Gravitropism in Higher Plants1

Since 1806, we have known that plant organs use gravity as a guide for growth (Knight, 1806). The gravity-directed growth process, called gravitropism, dictates upward shoot growth to ensure a proper positioning of the leaves for efficient photosynthesis and gas exchange. It also directs roots to grow downward in soil, where they can reach out to take up the water and mineral ions required for plant growth and development. Gravitropism dictates upward shoot growth, as illustrated by the trees shown at the end of the corn field. The corn plants, on the other hand, were recently prostrated by a heavy storm (a). However, in a few hours, their shoots curved upward, as a result of gravitropism (b). At the end of the 19th century, Ciesielski (1872) and Darwin (1880)demonstrated that a structure at the tip of the roots, the cap, is essential for root gravitropism. They postulated that the root cap could perceive a change in root-tip orientation within the gravitational field (gravistimulus). Graviperception would then produce a physiological signal that, upon transmission to the elongation zone, would promote a differential cellular elongation on opposite flanks, which is responsible for the development of a curvature. The resulting curvature would allow the root tip to resume growth along a gravitropically more acceptable vector. These important early observations along with the proposed model for gravity perception marked the beginning of numerous studies that extended throughout the entire 20th century and helped us to gain a better understanding of the various physiological and molecular processes underlying gravitropism. In this Update we discuss our current knowledge of the gravitropic response of higher plants, with a special emphasis on roots. Different root-tip cells are involved in the sensing of and response to a gravistimulus (reorientation within the gravity field). The root tip of a 5-d-old Arabidopsis seedling contains a root cap made of columella and peripheral cells, a promeristem, a DEZ composed of cells that expand both longitudinally and radially, and a CEZ consisting of cells that preferentially elongate. Gravity sensing occurs primarily in the columella cells, called statocytes, of the root cap, whereas the curvature response to a gravistimulus occurs in the DEZ and CEZ. The root shown in this picture was viewed under a light microscope using Nomarski optics. Root width = 160 μm. The primary mechanism for gravity sensing in roots involves the sedimentation of amyloplasts in the root-cap columella cells. a, In this light micrograph starch-filled amyloplasts (dark brown) are revealed in the root-cap columella cells of a vertically grown Arabidopsis seedling by staining with an iodine/potassium iodide solution (Kiss et al., 1996). One of these columella cells, chosen as an example, is identified by a white circle in a and is schematized in b. The columella cell contains a nucleus (N) at the top, a peripheral ER, and sedimentable amyloplasts (a) at the physical bottom. c, Upon gravistimulation (reorientation of the root to the horizontal), the statocyte amyloplasts sediment from their original position (punctuated, light gray circles) to the new physical bottom of the cell (darker gray circles). According to the amyloplast-statolith hypothesis, amyloplast sedimentation is the primary mechanism by which gravity sensing occurs. In all of the panels, the root tip and statocytes were drawn according to their orientation relative to the gravity vector, g, represented by an arrow. The statocytes are highly polarized cells that contain a peripheral ER, a nucleus positioned in the middle or at the top, and dense amyloplasts sedimented at the physical bottom (Fig. 3b). When a plant organ is tilted within the field of gravity, amyloplasts sediment to the new physical bottom of the statocytes (Fig. 3, b and c). Amyloplast sedimentation is believed to activate receptors that trigger a signal transduction pathway leading to the formation of a physiological signal, which is responsible for organ-tip curvature (Evans and Ishikawa, 1997). Both direct and indirect evidence support a role for amyloplast sedimentation in gravitropic sensing. First, decapped roots do not respond to gravistimulation despite wild-type rates of growth (Darwin, 1880). Hence, the root cap appears to be essential for root gravitropism (Sack, 1997). Second, centrifugation experiments in which lateral acceleration forces were applied to different regions of a plant positioned along the general radius of a centrifuge demonstrated