American Journal of 93(10): 1466–1476. 2006.

A UNIFIED HYPOTHESIS OF MECHANOPERCEPTION IN PLANTS1

FRANK W. TELEWSKI2

W. J. Beal Botanical Garden, Department of Biology, Michigan State University, East Lansing, Michigan 48824 USA

The perception of mechanical stimuli in the environment is crucial to the survival of all living organisms. Recent advances have led to the proposal of a plant-specific mechanosensory network within plant cells that is similar to the previously described network in animal systems. This sensory network is the basis for a unifying hypothesis, which may account for the perception of numerous mechanical signals including gravitropic, thigmomorphic, thigmotropic, self-loading, growth strains, , xylem pressure potential, and sound. The current state of our knowledge of a mechanosensory network in is reviewed, and two mechanoreceptor models are considered: a plasmodesmata-based cytoskeleton–plasma membrane– wall (CPMCW) network vs. stretch-activated ion channels. Post-mechanosensory physiological responses to mechanical stresses are also re- viewed, and future research directions in the area of mechanoperception and response are recommended.

Key words: ; gravity; mechanoperception; sound; thigmomorphogenesis; ; turgor pressure; wind.

The ability to sense and respond to physical stimuli is of key gravitational field is proposed to induce internal pressure importance to all living things. Among the common differences at the CPMCW interface, which can be considered environmental stimuli detected by living organisms are light, an external mechanical signal (Staves et al., 1997; MacCleery temperature, and a variety of chemical signals. A number of and Kiss, 1999; Balusˇka et al., 2005). Therefore, a more these stimuli appear to be closely related and can be considered broadly unifying mechanism may underlie graviperception in as physical–mechanical stimuli, that is differences in a plants than that evoked by a hypothesis that relies on how the mechanical force or pressure perceived by the living cell. A mechanical signal is initiated; an actual sensory structure cell may perceive gravity; strains caused by self-loading and within the cell may allow for mechanoperception as the plant is internal growth; mechanical loading by snow, ice, and fruit, reoriented with respect to gravity. Supporting the concept of a wind, rainfall, touch, sound; and the state of hydration within a unified hypothesis for mechanical sensing in the gravitropic cell (turgor pressure). All organisms appear to perceive these response is the work of Massa and Gilroy (2003) who reported mechanical signals, regardless of their taxonomic classification when a root cap came in contact with a horizontal glass plate or life habit (sessile vs. motile). The significant differences (inducing thigmotropic stimulus), the root cells behind the between taxonomic groups, specifically plants and animals, are growing tip began to grow horizontally. This allowed the root found in the individual molecular components of the cap to maintain contact with the plate, while the rest of the root microstructure of the internal cellular sensing network (Jaffe grew over and parallel to the obstacle with a step-like growth et al., 2002; Balusˇka et al., 2003) and in the response of an form. The authors suggested that the gravisensitive cells of the individual organism to each mechanical stimulus. root cap also sense the touch and signal the columella cells to alter their gravitropic response, so that they act together to Internal mechanical forces—The sensing of gravitropic redirect root growth to avoid obstacles while continuing a signals by plants has been studied for 200 years (Knight, 1806). general downward pattern of growth. Since the first study, the elucidation of the mechanism of In plants, gravitropism can occur in either primary or gravitropic perception has been researched in a broad array of secondary tissues. In primary growth, the gravitropic curvature plants from algae to trees and in a variety of plant organs. To results from differential cell elongation on opposite sides of the date, two compelling hypotheses exist regarding gravipercep- displaced organ. In the case of secondary growth, the tion in plants: the starch–statolith hypothesis and the gravitropic response includes the formation of reaction wood; hydrostatic model of gravisensing (for reviews, see Sack, tension wood in porous angiosperms and compression wood in 1991, 1997; Balusˇka and Hasenstein, 1997; Staves et al., 1997; nonporous angiosperms and (Timell, 1986a). MacCleery and Kiss, 1999; Boonsirichai et al., 2002; Drøbak et Tension wood forms on the upper side of a displaced stem and al., 2004). Both hypotheses ultimately rely on the sensing of a is characterized by the formation of gelatinous fibers with mechanical signal at the cytoskeleton–plasma membrane–cell lower lignin content, smaller diameter, and fewer vessels and wall interface (CPMCW) interface. In the case of statoliths, by a realignment of cellulose microfibrils into a vertical falling starch grains or other organelles impact the plasma orientation within the gelatinous layer, which forms inside a membrane thus inducing an internal mechanical signal (Sack, partially developed and lignified S2 layer of secondary cell 1991, 1997; Balusˇka and Hasenstein, 1997; Perbal et al., 2004). walls of gelatinous fibers. Compression wood forms in Similarly, the reorientation of a plant organ within a response to gravity on the lower side of displaced stems and is characterized by tracheids with a thickened secondary cell wall with higher lignin content, a round cross section, 1 Manuscript received 31 March 2006; revision accepted 16 August 2006. The author thanks J. S. Kilgore, L. Koehler, and two anonymous intracellular spaces at cell corners, and a realignment of reviewers for critically evaluating this manuscript. The research was cellulose microfibrils in the S2 layer to a 458 to 608 orientation supported by the National Research Initiative of the USDA Cooperative with respect to the axis of the stem. State Research, Education and Extension Service, grant nos. 2002-35103- The formation of reaction wood in stems, branches, and 11701 and 2005-35103-15269. roots is not an exclusive response to gravity in woody plants. 