IONIC SIGNALING IN PLANT GRAVITY AND TOUCH RESPONSES
Gioia D. Massa1, Jeremiah M. Fasano2 and Simon Gilroy1
1
Biology Department, The Pennsylvania State University, University Park, PA
2
U. S. Food and Drug Administration, College Park, MD
ABSTRACT
Plant roots are optimized to exploit resources from the soil and
as each root explores this environment it will encounter a range
of biotic and abiotic stimuli to which it must respond. Therefore,
each root must possess a sensory array capable of monitoring
and integrating these diverse stimuli to direct the appropriate
growth response. Touch and gravity represent two of the
biophysical stimuli that plants must integrate. As sensing both of
these signals requires mechano-transduction of biophysical
forces to biochemical signaling events, it is likely that they share
signal transduction elements. These common signaling
components may allow for cross-talk and so integration of
thigmotropic and gravitropic responses. Indeed, signal
transduction events in both plant touch and gravity sensing are
thought to include Ca2+- and pH-dependent events. Additionally,
it seems clear that the systems responsible for root touch and
gravity response interact to generate an integrated growth
response. Thus, primary and lateral roots of Arabidopsis respond
to mechanical stimuli by eliciting tropic growth that is likely
part of a growth strategy employed by the root to circumvent
obstacles in the soil. Also, the mechano-signaling induced by
encountering an obstacle apparently down-regulates the
graviperception machinery to allow this kind of avoidance
response. The challenge for future research will be to define
how the cellular signaling events in the root cap facilitate this
signal integration and growth regulation. In addition, whether
other stimuli are likewise integrated with the graviresponse via
signal transduction system cross-talk is an important question
that remains to be answered.
____________________________________________________
INTRODUCTION
The architecture of the plant root system develops to
promote anchorage and to optimize soil exploration and
resource exploitation. This highly plastic developmental
program is played out in an environment rich in a diverse
array of stimuli. These resources/stimuli are also often
patchily distributed meaning that the root system must
possess the capacity to locally exploit or respond to this
spatial heterogeneity. Therefore, factors as diverse as
gradients in water availability, the spectrum of microbial
flora and fauna, and even light, all impact on how the root
system must locally develop. Indeed, plants may even
perform better in response to patchily distributed minerals
in the soil with the plant able to sense the volume of the
patch and even the steepness of the nutrient gradient at the
patch’s edge (Wijensinghe and Hutchings, 1999). Each
root, be it primary or lateral, appears capable of
perceiving a wide range of environmental and internal
factors and responding with an integrated growth
response. The perception and signaling mechanisms
____________________
* Correspondence to: Simon Gilroy
Biology Department, The Pennsylvania State University
208 Mueller Laboratory, University Park, PA 16802
Email: [email protected]
Tel: 814-863-9626; Fax: 814-865-9131
associated with each stimulus likely allow extensive
cross–talk between stimulus/response pathways at the
level of signal transduction networks. Unfortunately, in
general, the signal perception/transduction events for
most of these soil stimuli are unknown. However, for two
of the factors that modulate root development, touch and
gravity, we are beginning to decipher the ionic basis of
signaling and develop models of how these two mechanoperception/response systems of the root might be linked at
the level of their signal transduction events. In this review,
we will therefore describe what is known about the
signaling systems linked to gravity and touch, and explore
the evidence that these signaling networks might actually
interact to mediate an integrated root growth response.
GRAVITY PERCEPTION IN THE ROOT
In plants, gravity sensing is generally thought to be
confined to specialized sensory cells called ‘statocytes’
(Kiss, 2000; Sack, 1991). These cells are found localized
to the columella of the root cap, the endodermis of the
shoot and the graviresponsive motor cells of the pulvinus.
The statocytes contain amyloplasts that, due to their dense
starch content, move within the cell in response to gravity
and so trigger graviperception events. These perception
events transform the gravity vector to biochemical
changes that in turn elicit the directional, or tropic, growth
response. This series of events initiated by amyloplast
sedimentation was proposed by Haberlandt (1900) and
Nemec (1900) as the “starch statolith hypothesis”. In
statocytes, including the bundle sheath cells of the
pulvinus, endodermal cells of the shoot, and columella
cells of the root cap, a mobile signal must be moved from
sensing to responding cells. This signal transmission is
perhaps most obvious in roots, as shown in Figure 1, with
gravisensing occurring in the root cap and the growth
response occurring as asymmetrical growth in the
elongation zone (reviewed in Sack, 1991), however
similar phenomena must be occurring in other organs. For
example, the sensing events in the endodermal cells must
coordinate growth in adjacent cortical and epidermal cells
during shoot gravitropism.