that the primary site of gravity sensing in roots overlaps with the root cap (for review, see Poff and Martin, 1989). Third, a good correlation exists between amyloplast density and gravitropic sensitivity in plants carrying amyloplasts, the starch content of which was reduced by physiological treatments or by mutations in genes required for its synthesis or accumulation (Sack, 1991; Kiss et al., 1996). Fourth, developmental mutants lacking a differentiated endodermis in their shoots and roots are shoot agravitropic. Because the shoot endodermis contains statocytes and the root counterpart does not, this observation is also compatible with the proposed model (Fukaki et al., 1998). Amyloplast displacement in statocytes is sufficient to promote organ-tip curvature. The application of high-gradient magnetic fields to Arabidopsis root tips promoted a lateral displacement of root-cap amyloplasts and a subsequent development of root-tip curvatures in the direction of statolith displacement (Kuznetsov and Hasenstein, 1996). The presence of starch in the plastids was necessary for amyloplast displacement and root-tip curvature in this system (Kuznetsov and Hasenstein, 1996). Furthermore, the displacement of amyloplasts in the starch parenchyma of barley coleoptiles and tomato hypocotyls promoted the development of a curvature in the direction opposite to that of statolith displacement, as expected for organs subject to negative gravitropism (Kuznetsov and Hasenstein, 1997). The role of amyloplast sedimentation in gravitropic sensing is also supported by recent laser-ablation experiments, which showed that Arabidopsis roots lose most of their gravitropic abilities when the central amyloplast-containing columella cells are ablated (Blancaflor et al., 1998). We should caution, however, that amyloplast sedimentation might not be the sole mechanism of gravity sensing. In fact, the laser-ablation experiments discussed above revealed that the decapped root tips are still able to respond to gravistimulation, albeit with altered kinetics and increased threshold stimulation times (Blancaflor et al., 1998). In addition, electrophysiological studies demonstrated that fast changes in proton flux occur on the physical topside of the DEZ upon gravistimulation. Such changes are too fast to derive from the transmission of a chemical signal from the root cap to the DEZ, suggesting that DEZ cells can directly sense the gravistimulation or that a fast electrical signal is transmitted from the root cap to the DEZ (Behrens et al., 1982; Monshausen et al., 1996). Other gravity-sensing mechanisms may also produce these interesting secondary responses. The gravitational pressure model proposes that plant cells perceive gravity by sensing their buoyancy within the surrounding medium (Staves, 1997). Hence, gravity may tend to displace the protoplast within the cell wall, exerting a tension between the plasma membrane and the extracellular matrix on the topside and compression at the bottom. The tension and compression may result in the activation of specific stretch-activated channels that trigger signal transduction pathways leading to the final cellular responses (Staves, 1997). According to that model, the dense amyloplasts contribute to gravity sensing by increasing the total weight of the cell, thus increasing the differential tension/compression exerted by the cell on its extracellular matrix. The gravitational pressure model seems adequate to explain the gravity sensing that directs cytoplasmic streaming in the large internodal cells of Chara corallina(Staves et al., 1997) and causes gravitaxis in Euglena cells (Konings, 1995). However, its involvement in gravitropic sensing by much smaller cells such as the statocytes in higher plants remains controversial (Sack, 1997). How amyloplast sedimentation is transduced into a physiological signal in the statocytes remains an enigma. Indirect evidence suggests that the sedimentable amyloplasts are enmeshed in a dense network of short and dynamic actin microfilaments connected to a region of the statocyte cortex rich in microtubules, ER, and membrane-bound cytoskeleton elements. This network appears to restrain the movement of amyloplasts at the statocyte periphery (Volkmann et al., 1991; Baluska and Hasenstein, 1997). The association of statoliths with microfilaments may be mediated by myosin-like motor proteins found in the vicinity of sedimenting amyloplasts in root-cap statocytes (Wunsch and Volkmann, 1993). Unfortunately, direct evidence for interaction between amyloplasts and microfilaments in statocytes