Journal of Experimental Botany, Vol. 60, No. 1, pp. 43–56, 2009
doi:10.1093/jxb/ern315 Advance Access publication 16 December, 2008
DARWIN REVIEW
Thigmomorphogenesis: a complex plant response
to mechano-stimulation
E. Wassim Chehab, Elizabeth Eich and Janet Braam*
Rice University, Biochemistry and Cell Biology, 6100 Main St. Houston, TX 77005, USA
Received 2 September 2008; Revised 14 November 2008; Accepted 17 November 2008
Abstract
In nature, plants are challenged with hurricane winds, monsoon rains, and herbivory attacks, in addition to many
other harsh mechanical perturbations that can threaten plant survival. As a result, over many years of evolution,
plants have developed very sensitive mechanisms through which they can perceive and respond to even subtle
stimuli, like touch. Some plants respond behaviourally to the touch stimulus within seconds, while others show
morphogenetic alterations over long periods of time, ranging from days to weeks. Various signalling molecules and
phytohormones, including intracellular calcium, jasmonates, ethylene, abscisic acid, auxin, brassinosteroids, nitric
oxide, and reactive oxygen species, have been implicated in touch responses. Many genes are induced following
touch. These genes encode proteins involved in various cellular processes including calcium sensing, cell wall
modifications, and defence. Twenty-three per cent of these up-regulated genes contain a recently identified
promoter element involved in the rapid induction in transcript levels following mechanical perturbations. The
employment of various genetic, biochemical, and molecular tools may enable elucidation of the mechanisms through
which plants perceive mechano-stimuli and transduce the signals intracellularly to induce appropriate responses.
Key words: ABA, auxin, brassinosteroids, calcium, ethylene, jasmonates, nitric oxide, ROS, thigmomorphogenesis, touch.
Introduction
When animals encounter adverse or life-threatening circumstances, they often react by relocating to a more favourable
environment. Plants do not have this luxury of high
mobility. They are non-motile organisms that are persistently challenged by a wide spectrum of environmental
stresses. These stimuli can be extremely detrimental to plants
if they had not evolved mechanisms to sense and respond to
their dynamic surroundings (Liscum, 2002). Examples of
challenges related to mechanical force include wind, physical barriers, and predation. Initially, plants have to sense
these stimuli and subsequently launch appropriate responses
by either avoiding obstacles, clinging to supporting structures, or producing toxic chemicals to fend off herbivorous
predators. In 1881, Charles Darwin reported on mechanostimulus-induced plant behaviour. In The power of movement in plants, Darwin described in detail directed plant
growth in response to external stimuli (Darwin and Darwin,
1881), including how roots of many plant species reorient
their growth direction upon making contact with barriers.
Such observations were fascinating to Darwin and continue
to be an active and intriguing area of research.
Mechanical perturbations are among the many environmental stimuli to which plants respond. Plants sense forces
ranging from very intense and physically damaging to more
subtle, moderate ones. Many studies have focused on plant
responses to wounding, a tissue-damaging mechanical perturbation often used to simulate insect and microbe attacks.
Plants sense and respond to mechanical stimuli immediately,
as well as over time, by synthesizing an array of phytohormones and other chemicals in addition to expressing defencerelated genes that decrease herbivore ability to colonize, feed,
and/or reproduce (Green and Ryan, 1972; Karban and
Baldwin, 1997; Chen et al., 2005; Chehab et al., 2006, 2008).
Similar responses occur in plants stimulated by more subtle
mechanical cues including touch. In addition to the production of the phytohormones and expression induction of
* To whom correspondence should be addressed: E-mail: [email protected]
ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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44 | Chehab et al.
defence-related genes, touched plants also respond physically
to the stimulus, but with varying degrees depending on the
plant species examined. Some responses are very rapid and
highly noticeable due to the presence of specialized cells
which constitute part of the plant touch response machinery
(Braam, 2005). For example, touching Mimosa pudica will
cause leaf folding within 1 s (Fig. 1) and disturbing the
trigger hairs on a Venus’ Fly Trap leaf will cause the trap to
close within the same time frame.
Thigmomorphogenesis
Plants without specialized sensory cells also respond to
mechanical perturbations. However, they react slowly over
time by altering their morphology as well as their growth rate.
Salisbury (1963) reported that repeatedly touching the leaves
of young cocklebur plants caused a 30% inhibition in growth
in addition to an increase in the rate of leaf senescence. Mark
Jaffe was the first to introduce the term ‘thigmomorphogenesis’ to describe these mechanically-induced responses
(thigma is the Greek word for touch) (Jaffe, 1973). Thigmomorphogenesis in higher plants is generally a slow response
occurring over time and, unlike the responses of Mimosa or
the Venus Fly Trap, touch-induced morphological changes
are not readily apparent immediately after the stimulus
(Jaffe, 1973). Among many plant species, a common thigmomorphogenetic response includes a decrease in shoot elongation coupled to an increase in radial expansion (Telewski and
Jaffe, 1986; Braam and Davis, 1990; Braam, 2005). An example of thigmomorphogenetic changes displayed by Arabidopsis
is shown in Fig. 2. Other responses include alterations in
chlorophyll content, hormone levels, biotic and abiotic stress
resistance, pithiness, flowering time, senescence, and stomatal aperture (Biddington, 1986). Some thigmomorphogenetic alterations cause strengthening whereas others result
in the relaxation of the perturbed tissue; both responses are
thought to help plants cope or withstand repeated force
stresses (Grace, 1977; Glidden, 1982; Jaffe et al., 1984;
Telewski, 1995; Biddington, 1986; Telewski and Jaffe,
1986). Therefore, thigmomorphogenesis may be an adaptive
response to help plants improve resistance to mechanical
perturbations (Grace, 1977; Glidden, 1982; Telewski and
Jaffe, 1986). Interestingly, different tissues show variations
in their magnitude of responses to mechanical stimuli.