The starch statolith hypothesis is generally supported
by current experimental data. Kiss and coworkers used
reduced-starch and starchless Arabidopsis mutant lines to
demonstrate a positive correlation between amyloplast
starch content and the sensitivity of the graviresponse
(Kiss et al., 1989; Kiss et al., 1996; Vitha et al., 2000).
If starch content was reduced by less than 50%, there was
no impact on gravitropism. However, more than 50%
reduction or complete elimination of starch from the
plastids in these mutants (confirmed by electron
microscopy) significantly reduced the graviresponse.
These observations led these authors to propose that the
plant gravisensor system is over-engineered by about 50%.
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S. Gilroy — Ionic Signaling in Plant Gravity and Touch Responses
Figure 1. Functional/developmental zones of a three-day old primary root of Arabidopsis thaliana. A. The regions of gravity sensing
and gravitropic response are highlighted. The distal elongation zone is centered on the region showing 30% maximal cell elongation, the
central elongation zone on the region showing maximum elongation rate. B. Schematic of root cap organization. RAM, root apical
meristem; S1-S3, the S1, S2 and S3 layers of the columella; pc, peripheral cell; tc, tip cell. C. Relative effect of laser ablation of specific
columella cells on reducing gravitropism. D. Amyloplast sedimentation as a percentage of maximal sedimentation rate (1.2 µm/min;
data modified from Blancaflor et al., 1998).
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S. Gilroy — Ionic Signaling in Plant Gravity and Touch Responses
Due to this apparent importance of amyloplasts for
sensing, their movements have been quantified both in
fixed and living tissue sections and intact plants
(Blancaflor et al., 1998; Clifford and Barclay, 1980;
Heathcote, 1981; Larsen, 1969; MacCleery and Kiss,
1999; Massa and Gilroy, 2003; Sack et al., 1984; Sack
et al., 1985; Sack et al., 1986; Sievers et al., 1989). These
kinetic studies have provided some insight into how the
initial sensing events are likely to occur. Therefore, most
plastids begin to reach the new lower side of the
columella on the order of five minutes after reorientation.
This observed time period is much longer than the few
seconds thought to be required for perception, implying
that gravity perception events are unlikely to be elicited
by large-scale sedimentation per se or by amyloplasts
impacting on the lower surface of the cell. Rather
gravitational forces on the amyloplasts are likely
transmitted via some cytoplasmic network, such as the
cytoskeleton, either in a coherent force transmissive
network (Blancaflor, 2002) or a more tenuous percolation
responsive array (Forgacs, 1995). Thus, force transmission and associated signal initiation, for example the
opening of mechanosensitive channels, would occur
essentially instantaneously when the amyloplasts begin to
sediment. The complex biophysical interactions acting on
these amyloplasts are hinted at in the observations that the
kinetics of their sedimentation also appear complex.
For example, Yoder et al. (2001) observed that in maize,
sedimentation appeared to be confined to the periphery of
the cell and amyloplasts did not readily enter the cell core
except through preferentially traversed cytoplasmic
channels. It is likely that amyloplast movements are
highly constrained by an as yet poorly characterized force
transmissive network.
Blancaflor et al. (1998) attempted to determine
whether the observed amyloplast motility might relate to
functional significance in graviperception. They correlated
the loss of graviresponse associated with selective removal
of each defined class of columella cell, using laser
ablation, to differential amyloplast motilities in these
cells. Previous workers had demonstrated the importance
of the cap in gravisensing by surgical removal of this
organ (e.g. Konings, 1968), but the small number of cell
layers and the simple pattern of cellular organization in
the Arabidopsis root cap made a cell-by-cell laser ablation
study feasible. Blancaflor et al. (1998) reported a strong
correlation between the degree of loss of gravicurvature
upon ablating defined cells and the amyloplast
sedimentation rates in those particular statocytes (Figure 1).
Consequently, the ablation of the central S2 cells of the
columella, cells with the most freely sedimenting amyloplasts, led to the greatest inhibition of graviresponse.
An important caveat about interpreting these ablation
experiments is that tissue integrity of the cap may be
important for functions other than perception and it is
even possible that the columella could serve as a relay
point for sensing occurring elsewhere, as suggested by
Wolverton et al. (2002). Even so, the correlation of
maximum amyloplast motility with cells apparently
generating the largest component of the graviperception
event does support the idea that the gravisignaling system
is tightly coupled to plastid movements.