is not available. Because the sedimentable amyloplasts lie on top of the peripheral ER in the statocytes, it is also possible that the peripheral ER restrains amyloplast movement (Sack, 1997). The molecules transducing the gravity signals in statocytes have not yet been characterized. The pulling action generated by amyloplast sedimentation could stretch specific plasma and/or ER membranes to activate mechanosensitive channels. The opening of these channels could then allow a local transient increase in cytoplasmic Ca2+ levels that would trigger a signal transduction pathway, leading to the production of physiological signals (Sievers et al., 1991). The involvement of cytosolic Ca2+ as a second messenger in the transduction of gravity signals in the statocytes has long been hypothesized. Gravitropism is eliminated by both blockers of stretch-activated channels (e.g. Gd3+ and La3+) and inhibitors of calmodulin or Ca2+-ATPase activities (Biro et al., 1982;Björkman and Leopold, 1987; Stinemetz et al., 1987; Sievers and Busch, 1992). Furthermore, high concentrations of Ca2+ were detected in statocyte amyloplasts (Chandra et al., 1982), and statocytes were found to contain higher levels of calmodulin than other cell types (Allan and Trewavas, 1985). Also, a Ca2+/calmodulin-dependent protein kinase may be involved in the light-dependent orthogravitropic response of roots in several corn cultivars (Lu and Feldman, 1997). Unfortunately, recent experiments with Ca2+ reporter systems failed to identify gravity-induced transient changes in cytosolic Ca2+ levels (Legue et al., 1997). The phosphoinositide pathway may also be involved in gravitropism. The phosphatidylinositol-4-phosphate-5-kinase activity responsible for the production of phosphatidyl-inositol-4,5-bisphosphate was found to increase in the lower side of grass pulvini within 10 min after gravistimulation and in the upper side in approximately 30 min (Perera et al., 1998). Phosphatidylinositol-4,5-bisphosphate is a biosynthetic precursor of inositol-1,4,5-trisphosphate, an intracellular second messenger that activates Ca2+ release from internal stores. This suggests that the phosphoinositide pathway is an important mediator of gravity signal transduction, at least in grass pulvini. Little is known about the molecular mechanisms involved in gravity signal perception and transduction. In fact, although several gravitropism genes have been identified, only ARG1 has been implicated in that phase (Sedbrook et al., 1999). This role forARG1 was proposed based on genetic and physiological studies of Arabidopsis, which indicated that arg1 mutations affect root and hypocotyl gravitropism but display no pleiotropic phenotypes. Molecular analysis indicates that the ARG1 gene encodes a DnaJ-like protein, which carries a coiled coil domain at the carboxy terminus and a putative transmembrane domain in the middle (Sedbrook et al., 1999). DnaJ-like proteins are encoded by large gene families in all of the species that were analyzed. They are reported to function in protein folding, protein trafficking, and the facilitation of multiple signal transduction pathways (Miyata and Yahara, 1991; Xu and Lindquist, 1993;Kimura et al., 1995). The J domains of several DnaJ-like proteins were found to interact with a conserved subdomain of HSP70, modulating its ATPase activity (Langer et al., 1992; Tsai and Douglas, 1996). It is interesting that some of these proteins can form large hetero-oligomeric complexes, which bind to actin filaments in a calmodulin-dependent fashion, and mediate specific signal transduction pathways (Nishida et al., 1986; Pickard et al., 1990;Miyata and Yahara, 1991; Xu and Lindquist, 1993; Kimura et al., 1995). The putative coiled coil domain found at the carboxy terminus of ARG1 is structurally similar to that found in a number of proteins that bind to cytoskeleton elements (Sedbrook et al., 1999). Taken together, these data suggest that the ARG1 protein may connect some components of the gravity signal transduction pathway to the cytoskeleton or connect the cytoskeleton to specific plasma or organelle membranes in the statocytes, mediating the reception of gravity signals. Alternatively, ARG1 may target proteins involved in gravity signal transduction to specific compartments within the statocytes (Sedbrook et al., 1999). Detailed analysis of ARG1 protein localization and function will shed more light on the molecular mechanisms underlying gravity signal transduction. Gravitropic curvature is a consequence of differential cell elongation on opposite sides of the organ (root or