2 E-mail: [email protected] The formation of reaction wood has also been observed to 1466 October 2006] TELEWSKI—MECHANOPERCEPTION IN PLANTS 1467 develop in branches and stems as a means of reshaping crowns further supported by a study on wood formation in Arabidopsis and as a possible phototropic response (Engler, 1924). Tension in which the application of weight, and thus a compressive wood has been reported to form in the vertical stems of rapidly force on the stem, induced secondary growth in a species that growing poplar (Populus) trees (for a review, see Telewski et only produces herbaceous growth under ‘‘normal’’ environ- al., 1996). Timell (1986c) suggested that the reaction wood mental conditions (Ko et al., 2004). Ko et al. suggested that the may form to keep woody plants in balance with their physical mechanical stimulus of self-weight is perceived by the stem environment (e.g., gravity, wind, and light), subsequently and induces the differentiation of a secondary vascular generating internal growth strains that result in the physical cambium and subsequent formation of secondary xylem and reorientation of woody plant organs. that self-weight may play a more important role in the The maturation of xylem cells in the cambial zone involves development of the woody plant growth habit. the alteration of individual cell lengths. In many instances, there The self-loading induced by the bearing of fruit has been is intrusive growth in which the cells elongate within the shown to influence growth in branches (Alme´ras et al., 2004; relatively rigid structure of the stem, inducing internal Vaast et al., 2005). The forces associated with the bearing of compressive forces (Boyd, 1985; Archer, 1987; Fournier et fruit are primarily perceived in branches and at branch bases al., 1991a, b; Larson, 1994). In other cases, the cells shrink upon and consist of an alternate compressive force on the lower side maturation inducing a tensile force within the stem. The of the branch and a tensile force on the upper side of the branch generation of these internal growth strains is responsible for the so that the branch acts like a cantilever (Wainwright et al., realignment of stems in the gravitropic response, with 1976; Niklas, 1992). The additional load will stimulate compression wood developing a compressive growth strain increased reaction wood formation in the branches. and tension wood forming a tensile growth strain (Wilson, Each living cell within a plant, with its organelles and 1981; Alme´ras et al., 2005). Growth strains also develop in protoplasm, functions mechanically like a water-filled balloon stems aligned vertically with respect to gravity and may (hydrostat), exerting a circumferential tensile force and radial function to maintain mechanical balance within woody plants as compressive force within the plasma membrane and pressing part of a phototropic response, self-loading, or from differential against the surrounding cell wall. The plasma membrane loading caused by crown asymmetry (Archer, 1987). controls turgor by regulating the flow of water and solutes Within a vertically aligned stem, there are two potential between the apoplast and symplast. Turgor pressure can be sources of compressive force loading. The most obvious is due increased (hypertonic) or decreased (hypotonic) by altering the to self-loading along the vertical axis of the stem as a result of osmotic potential of the apoplast or symplast, thus either the accelerating force of gravity. A second compressive force forcing water into a cell increasing turgor or by drawing water has been suggested to be induced by the constrictive nature of out of a cell and decreasing turgor (plasmolysis). Turgor is also bark tissues (referred to as bark pressure), resulting in a radial decreased by drought stress, sometimes resulting in the loss of compressive force that affects xylogenesis in the cambial zone mechanical strength of plant tissues, which results in wilting. (DeVries, 1875). In earlier studies, the radial compressive force Under these conditions, turgor pressure can contribute of a constricting outer bark was hypothesized to increase significantly to the mechanical properties of plants, especially during the growing season from the radial growth of the in soft, non- or low-lignified tissues and organs characteristic cambium and to be responsible for the formation of smaller, of primary growth (for reviews, see Niklas, 1989, 1991). denser latewood cells and the ultimate formation of annual Sensing changes in turgor is crucial to survival in plants. It is growth rings (for a review, see Larson, 1960). In subsequent possible that changes in turgor impart mechanical stresses on studies, this hypothesis was refuted, and annual growth rings the CPMCW, which serves as the mechanosensory network for were found to form in response to external environmental plant cells. Sensing water potential within the plant via internal stimuli including day length and changes in plant growth mechanical stresses at the cellular level could be the means for regulator content (for review, see Little and Savidge, 1987; what is termed hydraulic signaling, providing a faster signaling Roberts et al., 1988; Larson, 1994). Although the bark pressure and stomatal response to the onset of drought stress than could hypothesis appears to bear little on the formation of annual be predicted by a root-generated chemical signal such as growth rings, the application of a compressive force to cambial abscisic acid (Comstock, 2002). Hayashi et al. (2006) provided explants (tissue culture) appears to function in maintaining the data to support the role of both the CPMCW and Ca2þ- structure and organization of the vascular cambium in vitro, mechanosensitive stretch-activated ion channels in plant cells ensuring the