However, it is likely that multiple gravity perception
systems have appeared during plant evolution (Barlow,
1995) and alternate, non plastid-based models of gravisensing have also been put forward. For example, cells
may monitor the tensive and compressive forces elicited
by the mass of their own cytoplasm (Wayne and Staves,
1996) and a gravity sensor system has been proposed to
reside in the amyloplast free elongation zone of the root
(Moore et al., 1998; Wolverton et al., 2002). Pickard and
Ding (1993) provided one possible explanation of this
elongation zone gravity sensing system. They proposed
that plant cells are capable of perceiving mechanical
stress by means of a plasma membrane localized
mechano-sensory ion channel complex, or ‘plasmalemmal
control center’. Compression or tension of individual cells
would trigger ion fluxes through these channel
complexes. Horizontal elongating plant organs might
experience compression and tension on their lower and
upper flanks, respectively. Though relatively small, these
mechanical forces might be sufficient to trigger signal
transduction events through the amplifying effects of the
plasmalemmal control center.
Despite these alternate mechanisms for gravisensing,
the starch-statolith model is supported by most current
data as the principle mechanism for gravity perception in
both roots and shoots of higher plants (Kiss, 2000). It is
important at this point to note that while it seems that
amyloplast sedimentation does reflect an important feature
of the gravity sensing apparatus, it still remains unclear
what precise feature of this motility is responsible for
signal generation. Therefore, while amyloplast interaction
with a coherent cytoskeletal network remains a strong
candidate for the system translating amyloplast force to
biochemical signal, translocation of the plastid (Behrens
et al., 1985), disruption of internal structures by the
traveling amyloplast (Yoder et al., 2001), friction upon a
sensitive surface during travel (Iversen and Larsen, 1973),
or transmission of force through a fairly tenuous cytoskeletal
network (Baluska and Hasenstein, 1997) have all been
proposed as important links between statolith motility and
the initial perception events. Assuming one of these
features of amyloplast movements does reflect signal
generation, one question it immediately raises is what is
the nature of the biochemical signaling events that are
triggered? Recent evidence suggests that changes in ionic
fluxes may represent some of these initial gravisignaling
elements.
IONIC SIGNALING IN THE GRAVITROPIC
RESPONSE
Changes in cytoplasmic Ca2+ are recognized as ubiquitous
signal transduction events in a diverse range of plant
responses, making Ca2+ an attractive candidate as a
signaling element in the events of graviperception (Bush,
1995; Hepler and Wayne, 1985; Sanders et al., 1999).
There is also much circumstantial data implicating Ca2+ in
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S. Gilroy — Ionic Signaling in Plant Gravity and Touch Responses
the graviresponse. For example, polar transport of Ca2+ in
the apoplast has been detected in maize and pea root caps
following gravistimulation and appears to be a functional
component of the gravitropic response (Björkman and
Cleland, 1991; Lee et al., 1983a; Lee et al., 1984). Thus,
application of Ca2+ chelators such as EDTA, EGTA and
BAPTA to maize root caps abolished gravitropic
curvature (Björkman and Cleland, 1991; Lee et al., 1983b).
There appears to be a complex interplay of Ca2+ and auxin
transport in the cap, with auxin transport inhibitors
blocking cap-based polar Ca2+ transport (Lee et al., 1984)
and Ca2+ chelation blocking gravity-induced auxin
redistribution (Young and Evans, 1994). There is
extensive evidence that redistribution of auxin in the root
apex mediates the tropic growth control in the elongation
zone (Muday, 2001) implying that root cap Ca2+ dynamics
might play an important role in controlling this regulatory
auxin flux.
There is also substantial evidence that the Ca2+dependent regulatory protein calmodulin is involved in
gravity perception. For example, Heilmann et al. (2001)
demonstrated selective recruitment of calmodulin and
calreticulin transcripts into polyribosomes on the lower
side of gravistimulated maize pulvini. Similarly, in roots,
calmodulin protein levels are elevated in the root apex
(Allan and Trewavas, 1985; Stinemetz et al., 1987) and
these levels are enhanced upon gravistimulation (Sinclair
et al., 1996). In addition, calmodulin levels in the root
apex were found to correlate with the degree of red-lightinduced graviresponsiveness in Merit roots (a maize
cultivar requiring light to become gravitropic; Stinemetz
et al., 1987). Work with calmodulin antagonists suggests
that this calmodulin is required for gravity perception.
Hence, treatment of roots with calmodulin antagonists at
levels that did not inhibit root elongation still inhibited
gravitropic curvature (Sinclair et al., 1996; Stinemetz et al.,
1992). These calmodulin antagonists also affected
gravistimulation-induced polar transport of Ca2+ (Lee et al.,
1983a; Lee et al., 1984) and the development of an
asymmetric proton current (Björkman and Leopold, 1987)
that may be an important element of gravisignaling, see
below. The development of this proton current precedes
polar Ca2+ transport, and it is not blocked by auxin
transport inhibitors (Björkman and Leopold, 1987),
suggesting that Ca2+/calmodulin may play a role in a very
early step in graviperception that involves the activation
of proton pumping by the columella cells.