Specifically, young tissues have a stronger thigmomorphogenetic response than older ones (Biddington, 1986). A
possible explanation is that young tissues may be more
fragile and susceptible to stresses and thus must respond
strongly to survive harsh environmental stimulations.
Although thigmomorphogenesis is generally perceived as
a slow response to mechanical perturbation, very fast
physiological responses, not observable to the naked eye,
have been reported. For example, Jaffe (1976) detected
changes in electrical resistance within a few seconds of
touch. Furthermore, Jaeger et al. (1988) documented the
inhibition of phloem transport within 2 min following
mechanical perturbation.
Cellular signalling
Plant thigmomorphogenetic responses to mechanical stimulations have been reported to be saturable (Beryl and
Mitchell, 1977), dose-dependent (Jaffe, 1976), and systemic,
i.e. the stimulus and its subsequent response translocate
from plant regions directly stressed to non-disturbed distal
regions (Erner et al., 1980). Furthermore, perturbed plant
symptoms of altered morphology and growth rate can be
mimicked or antagonized by applications of specific chemical compounds (Erner and Jaffe, 1982; Boyer et al., 1983;
Biro and Jaffe, 1984). Altogether, these data suggest that
plant responses to mechanical perturbations are mediated
by signalling molecules. Hormones, secondary messengers,
nitric oxide (NO), reactive oxygen species (ROS), as well as
lipid-derived metabolites have been implicated as potential
signalling factors. These factors and the potential implications in plant responses to mechanical perturbations are
discussed here.
Calcium
Calcium (Ca2+) is a universal signal transduction molecule.
Its signalling capabilities are implicated in plant responses
Fig. 1. Mimosa pudica (sensitive plant). (A) Opened leaves before stimulation. (B) Folded up leaves after touching.
Plant thigmomorphogenesis | 45
Fig. 2. Thigmomorphogenesis in Arabidopsis. Mechanically stimulating Arabidopsis plants by touching them three times daily
results in thigmomorphogenetic responses characterized by
stunted growth, inhibition of inflorescence elongation, as well as
a delay in the timing of the transition to flowering as compared to
non-stimulated plants.
to diverse stimuli. Plant cells cannot tolerate the levels of
Ca2+ found outside the cells (10 3 M), so cytosolic Ca2+ is
kept at low concentrations (<1 lM) by active removal to the
extracellular space or to intracellular organelles such as the
endoplasmic reticulum (ER) and vacuole. Cells capitalize on
the consequent Ca2+ gradient across these membranes and
can rapidly and efficiently generate cytosolic Ca2+ influx
through the opening of Ca2+ channels present in the plasma
membrane (Ding and Pickard, 1993) and intracellular
organellar membranes, such as that of the ER and vacuole
(Knight et al., 1992; Klusener et al., 1995).
The immediate, transient, and dose-dependent increase in
cytosolic Ca2+ following mechanical perturbations has led
scientists to propose that Ca2+ may play a role in plant sensing and subsequent responses to such stimuli (Toriyama and
Jaffe, 1972; Knight et al., 1991, 1992; Trewavas and Knight,
1994; Batiza et al., 1996; Legue et al., 1997; Calaghan and
White, 1999; Berridge et al., 2000; Fasano et al., 2002). For
example, Allen et al. (1999) reported that mechanically
stimulating Arabidopsis roots causes a transient and local
increase in intracellular Ca2+. Ca2+ sensors have also been
implicated in gravitropic and mechanical stresses (Sinclair
et al., 1996), further suggesting the involvement of Ca2+ as
a second messenger in the roots’ signalling mechanisms in
response to a range of stimuli (reviewed by Fasano et al.,
2002). Intriguingly, the intracellular Ca2+ levels in animal
cells also increase following mechano-stimulation. Opening
of stretch-activated Ca2+ channels has been proposed to be
responsible (Sachs, 1986). Similar channels have been
identified on plant plasma membranes (Gens et al., 2000).
Calcium-induced calcium release channels are also present
on vacuolar membranes (Ward and Schroeder, 1994).
Mechanically stimulated transgenic plants expressing
aequorin, a Ca2+-dependent bioluminescent protein, show
rapid and transient intracellular Ca2+ increases derived
from internal pools (Knight et al., 1992). However, recent
studies support a role for plasma membrane stretchactivated channels in plant responses to mechano-stimulations
(Hayashi et al., 2006). One possibility is that, following
mechanical perturbations, small bursts of Ca2+ may enter the
cytosol from the extracellular space through plasma membrane stretch-activated channels; the resulting overall increase
in Ca2+ may be below the level of aequorin detection.
However, these Ca2+ bursts may be sufficient to gate calciuminduced calcium release channels, causing the influx of large
amounts of Ca2+ from intracellular stores.
