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Home , Electrophysiology, Gravitropism

Electrophysiology of gravitropism

Bratislav Stanković

Brinks Hofer Gilson & Lione, 455 N. Cityfront Plaza Drive, Chicago, Illinois 60611, U.S.A.

Introduction

Earth’s gravitational field influences plant growth, morphology, and development. The vector of the gravity force is powerful enough to largely dominate the other directional tropic stimuli to which respond. Both and shoots respond to gravistimulation with differential, directional growth, through processes known as positive and negative gravitropism, respectively (Darwin, 1986). Some of the cellular events that underlie gravitropic responses are known (recently reviewed by Morita and Tasaka, 2004); the completion of a mechanistic model of plant gravity responses remains an elusive objective. In roots, the gravity-sensing cells are the columella cells located in the cap. These cells, known as stacocytes, contain starch-filled amyloplasts that sediment in the direction of gravity (Masson, 1995). In shoots, the gravity-perceptive tissue is the endodermis, which contains sedimentable amyloplasts (Tasaka et al., 1999). In both roots and shoots, the perceived gravitropic stimulus is transduced to cells that start exhibiting differential growth, resulting in organ bending and reorientation. Little is known about the signaling pathway linking gravity perception to differential growth responses, either in the root, or in the shoot. Plant cells exhibit a spectrum of bioelectric characteristic including electrical potentials, conductance, impedance, and permeability. Plant physiological functions are closely intertwined with cells’ electric properties, through processes that involve energy maintenance and ion exchange with the environment. Steady-state electrical potentials can be measured across the plasma membrane, using microelectrodes and patch-clamping techniques. On the external plant surface, electrical potentials and ion fluxes are monitored using surface-contact electrodes and vibrating probes. A variety of abiotic stimuli induce electrical activity in plants. The best- characterized electrical signals in plants are the action potentials and the variation potentials (“slow waves”). -gated, mechanosensitive, and ligand-activated ion channels, as well as proton pumps, are involved in generation and maintenance of these bioelectric potentials. Action potentials and variation potentials are both local and intercellularly propagated electrical signals. Transmitted to distant regions, these signals trigger an array of systemic molecular and cellular responses (reviewed by Davies and Stanković, 2005). The information that the electrical signals carry and the responses that they evoke depend on either the ions traversing the membrane, the change in , or both. Despite the long-documented existence of gravity-induced electrical activity in plants, this field is marked with a surprising dearth of investigation. Discovered a century ago in the petioles of Tropaeolum as a differential change in extracellular electric potential (Bose, 1907), the phenomenon of gravielectricity has been rarely studied by either electrophysiologists or by researchers studying gravitropism. Therefore, coming forth with a hypothesis to describe gravielectrical responses in plants is a speculative endeavor. This review summarizes the state of knowledge related to the role of extra- and intra-cellular electrical activity in gravistimulated higher plants. The seminal studies concerning the electrophysiology of plant gravitropism are highlighted. A few ideas on the correlation of electrical activity with responses to gravity are presented. In conclusion, a prospect for future research on the electrophysiology of gravitropism is suggested.

Extracellular gravielectric potentials

The early studies on involvement of electrical potentials in plant gravitropism involved measurements of extracellular electric potentials. In the heroic age of discovery of plant electrical activity, measurements were done using extracellular, surface contact electrodes, using ionic bridges typically consisting of diluted potassium chloride. Following the studies of Bose (1907), pioneering investigations in the field were conducted by Brauner (1927), Clark (1937), and Schrank (1947). These studies provided evidence that reorientation of plants induces transient electrical activity, a phenomenon that was dubbed “geolectric effect”. Decades had to pass before that phenomenon received further attention from plant biologists.