Further circumstantial evidence for a role of Ca2+ in
gravisignaling comes from studies on a Ca2+-related
second messenger, inositol-1,4,5-triphosphate (IP3).
Inositol-1,4,5-triphosphate is a Ca2+ mobilizing second
messenger, most likely releasing Ca2+ from an intracellular storage compartment such as the vacuole or ER to
trigger subsequent Ca2+-dependent signaling events
(Munnik et al., 1998). Inositol-1,4,5-triphosphate has
been implicated in the gravitropic response of grass
pulvini with a transient increase in IP3 levels occurring
within minutes of gravistimulation of maize and oat
pulvini cells (Perera et al., 1999, Perera et al., 2001).
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Although these initial changes in IP3 showed no
asymmetry, a longer term, sustained increase appeared on
the lower side of the pulvinus after several minutes of
gravistimulation. Phosphatidyl-inositol-phosphate (PIP)
kinase activity also increased in these regions of the
pulvinus, suggesting a possible mechanism for generating
the asymmetry in IP3 through increased production of
phosphatidyl-inositol-bisphosphate, the substrate for the
phospholipase C which forms IP3. At present however,
whether this IP3 based system is linked to gravisignalingrelated Ca2+ changes remains undefined.
Direct evidence for a Ca2+ change elicited in gravity
sensing cells in response to gravistimulation remains
equivocal. When cress roots were treated with
cyclopiazonic acid, an inhibitor of ER Ca2+-ATPases,
gravicurvature was inhibited whereas growth per se was
unaffected (Sievers and Busch, 1992), providing tentative
support for the involvement of an internal calcium store in
gravity perception. Gehring et al. (1990) have reported a
cytosolic Ca2+ change in maize coleoptiles upon
reorientation. However, it is difficult to clearly attribute
the reported changes to gravity-related alterations in
cytosolic Ca2+ (Digby and Firn, 1990). Plieth and
Trewavas (2002) have also reported gravistimualtioninduced Ca2+ changes, shown in Figure 2. These authors
used Arabidopsis seedlings expressing the Ca2+ sensing
protein aequorin targeted to the cytosol and monitored a
Ca2+ change upon seedling reorientation. The changes
were likely either small in amplitude or localized to a very
few cells as to detect them required simultaneous
measurement of 500–1000 seedlings coupled to the use of
the most sensitive form of aequorin cofactor, cp-coelentrazine,
available (Pleith and Trewavas, 2002). Unfortunately the
aequorin measurement approach precluded any spatial
localization of the change. Figure 2 also shows the
cytoplasmic Ca2+ changes measured in response to
mechanical perturbation highlighting the potential for
measuring Ca2+ changes associated with the physical
manipulations of the reorientation used in gravitropism
experiments. As described in more detail below, touch
signaling is well characterized as eliciting Ca2+-dependent
events in plants and it is often extremely difficult to
divorce the influence of touch responses in
gravistimulation protocols. In addition, touch appears to
modify the graviresponse (Massa and Gilroy, 2003;
Wolverton et al., 2000) further complicating the design of
experiments to test events specifically associated with
gravistimulation.
Legué et al. (1997) imaged cytosolic Ca2+ in root
statocytes and although clear touch related Ca2+ transients
were observed, these researchers failed to detect any Ca2+
changes upon reorientation. These results suggest that
sustained, steady state changes in cytosolic Ca2+ are
unlikely to mediate gravity signaling events in roots.
However, highly localized changes in Ca2+, especially if
these changes occur in membrane delimited
microdomains, are beyond the resolution of all the Ca2+
measurement techniques that have so far been applied to
monitor gravisignaling in plants. In addition, the elevated
S. Gilroy — Ionic Signaling in Plant Gravity and Touch Responses
calmodulin bound to the channel mouth on the cytosolic
face of the membrane. Only voltage gated Ca2+ flux
through these specific channels will trigger this
calmodulin activity. General increases in cytoplasmic
Ca2+ do not have access to this calmodulin pool and so
fail to elicit a CREB response. Similarly, monitoring the
bulk cytoplasmic Ca2+ level would not reveal the relevant
Ca2+ changes. Thus, the microstructure of the signaling
system could have profound effects on the subcellular
signaling ‘signature’. In addition to this spatial component
to signaling, it is becoming recognized that information
may also be encoded by the temporal patterning of the
Ca2+ change. For example, the frequency of Ca2+
transients has been shown to determine the extent of the
closure response of stomatal guard cells (Allen et al.,
1999). Due to such potentially complex spatial and/or
temporal characteristics of signaling, problems of Ca2+
measurement resolution, and difficulties in separating
touch- from gravity-related signaling events, at present
the question of whether Ca2+ changes represent early
gravisignaling events in statocytes of higher plants
remains open.