Increases in intracellular Ca2+ need to be sensed to
function in signal transduction. Braam and Davis (1990)
discovered an intimate connection between Ca2+ sensor
proteins and mechano-stimulation. Arabidopsis plants
mechanically perturbed by wind, rain or touch induce the
expression of at least four touch (TCH) genes within 10–30
min post-stimulation. Three of the TCH genes encode
potential Ca2+ sensors: calmodulin (CaM) and calmodulinlike (CML) proteins. More recently, Lee et al. (2005)
performed a comprehensive transcriptome analysis in Arabidopsis and identified 12 CML genes whose expression is
up-regulated in response to touch. Consistent with the
possibility that CaM and CMLs may have roles in touch
responses, inhibitors of CaMs and possibly a subset of CMLs
block mechano-stimulated thigmomorphogenetic responses
(Jones and Mitchell, 1989). Therefore, CaMs as well as
CMLs are potential sensors of cytosolic Ca2+ changes
induced by mechanical perturbations. Following increases
in intracellular Ca2+, these proteins may be activated by
binding Ca2+ and, consequently, modulate target enzyme
activities to mediate the appropriate physiological responses.
Our laboratory has previously performed a genome-wide
analysis to identify and classify Arabidopsis CaM and CML
genes. The presence of 50 CMLs and 7 CaMs has been
reported (McCormack and Braam, 2003; McCormack
et al., 2005). Some of the encoded products have altered
Ca2+ binding EF-hand motifs. For example, CaM1 potentially has four functional EF hands whereas CML1,
CML28, CML13, and CML12 have 1, 2, 3, and 6 predicted
EF hands, respectively (McCormack and Braam, 2003). EFhand motifs are central to the function of Ca2+ sensors.
Modifications in such a vital region of the protein may
reflect potential variations in binding affinity for Ca2+. One
possibility is that the intracellular Ca2+ levels may selectively
activate specific CaMs and/or CMLs depending on their Ca2+
affinity. As a consequence of Ca2+ binding, CaMs and/or
CMLs are predicted to undergo an alteration in structure
(Babu et al., 1985; Wriggers et al., 1998, Delk et al., 2005;
Tsai et al., 2007) and reveal hydrophobic surfaces with
which they interact with target proteins. McCormack and
colleagues further compiled data from Expressed Sequence
Tag (EST) databases as well as from 1900 Arabidopsis
Affymetrix microarray chip experiments and concluded that
these potential Ca2+ sensor encoding genes are expressed
under different stimuli and have distinctive developmental
as well as spatial expression patterns (McCormack and
Braam, 2003; McCormack et al., 2005). For example,
CaM1, 2, 3, and 4 are expressed in siliques and leaves,
46 | Chehab et al.
whereas CaM1 transcripts are in roots. CML7, 8, and 9 are
expressed in flowers, leaves and developing siliques; CML10 is
expressed in leaves (Ling et al., 1991; Perera and Zielinski,
1992; Gawienowski et al., 1993; Ling and Zielinski, 1993;
Zielinski, 2002; McCormack and Braam, 2003; McCormack
et al., 2005). CML23 and CML24, which encode proteins
that are 78% identical, are both expressed in root tissue;
however, CML23 is more restricted to the outer epidermal
regions and the extreme root tip; whereas CML24 expression is absent from the distal tip but is in the meristematic
and elongation zones (Delk et al., 2005; Tsai et al., 2007).
Furthermore, Chiasson et al. (2005), using GUS staining
analyses, showed that exogenous application of methyl
jasmonate (MeJA) on Arabidopsis plants results in activating the promoter of CML39 but not that of CML37 or
CML38. Therefore, the differences in the predicted CaM
and CML structures, in addition to the variations in their
transcript expression patterns, indicate that these Ca2+
sensors are likely to have evolved distinct in vivo functional
roles, some of which might be involved in tissue-, developmental-, and/or dose-specific thigmomorphogenetic
responses to mechano-stimulations.
Jasmonates
Jasmonates (JAs) are a family of cyclopentanone derivatives
synthesized from linolenic acid via the octadecanoid pathway.
These lipid-derived metabolites, which include jasmonic acid
(JA), its methyl ester (MeJA), and 12-oxo-10,15-phytodienoic
acid (12-OPDA) have been implicated in plant thigmomorphogenetic responses to mechano-stimulations. Recently,
Tretner et al. (2008) reported that touch-treated Medicago
truncatula plants show an increase in chlorophyll content and
stunted shoot growth in addition to an increase in biomass.
These thigmomorphogenetic alterations reflect similar phenotypes observed in Arabidopsis cev1 mutant plants producing constitutively high levels of JA and 12-OPDA (Ellis
et al., 2002). Stelmach et al. (1998) have also reported that
application of coronatine, a 12-OPDA analogue, on Phaseolus vulgaris elicits physiological changes reminiscent of
thigmomorphogenesis. Furthermore, mechanically impeding root growth causes temporary inhibition of root
elongation coupled to an increase in JA (Staswick, 1992;
Berger, 2002; Yan et al., 2007). JAs are also implicated in
sensing and subsequent coiling of the Bryonia dioica tendrils
(Weiler et al., 1993), a physiological response produced by
touch. Intracellular MeJA levels in coiling tendrils are more
elevated compared with those from already coiled ones.