Shoots

Plants are electrically active. They generate characteristic steady-state transmembrane potential differences and extracellular ionic current patterns. may be involved in the establishment of plant and organ polarity (Nechitailo and Gordeev, 2001). For example, electrical current flows along the surface of upright-growing epicotyls (Toko et al., 1989, 1990). On the physically lower end of the organ, the plasma membrane is hyperpolarized by 1-2 mV. This hyperpolarization is presumably correlated to spatial information (Etherton and Dedolph, 1972). Horizontal reorientation induces electrical activity in both lower and higher plants (reviewed by Weisenseel and Meyer, 1997). Gravistimulation alters the patterns of extracellular ionic currents, causing current asymmetry. Gravistimulation also changes the patterns of electrical potential along roots and shoots. For example, within minutes of horizontal reorientation of , the electrical potential became more positive on the lower side than on the upper side. The maximal change in potential was 20-25 mV, and it was reversible when the coleoptiles were rotated back to vertical (Grahm and Hertz, 1962). In soybean hypocotyls, directional change in the gravity vector induced fast electrical field changes (Tanada and Vinten-Johansen, 1980). These changes were

2 reflected as increase in positive electrical potential in the lower side of the hypocotyl, occurring rapidly, approximately one minute after horizontal placement. The increase in positive electrical potential was maximal in the region undergoing gravity-induced curvature, in a zone 1-2 cm below the hook. The maximal amplitude of the transient change in electrical potential was about 17 mV (Tanada and Vinten-Johansen, 1980). Similarly, in bean epicotyls, electrical potential on the lower side increased, whereas the potential on the upper side decreased for about one hour after gravistimulation (Imagawa et al., 1991). The electrical activity was monitored as soon as the plants were rotated. The amplitudes of the electrical potential changes were 10-25 mV, and were dependent on the position of the electrodes attached to the epicotyls (Imagawa et al., 1991). More recently, Shigematsu et al. (1994) monitored rapid surface potential changes in gravistimulated bean epicotyls. In a limited region on the upper side of the epicotyl, surface electrical potential decreased soon after gravistimulation. The magnitude of the transient potential change was about 10 mV, and it occurred 30-120 seconds following gravistimulation. At the same time, the surface potentials on the lower side of the epicotyls scarcely changed. The rapid change in potential on the upper side was highly correlated to the early downward curvature (transient positive gravitropic response) that was simultaneously monitored (Shigematsu et al., 1994). It has been suggested that the electrical asymmetry across the epicotyl is related to asymmetric distribution and contributes to induction of H+ secretion, which is thought to initiate differential growth in shoot gravitropism (Wright and David, 1983). Indeed, because the electrical changes occur prior to the plausible movement of auxin, it was postulated that the signals mediate the asymmetric auxin distribution or asymmetric Ca2+ distribution during gravitropism (Imagawa et al., 1991).

20 Upper side 15 Lower side 10

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Figure 1. Changes in extracellular potentials in gravistimulated shoots (approximate data are redrawn from Imagawa et al., 1991). Kinetics of the change in surface electrical potential in the upper and lower sides of gravistimulated adzuki bean (Phaseolus angularis) epicotyl is shown. The measured area was approximately 20 mm from the base of the first leaves. At zero time, the epicotyl was rotated to horizontal position.

Roots

The growing root tip also exhibits strong electrical activity. Detailed measurements of endogenous currents surrounding roots were reported over 40 years ago (Scott and Martin, 1962). Since then, several related studies have indicated that the pattern of endogenous ionic currents around vertically growing roots is consistent across species. Ionic currents typically enter the and the younger parts of the elongation zone, and leave the remainder of the elongation zone and the more mature parts of the root (Behrens et al., 1982; Weisenseel et al., 1992). An area of variable current exists around the root cap; an area of large inward current is present around the meristem and apical part of the elongation zone; and an area of moderate outward current is present in the remainder of the elongation zone and the mature root tissue (Collings et al., 1992). Horizontal reorientation of plants induces changes in the current pattern around the root. The change in current pattern is rapid, and is initiated at the root cap; it also appears at the meristem and the elongation zone. Along cress rots, differential current pattern was monitored with just 3-5 min of reorientation (Behrens et al., 1982; Iwabuchi et al., 1989). Also in cress roots, the shifts in electrical current pattern were correlated to changes in the growth rate following gravistimulation (Iwabuchi et al., 1989). Using a vibrating probe, Björkman and Leopold (1985, 1987) studied the gravistimulus-induced electrophysiological asymmetry in maize roots. They discovered that gravistimulus induced a transient increase in current flow on the upper side of the root. The increase began 2-6 min following gravistimulation, had a magnitude of approximately 1 µA cm-2, and lasted approximately 10 min. A consistent change in current was observed only in the region adjacent to the statocytes (columella cells). Gravistimulation had little effects on currents in the elongation zone and at the tip of the root cap (Björkman and Leopold, 1985). Gravity-induced current changes along maize roots were also monitored by Collings et al. (1992). Within 10-15 min upon reorientation, currents above the root changed from inward to outward, resulting in asymmetries of up to 1.5 µA cm-2. That asymmetry is significant, considering that the maximum average current density in vertical roots was approximately 1.62 µA cm-2. In similar studies, the lag of the onset of current change occurred prior to the initiation of bending. This lag has been correlated to the so-called presentation time for gravity sensing (Behrens et al., 1982; Monshausen and Sievers, 2002).