pH-DEPENDENT SIGNALING IN THE
GRAVIRESPONSE
Figure 2. Changes in cytoplasmic Ca2+ induced by
gravistimulation or touch in Arabidopsis thaliana seedlings
expressing aequorin. A. Gravistimulation with dishes of
seedlings either rapidly rotated to 135˚ and back to vertical
(±135˚) or maintained at 135˚ for 25 minutes and then
reoriented to vertical for 25 minutes (repeated twice).
B. Seedlings were mechanically stimulated by pulsing 50 mL/sec
air for 1 second on a vertically oriented dish of seedlings. Pulses
applied at t = 25, 50, 75, 100, and 125 minutes. Each dish
contained 500–1000 seedlings. (Reprinted with permission from
Plieth and Trewavas, 2002).
levels of calmodulin (Dauwalder et al., 1986) and
calmodulin-like proteins (e.g. TCH3, Antosiewicz et al.,
1995) found in statocytes should sensitize these cells to
small changes in Ca2+ perhaps compounding problems of
detection. Microdomain signaling and enhanced response
sensitivity can lead to dramatic increases in signal
specificity through structurally delimited signal
processing, a feature well documented in Ca2+-dependent
signal transduction in mammalian cells. For example,
despite the range of voltage-gated Ca2+ channels in the
plasma membrane of neurons, only Ca2+ flowing through
L-class channels can elicit activation of the CREB
transcription factor and changes in transcription
(Dolmetsch et al., 2001). This specificity occurs through
In contrast to a role for Ca2+, there is much direct
evidence that pH changes constitute an early and
functional element of gravisignaling events. Rapid
changes in proton fluxes have been monitored around the
gravistimulated root cap with a substantial proton efflux
appearing from the upper flank (e.g. Collings et al., 1992;
Zieschang et al., 1993). Gravistimulus-induced membrane
potential changes monitored in the columella
(Monshausen et al., 1996; Sievers et al., 1995) are also
consistent with the activation of a plasma membrane
H+-ATPase as an early element of the graviperception
machinery. These elevated and asymmetric proton fluxes
correlate with a sustained cell wall acidification and
transient (10 minute) cytoplasmic pH increase within the
columella (Fasano et al., 2001; Scott and Allen, 1999).
The largest and longest cytoplasmic pH increases, shown
in Figure 3, occur in cells with the fastest amyloplast
sedimentation rate and the largest functional significance
in gravisignaling as inferred from the laser ablation
studies shown in Figure 1. These cytoplasmic pH changes
observed in the statocytes appear to be a functional
element in graviperception since disturbing the pH
dynamics of the whole cap (Scott and Allen, 1999), or
specifically in the columella cells (Fasano et al., 2001),
alters subsequent tropic curvature. Similar observations of
gravistimulation-induced pH changes in the maize pulvinus
(Johannes et al., 2001) suggest pH fluxes may play a
widespread role in gravisignaling.
Although these pH fluxes in the cap are likely
specifically related to signaling events, pH, especially
wall pH, also appears to have a prominent regulatory role
in the growth component of the tropic response.
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S. Gilroy — Ionic Signaling in Plant Gravity and Touch Responses
Figure 3. Changes in root cap cytosolic pH of different root cap zone cells (see Fig. 1B) while undergoing gravistimulation induced
by 90˚ reorientation at 0 minutes. A. The pH sensing fluorescent dye BCECF-Dextran was microinjected into defined root cap cells to
allow ratio imaging of changes in pH upon gravistimulation. Representative S3 and S2 columella cell injections are shown. V, vacuole.
Scale bar 20 µm, 5 µm in insets; (B.-D.). Average cytosolic pH was monitored in different BCECF-dextran microinjected cells upon
gravistimulation by 90˚ reorientation at 0 minutes. Data represent mean ± SEM, n > 10 (Reprinted with permission from Fasano et al., 2001).