Furthermore, the application of MeJA, or its precursor 12OPDA, on B. dioica also elicits a tendril response (Weiler
et al., 1993).
What might trigger the increase in the jasmonate levels
following such perturbations? Touch causes an increase in
lipoxygenase (LOX) transcripts as well as those of other
genes constituting part of the JA biosynthetic pathway
(Mauch et al., 1997; Lee et al., 2005; Tretner et al., 2008).
LOX is responsible for the production of jasmonate
precursors from free polyunsaturated fatty acids. As pre-
viously discussed, mechano-stimulation also increases cytosolic Ca2+ levels. Therefore, the elevated Ca2+ levels may be
responsible for the localization of phospholipase D (PLD)
to membranes. Membrane-associated PLD is active and
releases free polyunsaturated fatty acids from membrane
phospholipids (Vick, 1993; Ryu and Wang, 1996; Creelman
and Mullet, 1997). These unesterifed fatty acids are acted
upon by LOX to initiate the jasmonate biosynthetic
pathway. This scenario provides a potential mechanism for
the increase in MeJA and OPDA levels in response to
mechano-stimulations (Stelmach et al., 1998; Chehab et al.,
2008). A similar mechanism has been reported in animal
cells whereby Ca2+ stimulates eicosanoid biosynthesis by
activating LOX and phospholipase (Taylor et al., 1990).
Altogether these findings suggest that subsequent to
mechanical stimulations, JAs might be playing a role in the
transduction pathway, thus coupling the stimulus to the
observed thigmomorphogenetic responses. However, to identify the specific function(s) of these lipid-derived metabolites in the touch-induced responses, it is necessary to
perform further investigations. Mutants defective in their
ability to synthesize JAs such as aos (Park et al., 2002;
Chehab et al., 2008) and opr3 (Stintzi and Browse, 2000) or
that are insensitive to these compounds, such as coi1 (Feys
et al., 1994), will be valuable in further investigating the
role(s) JAs play in the mechano-responsive pathway.
Ethylene
Ethylene is a gaseous hormone that regulates a variety of
plant processes including aspects of metabolism and development as well as defence (Ecker and Davis, 1987; Abeles
et al., 1992; Bleecker and Kende, 2000). Ethylene is the
hormone that has been studied for longest with respect to its
involvement in plant responses to touch (Mitchell and
Myers, 1995). Thigmomorphogenetic changes mimic those
that occur subsequent to ethylene exposure (Goeschl et al.,
1966; Brown and Leopold, 1972; Jaffe and Biro, 1979; Erner
and Jaffe, 1983; de Jaegher et al., 1987; Abeles et al., 1992;
Telewski, 1995). For example, frequently rubbing tomato
plant internodes causes bending of the petiole, whereas
repeatedly touching or physically impeding the growth of
bean plants results in a significant increase in stem diameter
as well as stunted growth (Jaffe, 1973, 1976), all of which are
thigmomorphogenetic characteristics resembling aspects of
ethylene exposure. Ethylene release also increases in many
different plant species following mechanical perturbation
(Goeschl et al., 1966; Brown and Leopold, 1972; Robitaille
and Leopold, 1974; Biro and Jaffe, 1984; Takahashi and
Jaffe, 1984; Telewski, 1995; Onguso et al., 2006). Physically
impeding maize roots results in an increase in root ethylene
production coupled to aerenchyma formation (Sarquis et al.,
1991; He et al., 1996). Furthermore, dark-grown pea plants
produce large amounts of ethylene as they push up through
glass beads compared to unimpeded plants (Goeschl et al.,
1966). Goeschl et al. (1966) also found that ethylene application on pea plants acts as a substitute for mechanical
stimulation and elicits a thigmomorphogenetic dwarf
Plant thigmomorphogenesis | 47
response. Intriguingly, 1-aminocyclopropane-1-carboxylate
synthase (ACS), encoding a key enzyme in the ethylene biosynthetic pathway, is rapidly up-regulated following mechanical stimulation (Biro and Jaffe, 1984; Botella et al., 1995;
Arteca and Arteca, 1999). If increased ACS expression leads
to enhanced ethylene production, this transcriptional change
may be an important regulatory step in thigmomorphogenesis induction.
A key question to answer is whether ethylene plays an
early transduction role in the mechanical perturbation
response. Whereas transcriptional induction of mechanoresponsive genes occurs within 5–30 min post-stimulation,
ethylene production peaks at ;2 h in beans (Jaffe and Biro,
1979) and 9 h in Pinus taeda (Telewski and Jaffe, 1986)
following stimulation. This delay in ethylene production
suggests that ethylene is unlikely to be a primary factor in
the mechano-response signal transduction pathway (Boyer
et al., 1983; Biro and Jaffe, 1984, 1986; Biddington, 1986;
Johnson et al., 1998).
Inhibitor and mutant studies are valuable in order to reveal
potential roles for ethylene in mechano-responses. Mutants
defective in the ethylene response pathway show touchinducible decreases in elongation growth (Johnson et al.,
1998; Coutand et al., 2000). Therefore, the ethylene pathway
is not required for this particular response following
mechano-perturbations (Johnson et al., 1998; Coutand et al.,
2000). Furthermore, increases in touch-inducible expression
of at least the subset of genes examined still occur in the
ethylene response mutants (Johnson et al., 1998). Therefore,
although aspects of thigmomorphogenesis, such as increases
in radial expansion, may be regulated through ethylene,
many features of plant touch responses are independent of
this phytohormone’s regulation.