stem); it is believed to be mediated by an auxin gradient, as originally proposed in the Cholodny-Went theory (for review, see Lomax, 1997). This model is supported by experiments that revealed a correlation between the gravitropic response and the redistribution patterns of exogenously applied radiolabeled IAA across gravistimulated organs (Lee et al., 1983; Young et al., 1990; Lomax, 1997) and by the observation that several auxin-responsive genes are asymmetrically activated upon gravistimulation (Li et al., 1991; Luschnig et al., 1998). The statocyte-containing endodermal tissue in shoots transports auxin from its site of synthesis in the shoot apex to its site of action (Lomax et al., 1995; Gälweiler et al., 1998). Hence, it is plausible that amyloplast sedimentation upon gravistimulation activates membrane-associated, auxin-efflux carriers in cells on the bottom side, promoting the lateral transport of auxin to adjacent cortical and epidermal tissues. Auxin accumulation at the bottom side would promote a differential cellular elongation between upper and lower flanks, leading to upward shoot curvature (Lomax, 1997). Accordingly, a change in the polarity of lateral auxin transport across gravistimulated shoots was shown to correlate with the changes in the direction of gravitropic curvature induced in lazy-2 tomato shoots by nondirectional red-light treatment (Lomax, 1997). In roots the physiological signal(s) generated upon gravity-receptor activation in the root-cap statocytes must be transmitted to the DEZ and the CEZ for a curvature response to develop (Evans and Ishikawa, 1997). Careful time-lapse video analyses of graviresponding roots suggest that the curvature response is rather complex. Soon after stimulation, there is a transient cessation in cell expansion on both sides (upper and lower) of the CEZ. Simultaneously, a small group of cells on the upper side of the DEZ elongate more rapidly than they would in the absence of a gravistimulus, resulting in root-tip curvature. Then cellular elongation proceeds on the upper side of the CEZ, whereas cellular elongation remains inhibited on the lower side. As a result, the root tip reorients, tending to return to its original growth vector, and the site of curvature moves basipetally toward the mature zone. When the root tip reaches a gravitropically acceptable growth direction, the rate of curving decreases and then reverses. An of the root tip that occurs with at and the root growth and 1991; and 1993). The application of auxin to roots at levels sufficient to growth does not the root In fact, a a small group of DEZ cells under these and 1993). the phase of that involves an increased rate of cellular elongation at the topside of the DEZ appears to be auxin of Ca2+ is generated across the root cap in response to gravistimulation (Lee et al., and 1991). no data have thus demonstrated that this is transmitted to the DEZ, it is possible that it the phase of the of extracellular in of root gravitropism. Furthermore, application of Ca2+ to side of the DEZ in the development of a curvature toward the site of application (for review, see and Ishikawa, 1997). Ca2+ is believed to an important role in the of cell by molecules and 1992). Hence, the increased Ca2+ in the cell may increase and cellular It is also possible that changes in are responsible for changes in intracellular Ca2+ levels and Trewavas, 1997). The Ca2+ may auxin transport gravitropism, and Ca2+ may the sensitivity of root cells to auxin action (for review, see and Ishikawa, 1997). the phase of root appears to be to concentrations of it was in and mutants (Evans and Ishikawa, 1997). This may suggest a role for auxin that is of growth In that it is interesting to that the development of an Ca2+ across the root cap auxin transport (Lee et al., and 1991). is evidence the involvement of auxin in the second phase of root in which a differential cellular elongation occurs on opposite of the CEZ. only is the second phase to concentrations of auxin but inhibitors of auxin transport also root gravitropism and Accordingly, mutations in genes involved in auxin transport or response affect this in the redistribution of auxin in the root a, The model of auxin transport in roots that auxin in the shoot apex is to the root tip the (Evans and Ishikawa, 1997). there it is to more peripheral and is basipetally to the DEZ and CEZ, where it cellular The transport of auxin epidermal cells upward involves and carriers represented by a and represented by a epidermal cells of the CEZ are schematized on side of the They a localization