike the specialized gravisensing cells of the endodermis
and columella, nonspecialized cells throughout the plant can
respond to such mechanical stress (Fig. 3).
The touch-induced [Ca2+] spike is blocked by ruthenium red,
an endomembrane Ca2+ permeable channel inhibitor that is
known to inhibit the Ca2+ permeable channels in the plant endo-
plasmic reticulum (Bauer et al., 1998) and the SV channel in the
vacuole (Pottosin et al., 1999). Early reports also suggested that
these touch-induced changes were unaffected by the putative
plasma membrane Ca2+ channel inhibitors Gd3+ or La3+ (Knight
117
et al., 1992; Haley et al., 1995; Legue et al., 1997). However,
more recent work shows that high extracellular Ca2+ can sup-
press the action of these inhibitors and that when assayed at
lower Ca2+ levels in the medium, the mechanically induced
Ca2+ increase is blocked by their action, at least in roots
(Monshausen et al., 2009; Richter et al., 2009). These results
suggest the presence of mechanoresponsive channel activity at
both the plasma and internal membranes or that [Ca2+] influx
through channels in the plasma membrane induces Ca2+ release
from intracellular stores in a phenomenon known as Ca2+-
induced Ca2+ release (CICR) (Ward and Schroeder, 1994;
Bewell et al., 1999).
The wind- and touch-induced [Ca2+] spikes resemble the first
[Ca2+] spike induced by gravistimulation in terms of their kinet-
ics (Fig. 3) (Plieth and Trewavas, 2002; Toyota et al., 2008b).
However, the first [Ca2+] spike in gravitropism-related experi-
ments is inhibited by Gd3+ and La3+ but not by ruthenium red
under conditions in which the touch-related changes showed
the opposite pharmacological characteristics (Toyota et al., 2008b).
Based on this result, it seems less likely that the gravisensor simply
reflects a specialized use of a general plant touch-sensing system.
MSL PROTEINS: PLANT MECHANORESPONSIVE
CHANNELS
Mechanosensitive ion channels have been widely postulated
as plant mechanoreceptors, paralleling their known role in
mechanoperception in other kingdoms (Kung, 2005), and much
electrophysiological evidence has accumulated for the exis-
tence of such channels in plants and algae (Table 1). These
channels generally show rapid adaptation to a steady stimulus
(Hamill and Martinac, 2001). As such, they should generate
rapid but short-lived signals in response to mechanical stimula-
tion. A key question arising from these observations is what
is/are the molecular identity of these plant mechanosensory
channels? The molecular identification of at least one class of
these channels came from comparison to perhaps the best char-
acterized osmo/mechanosensitive channels known, the mecha-
nosensitive channel of large/small conductance (MscL/S) from
bacteria.
Bacteria release cytoplasmic low-molecular-weight contents
to the extracellular medium under hypoosmotic stress to resist
uncontrolled increases in turgor (Ruffert et al., 1997). Thus,
MscL/S are activated by hypoosmotic stress (Cui et al., 1995)
and protect the bacteria against cell lysis (Blount et al., 1997;
Levina et al., 1999). The gating mechanism is well defined for
these channels, with tension in the membrane leading to phos-
pholipids pulling on the transmembrane domains of the channel,
causing it to open in an iris-like fashion (Kung et al., 2010).
There are 10 proteins that are homologous to the bacterial
MscS in Arabidopsis thaliana (Pivetti et al., 2003), named the
MscS-like (MSL) proteins (Haswell and Meyerowitz, 2006),
and there are now many identified homologs throughout the
plant kingdom. AtMSL3 rescued the hypoosmotic stress-
induced cell lysis of E. coli lacking its intrinsic mechanosensitive
channels (Haswell and Meyerowitz, 2006), suggesting this gene
can function as a mechanosensitive channel, at least in E. coli
(Levina et al., 1999). Both MSL2 and -3 have an N-terminal
chloroplast transit peptide, and as predicted, the subcellular lo-
calizations of their GFP-fusion proteins were the plastid enve-
lope (Haswell and Meyerowitz, 2006) and overlapped with the
plastid division protein, AtMinE (Maple et al., 2002). Although
118 AMERICAN JOURNAL OF BOTANY [Vol. 100

TABLE 1. Examples of stretch-activated channel activities in plants and algae.