The in vivo interactions of ethylene are complex, as
ethylene can cross-talk with other growth regulators, such as
auxin (Rahman et al., 2001), abscisic acid (Beaudoin et al.,
2000; Ghassemian et al., 2000), cytokinins (Cary et al., 1995),
and gibberellins (Rahman et al., 2000). Therefore, a concerted
action of ethylene, in addition to other signalling molecules,
may constitute the machinery that is important for the full
morphological responses observed in plants subjected to
mechanical perturbation.
are more drought resistant as compared to untreated plants.
The authors attributed these developmental and physiological changes to elevated in vivo ABA levels (Whitehead, 1962;
Weyers and Hillman, 1979).
Although some evidence suggests that ABA is involved in
the plant thigmomorphogenetic responses, further experiments are needed to unravel ABA function in touchinduced changes. ABA-insensitive and biosynthetic mutants
are powerful tools to reveal potential role(s) for ABA in
plant responses to mechanical force.
Auxin
The functional role of auxin in plant tropisms has been an
active area of research for at least the past eight decades
(Esmon et al., 2006; Lucas et al., 2008). Auxin has been
implicated in plant thigmomorphogenetic responses. Mechanical stimulation of soybean and pea plants reverses
auxin-promoted shoot elongation (Victor and Vanderhoef,
1975; Mitchell, 1977). Furthermore, Hofinger et al. (1979)
demonstrated that auxin, normally present in the lower
internodes of the wild cucumber, Bryonia, disappears when
the plants are mechanically stimulated. A major mechanism for auxin turnover is peroxidase-mediated oxidative
decarboxylation (Normanly et al., 1997; Ostin et al., 1998).
An increase in the peroxidase activity occurs in mechanically perturbed plants (Hofinger et al., 1979; Boyer et al.,
1979). Therefore, Boyer and colleagues proposed that
peroxidase-driven catabolism of auxin is stimulated in the
lower internodes of Bryonia upon the mechanical application of force (Boyer et al., 1979).
The potential Ca2+ sensor, CML12, first identified as
TCH3 (Braam and Davis, 1990; Braam, 1992; Sistrunk
et al., 1994; Antosiewicz et al., 1995) interacts with and
regulates the activity of pinoid (PID) (Benjamins et al.,
2003). PID is a serine/threonine protein kinase that potentially acts as a switch in regulating the activity of the PIN
family of auxin regulators (Bennett et al., 1996; Christensen
et al., 2000; Benjamins et al., 2003; Friml et al., 2004). This
relationship between a Ca2+ sensor encoded by a touchinducible gene and a protein involved in auxin transport
represents an intriguing connection between auxin signalling
and thigmomorphogenesis.
Abscisic acid
Abscisic acid (ABA) is another plant hormone that regulates
stress responses and developmental processes (Giraudat,
1995). The in vivo accumulation of ABA retards and/or suppresses plant growth, phenotypes also observed in thigmomorphogenesis. The mechanical perturbation of different
plant species results in thigmomorphogenetic responses
coupled with the accumulation of ABA (Jeong and Ota,
1980; Erner and Jaffe, 1982). For example, mechanostimulation of bean plants results in increased ABA levels
followed by growth retardation (Erner and Jaffe, 1982). A
similar growth response is observed subsequent to exogenous
ABA application (Erner and Jaffe, 1982). Furthermore,
mechanically stimulated rice plants have closed stomata and
Brassinosteroids
Brassinosteroids (BRs), comprising brassinolide as well as
other related compounds, have been implicated in regulating vegetative growth, seed germination, as well as other
physiological processes (reviewed by Haubrick and
Assmann, 2006). BRs have also been linked to plant
thigmomorphogenesis. Exposing Arabidopsis plants to 24epibrassinolide, a highly active BR (Mandava, 1988), results
in the up-regulation of TCH4 (Xu et al., 1995; Iliev et al.,
2002). TCH4 encodes a xyloglucan endotransglucosylase/
hydrolase (XTH) (Xu et al., 1995; Purugganan et al., 1997;
Campbell and Braam, 1998; Rose et al., 2002), an enzyme
predicted to have a role in cell wall modification.
48 | Chehab et al.
Touch-inducible genes, such as TCH4 (also known as
XTH22), that may affect cell wall properties have been
suggested to play a role in Arabidopsis thigmomorphogenesis
(Xu et al., 1995; Purugganan et al., 1997). Recently, Arteca
and Arteca (2008) reported that the application of brassinolide in combination with auxin on Arabidopsis plants results
in the increased production of ethylene, a phytohormone
suggested to play a role in aspects of thigmomorphogenesis.
Therefore, the 24-epibrassinolide-mediated induction of
TCH4/XTH22 expression, as well as the involvement of
brassinolide in increasing the production levels of ethylene,
suggest the involvement of BRs in thigmomorphogenesis.