4 Intracellular gravielectric potentials

Despite the suggested role for cytosolic ions in responses to gravity, the nature of membrane potential changes in response to gravistimulation is not well understood. Different types of ion fluxes have been correlated to gravitropism and to the concomitant electrical responses. Most frequently, the gravity-induced fluxes of Ca2+ and H+ have been investigated. A few studies have suggested that calcium may not be involved, or may not be necessary in gravitropism. Examining the gravity-induced bending of maize roots, Collings et al. (1992) discovered that lanthanum had little effect on either the current asymmetry or the growth response. Even a thorough imaging study failed to identify a change in cytosolic Ca2+ in gravistimulated roots (Legue et al., 1997). Some researchers have opined that much of the differential upon gravistimulation may be carried by protons instead of Ca2+ (Behrens et al., 1982; Collings et al., 1992).

Shoots

Adding to the controversy as to the possible role of Ca2+, several pharmacological studies using Ca2+ inhibitors suggest involvement of Ca2+ in gravitropism (Lee et al., 1983a; Philosoph-Hadas et al., 1996; Belyavskaya, 1996). For example, maize roots cultured in EDTA and EGTA lost their ability to respond to gravity; the response was restored by addition of CaCl2 but not MgCl2. Furthermore, asymmetric application of Ca2+ solution to vertical roots induced curvature toward the side of high Ca2+ concentration (Hepler and Wayne, 1985). Significant trans-organ fluxes of Ca2+ are triggered by gravitropic stimulation. In oat coleoptiles, Ca2+ fluxes were monitored within 10 min following gravistimulation, preceding the initiation of organ bending (Roux et al., 1983). Calcium ions were asymmetrically distributed in oat coleoptiles during and after gravistimulation (Daye et al., 1984). Calcium redistribution was also monitored in the graviresponding pulvini of Mimosa (Roblin and Fleurat-Lessard, 1987). Studying transgenic Arabidopsis expressing aequorin, a role for cytoplasmic Ca2+ was recently affirmed in the gravity transduction mechanism. In these plants, distinct calcium signaling was observed in response to gravistimulation, with kinetics of increases in intracellular Ca2+ being very different from Ca2+ transients evoked by other abiotic stimuli. The cytoplasmic Ca2+ transients had duration of many minutes, and were correlated to the strength of the displacement stimulus (Plieth and Trewavas, 2002). Extracellular Ca2+ is also needed for gravitropism (Björkman and Cleland, 1991). Oat coleoptiles incubated in 1 mM EGTA did not exhibit a gravitropic response. However, when the EGTA solution was displaced with a solution containing Ca2+, gravitropism was restored (Daye et al., 1984). Similarly, elevated concentration of apoplastic Ca2+ was observed in the slower growing parts of gravistimulated organs (Lee et al., 1983b). Thus, increases in apoplastic Ca2+ concentration are correlated with the gravitropic response. In addition to calcium, fluxes of other ions occur upon reorientation. Redistribution of other ions such as potassium and phosphorus was observed during gravity-induced curvature in sunflower hypocotyls and maize coleoptiles (Goswami and