Plant species Permeability Physiological properties References

Allium cepa
Epidermis of leaf sheath and bulb Ca2+, K+ Inhibited by Gd3+ and La3+ a, voltage- and temperature dependent, Ding and Pickard, 1993a, b
scale mediated by cytoskeleton
Fucus spiralis or F. serratus
Rhizoid Ca2+, K+ Inhibited by Gd3+, voltage-gated Taylor et al., 1996
Lilium longiflorum
Pollen grain and tube Ca2+, K+ b Inhibited by Gd3+ and a spider venom Dutta and Robinson, 2004
Vicia faba
Guard cell Ca2+, K+, Cl− c Cosgrove and Hedrich, 1991
Ca2+ Inhibited by Gd3+, voltage-dependent, osmosensitive; mediated by Zhang et al., 2007
actin filament
Ca2+ Activated by amphipath trinitrophenol (TNP) Furuichi et al., 2008
K+ Non-osmosensitive Liu and Luan, 1998
Zostera muelleri
Epidermis K+ Garrill et al., 1994
Arabidopsis thaliana
Hypocotyl Cation Inhibited by La3+ Lewis and Spalding, 1998
Valonia utricularis
Mother cells Cl− Heidecker et al., 1999
Nicotiana tabacum
Suspension-cultured cell Anion Falke et al., 1988
Chlamydomonas reinhardtii
Chloroplast (MSC1) d Anion Hysteretic behavior upon gating Nakagawa et al., 2007
Arabidopsis thaliana
Mesophyll cell Anion Activated by TNP Qi et al., 2004
Root (MSL9 and MSL10) (Ca2+) e, Cl− Cooperative or independent stretch-activated channel activities of Haswell et al., 2008
MSL9 and MSL10
Mesophyll cell K+, Cl− Not affected by ATP, voltage-dependent Spalding and Goldsmith, 1993
Beta vulgaris
Root cell vacuole K+, Cl− Inhibited by Gd3+, slightly voltage-dependent, osmosensitive Alexandre and Lassalles, 1991
a Gd3+ and La3+ initially enhanced the SA channel activity after their application and subsequently inhibited it.
b Two SA channels permeable to Ca2+ and K+, respectively.
c Three SA channels permeable to Ca2+, K+ and Cl−, respectively.
d MSC1 was functionally reconstituted in E. coli and electrophysiologically investigated.
e Permeability for Ca2+ is much lower than that for Cl−.

msl2-1 and msl3-1 single mutants did not have obvious pheno-
types, msl2-1 msl3-1 double mutants have greatly enlarged,
spherical chloroplasts when compared with wild type. These
results support the model that MSL2 and MSL3 regulate the
size and shape of plastids by changing ion flux in response to
membrane stretch, protecting them from hypoosmotic stress
that occurs during normal plant growth (Veley et al., 2012).
Consistent with these ideas, a homolog of MscS, termed MSC1,
was also cloned in Chlamydomonas and functionally reconsti-
tuted in E. coli; results from its RNAi suppression are consis-
tent with a role in plastid function (Nakayama et al., 2007).
MSC1 appears to be an anion-selective, stretch-activated chan-
nel, most probably passing Cl−.
The role of the other MSLs in Arabidopsis remains unclear.
Two of the MSL proteins, MSL9 and MSL10, localize to root
cell plasma membrane and have been demonstrated to function
as stretch-activated channels (Haswell et al., 2008). Patch clamp
analyses indicate that MSL9 and MSL10 form a heteromeric
complex that generates a 10-pA stretch-activated channel, with
the three other root-expressed MSLs (MSL4, -5, and -6) playing
a similar but minor role. However, the quintuple knockout of all
these genes showed no obvious phenotype in a range of abiotic
stress, osmotic challenge, and touch-related screens (Haswell
et al., 2008); G. Monshausen (Penn State University) and S. Gilroy,
unpublished data). Much like MSC1 in Chlamydomonas, MSL9
and MSL10 have higher permeability to Cl− than Ca2+. There-
fore, the likelihood of a contribution of MSL9 and MSL10 to
mechanical stress- or gravistimulation-induced [Ca2+] increases
is small. The msl4;msl;5msl6;msl9;msl10 quintuple mutant
shows no detectable lesion in root gravitropic response.