Nitric oxide
Nitric oxide (NO) is involved in regulating various developmental and physiological processes in plants, including seed
germination, cell differentiation, transition to flowering, and
senescence (Stamler et al., 1992; Leshem, 1996; Keeley and
Fotheringham, 1997; Gouvea et al., 1997; Kojima et al.,
1998; Van Camp et al., 1998; Beligni and Lamattina, 2000;
He et al., 2004). NO is also important in plant defence
responses against invading pathogens (Dangl, 1998; Durner
and Klessig, 1999; Ma et al., 2008). The biosynthesis and
function of NO has been well studied in animals. NO production in animal cells is regulated by Ca2+/CaM. The
mechanism of NO production in plants remains unclear
(Zemojtel et al., 2006; Moreau et al., 2008). However, Garces
et al. (2001) reported that mechanically stressing Arabidopsis
cells results in high NO production. Since mechano-stress
causes intracellular Ca2+ fluctuations as well as transcriptional induction of CaM and CML genes in plant cells, it
suggests that, similar to animal cells, these Ca2+ sensors may
also have roles in NO production/regulation in plants (Braam
and Davis, 1990; Knight et al., 1992; McCormack and
Braam, 2003; Delk et al., 2005; Lee et al., 2005; McCormack
et al., 2005). Our laboratory has previously shown that
CML24, also known as TCH2, is transcriptionally induced
following touch (Braam and Davis, 1990). Tsai et al. (2007)
report that Arabidopsis CML24 mutants have aberrant NO
levels. More recently, Ma et al. (2008) show that applying the
CaM antagonist (W7) to Arabidopsis plants impairs NO
generation in the innate immune response. A CML24 loss-offunction mutant is also defective in pathogen-induced NO
accumulation (Ma et al., 2008); thus the function of CML24
is required for appropriate NO production and accumulation
in the innate response to pathogens. Altogether, these findings implicate CML24 in transducing Ca2+ signals important
for regulating NO accumulation.
Reactive oxygen species
Plants subjected to various environmental stresses, including
mechanical stress,
accumulate hydrogen peroxide (H2O2) and
superoxide O2 , reactive oxygen species (ROS) (Legendre
et al., 1993; Yahraus et al., 1995; Legue et al., 1997; GusMayer et al., 1998; Bergey et al., 1999; Bolwell, 1999;
Orozco-Cardenas and Ryan, 1999; Jih et al., 2003). For
example, the mechanical stimulation of Mesembryanthemum
crystallinum leaves causes an increase in the in vivo levels of
H2O2 (Slesak et al., 2008). Furthermore, the application of
physical stress on soybean cell cultures results in elevated
ROS levels (Yahraus et al., 1995). Cells may utilize ROS as
signalling molecules to regulate the expression of genes (Van
Breusegem et al., 2001). Recently, Mori and Schroeder (2004)
reported that ROS may control Ca2+-permeable channel
activity, thus suggesting a role for ROS in intracellular Ca2+
regulation. As previously discussed, Ca2+ potentially plays an
important role in mediating the touch-induced responses
following mechanical stimulation. The coincidence of ROS
and Ca2+ changes following mechano-stimuli suggests that
these molecules may be functionally linked and that both
may play a role in plant touch responses.
It is unlikely that a single signalling factor controls the full
suite of the plant touch responses. More likely, it is the
concerted action of ROS, Ca2+, and plant hormones that
function interdependently to generate the observed touchinducible morphological and physiological responses.
Identifying touch-inducible genes
Thigmomorphogenesis, characterized by the many physiological and morphological changes that occur with
mechano-stimulation, probably requires alterations in gene
expression. Braam and Davis (1990) first reported the
unexpected existence of touch-inducible genes in plants. A
differential cDNA library screen led to the discovery of
touch-inducible (TCH) genes, genes that are rapidly upregulated in expression following touch and other forms of
mechano-stimulation (Braam and Davis, 1990). TCH1
encodes one of the Arabidopsis CaMs (CaM2) (Braam and
Davis, 1990; Lee et al., 2005), TCH2 and TCH3 encode
CML proteins (CML24 and CML12, respectively) (Braam
and Davis, 1990; Sistrunk et al., 1994; Khan et al., 1997;
McCormack and Braam, 2003; Delk et al., 2005;
McCormack et al., 2005) and TCH4, described earlier in
this report, encodes a xyloglucan endotransglucosylase/
hydrolase (Xu et al., 1995; Purugganan et al., 1997; Campbell and Braam, 1998; Rose et al., 2002). Additional genes
were subsequently identified to have mechano-sensitive
expression, such as ACC synthase (Arteca and Arteca,
1999; Tatsuki and Mori, 1999) as well as others encoding
protein kinases (Botella et al., 1996; Mizoguchi et al., 1996).
These studies were not conducted to saturation and thus
identified only a limited number of the touch-inducible
genes. A genome-wide screen to reveal the extent of touch
inducibility among all Arabidopsis genes was reported by
Lee et al. (2005). Over 2.5% of the Arabidopsis expressed
genes are up-regulated at least 2-fold in response to simple
touch stimulation. Lee et al. (2005) identified Ca2+-binding
protein genes and cell wall-associated protein genes as the
most highly represented functional classes of the touchregulated genes. Twelve out of 48 CML genes present on
the Affymetrix chip are up-regulated in response to touch.
Transcriptional increases for genes encoding Ca2+-binding
Plant thigmomorphogenesis | 49
proteins are perhaps not expected because Ca2+ sensors are
thought to be regulated post-translationally through the
perception of cytosolic Ca2+ changes. Thus, both transcriptional and post-translational regulation mechanisms are
likely to impact CaM and CML functions.