5 Audus, 1976). Redistribution of K+ and Cl- was also monitored during the gravitropic response of Mimosa pulvini (Roblin and Fleurat-Lessard, 1987). These findings are particularly interestingly, because the current belief is that both action potentials and variation potentials in plants involve calcium influx followed by chloride and potassium efflux. Transient fluxes of Ca2+, Cl-, and K+, and the concomitant electrical signals, might play a significant role in transducing the change in the gravity vector into a cellular response leading to differential growth. Earlier it was suggested that, even if the same ions are involved in transducing the information about a multitude of abiotic stimuli, the downstream signaling events might not be the same, since the flux of any ion will depend on the kind, location, number, activity, connections, and other properties of the channel through which it passes (Davies and Stanković, 2005). Therefore, different channel and pump properties might create selectivity and specificity of the cellular responses.

Roots

Rapid changes in membrane potential in gravistimulated root cap cells have been monitored in several model systems (Behrens et al., 1985; Björkman and Leopold, 1987; Sievers et al., 1995). Where transient depolarization of columella cells on the lower side of cress roots was observed, the initial changes in membrane potential were measured within a few seconds following gravistimulation. At the same time, statocytes on the upper side of the root cap gradually became hyperpolarized (Behrens et al., 1985). In roots of mung beans, gravistimulation changed the intracellular potentials of the elongating cells. Differential membrane potentials between upper and lower flanks of the root cap were recorded within 30 seconds following gravistimulation (Ishikawa and Evans, 1990). The rapidity of such gravistimulus-induced changes in electrical parameters has been used as an argument for assigning a role to electrical potentials as possible signal transmitters or transducers in the gravity response. Distinct spatial and temporal patterns of pH fluxes are also correlated with gravistimulation. These include both apoplastic proton fluxes, as well as cytoplasmic pH changes. Gravity was found to induce rapid pH changes in Arabidopsis columella cells, causing rapid acidification of the apoplast (Scott and Allen, 1999). In Arabidopsis, Fasano et al. (2001) observed that the root cap apoplast acidified from pH 5.5 to 4.5 within 2 min of gravistimulation. Furthermore, cytoplasmic pH increased only in the columella cells from 7.2 to 7.6, but was unchanged elsewhere in the root. Similar gravity-induced changes in cytoplasmic pH occur in the maize pulvinus, shoot tissue that is specialized in gravity perception (Johannes et al., 2001). Such pH changes may be a component of the changes in membrane potential that occur during gravitropism.

6 5 110 Angle (degrees) Intracellular potential (mV) 0

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Figure 2. Changes in curvature and intracellular potential in gravistimulated mung bean root (approximate data are redrawn from Ishikawa and Evans, 1990). Kinetics of the change in intracellular potential of an upper cortical cell within the elongation zone upon reorientation from 0º (vertical) to 90º (horizontal) and back to 0º. The cell position was 2 mm from the root tip, and 0.15 mm from the upper surface in horizontal orientation. When the root was in vertical orientation, the intracellular potential value was approximately -115 mV. The root was turned horizontally at zero time, and turned vertically at approximately 45 seconds.

Physiology of the gravielectric phenomena

The bioelectric polarity of plants begins at the molecular level. Cells contain a multitude of structural and functional molecules that electrically behave as dipoles. These molecules exhibit defined reactive capacities. In addition, cells contain highly conductive aqueous electrolytes that are separated by low-conductivity membranes populated with electrically active macromolecules. The plant’s bioelectric cellular heterogeneity and polarity are intrinsic features of an organism that is evolutionary adapted to gravity. Microgravity causes abnormal and abnormal (Nechitailo and Gordeev, 2001; Link, Stanković et al., unpublished data). However, establishing causal relationships between the changes in electrical activity and the known gravitropic responses is a challenging and speculative task. The heterogeneity of