continued production of apparently normal xylem under hypotonic and hypertonic conditions. They report (Brown and Sax, 1962; Brown, 1964; Makino et al., 1983). stretch-activated ion channels function in sensing both Additionally, self-loading along a vertical axis contributes hypotonic and hypertonic conditions, where as the CPMCW significantly to development in plants. The ability of a tree to is only involved in sensing hypertonic conditions. perceive its own weight must play a significant role in determining overall allometry and mechanical properties of External mechanical forces—Numerous external mechan- wood (density and elastic modulus) produced by a mechani- ical stimuli can be perceived by an organism. Some are induced cally loaded vascular cambium, in the absence of any lateral by gradients in pressure within the atmosphere and are loading induced by wind or other external mechanical forces responsible for wind, while pressure gradients in aquatic (McMahon, 1973; Wainwright et al., 1976; Niklas, 1992, systems are created by currents or tidal flows. Pressure waves 1994). In a few studies, the application of a compressive force that form sound waves are transmitted in both aerial and induced differentiation of cambium initials within a mass of aquatic environments. Other mechanical stimuli are induced by dedifferentiated callus cells and within graft unions (Lintilhac gravity, such as the accumulation of ice or snow, but do not and Vesecky, 1981; Barnett and Asante, 2000). The notion that necessarily induce a gravitropic response. A third category can self-loading will impact the formation of a vascular cambium be classified as touch, such as that induced by the impact of and subsequent secondary xylogenesis has recently been raindrops, hailstones, other inanimate objects, or by other 1468 AMERICAN JOURNAL OF BOTANY [Vol. 93 organisms. These mechanical stimuli have collectively been If a tree be placed in a high and exposed situation, where it is termed touch or thigmo stimuli and produce a number of much kept in motion by winds, the new matter which it thigmo responses in plants, including thigmomorphogenesis, generates will be deposited chiefly in the roots and lower parts thigmotropism, thigmonasty, and the thigmotactic response of the trunk; and the diameter of the latter will diminish rapidly (Jaffe et al., 2002; Braam, 2005). Once again, the perception of in its ascent. . . . the growth of the insulated tree on the an external touch or mechanical signals depends upon the mountain will be, as we always find it, low and sturdy, and well CPMCW mechanosensing network. calculated to resist the heavy gales to which its situation Vogel (1994) outlined the parameters influencing life in constantly exposes it. moving fluids and provided the fundamentals needed to The alteration in growth and stem allometry in response to understand the physics of fluid dynamics in aquatic systems wind first described by Knight as an increase in radial growth and the atmosphere. As mentioned previously, differentials in and a decrease in height growth would be defined 170 years atmospheric pressure result in pressure waves defined as later by Jaffe (1973) as thigmomorphogenesis and later as the currents of air or wind. Currents in aquatic ecosystems are also thigmomorphogenetic theory (Jaffe, 1984). This term is now pressure waves. Waves in aquatic systems or wind can be used to describe the response of plants to wind and other laminar or turbulent (Vogel, 1994). Due to the greater density mechanical perturbations, including mechanical bending or and viscosity of water, the force imposed by a wave at a given flexing or by touching or brushing by passing animals. Similar velocity on a structure is much greater than by wind. Denny to the topic of gravitropism, the intervening 203 years since the and Gaylord (2002) gave the example of a 2 m s1 (4.5 miles 1 1 printing of Knight’s thigmomorphogenetic study have seen a h ) velocity wave as being roughly equivalent to a 58 m s multitude of papers reporting the influence of wind or other (130 miles h1 or between a category 3 and 4 hurricane) wind 1 mechanical perturbations on plant growth, many of them in terms of applied force. They go on to state that 25 m s summarized in the reviews by Grace (1977), Jaffe (1984, wave velocities are not uncommon in shoreline environments 1985), Biddington (1986), Vogel (1994), Telewski (1995), and that such a wave exerts the equivalent force of that nearly Mitchell (1996), Jaffe et al. (2002), and Braam (2005). The equal to a wind in excess of Mach 2. One of the complexities of perception of a thigmomorphogenetic stress by the mechano- studying the response of plant mechanical stresses imposed by sensing network is rapidly followed by a mechanoresponse wind or wave is dissecting the individual forces acting upon the cascade, which has been shown to be dose dependent (Erner et structure of a plant. Wind places an asymmetric pressure on the al., 1980; Jaffe et al., 1980; Braam and Davis, 1990; Knight et side of a plant creating a cantilever with the rotation point al., 1992; Telewski et al., 1997; Telewski and Pruyn, 1998; located in the root plate (Vogel, 1994). Wind induced sway is Hepworth and Vincent, 1999; Telewski, 2000). The mechano- considered the primary mechanical stress inducing an physiological-response cascade will be addressed later in this alternating compressive and tensional force, with some torsion manuscript. Characterization of the forces within a bending or applied in stems and roots (Telewski, 1995). Pressure waves flexing plant organ can be difficult to characterize and quantify can also displace the stem within the gravitational field long due to the differing nature of the applied force, geometry, and enough to induce a gravitropic response, which should be morphology of the organ and the anisotropic nature of different considered as a secondary wind-induced mechanical stress tissues, which comprise the plant organ as a cellular composite (Telewski, 1995). Due to the neutrally buoyant nature