In addition to potential Ca2+ sensor genes, genes encoding
cell wall modification enzymes are also highly represented
among those up-regulated in expression by touch stimulation.
Nearly half of the 33 XTH genes are induced in expression
by touch. XTHs catalyse the cleavage and religation of
xyloglucan polymers in the plant cell wall, thus possessing
the potential to modify wall architecture (Campbell and
Braam, 1998; Steele et al., 2001). Xyloglucans hydrogen
bond to cellulose microfibrils, thus cross linking them. The
cellulose/xyloglucan structure provides cell wall integrity
(Rose et al., 2002). Plant cell shape, size, and form are
largely determined by the plant cell wall. Thus, enzymes that
regulate wall architecture are central to the determination of
plant morphology. Altered expression of cell-wall modifying
genes, such as XTHs, in response to mechanical perturbation may, therefore, be critical for the consequent morphological changes that manifest in thigmomorphogenesis.
The third most represented gene class among touchregulated genes is the one with potential roles in disease
resistance (Lee et al., 2005). JA signalling as well as JArelated compounds play roles in plant defence responses by
activating defence-related genes (Chehab et al., 2008; Howe
and Jander, 2008). Therefore, the up-regulation of these
defence-relevant genes in mechano-stimulated plants might
be due to the increased production of molecules involved in
the defence-related signal transduction pathway. Furthermore, the expression induction of about 10% of kinase and
transcription factor genes following touch suggests an active
role for the kinase transduction pathways and downstream
transcriptional activities in launching the appropriate
responses to such perturbations (Lee et al., 2005).
As stated previously, roots also respond to mechanical
stimulations. In an attempt to identify the genes specifically
involved in such underground stresses, Kimbrough and
colleagues performed expression profiling studies on
mechanically perturbed roots (Kimbrough et al., 2004). Out
of 22 744 genes present on the Affymetrix GeneChip, 1696
had altered transcript abundance. Interestingly, 96% of
these genes are also transcriptionally regulated by gravitropism (Kimbrough et al., 2004). This observation is consistent with evidence that suggests the sharing of common
mechano-transduction elements between gravitropic- and
mechano-responses (Mullen et al., 2000; Moseyko et al.,
2002; Massa and Gilroy, 2003). Genes specifically induced
following root mechanical perturbations are only 26 in
number. The presence of such root-induced mechanospecific regulated genes that are distinct from those
controlled by gravity might serve initially to perceive the
distinct mechano-perturbation events and possibly initiate
a general signal transduction pathway shared by gravitropic
stimulations ultimately to result in root reorientation.
Recently, Walley and colleagues performed whole genome microarray analyses to monitor Arabidopsis gene
expression changes within 5 min post-wounding, a stimulus
that shares mechanical properties with touch (Walley et al.,
2007). The aim of this work was to begin identifying the
primary stress signal transduction pathway components
following mechanical perturbation. Approximately 160
Rapid Wound Responsive (RWR) genes were identified.
Analyses of sequences 5’ to predicted translational start
sites revealed an over-represented Rapid Stress Response
Element (RSRE) motif. This motif, when used with
a minimal transcriptional promoter, is sufficient to confer
an in vivo rapid transcriptional response to a wide range of
stresses including wounding. Touch was not investigated.
However, transgenic plants with a multimerized RSREcontaining 5’ region driving LUCIFERASE (LUC) also
show an apparent touch response. Plants that were not
wounded but to which a drop of water, oligouronides
(OGA), or insect regurgitate was added, show a response
higher than the background but lower than when plants are
mechanically wounded. Furthermore, 23% of the upstream
regions of touch-inducible genes reported by Lee et al.
(2005) contain the RSRE cis-element (EW Chehab and J
Braam, unpublished results). The presence of the RSRE in
touch-inducible genes suggests that this motif might also
play a role in the signal transduction of mechano-stimulation leading to gene expression changes.
Comparison of the RWR genes with the transcript profile
of the touch-induced genes obtained by Lee et al. (2005)
reveals 47 common ones (EW Chehab and J Braam, unpublished results). These common genes include ones involved in the ethylene pathway, such as ACC SYNTHASE 6
and ETHYLENE RESPONSE FACTORS (ERF2 and
ERF4), auxin signalling such as AUXIN RESPONSE PROTEIN gene (ARP), and finally Ca2+ signalling such as TCH3
(CML12), CALCIUM-DEPENDENT PROTEIN KINASE
32 (CDPK32) and CML38. These data indicate that there is
an overlap in genes up-regulated in expression by touch and
wound stimuli. Thus, the initial response to touch or
wounding may be mediated through similar signal transduction factors and machinery, potentially involving Ca2+
and/or phytohormones. However, the distinctive gene expression patterns between the two microarrays might be attributed to the magnitude of the stimulus, with touch being
milder than wounding, and/or to the temporal expression
difference following stimulation.
As previously discussed, many genes have been suggested
through microarray analyses to play roles in the plants’
responses to mechano-stimulation. Recently, Perrin et al.
(2007) used activation tagging to identify the microtubuleassociated WAVE-DAMPENED 2 (WVD2) protein as
potentially involved in Arabidopsis thigmomorphogenesis.
Plants constitutively expressing WVD2 exhibit phenotypes
characterized by short and thick stems and roots as well as
reduced cell elongation. The transgenics also show a more
dramatic thigmomorphogenetic response to touch compared
with the wild type.