Gravistimulation elicits proton efflux in the elongation
zone of the root (Fasano et al., 2001; Monshausen et al.,
1996; Mulkey and Evans, 1981; Mulkey et al., 1982; Pilet
et al., 1983; Taylor et al., 1996; Versel and Pilet, 1986;
Zieschang et al., 1993) that likely regulates asymmetrical
organ elongation through the phenomenon of acid growth
(Büntemeyer et al., 1998; Collings et al., 1992; Cosgrove,
2000; Edwards and Scott, 1974; Evans, 1976; Felle, 1998;
O’Neill and Scott, 1983; Peters and Felle, 1999; Taylor
et al., 1996). Recently, Monshausen and Sievers (2002)
demonstrated that the surface pH of Lepidium sativum
roots showed asymmetric changes upon gravistimulation,
with pH decreasing on the upper flank and increasing on
the lower. This asymmetry developed first at the tip and
moved to the elongation zone with kinetics similar to
those of polar auxin transport further suggesting a link
between pH flux and auxin transport
The picture emerging from these studies on
gravisignaling and perception is that proton fluxes in the
root cap, perhaps mediated by a Ca2+/calmodulindependent activation system are some of the earliest
functional signaling elements triggered by statolith
movements. These pH fluxes may either encode the
direction of gravity for the root or, perhaps more likely,
reflect activation of a very early part of the gravity
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Gravitational and Space Biology Bulletin 16(2) June 2003
signaling machinery, such as auxin transport, that regulates
information export to the elongation zone. However, it is
important at this point to note that there is no case in
nature where this gravitropic signaling system will be
operating in isolation from myriad other sensory inputs to
the root. The entire stimulus environment of the root may
well alter the precise signal transduction events to a single
stimulus. Therefore, it becomes of critical importance to
place the gravisignaling system in the context of its role in
the multisensory signaling network of the root. We have
perhaps the best understanding of these kinds of sensory
interactions between the two mechano-sensory systems in
the root cap, the gravisensing and touch perception systems.
GRAVISIGNALING IN THE SOIL,
THE INFLUENCE OF TOUCH
There is accumulating evidence that the touch and gravity
sensing/response systems interact. For example, root
waving and coiling are thought to represent the
interactions of thigmotropism caused by growing on a
hard surface, coupled to gravitropism and circumnutation
(Mullen et al., 1998; Okada and Shimura, 1990; Simmons
et al., 1995). Similarly, Mullen et al. (2000) have shown
that general mechanical stimulation of roots increases the
S. Gilroy — Ionic Signaling in Plant Gravity and Touch Responses
latent period prior to the onset of gravitropism and Massa
and Gilroy (2003) reported that mechanical stimulation of
the peripheral cells of the root cap led to a reduction in
gravisensing and subsequent gravitropic response. These
kinds of interactions are shown in Figure 4, where the
gravitropic growth of the root (Fig. 4A) is modified by
mechanical contact with a barrier. In this experimental
design, the root hits a horizontal barrier, and immediately
begins reorientation, with the root forming a convex bend
in the central elongation zone (CEZ), and later a concave
bend in the distal elongation zone (DEZ). These tropic
responses allow the root tip to track along the obstacle at
a constant angle of 136˚ to the horizontal (Massa and
Gilroy, 2003). The root cap is maintained in contact with
the obstacle throughout this response, apparently to allow
the root to continuously sample the mechanical stimulation
afforded by the barrier to maintain lateral tropic growth
until the object is circumvented. It appears that as the root
cap receives mechanical stimulation it down-regulates
gravitropism, allowing the formation of a new tropic
response (Massa and Gilroy, 2003). Considering these
apparent interactions between touch and gravity signaling,
the physical nature of both stimuli and the idea that
gravity sensing might even have evolved from a primitive
touch perception apparatus (Trewavas and Knight, 1994)
it is tempting to speculate that such touch/gravity system
interactions could reflect cross-talk between their respective
cellular signal transduction elements. Such interactions
are especially likely as both gravisignaling and touch
perception seem intimately linked to ionic signaling events.
Figure 4. Kinetics of the response of a primary root of Arabidopsis thaliana to A. an obstacle to downward growth, and
B. horizontal gravistimulation. 0 minutes in A. is the point of touching the barrier and in B. is the time of rotation from vertical.
CEZ, central elongation zone; DEZ, distal elongation zone. Scale bars are 500 µm.