MCA1 AND 2 AS REGULATORS OF
MECHANOSENSITIVE CA2+ FLUX
Further candidates for elements of a mechanically responsive
Ca2+ regulatory channel come from using Arabidopsis cDNA
to functionally complement a yeast mutant knocked out in a
stretch-activated channel component named mid1 (Kanzaki
et al., 1999; Nakagawa et al., 2007). The resulting clone was
named MID1-COMPLEMENTING ACTIVITY1 (MCA1), and
its expression led to both increased Ca2+ uptake when expressed
in the mid1 mutant and rescuing of this mutant’s lethal pheno-
type. In plants, MCA1 mRNA is expressed in multiple organs
including roots, leaves, stems, flowers, and siliques. The sub-
cellular localization of GFP-fused MCA1 in root cells was in
the plasma membrane, which was consistent with the expres-
sion pattern in yeast. MCA1 overexpression in Arabidopsis
seedlings showed enhanced [Ca2+] increases induced by hy-
poosmotic stress, a result confirmed with the tobacco MCA1
homolog in tobacco BY2 cells (Nakagawa et al., 2007; Kurusu
et al., 2011).
In an MCA1-overexpressing line, the classic touch-induced
gene TCH3 was constitutively upregulated, implying that
January 2013] TOYOTA AND GILROY—GRAVITY AND SIGNALING
MCA1 overexpression lines had become hypersensitive to me-
chanical stress (Nakagawa et al., 2007), although it is important
to note TCH3 expression is now known to be responsive to
many signals in addition to mechanical stress such as darkness
(lee et al., 2005) or ozone treatment (Short et al., 2012). Inter-
estingly, primary roots of a mca1 null mutant failed to penetrate
a hard agar layer, whereas wild-type roots could, suggesting
that the mca1 null mutant could not sense and adapt to differ-
ences in hardness of its surroundings. Taken together, these
data seem consistent with MCA1 encoding a component of a
Ca2+-permeable, stretch-activated channel involved in touch
sensing in Arabidopsis roots. However, the precise role of this
protein remains to be defined. MCA1 shows only 10% amino
acid identity to mid1. It does contain a classic EF-hand Ca2+-
binding motif and is predicted to have 1–3 transmembrane do-
mains. MCA1 is also known to form homodimers and tetramers
(Yamanaka et al., 2010; Nakano et al., 2011). Heterologous ex-
pression of Arabidopsis MCA1 increases mechanosensitive chan-
nel activity in the plasma membrane of Xenopus laevis oocytes
(Furuichi et al., 2012). However, expression of the N-terminal
part of both MCA1 (and MCA2, see below) containing a single
predicted transmembrane domain is sufficient to induce accel-
erated Ca2+ uptake in yeast (Nakano et al., 2011), suggesting a
possible regulatory activity rather than direct channel-forming
role.
In Arabidopsis, MCA1 has a single paralog, MCA2 (72.7%
identical at the amino acid level), and here the function seems
clearer. MCA2 expression is restricted to the endodermis and
vascular bundle, and this localization along with the reduced
root Ca2+ uptake seen in the mca2 null is consistent with a role
in mediating symplastic Ca2+ uptake as part of the root’s min-
eral transport system (Yamanaka et al., 2010). MCA2 knock-
outs do not have an agar penetration phenotype, and the double
mutant between mca1 and mca2 acts like mca1, suggesting
these proteins have distinct roles within the plant. Until an elec-
trophysiological study on MCA1 and MCA2 at the single chan-
nel level has been done, whether these proteins function directly
as stretch-activated channels or represent regulatory compo-
nents of a larger channel complex will remain unknown.