of material (Niklas and Moon, 1988; Beusmans and Silk, 1988; aquatic systems, macrophytes may not require a significant Niklas, 1992; Vogel, 1992; Moulia et al., 1994; Moulia and gravitropic response upon displacement by waves. The weak Fournier, 1997; Coutand et al., 2000; Coutand and Moulia, and highly compliant stems of large algae allow for them to be 2000; Telewski, 2000). In a detailed analysis of the mechanical highly flexible rather than protective and rigid like the stems of stimulus of bending and resulting growth response in tomato most terrestrial plants (Denny and Gaylord, 2002). This highly (Lycopersicon esculentum Mill. Var. VFN8), Coutand and flexible structure allows algae to float back to the vertical Moulia (2000) reported that mechanosensing is both local and orientation when wave action ceases, without the need to scattered through the stem and explained the variability of the induce specialized strain-generating tissues such as reaction growth response by the integrals of the longitudinal strain field wood that is needed to re-orient a terrestrial plant within a within the bending stem. Jaffe et al. (1980), Telewski and gravitational field. Puryn (1998), and Coutand and Moulia (2000) reported a The ability of a plant to respond to wind (for reviews, see logarithmic relationship of the sensory function between the Jaffe, 1985; Biddington, 1986; Vogel, 1994; Telewski, 1995) dose of mechanical stimulus and growth response. Coutand et or waves (Vogel, 1994; Koehl, 1999; Puijalon and Bornette, al. (2000) reported that increasing the force applied to tomato 2004; Puijalon et al., 2005) by altering morphology, anatomy, stems from 0 to 175 g or stem displacement from 108 to 258 did and biomechanical properties enables the plant to withstand not influence the duration of the growth response. Therefore, additional mechanical loading. People have long observed that the dose response appears to be sensitive to the number of wind influences the morphology and growth of plants, perturbations and not sensitive to the amount of force applied especially trees, creating metaphors and lyrics about trees in each individual perturbation (Coutand et al., 2000). growing in windy environments being ‘‘tougher’’ and able to By definition, sound is acoustic energy in the form of an endure hardship. The first scientific study to document the oscillatory concussive pressure wave transmitted through influence of wind on tree growth was published by Knight gases, liquids, and solids. It is audible to the human ear and (1803) in which staked apple trees (Malus) produced less radial falls into the frequency range of 20–2000 Hz. Sound above this growth than trees allowed to sway freely in the wind. When range is classified as ultrasound, and sound below this range is Knight restricted wind-induced motion to the bilateral (north to infrasound. One only needs to hold one’s hand in front of a south), the stem formed an oval with the proportion of 13:11. base speaker or be next to a car in which the volume is turned Knight went on to state (p. 281): up to feel the pressure wave in the near infrasound range. October 2006] TELEWSKI—MECHANOPERCEPTION IN PLANTS 1469

Vegetation is known to absorb acoustic energy (Eyring, 1946; in the plant kingdom, thigmonastic movements, most com- Martens and Michelsen, 1981; Price et al., 1988) and has been monly associated with the rapid movement in plants in employed to deaden the noise of urban environments (Huisman response to touching. Numerous plant traps are thigmonastic and Attenborough, 1991; Attenborough, 2002). However, in nature and were described by Darwin (1893). These include unlike wind or waves, the level of sound energy normally the Venus’ flytrap (Dionaea muscipula Ellis ex L.), first experienced by plants in the environment does not appear to described by Curtis (1834), which produces a trap from a invoke a significant compromising mechanical stress to plant modified . On the abaxial surface of each half of the leaf structure. Mechanical energy imparted to a can traps are three mechanosensing trigger hairs, which, when induce it to sway to its resonant frequencies, usually in the stimulated, close the trap within a second (Brown, 1916). The infrasound range, which will be a function of the height and movement by the modified leaf trap and the tentacles of the mechanical properties of its tissues. These multiple resonance sundew ( rotundifolia L.) incorporates both thigmo- frequencies can be used, in turn, to calculate the height, flexural nastic and thigmotropic responses (Lloyd, 1942). Darwin stiffness, and modulus of elasticity of the stem (for a review, see (1880, 1893) reported that the force imparted to the tentacles Niklas and Moon, 1988). As discussed, plants need to perceive covering the surface of the leaf trap by the weight of a human and respond to wind or wave induced movement to acclimate to hair was sufficient to supply the mechanostimulus to induce a a given environment. They will even sway in a harmonic motion response, yet neither wind nor rain triggered a response. The at low resonant frequencies. Although plants can effectively sensitive plant ( L.) also has a rapid absorb sound and even generate sound via wind-induced thigmonastic response with the rapid folding of its leaflets resonance of various structures such as needles and spines (for and movement of the entire compound leaf at the example, the whispering winds in a pine forest), can they upon touch. Similar thigmonastic responses have been reported perceive sound, and, if perceived, will they respond to sound? Is in Blopytum sensitivum (L) DC (syn. Cassia sensitivum L.) and there a developmental advantage to responding to sound leading in certain Oxalis species (Umrath, 1958). to acclimation to sonic stresses in the environment? Thigmotropic movements, induced by unilateral contact with Ultrasound has been shown to have the greatest effect on another living organism such as a pollinator or mechanical plants, specifically on seed germination (for reviews, see