CALCIUM SIGNALING IN THE TOUCH
RESPONSE
Ca2+ has been shown to be an important secondary
messenger in a range of gravity and mechanical signaling
events in animals (Gillespie and Walker, 2001) and an
array of experimental evidence supports a similar role for
Ca2+ in plants. The bulk of the experimentation involves
using transgenic plant material expressing the Ca2+
sensitive photoprotein aequorin (Knight et al., 1991). Ca2+
signatures have been detected in response to mechanical
stimulation in a variety of such transgenic plants including
tobacco (Knight et al., 1991; Knight et al., 1992; Knight
et al., 1993), Arabidopsis (Plieth and Trewavas, 2002)
and the moss Physcomitrella patens, (Russell et al.,
1996). These transients are likely from intracellular
sources rather than trans-plasma membrane influx as they
can be inhibited by ruthenium red, which is thought to
block the movement of Ca2+ from internal stores (Knight
et al., 1992). Due to the fairly low intensity of aequorin
luminescence, however, it has been difficult to monitor
Ca2+ changes on a cellular level. Other evidence for the
involvement of Ca2+ in mechanical signaling comes from
experiments using Ca2+ sensitive fluorescent dyes. Legué
et al. (1997) loaded Arabidopsis roots with the fluorescent
Ca2+ indicator Indo-1 and showed that touch would
induce cell-to-cell transduction of Ca2+ transients. This
response had its lowest threshold for mechanical induction
in the root cap and also could be inhibited with ruthenium
red. In addition to touch however, Ca2+ transients have
been detected from many other stimuli, including cold
shock (Knight et al., 1992), pathogen elicitors (Blume
et al., 2000), and Nod factors acting on legume root hairs
(for example see Wais et al., 2002). It is not yet understood
if these observed Ca2+ transients are a mechanism of
‘priming’ the cell which then responds to a second signal
which specifies the response, or rather if the spatial and/or
temporal footprint, the so-called ‘Ca2+ signature’ (Blume
et al., 2000; Wood et al., 2000) itself encodes specificity.
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S. Gilroy — Ionic Signaling in Plant Gravity and Touch Responses
In addition to mediating this ‘general’ touch
sensitivity seen in most plant cells, Ca2+ also appears
involved in transducing events in cells highly specialized
for mechano-perception. For example, touch-induced
tendril coiling of Bryonia dioica can be inhibited by the
application of Gd3+ and erythrosin B, putative inhibitors
of the voltage-gated Ca2+ channel and organellar Ca2+ATPase characterized in these cells (Klüsener et al., 1995;
Liß et al., 1998). However, we must await direct
measurements of tendril-related Ca2+ changes to place
these transporters in a functional touch-related Ca2+
signaling
network.
In
addition,
touch-induced
thigmomorphogenesis in soybean hypocotyls can be
stimulated by Ca2+ or Ca2+ ionophore and inhibited by
either the Ca2+ chelator EGTA or calmodulin antagonists
(Jones and Mitchell, 1989). Thus there is mounting
evidence for a role of Ca2+ signaling in the diverse array
of plant touch-related responses from specialized sensory
events to a more generalized and widespread response
such as whole plant thigmomorphogenesis.
Experiments where Ca2+ was applied to roots also
support the involvement of Ca2+ in root tropistic
responses. Work by Ishikawa and Evans, (1992) and
Takahashi et al. (1992), showed that agar blocks could
cause roots of Z. mays to bend if placed in the root cap
region and this response was enhanced when Ca2+ was
present in the blocks. When blocks were placed on the
elongation zone, no bending occurred unless Ca2+ was
present in the agar (Ishikawa and Evans, 1992, but see
Takahashi et al., 1992 for alternate perspective), perhaps
indicating that Ca2+ can induce modification of the growth
process. These experiments suggest that Ca2+ is important
in both the signaling aspects of touch and more generic
downstream events related to growth via either cytoplasmic signals (possibly changes to auxin sensitivity) or
apoplastic modifications (e.g. modifications of pectin
crosslinking).
As noted above for gravisignaling, calmodulin is one
of the best-characterized mediators of Ca2+ signaling and
it also appears an important part of the touch response.
Several researchers have detected increases in calmodulin
and calmodulin-like gene expression in various plant
species in response to mechanical stimulation
(e.g. Botella and Arteca, 1994; Braam and Davis, 1990;
Ito et al., 1995; Takezawa et al., 1995). The time-course
of gene induction precludes these changes being involved
in any initial touch-signaling pathway, however increases
in calmodulin level might be designed to prolong initial
signaling events. Changing calmodulin levels or
localization could effectively tune the specificity of the
Ca2+ response, especially with the isoform specific
differences in enzyme activation that are seen in the
various members of the calmodulin family (Lee et al.,
2000; Liao et al., 1996; Zielinski, 1998). Also, changes in
calmodulin microdomain localization have been shown to
modulate Ca2+ signaling in animal cells (Persechini and
Cronk, 1999), and this alteration of cellular microenvironment is a mechanism that plant cells might utilize
in mechanosensing. Consequently, changes in expression
78
Gravitational and Space Biology Bulletin 16(2) June 2003
levels might contribute to the specificity of future
response, possibly either by isoform-specific signal
induction or by changing the preparedness of the cell for
future signals. Alternatively, the calmodulin increases
may be involved in mediating downstream growth
responses, i.e. acting more like a response factor than an
initial signaling element.