7 resistance and , as well as the existence of both passive and active transmembrane ion channels and pumps, creates considerable macromolecular complexity. The Cholodny-Went theory of plant gravitropism is alive and well. According to this theory, a lateral gradient of auxin induces differential growth leading to organ curvature. The upward growth of horizontally positioned stems occurs due to asymmetric auxin distribution, and its increased concentration in the lower side. Inhibitors that prevent also inhibit gravitropic responses (Katekar and Geissler, 1980). Half a century ago, the then-discovered gravistimulus-induced positive electrical potential in cells of the lower side of plant tissue was viewed as the driving force leading to differential auxin accumulation (Wilkins, 1966; Woodcock and Wilkins, 1971). The differential changes in auxin might be correlated with gravistimulus-induced proton fluxes. Recent elegant studies suggest that the H+ dynamics in the root cap during gravistimulation is highly complex (Scott and Allen, 1999). Protons are known carriers of current. In plant cells, the plasma membrane H+-ATPase is primarily responsible for generating the membrane potential (Assmann and Haubrick, 1996). The H+-ATPases on the lower side of gravistimulated shoots transport protons into extracellular space. These positive charges might attract auxin, leading to differential growth. Indeed, auxin movement has been correlated to amyloplast sedimentation. In an electrophysiological context, intracellular increase in positive charge could lead to accelerated influx and accumulation of negatively charged growth substance, resulting in increased differential growth during the gravitropic response. Exactly how these changes in electrical activity might signal differential growth is unclear. The electrical asymmetry may provide positional (up/down) information, intracellularly, or trans-organ. The overall scheme might be complex beyond our current understanding, as different ion fluxes might be counterbalanced. For instance, while H+ secretion results in membrane hyperpolarization, simultaneous K+ uptake of positive charge via a H+/K+ symporter (Philippar et al., 1999) will lead to acidification of the apoplast at unchanged membrane potential. Components of the plasmalemma may be sensing the changes in the force of gravity, and may be subsequently regulating the activity of plasmalemmal ion channels and pumps. Actin (microfilament) networks could be absorbing the force of sedimentation of amyloplasts, and could be used to amplify the signal via connected ion channels (Figure 3). Feedback regulation might be involved. It has been suggested that extended, perhaps continuous signaling occurs between the root cap area and the root elongation zone through active maintenance of the resting membrane potential in statocytes (Monshausen et al., 1996). Alternatively, the electrical responses could be merely causal effects of other subcellular responses that act as primary transducers of the gravitropic stimulus. For instance, changes in cytoskeletal tension upon reorientation of the cells might induce the activity of mechanosensitive ion channels. The resulting changes in membrane potential could thus be secondary consequences of some primary signaling mechanism that functions during gravitropism. The purpose of signal transduction is conductance of information and control. Nature uses electrical signals as convenient means for information transfer, amplification,

8 and control in biological systems. In cellular context, electrical transducers are coupled to actuators that stimulate other transducers, becoming integrated into a myriad of control processes that constitute cellular homeostasis. Upon gravistimulation, ion channels, ion pumps, H+-ATPases, actin microfilaments, amyloplasts, and other molecular components create a network that may use electrical transients as a means of transducing the information about the perceived reorientation (gravity stimulus). Such a network can be likened to an electronic circuit, where changes in ionic amplitudes, frequency, and duration of transients will translate into a physiological response to gravity. This cellular network (circuit) might be characterized with cardinal design principles such as feedback stabilization, where the precise characteristics of the individual transducers and actuators are relegated to secondary importance. A design where the variations are compensated by the feedback received would allow the reliability of the transduction process to be inherent in the circuit design rather in the exactitude of its components. That circuit would exhibit tolerance regarding variations in the transducers, actuators, and communication links. If cytosolic ions are involved as electrical signals in transduction of the gravistimulus intro graviresponse, their precise conduction pathways remain elusive. Hejnowicz et al. (1991) proposed a signaling pathway via an intrasymplasmic system composed of desmotubules and the endoplasmic reticulum (1991). This is a plausible proposition; some studies suggest involvement of the endoplasmic reticulum in the response to gravity. Further studies, particularly in relation to sedimentation of amyloplasts onto the endoplasmic reticulum, are required to test this hypothesis.