structure, result in alterations in plant growth that include the Davidov, 1961; Timonin, 1966; Halstead and Vicario, 1969; bending of floral parts towards pollinators, the twining of stems Hageseth, 1974; Weinberger and Burton, 1981; Miyoshi and or roots for physical support, and the coiling of tendrils (for Mii, 1988). Timonin (1966) reported that ultrasound treatment review, see Jaffe et al., 2002). altered the viscosity of macromolecule solutions in seeds. Near ultrasound (1.4 kHz, 0.095 kdb) was reported to increase Structure of the mechanosensing network in plant cells— metabolism in chrysanthemum roots, characterized by increas- Morris and Homann (2001) reviewed the concepts of cell es in amylase activity, soluble sugar, and protein (Yi et al., surface area regulation and membrane tension, providing 2003). The treatment of chrysanthemum callus with 1.4 kHz insight into the role of membrane tension in cell biomechanics sound increases indoleacetic acid levels while decreasing and the potential role of membrane tension in mechanoper- abscisic acid levels (Wang et al., 2004). ception in both plants and animals. To provide scale, the The perception and response of plants to sound, more resting tension of a plant protoplast membrane was reported to specifically music, has been a part of folklore (see Weinberger be 0.12 mN m1 (Kell and Glaser, 1993), the force required to and Graefe, 1973) and the source of inspiration for countless activate mechanosensitive channels is approximately 1 mN primary and secondary school student science fair projects m1 (Sachs and Morris, 1998), and the lytic tension for plant beginning in the 1940s (Klein and Edsall, 1965; personal protoplasts is 4 mN m1 (Kell and Glaser, 1993). The observation). The influence of music, a complex mixture of stretching and relaxation of the cell membrane in response to notes, tones, amplitudes, and harmonics, on plant growth has changes in the mechanical environment of cells as a component been the subject of scientific debate for decades. Singh and of mechanosensing fits well with earlier reports of the role of Ponniah (1955a, b, 1963) reported on the stimulatory influence stretch-activated membrane channels in the response of plants of music on plant growth in a number of species. Klein and to mechanical stresses (Edwards and Pickard, 1987; Ding and Edsall (1965) reported no influence of a diverse selection of Pickard, 1993). The perception of a mechanical signal by cells music, from classical to rock and roll, on the growth of Tagetes is a rapid process with a rapid translation of the mechanical erecta L. Weinberger and colleagues conducted a number of force into a biochemical or bioelectric message (Balusˇka et al., studies on the influence of both music and single frequency 2003; Ingber, 2003a, b). Significant progress has been reported sound, both in the audible and ultrasound range, on plant in the elucidation of the molecular basis of mechanosensory growth and seed germination and reported that sound can perception and transduction in animal systems, particularly the influence plant growth (Weinberger and Measures, 1968; physical coupling between the cytoskeleton and cell mem- Measures and Weinberger, 1970; Weinberger and Das, 1971; brane, which provides a continuous structural/mechanical Weinberger and Graefe, 1973; Weinberger et al., 1979; network throughout the cell (for reviews, see Janmey, 1998; Weinberger and Burton, 1981). Most recently, Creath, and Gillespie and Walker, 2001; Balusˇka et al., 2003; Ingber, Schwartz (2004) reported music increased the rate of seed 2003a, b). Jaffe et al. (2002) proposed that a similar germination in zucchini ( pepo L.) and okra mechanosensing network exists within plant cells, linking the (Abelmoschus esculentus (L.) Moench). cytoskeleton–plasma membrane–cell wall structures. Their Touch, defined as the act of making physical contact with model proposed linker molecules within plant cells as RGD another solid object and inducing a mechanical stimulus, leads (arg-gly-asp)-containing peptides, similar to the integrins, to two other thigmoresponses in plants (for reviews, see Jaffe et RGD-containing proteins in multicellular eukaryotes that lack al., 2002; Braam, 2005). This group of two responses to a cell wall. In non-plant and non-fungal systems, the integrins mechanical stimuli includes one of the most dramatic responses facilitate bidirectional signaling and bind to actins (for review 1470 AMERICAN JOURNAL OF BOTANY [Vol. 93 see Balusˇka et al., 2003). In plants, the RGD integrin-like guru et al., 2000). These studies support a role for callose peptide linkages were proposed to connect microtubules to the synthesis in mechanoperception. plasma membrane, which contains Caþþ ion channels. Hechtian The complex structure and function of the actin cytoskeleton strands [plasma membrane sleeves containing endoplasmic in plants and its linkage to the cell membrane and cell wall reticulum, actin microfilaments, and microtubules (Lang et al., continues to be elucidated. One of the roles appears to be in 2004)] then bind the plasma membrane to the cell wall via the endocytosis as well as in signaling (Sˇamaj et al., 2004, 2005). actin-binding, integrin-like peptides attached to the interior of Balusˇka et al. (2005, p 106) proposed that the combined role of the membrane, which facilitated the opening and closing of the actin cytoskeleton in both signaling and endocytosis, stretch-activated membrane channels (Jaffe et al., 2002). combined with the actin- and pectin-rich adhesive domains of Balusˇka et al. (2003) compared and contrasted the linker cross walls at the polar ends of cells, comprise a ‘‘. . .‘plant molecules between the cytoskeleton and cell membrane in developmental synapse’ in which auxin and pectin-derived animal and plant systems. They argued that the failure to find signaling molecules act as plant-specific transmitters for cell- true integrin homologs in plants or fungi precluded their role in to-cell communications.’’ In their review of cell surface area the plant or fungi mechanosensing network (Hussey et al., 2002) and membrane tension, Morris and Homann (2001) reported and that plants may have their own unique set of actin-binding that high tension of the cell membrane promotes exocytosis and proteins. Among the molecules functioning as linkers between low tension promotes endocytosis. These observations lead the cytoskeleton and cell wall, proposed by Balusˇka et al. (2003) Balusˇka et al. (2005) to further propose a mechanical in their model, are cell-wall-associated kinases (WAKs), mechanism to explain the perception of a gravitational signal pectins, arabinoglactan proteins (AGPs), cellulose synthase, in plants. Displacement of a plant cell from the vertical position formins, plant-specific myosins of class VIII, phospholipase D, alters the gravitational loading imposed upon its plasma and callose synthase. In the past three years since Balusˇka et al. membrane, increasing tension or stretching the upper side of (2003) proposed their model of a mechanosensing network the plasma membrane and decreasing tension on the lower side. linking the CPMCW at cross-walls, further research indicates This, the authors propose, will shift the position of the synaptic that plant-specific myosins of class VIII and formins are the domains that secrete auxin, resulting in the observed strongest candidates as the elusive adhesive molecules (Balusˇka accumulation of auxin at the bottom of the displaced cell and Hlavacˇka, 2005). Cross-walls, located at the nongrowing (Friml et al., 2002; Ottenschla¨ger et al., 2003) and the axial end of cells are enriched with plasmodesmata, actin, induction of a gravitropic response. myosin VIII, and profilin (reviewed by Balusˇka and Hlavacˇka, Balusˇka et al. (2003) never included a role for stretch- 2005). Recently, two studies confirmed the role of formins in activated membrane channels in their mechanosensing network nucleation and bundling of F-actin (Michelot et al., 2005; Yi et model. A second model for mechanosensing, based on the adhesion of the cell membrane to the cell wall involves al., 2005). Deeks et al. (2005) reported that the cross walls of arabinogalactan proteins (AGPs), and wall-associated kinase roots, hypocotyls, and shoot cells of Arabidopsis were rich in (WAK), does include stretch-activated channels (Ding and group 1 formins (proteins AtFH4 and AtFH8) and that AtFH4 Pickard, 1993; Pickard and Fujiki, 2005). Gens et al. (2000) binds to profilin, influencing the polymerization of actin. provided evidence from BY-2 tobacco cells that mechanosen- Strengthening the putative role of formins as adhesive sory (stretch-activated) calcium-selective cation channels are molecules linking the cell wall and cytoskeleton is the grouped on the cell membrane and are associated with AGPs extensin-like domain of the group 1 formins predicted to insert and WAKs, which appear to fasten the cell membrane to the into the cell wall (Cvrcˇkova´, 2000; Cvrcˇkova´etal., 2004). cell wall. The grouping of these molecules and channels was Myosin VIII, also located at cross walls and bound to actin termed the plasmalemmal reticulum (Gens et al., 2000). filaments within the cytoplasm, is reported to be involved in Mechanosensory calcium-selective cation channels also have callose synthesis, possibly binding with a membrane-spanning been reported in the guard cells of Vicia (Cosgrove and callose synthase subunit and providing a callosic crosslink Hedrich, 1991) and lily pollen tubes (Dutta and Robinson, between the cell wall and cytoskeleton at cell plates, pit fields, 2004); however, the plasmalemmal reticulum has only been and plasmodesmata (Balusˇka et al., 2003; Balusˇka and observed in the BY-2 cells (Pickard and Fujiki, 2005). The Hlavacˇka, 2005). Lang et al. (2004) reported callose was putative role of WAKs and AGPs in the cytoskeleton-plasma localized along the fibrous meshwork covering Hechtian membrane-cell wall continuum and mechanosensing was strands, Hechtian recticulum at the cell wall, and protoplast 4 reviewed by Balusˇka et al. (2003). h after plasmolysis. Although the meshwork fibers joined the cell wall, Lang et al. (2004) failed to observe callose directly on Physiological memory of mechanoperception—Most stud- the plasma membrane. Callose synthesis is associated with a ies designed to investigate the growth response associated with number of wound, thermal, and mechanical stress responses as mechanoperception have been conducted on actively growing well as with fungal attack and pollen tube elongation. Jaffe and plants. Very little is known regarding the ability of a plant to Telewski (1984) reported callose deposition increased in the perceive a mechanical signal during a ‘‘nongrowing’’ or phloem of bean (Phaseolus vulgaris L.) and loblolly pine dormant period and to respond in the subsequent growing (Pinus taeda L.) stems 1 h after mechanical stimulation, season. Valinger et al. (1994) provide the first evidence of a peaking after 9 h, and was completely reabsorbed by 25 h. Jaffe possible memory in plants to record mechanical loading during and Leopold (1984) similarly reported callose deposition winter dormancy. They reported in their study on Pinus within 5 min of gravity stimulation in response to gravity in sylvestris L., that trees can perceive a periodic bending stress Zea mays L. and Pisum sativum L. Callose and laricinan, which when kept at 68C. When the trees were moved to conditions is similar to callose, are components of compression wood cell favorable for growth, the bent trees had a thigmomorphoge- walls (Hoffmann and Timell, 1970). The deposition of callose netic response when compared to nonbent control trees. Past inhibits cell-to-cell communication at plasmodesmata (Siva- studies on dormancy and seasonal mitotic activity in apical October 2006] TELEWSKI—MECHANOPERCEPTION IN PLANTS 1471 buds (Carlson et al., 1980; Carlson, 1985) and more recent across the cell membrane and into the cell in response to genomic studies (see for example Ko et al., 2006) have shown mechanical stress (Ding and Pickard, 1993; Pickard and Fujiki, that even during so-called periods of dormancy, plants 2005). An increase in cytoplasmic calcium in response to maintain an active level of metabolism. However, the nature mechanical stress has been documented in several plant of the stored mechanical message and how it stimulates growth systems (Toriyama and Jaffe, 1972; Knight et al., 1991, after dormancy is still unknown. 