pH AND TOUCH SENSING
While there is strong evidence for a role of pH changes in
gravisensing (Fasano et al., 2001; Johannes et al., 2001;
Scott and Allen, 1999) and a host of other cellular
responses (see Fasano et al., 2002 for summary), there is
no direct evidence to date of the involvement of pH
changes in touch signaling in plants. Very tentative
evidence for such a role arises from observations of
Bryonia tendrils, where there appears to be touch-related
modification of H+-ATPase function (Bonnin et al., 1996;
Bourgeade and Boyer, 1994). Interestingly, BCC1 also
appears to be responsive to changes in cytoplasmic pH in
these cells (Klüsener et al., 1997). Therefore, if pH
changes do occur in response to mechanical perturbation
they may play a role in a pathway where pH and Ca2+
interact to specify and modulate the response.
IONIC INTERACTIONS: SIGNAL PROCESSING
IN THE ROOT CAP
As can be seen from the discussions above, we are only
just beginning to decipher the molecular events associated
with early ionic signaling in the root cap in response to
mechanical signals, either at the level of gravitational
effects on intracellular amyloplasts or the effects of
extracellular touch at the surface of root epidermal cells.
However, we obviously lack an understanding of many of
the key elements of these signaling systems and how they
might interact. For example, although current evidence
strongly implicates ionic signaling in the earliest transduction events of mechano-perception, and highlights
Ca2+ and calmodulin as potentially important elements in
both touch and gravity signaling, we do not yet have a
clear picture of how these elements either separately
encode information about each stimulus or might combine
this information to elicit an integrated response. These
signaling systems are able to elicit a complex cascade of
cellular events and these are likely composites of specific
gravity and touch response elements, as well as the many
common cellular factors these stimuli utilize. The
complexity of such multiple responses is well
demonstrated by the results seen from micro-array
analysis of roots undergoing rotation to different angles
for different time periods. An almost bewildering array of
transcriptional changes are seen under these
circumstances, and importantly, a large number of the
transcriptional changes appear common to treatments that
involve either mechanical or gravitropic stimulation.
Hence, 39% of the genes that were turned on or off by
gravistimulation (90˚ reorientation) were also regulated
by mechanical stimulation (360˚ rotation; Moseyko et al.,
2002). Interestingly, many of the genes identified as being
S. Gilroy — Ionic Signaling in Plant Gravity and Touch Responses
graviresponsive were from functional groups not normally
associated with gravity signaling or response. For
example, the largest category of transcriptionally
regulated genes consisted of oxidative burst/plant
defense-related genes such as Cytochrome P450. At the
same time, despite the current models of an actin-based
gravisensor and microtubule-related regulation of tropic
growth, expression of only one cytoskeletally related
gene, a kinesin-like protein, was altered in response to
gravistimulation (Moseyko et al., 2002). Hopefully,
characterization of the many unknown genes whose
expression was altered by reorientation and future work
comparing such responses to those of gravitropically
impaired mutants will help shed more light on the
complexities gravity and mechanical interaction.
All plant organs possess a complex sensory array
capable of processing multiple environmental signals to
an appropriate integrated response. We have concentrated
on discussing the interaction of two closely related
biophysical signals, touch and gravity, but evidence for
signal integration abounds. The well-documented,
interactions of gravitropism and phototropism (Okada and
Shimura, 1992; Ruppel et al., 2001; Vitha et al., 2000),
touch, gravitropism, and obstacle avoidance (Figure 4;
Massa and Gilroy, 2003; Mullen et al., 1998; Okada and
Shimura, 1990; Simmons et al., 1995) and hydrotropism
and gravitropism (Takahashi et al., 2002) all highlight
that a critical element in our understanding of the
transduction of any one tropic signal will be to determine
how all these other stimuli shift the suites of signaling
elements each cell will utilize.
Behrens, H.M., Gradmann, D. and Sievers A. 1985.
Membrane-potential responses following gravistimulation
in roots of Lepidium sativum L. Planta 163:463–472.
ACKNOWLEDGEMENTS
Bourgeade, P. and Boyer, N. 1994. Plasma membrane H+ATPase activity in response to mechanical stimulation of
Bryonia dioica internodes. Plant Physiology and
Biochemistry 32:661–668.
The authors gratefully acknowledge the support of NASA
and NSF (Simon Gilroy and Gioia D. Massa). Jeremiah
M. Fasano was funded through the Plant Responses to the
Environment training grant from NSF. We are indebted to
Dr. Tatiana Bibikova for critical reading of the manuscript.
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Gravitational and Space Biology Bulletin 16(2) June 2003