CANDIDATE RESPONSE GENES INVOLVED IN THE
EARLY IONIC MECHANORESPONSE
The Arabidopsis TCH genes were the first to be characterized
as being upregulated in response to mechanical stress, being
induced within 30 min after mechanical stimuli (Braam and
Davis, 1990). TCH1 encodes an Arabidopsis calmodulin (CaM),
CAM2 (Braam and Davis, 1990), TCH2 and TCH3 encode
calmodulin-like (CML) proteins, CML24 and CML12, respec-
tively (Braam and Davis, 1990; Sistrunk et al., 1994; Delk et al.,
2005) and TCH4 encodes a xyloglucan endotransglycosylase,
an enzyme involved in cell wall remodeling (Xu et al., 1995;
Purugganan et al., 1997). Calmodulin is one of the most con-
served Ca2+-binding proteins in eukaryotes and acts as a Ca2+-
sensor (Yang and Poovaiah, 2003). In Arabidopsis, seven CaM
and 50 CML genes have been identified (McCormack et al.,
2005). If CaM or CMLs encoded by genes such as TCH1, -2,
and -3, act downstream of plant mechanoresponses, multiple
mechanical stress-induced [Ca2+] increases may well be de-
coded by these proteins and transduced into a sustained bio-
chemical response. Indeed, CML24 has been implicated in the
control of root growth in response to mechanical stimulation,
119
possibly through regulation of microtubule dynamics (Wang
et al., 2011). Interestingly, CaM is known to be involved in
gravitropic signal transduction and is assumed to be a down-
stream response element of gravistimulation-induced [Ca2+]
increases (Poovaiah et al., 1987). CaM antagonists inhibited root
gravitropism in maize (Stinemetz et al., 1992) and Arabidopsis
(Sinclair et al., 1996), and the concentration of CaM in the root
tip was also much higher than other regions of roots in pea
(Allan and Trewavas, 1985) and maize (Stinemetz et al., 1987).
Furthermore, mechanical stress upon gravistimulation may in-
duce an upregulation of TCH genes because CaM activity in the
root tip of maize increased approximately 3-fold during gravit-
ropism. In cells of the gravity-perceptive maize pulvinus, tran-
scripts of Ca2+ binding proteins such as calreticulin and CaM
increased 5-fold within 15 min of gravistimulation (Heilmann
et al., 2001). The upregulation of Ca2+ responsive proteins such
as CaM and CML by gravistimulation might make cells more
sensitive to slight [Ca2+] changes (Sinclair et al., 1996) and so
contribute to signaling during the extended component of the
gravitropic growth response. TCH2, -3, and -4 are also known
to be upregulated by a [Ca2+] increase (Braam, 1992; Nakagawa
et al., 2007) providing a feed-forward loop that may also help to
extend the initial Ca2+-triggered signal.
It is important to note here that TCH1–TCH4 do not repre-
sent the only genes whose expression is affected by mechanical
stimulation. For example, microarray analysis has shown that
up to 1600 genes can be rapidly up- or downregulated by me-
chanical stimulation (Kimbrough et al., 2004; Lee et al., 2005).
Interestingly, there is a large overlap in the patterns of tran-
scriptional changes after gravitropic stimulation (Kimbrough
et al., 2004). However, the transcriptional response to mechanical
stimulation and gravitropic response are complex, and distin-
guishing the primary signaling-related responses from the sec-
ondary effects remains a significant challenge.
THE CYTOSKELETON AND EXTENDED
MECHANOPERCEPTION/RESPONSE
At present, the available data on signals such as Ca2+ changes
in response to gravistimulation suggest activation of a rapid sig-
naling system to initiate reorientation. However, the gravitropic
response can play out over many hours, and so plants need a
regulatory system capable of generating directional information
and controlling, e.g., auxin trafficking, over these extended pe-
riods. For example, recent evidence suggests that during the
600 min that an Arabidopsis root can take to complete its gravi-
tropic response (to 90° rotation), the lateral auxin gradient
(thought to drive growth) persists for only 100 min (Band et al.,
2012). This observation led to the proposal that the starch stato-
liths in the columella act as “tilt switches” and that, once the
root cap reorients ~40°, the sliding of the amyloplasts to the
rootward end of the columella cell attenuates the tropic signal
responsible for directing asymmetrical auxin signaling. The
rest of the reorientation response would then be mediated by
persistent responses already set in motion by the 100 min of
auxin export. Yet, even in this case, the graviperception ma-
chinery must be sustaining directional signals capable of regu-
lating initial asymmetrical auxin trafficking for nearly 2 h.