1992; Trewavas and Knight, 1994; Legue´ et al., 1997; Pickard and Fujiki, 2005). Physiological responses to mechanoperception—A gener- Hydrogen peroxide (H2O2) and other reactive oxygen alized flow chart of physiological responses to thigmomecha- species (ROS) are part of the defense response of plants, for noperception is presented in Fig. 1. The first detectable example, in fungal attack involving mechanical insertion via response to a mechanical signal is a change in action potentials growth of a fungal penetration peg through the host cell wall and electrical resistance, which occurs within seconds after (for review, see Sutherland, 1991; Mehdy, 1994). Yahraus et perturbation (Sibaoka, 1966; Pickard, 1971; Jaffe, 1976), while al. (1995) induced an oxidative burst in cultured soybean mechanical shaking of stems blocks phloem transport within 1 [Glycine max (L.) Merrill] cells in response to osmotic stress to 2 min after perturbation (Jaffe and Telewski, 1984; Jaeger et (altered turgor pressure) and direct physical pressure (mechan- al., 1988). The next measurable change occurs as an increase in ical stress). The induction of ROS and an increase in cytosolic intracellular Ca2þ (for review, see Knight, 2000). For this Ca2þ appear to be concurrent, and ROS have been suggested to reason, it is currently unclear if stretch-activated Ca2þ channels regulate Ca2þ channel gating (Mori and Schroeder, 2004). (Ding and Pickard, 1993; Pickard and Fujiki, 2005) function in Jaffe (1976) reported mechanical stress caused a complete the primary perception or are triggered by the mechanosensing cessation of elongation growth 6 min after force application, with network proposed by Balusˇka et al. (2003). The function of the growth rate resuming after 15 to 30 min in Phaseolus stretch-activated channels is to facilitate the transport of Ca2þ vulgaris. Coutand et al. (2000) reported similar results with a cessation of elongation growth 8.3 6 3.7 min after induction of a mechanical stress in the basal stem of tomato plants; elongation ceased and subsequently required 60.3 6 3.5 min of recovery time before a normal rate of elongation growth resumed. They concluded that this is evidence for a rapid, acropetally transmitted signal from the point of flexure to the actively elongating zone directly below the apical meristem. The existence of an acropetally transmitted thigmomorphogenetic signal was first reported by Erner et al. (1980), who also concluded the transportable factor was not ethylene. Subse- quently, Takahashi and Jaffe (1984) reported the presence of phytoalexin-like substances in extracts of mechanically perturbed plants, which peaked in concentration 1 h after a application of a mechanical stress and, when applied to nonstressed plants, elicited a thigmomorphogenetic-like response. Apparently, cessation of elongation growth may actually precede the rapid up regulation (expression) of calmodulin and calmodulin-related genes (TCH1, TCH2, TCH3), which was observed in Arabidopsis 10 to 30 min after mechanoperception of a touch, wind, or rain stimulus and returned to base levels by 1 to 2 h (Braam and Davis, 1990). Arabidopsis TCH3 encodes for a Ca2þ binding protein which is expressed in response to both externally applied mechanical forces and internally generated growth strains during tissue development in the absence of an external mechanical stress (Sistrunk et al., 1994). Arabidopsis TCH4 encodes for a xyloglucan endotransglyco- sylase and was co-expressed with the other TCH genes (Xu et al., 1995). The expression of xyloglucan endotransglycosylase in response to wind was located in cells undergoing expansion (Antosiewicz et al., 1997). Touch, wind, and wounding all induced increased lipoxy- genase (LOX) mRNA transcription in wheat (Triticum aestivum L.) seedlings (Mauch et al., 1997). The mechanical stress induced response occurred within 1 h after treatment, and the amount of transcript was reported to be strongly dose- dependent. LOXs are involved or implicated in a number of metabolic pathways associated with plant growth and devel- opment, ABA biosynthesis, senescence, mobilization of lipid reserves, wound responses, resistance to pathogens, formation Fig. 1. Flow chart of the time course of physiological and growth of fatty acid hydroperoxides, and synthesis of jasmonic acid response to mechanical stress. See text for citation information. and traumatic acid (for review, see Mauch et al., 1997). 1472 AMERICAN JOURNAL OF BOTANY [Vol. 93

As previously mentioned, callose synthesis and deposition 2005) and the presence of mechanosensory calcium-selective are induced by flexing mechanical stress in the phloem of bean cation channels (Cosgrove and Hedrich, 1991; Ding and (Phaseolus vulgaris) and loblolly pine (Pinus taeda) stems 1 h Pickard, 1993; Dutta and Robinson, 2004) and the plasma- after mechanical stimulation, peaking after 9 h and being re- lemmal reticulum (Gens et al., 2000; Pickard and Fujiki, 2005). absorbed by 25 h (Jaffe and Telewski, 1984; Jaffe et al., 1985). Are they competing models for a mechanosensory systems in Callose deposition also occurs within 5 min of gravity plants, or are they interconnected and co-functional, and what stimulation in Zea mays and Pisum sativum. Deposition of is the specific signal that is transmitted by the network? callose occurred first on the upper side of displaced stems, and Another area of interest focuses on how plants differentiate after 2–3 h, the pattern was reversed. The callose inhibitor, 2- between the various mechanical signals. At the most simplistic deoxy-D-glucose (DDG), blocked callose formation