9 Figure 3. Structural overview of a columella cell showing the known subcellular components that might be involved in gravielectric responses. Note the presence of both voltage-activated (+/-) and mechanosensitive (double arrow) ion channels. Microfilaments (MF) are in contact with these channels and with proton pumps (H+), and with sedimenting amyloplasts (A). Mature columella cells exhibit distinct polarity, with the nucleus (N) being positioned at the upper end of the cell. The endoplasmic reticulum (ER) is particularly dense at the lower end of the cell. Desmotubules (D) might also be involved in communication with adjoining columella cells.

Early workers postulated that the gravielectric effect is a consequence of auxin distribution in shoots curving upward after gravistimulation (Grahm, 1964; Wilkins and Woodcock, 1965). Support for that hypothesis does exist. Gravistimulated cress roots exhibit different surface acidification in the root cap (Monshausen and Sievers, 2002). Through the use of proton-selective microelectrodes, gravity-induced surface pH changes were monitored across all root zones. The differential surface acidification progressed toward the elongation zone at a rate similar to the rate of auxin transport (250-350 µm min-1). The kinetics suggests a correlation of the gravity-induced pH changes (also correlated to changes in membrane potential) and the development of a lateral auxin gradient in the root gravitropic response (Monshausen and Sievers, 2002).

Prospects

The phenomenon of plant gravielectricity is understudied, and consequently poorly understood. Discrepancies exist in the reporter parameters of electrical activity following gravistimulation, and in interpretation of the results obtained. These discrepancies can be attributed to the heterogeneity of use of different model systems, measurements of potentials at incorrect locations, absence of uniform techniques, etc. Molecular genetics keeps providing substantial evidence to support the two fundamental hypotheses of gravitropism: the Cholodny-Went hypothesis and the starch- statolith hypothesis. Transgenic plants open the doors for dissecting the role of electrical potentials in gravitropism. Interesting discoveries have already been made. Using transgenic maize overexpressing K+ channels, it was discovered that the auxin-induced K+ channel expression is an essential step in the gravitropic responses of maize coleoptiles (Philippar et al., 1999). Upon gravistimulation of coleoptiles, differential expression between the upper and the lower halves of K+ channel was observed. This differential expression closely followed the gravitropic-induced auxin redistribution; it preceded any detectable bending of the (Philippar et al., 1999). More recently, it was also shown using transgenic plants that auxin activates KAT1 and KAT2, two K+- channel genes (Philippar et al., 2004). These observations led to the hypothesis that in maize coleoptiles transcriptional regulation of K+ channels represents a key step in response to physiological growth stimuli such as gravitropism (Philippar et al., 1999, 2004). Studies similar to these will help decipher the molecular bases underlying the nature and the significance of the electrophysiological responses during gravitropism.

10 A dose of cautious optimism is in order, as interesting work awaits those interested in studying plant gravitropism and the related plant space biology questions (Stanković, 2001). At present, there is no data about the effects of microgravity on plant electrophysiology. Measurements of electrical potentials and ion fluxes in microgravity will help reveal the influence of gravity on ion fluxes and electrical potentials. It was already hypothesized that microgravity-grown plants might become hypersensitive to gravity, having more activated Ca2+ channels that might fire action potentials upon return from orbit (Weisenseel and Meyer, 1997). Back on Earth, the process of signal transduction in plant gravitropism needs to be dissected. The molecular identity of the mechanosensing receptor (channel/pump?) that changes in the gravity vector is unknown. The timing, duration, amplitude, and frequency of electrical potentials associated with graviresponses need to be determined. The ions involved in the process need to be precisely identified in terms of a signaling cascade triggered by gravistimulation. Causal relationships of the electrophysiological activity with known molecular, cellular, and organ responses to gravity need to be established. Combined methodological approaches using electrophysiology, microscopy, and transgenic plants will yield new information that will advance our understanding of the role of electrical signals in plant gravitropic responses.

11 References

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