Many touch-inducible genes are also increased in expression following other stimuli, such as darkness, temperature
extremes, and hormones (Braam and Davis, 1990; Braam,
50 | Chehab et al.
1992; Sistrunk et al., 1994; Antosiewicz et al., 1995; Xu
et al., 1995; Polisensky and Braam, 1996; Delk et al., 2005;
Lee et al., 2005). Although these stresses do not appear to
share mechanical properties with those of touch or wounding, these stimuli induce expression of similar sets of genes.
For example, Lee et al. (2005) reported that ;53% of the
touch-inducible genes showing at least a 2-fold increase in
response to touch are also up-regulated by darkness. TCH2
(CML24) transcripts, for example, increase in abundance
by touch, heat, and cold (Braam and Davis, 1990; Braam,
1992; Polisensky and Braam, 1996). Consistent with the
RNA behaviour, transgenic Arabidopsis plants with the
putative TCH2 regulatory region regulating LUC reporter
expression have increased luminescence following not only
touch stimulation, but also exposure to cold or heat (Fig. 3).
Arabidopsis transgenics containing the ubiquitin regulatory
region driving LUC expression as controls show no
significant changes following similar stimuli (data not
Fig. 3. Activity of TCH2 promoter in response to touch, cold and heat. Soil-grown plants were imaged for 5 min prior to receiving stimuli
(0 min). Plants were then given treatments of cold (4 h at 4 C), heat (1 h at 37 C), or a brief touch. Control plants were treated identically
except for the application of the stimulus. Imaging following stimulation began 5 min after placing plants into the camera chamber and 5
min exposures were acquired every 6 min over 3.5 h. The first image after stimulus (5 min), the average ‘Peak’ activity image (107 min for
cold, 53 min for heat, and 83 min for touch), and the final image (209 min) are shown. Pseudo-colouring of images was performed on
commonly scaled images for each stimulus in WinLight (Berthold). A scale bar representing levels of activity from low (blue) to high (pink)
is shown at the right.
Plant thigmomorphogenesis | 51
shown). These data indicate that temperature stresses, in
addition to mechanical ones, can cause an increase in TCH2
regulatory region activity. However, the LUC reporter
experiment illustrates that expression characteristics, more
specifically spatial patterning, is distinct for the different
stimuli. Whereas the touch and heat responses are strongly
prominent in the plant apex, cold-induced expression is
more widely distributed with luminescence detected
throughout the rosette leaves. The finding that distinct
stimuli result in shared gene expression responses suggests
that either the mechanisms of perception of these diverse
stimuli are shared or that the signal transduction pathways
activated by distinct receptors converge at some step prior
to gene regulation.
A possible mechano-sensing network
How do plant cells sense touch? One hypothesis suggests
that intracellular pressure may play a role in perceiving
mechano-stimulation (Morris and Homann, 2001). A
mechano-stimulus received at the cell wall may cause
alterations in the intracellular pressure. This pressure stimulus may lead to the translocation of subcellular organelles
(Gus-Mayer et al., 1998), thus transducing the stimulus to
a cellular alteration with the potential for downstream
effects. This hypothesis is corroborated by Sato et al.
(1999) who observed that stimulation of a leaf with a glass
capillary induces chloroplasts to migrate away from the site
of contact, possibly due to changes in the intracellular
turgor. Nuclei have also been observed to move closer to
the site of micro needle contact with the cellular surface as
well as to the distorted cell wall (Kennard and Cleary, 1997;
Gus-Mayer et al., 1998).
Jaffe et al. (2002) propose a second hypothesis which
suggests that changes in membrane surface tension transduce the touch stimulation into stretch-activated channel
activity. In addition to membrane stretching, touch may result
in perturbation of connections among the cell wall, plasma
membrane, and the cytoskeleton, thus conducting the extracellular stimulus into an intracellular signal. The plant plasma
membrane has specialized extension regions called the
Hechtian strands composed of actin microfilaments, microtubules, endoplasmic reticulum as well as RGD (Arg-Gly-Asp)containing peptides (Lang et al., 2004). These peptides are
part of integrin-like proteins proposed to connect the plasma
membrane to the cell wall via the cytoskeleton (Lang and
Gunning, 2000; Lang et al., 2004). The stretching and
relaxation of the cell membrane in response to mechanical stimulation of the cell wall is thought to perturb the cell
wall-plasma membrane-cytoskeleton network and relay this
information to the interior of the cell. The presence of
stretch-activated channels as well as calcium-induced calcium
release channels (Edwards and Pickard, 1987; Ding and
Pickard, 1993; Ward and Schroeder, 1994) in plants suggests
the possibility that mechano-perturbations cause increases in
intracellular Ca2+ due to gating of these channels. These
intracellular Ca2+ increases are possibly sensed by Ca2+
sensor proteins, such as CaMs and CMLs, which have the
potential to bind these ions and subsequently interact with
their target enzymes to activate the downstream signal
transduction machinery leading to the mechano-induced
responses characterized by thigmomorphogenesis.
A substantial amount of work remains before unraveling
the signal transduction mechanism(s) through which plants
sense and respond to mechanical stimuli. Bioinformatics,
genetics, biochemistry, physiology, as well as molecular
approaches will aid the elucidation of this intriguing plant
communication between its external world and its intracellular microenvironment.
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