and level, from mechanoperception of a mechanical signal by a considerably reduced gravitropic bending in both species (Jaffe plant cell to the cascade of initial physiological responses, there and Leopold, 1984). Sound in the ultrasonic range was reported appears to be very little difference in the response pathway that to induce transient callose formation in cotton seed (Currier would allow for discrimination in programmed reaction to the and Webster, 1964). variety of mechanical stresses present in the environment. An Ethylene biosynthesis has been reported to be a fairly example is the differentiation of response between gravitropism ubiquitous response to a number of environmental stresses, vs. thigmomorphogenesis in woody plants. Reaction wood including mechanical stresses (for review, see Abeles et al., formation is a well-documented gravitropic response in woody 1992; Bleecker and Kende, 2000), and several researchers have plants (for review, see Timell, 1986a–c). A multitude of field suggested ethylene serves as a signaling molecule. Wind, observations suggest that wind can stimulate the vascular touch, and dynamic flexing have all been shown to induce cambium to produce reaction wood in the tree trunk, even if the ethylene formation in vascular plants (for review, see Telewski, tree shows no signs of having been displaced with respect to 1995), and ethylene has been reported to be involved in the gravity (see Timell, 1986c). Experimentally, Larson (1965) gravitropic response, a response to displacement resulting in also reported the formation of compression wood in Larix static bending with respect to the gravitational force vector seedlings exposed to an artificial unilateral wind. Although it (Savidge et al., 1983; for review, also see Steed et al., 2004). appears that mechanoperception in plants can be very rapid, the Ethylene production in response to mechanical stress peaks 2 h threshold of exposure or presentation to a gravitropic stimulus in Phaseolus vulgaris (Biro and Jaffe, 1984), and 9 h in Pinus may require several minutes to hours (the presentation time) taeda (Telewski and Jaffe, 1986) after force application. (for review, see Timell, 1986c). If a plant is displaced with Increased cell divisions by the vascular cambium occurred respect to gravity and returns to its vertical orientation before within 6 h after the application of mechanical stress in P. the presentation time is met, the plant will not exhibit vulgaris (Biro et al., 1980). The role of ethylene in response to gravitropic curvature. In order to isolate a gravitropic response mechanical stresses appears to affect secondary growth and from a thigmomorphogenetic response, Telewski (1989) subsequent development and differentiation of the vascular carefully flexed stems of Abies fraseri (Pursh.) Poir. and cambium and not impact primary growth (elongation or height returned them to the vertical orientation before the required growth) associated with apical meristems (Coutand et al., 2000; presentation time for initiation of a gravitropic response. The for review, see Braam, 2005). wood produced in response to this dynamic flexure possessed The role of auxin in gravitropism has been studied for almost characteristics that were intermediate between normal wood 80 years, and its role in plant leading to the postulation and compression wood. It would be interesting and useful to of the Cholodny–Went hypothesis (Went and Thimann, 1937) sort out how the mechanoperceptive system of plant cells has stood the test of time (Gutjahr et al., 2005; Esmon et al., discerns and differentiates between the various mechanical 2006). Surprisingly, little information exists on the role of forces imposed upon the plant. What is the timing mechanism auxin and other plant growth regulators in the thigmomorpho- required in meeting the presentation time of the gravitropic genetic response. Erner and Jaffe (1982) reported the response in response to static displacement and how does this accumulation of auxin-like substances and higher levels of differ from the perception of dynamic swaying? For example, abscisic acid (ABA) in response to mechanical bending. These how does the mechanosensory system distinguish between authors hypothesized the accumulation of these plant growth gravity, wind sway, vibration, and sound? regulators resulted from ethylene production earlier in the The application of current state-of-the-art analytical tools, thigmomorphogenetic response and was responsible for the such as in situ hybridization and visualization methods for reduction in internode (shoot) elongation. However, Johnson et locating gene expression and plant growth regulators, would al., (1998) challenged this hypothesis when they observed provide more detailed information on the putative roles of ethylene mutants still respond to mechanical perturbations with auxins and other plant growth regulators to further characterize a reduction in shoot elongation. The role of plant growth the thigmomorphogenetic response pathway. Finally, as regulators in the post mechanoperception-thigmomorphoge- mentioned by Braam (2005), the application of molecular netic response is still wide open for investigation. biological methods, including microarrays and bioinfomatics, will further elucidate the array of plant responses to mechanical Future areas for investigation —As is evidenced in this stresses and may help to identify the different physiological review, the field of study on mechanosensing and mechano- responses that occur after mechanoperception and to define perception in plants is progressing rapidly and supports the developmental changes specific to a specific mechanical signal. proposal of a unified hypothesis of plant perception of the mechanical environment. One area stands out, which requires LITERATURE CITED further elucidation, specifically integrating the role of the sensing network at the cytoskeleton–plasma membrane–cell ABELES, F. B., P. W. MORGAN, AND E. SALTVEIT JR. 1992. 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