Do plants have tonic receptors similar to those already char-
acterized as generating long-term mechanical signaling in the
nervous system of animals (Janig et al., 1968; Madrid et al., 2003)?
To our knowledge, there are few reports of mechanosensitive
120 AMERICAN JOURNAL OF BOTANY
channel activities that have these characteristics in plants. How-
ever, the cytoskeleton has emerged as a candidate element in
this long-term perception system as detailed next.
As noted previously, the microtubule cytoskeleton plays an
intimate role in the regulation of cell expansion through its in-
terplay with cellulose synthesis. Therefore, microtubules must
play an integral role in the regulation of asymmetrical growth
response during gravitropism (Taiz, 1984; Carpita and Gibeaut,
1993; Baskin et al., 1994; Baskin, 2005). Indeed, gravitropic
stimulation is well characterized as inducing changes in the ori-
entation of cortical microtubules but only after graviperception
has occurred. For example, gravistimulation resulted in changes
in the orientation of cortical microtubules to longitudinal and
transverse directions at the epidermis of upper and lower flanks
respectively, in maize coleoptiles or sunflower hypocotyls, but
the timing indicated a role in growth control rather than gravi-
perception (Nick et al., 1990; Blancaflor and Hasenstein, 1993;
Himmelspach et al., 1999).
Interestingly, Fischer and Schopfer (1998) proposed a differ-
ent model for this cytoskeletal response based on their studies
(Zandomeni and Schopfer, 1994; Fischer and Schopfer, 1997).
Here, gravistimulation also induced similar changes in orienta-
tion of cortical microtubules within several hours, consistent
with the above reports (Nick et al., 1990; Himmelspach et al.,
1999). However, when the horizontally placed (gravistimulated)
coleoptile could not develop a gravitropic bend when a weight
was hung onto its tip, the asymmetrical reorientation of cortical
microtubule was not observed. When the horizontally placed
coleoptiles were bent downward by a heavier weight, the orien-
tation of the cortical microtubules changed to the transverse and
longitudinal directions at the upper (extended) and lower (com-
pressed) flanks, which is the opposite with respect to the gravity
vector. These results suggest that the change in orientation of
cortical microtubules is caused by the mechanical bending but
not by the gravistimulation itself. Similar experiments were
carried out in azuki epicotyls with similar results (Ikushima and
Shimmen, 2005). Mechanical force supplied by hypergravity
(centrifugation) was also shown to be capable of directing mi-
crotubule orientation and subsequent growth in isolated proto-
plasts (Wymer et al., 1996), azuki bean epicotyls (Soga et al.,
2006), and Arabidopsis hypocotyls (Matsumoto et al., 2010).
These results support the model that cortical microtubules are
capable of responding to a continuous directional mechanical
stress. Furthermore, the mechanical bending-induced reorienta-
tion of cortical microtubules was inhibited by extracellularly
applied Gd3+, suggesting the possible involvement of stretch-
activated channels in this slow mechanoresponse (Ikushima and
Shimmen, 2005).
Endogenous mechanical forces due to turgor pressure and
growth may also alter microtubule orientation. Recently, a sim-
ulation based on experimental data revealed that mechanical
stress may pattern the shoot apical meristem through feedback
via reorientation of microtubules (Hamant et al., 2008). Hamant
et al. (2008) postulated that microtubules align along the direc-
tion of mechanical stress in a surface. Based on their model of
the meristem as a pressure vessel, i.e., an elastic shell inflated
by an inner pressure, the predicted direction of principal stresses
was consistent with their experimental data of microtubule ori-
entations. These results suggest that the microtubule orientation
to maximal stress directions is critical to regulate morphogen-
esis of meristems possibly via modulation of newly deposited
cellulose microfibrils. Thus, it may well be that long-term me-
chanical response is hard-wired into the microtubule-dependent
[Vol. 100
systems that modulate cell elongation through their sensitivity
to mechanical stress.