of the auxin-efflux which is responsible for the polarity of transport in the When a root is within the gravity field indicated by an activation of the gravity signal transduction pathway in the root cap in the formation of an auxin across the root more auxin at the bottom than at the top of the as illustrated by the width of the The resulting auxin is transmitted from the root cap into the DEZ and CEZ, where it a differential rate of cellular elongation on opposite flanks, which is responsible for the curvature. Root width = 160 μm. The patterns of and are with their involvement in auxin transport and in gravitropic signal transmission in roots. Both genes are in the DEZ and CEZ of Arabidopsis roots et al., and the protein is in the membranes of DEZ and CEZ epidermal cells and in cortical cells et al., 1998). However, of these genes is in the root cap, where an auxin is believed to be generated in response to gravistimulation. Hence, we must that other gene mediate the lateral of auxin in the This could be the function of other of the gene et al., 1998). Alternatively, the electrical signals discussed could directly the activity of auxin-efflux carriers in the DEZ and CEZ of the of auxin concentrations in different regions of the root tip gravistimulation and analyses of all of the gene are to these As in this review, auxin cell elongation in shoots and it in roots. the increased auxin on the bottom side of gravistimulated organs an upward curvature in shoots and a downward curvature in roots. does auxin cellular elongation in It appears that auxin cellular elongation by modulating the activity of the plasma membrane proton by cell and cellular and by the of a number of auxin-responsive genes a number of cell wall, plasma and proteins have been found to bind auxin at only has been proposed to the role of auxin in the of cellular expansion et al., 1998). is an protein found in the ER, although some of it was also found the It appears to the activity of the plasma membrane proton et al., and to promote cell expansion when in plants and cell et al., 1998). these suggest that as an auxin in cellular it remains possible that other proteins similar In to the plasma membrane proton and a essential for the of new cell components auxin also intracellular signal transduction pathways that result in changes in gene components of the auxin signal transduction pathway have recently been characterized. The Arabidopsis essential for root gravitropism and auxin encodes a protein that with to activate of the of proteins et al., 1998). the Arabidopsis gene encodes an protein, which has also been proposed to function in processes et al., 1998). These suggest that auxin activates the of resulting in the of several auxin-responsive was also shown to be essential for root gravitropism et al., 1998). understanding of the molecular processes that the various of gravitropism in higher plants has in the few The involvement of amyloplasts as primary gravitropic has been and various components of the gravity signal transduction pathway have been identified and are characterized. The role of auxin in the has also been and several molecules involved in its transport and action have been identified and are characterized. However, we still about the molecular and function of the putative gravity receptors and activation in the formation of a physiological the of that physiological signal has not been although auxin is of its other are involved in the gravitropic and are believed to root and shoot gravitropism by auxin transport (Lomax, et al., 1998). of action and involvement in the of the response in different plant organs have yet to be The gravitropic response is only of several responses that the direction of plant growth in in and also the patterns of growth 1995). The to multiple and may result in growth such as the growth of roots to a of gravity and stimulation and The may also be by other the roots of several cultivars orthogravitropic when light is by the and a Ca2+/calmodulin-dependent protein kinase is activated (Lu and Feldman, 1997). the gravitropic response of Arabidopsis hypocotyls is altered when are to This response is mediated by and and is inhibited by application of et al., 1996). the molecular mechanisms responsible for the of these processes have yet to be An of new based on and and and is and can be applied to the development of an to the of the processes we have They to important and in our understanding of the molecular and physiological processes that plant growth responses to We of of for the shown in central elongation elongation