Interestingly, although disruption of the microtubule cy-
toskeleton affects the elongation growth required for extended
gravitropic response, it appears to be the actin cytoskeleton that
plays a role in initial graviperception. Thus, gross disruption of
the actin cytoskeleton leads to inhibition of growth, but when
the actin depolymerizing drug latrunculin B is added at levels low
enough to support growth but disrupt actin structure, enhanced
gravitropic response in both roots and shoots is observed (Hou
et al., 2003, 2004; Palmieri and Kiss, 2005; Nakamura et al.,
2011). These observations imply that actin is acting as a nega-
tive regulator of tropic signaling. Precisely how actin is exert-
ing its effects remains unknown but depolymerizing actin is
known to alter amyloplast positioning in statocytes and to affect
their characteristic motility (Hou et al., 2004; Palmieri and Kiss,
2005; Saito et al., 2005; Nakamura et al., 2011), suggesting that
actin may be forming a dynamic network that is interacting
with the amyloplasts to modulate signal generation. In animal
cells, actin is well known as a component of a biochemical
mechanosensory network where, e.g., tension in the actin fila-
ment can modulate its interaction with regulatory proteins such
as cofilin (Hayakawa et al., 2011), providing one model for how
plant actin could operate to transduce even long-term mechani-
cal stress to biochemical activity. Recently an Arabidopsis
E3 ubiquitin ligase has also emerged as an unexpected po-
tential link between amyloplast and actin network in shoot en-
dodermal cells (Nakamura et al., 2011), providing a potentially
key step toward defining the components of this network in
plants.
CONCLUSION
Mechanical forces represent ubiquitous and fundamental sig-
nals in plant growth and development. All organisms living on
Earth are exposed to mechanical stress due to their own weight
regardless of their size. However, plant biologists are still tak-
ing their first steps toward defining the plant mechanoresponse
system with molecular precision. We have focused this review
on dividing mechanical stress and mechanoreceptor actions in
terms of their time course. What emerges from this discussion
is that our understanding of the rapid signaling events to me-
chanical/gravitropic stimulation is much more complete than of
systems that must monitor and then modulate the extended
growth responses that follow. This focus on short-term signal-
ing and response is likely inherent in the difficulties of assign-
ing cause and effect to processes occurring over many hours.
Thus, although mechanosensitive ion channels have been a fa-
vorite target of mechanobiology, the duration of channel activ-
ity is much shorter than the time needed to exhibit a long-term
growth response such as gravitropism. A plausible answer as to
how transient signals can regulate such extended response is
that the short signals are transduced into subsequent signals that
persist until the termination of the growth response. For gravit-
ropism, a series of poorly defined transduction cascades need to
fill in the gaps between activation of the initial mechanosensi-
tive channel (seconds to minutes), establishment of a lateral
auxin gradient (5 min in Arabidopsis roots), sustaining of the
lateral auxin gradient (1–2 h), and sustaining of the growth re-
sponse (many hours). However, the precisely maintained gravi-
tropic set-point angles seen in many organs hints at a much
more dynamic system capable of continuously monitoring and
January 2013] TOYOTA AND GILROY—GRAVITY AND SIGNALING
regulating an extended gravitropic stimulation/response. Al-
though strain-induced remodeling of cytoskeleton may provide
one component of the long-term mechano/graviresponse, a ma-
jor goal for the gravitropism field remains defining both the
rapid and extended perception machinery with molecular preci-
sion. New probes such as the DII-Venus auxin monitoring system
(Band et al., 2012; Brunoud et al., 2012) and vastly improved
bioprobes for signals such as Ca2+ (Choi et al., 2012) are pro-
viding us with an increasingly detailed picture of cellular events
during the tropic response in which to place these elusive miss-
ing pieces of the molecular puzzle of graviperception.
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