Root Tropism

Root Tropism: Its Mechanism and Possible Functions in Drought Avoidance

YUTAKA MIYAZAWA,1 TOMOKAZU YAMAZAKI TEPPEI MORIWAKI AND HIDEYUKI TAKAHASHI

Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai, Japan

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Plant Responses to Water Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Water Stress and Osmotic Adjustment Cause Growth Inhibition ...... B. Severe Drought Conditions Damage Plant Cells .......................... C. Avoidance and Tolerance of Severe Drought Conditions................ III. Mechanisms for Root Hydrotropism and its Possible Functions in Drought Avoidance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mechanism for Sensing Hydrostimulation ................................. B. Mechanism for Hydrostimulation Signal Transmission .................. C. Mechanism for Hydrotropic Root Bending ................................ D. Molecular Identification of Genes Responsible for Hydrotropism in Arabidopsis Roots .............................................................. IV. Mechanisms for Other Root Tropisms Related to Drought Avoidance. . . . A. Root Gravitropism ............................................................. B. Regulation of Gravitropism by Water Stress .............................. C. Root Phototropism............................................................. D. Ecological Function of Phototropism ....................................... V. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: E-mail: [email protected]

Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.

0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00010-2

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ABSTRACT Land plants have evolved various mechanisms for responding to unfavourable environmental signals, which allows them to tolerate or avoid environmental stresses such as water deficit. To date, physiological and molecular mechanisms that contribute to drought tolerance have been intensely studied, however the mechanisms that confer drought avoidance have been less understood. To avoid drought conditions roots must sense environmental stimuli and respond by regulating growth away from water scarce areas or toward wet areas. Indeed, roots respond to numerous environmental stimuli, such as gravity, light and moisture gradient, and exhibit gravitropism, phototropism and hydrotropism, respectively. Of these root tropisms, hydrotropism can be considered to contribute directly to drought avoidance. As soil water status is affected by gravity or intense light, positive gravitropism and negative phototropism are assumed to contribute to drought avoidance. In this chapter, we describe what happens to cells faced with a water deficit and then outline the molecular mechanisms underlying different tropisms, with particular emphasis on the molecular mechanism contributing to root hydrotropism.

I. INTRODUCTION All organisms depend upon water availability. Unlike mobile organisms, land plants are sessile in nature and must complete their life cycles where they germinate. Accordingly, land plants have evolved various mechanisms for responding to unfavourable environmental signals, allowing them to tolerate or avoid environmental stresses such as water scarcity. The physiological and molecular mechanisms contributing to drought tolerance have been studied extensively. Such mechanisms include stomatal closure, the synthesis and activity of the phytohormone abscisic acid (ABA), the synthesis of compatible solutes and the functions of regulatory genetic elements. These drought tolerance mechanisms are detailed in other chapters. In contrast, the mechanisms underlying drought avoidance are much less well understood. The root is the primary organ for water absorption. To avoid drought conditions, roots must sense environmental stimuli and respond by regulating growth away from dry areas or towards wet areas. To regulate growth in a directional manner, land plants have evolved tropisms, whereby organ growth is redirected in response to an environmental stimulus, such as gravity, light, a moisture gradient or touch. Some of these environmental stimuli, namely, gravity, light and a moisture gradient, are closely related to soil water status. As gravity affects water, positive gravitropism is assumed to be one mechanism for drought avoidance. Similarly, negative phototropism also plays a role in drought avoidance, as soil is dried by exposure to intense light. Further, plant roots can sense a moisture gradient and grow towards the wet area, a process termed positive hydrotropism. In this chapter, we will

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describe what happens to cells faced with a water deficit and then outline the molecular mechanisms underlying different tropisms, with particular emphasis on the molecular mechanism contributing to root hydrotropism.

II. PLANT RESPONSES TO WATER STRESS Environmental factors, such as water, light and temperature, will change across a day, season and year. Plants use these changes as cues for controlling the timing of developmental transitions such as germination and flowering. These environmental factors also provide plant cells with stresses, which result in the inhibition of cellular processes. Water is the most important environmental factor for plants. It is integrally linked to biophysical and biochemical process such as cell elongation and enzyme activity, respectively. Consequently, water shortages cause water stress in plants. A. WATER STRESS AND OSMOTIC ADJUSTMENT CAUSE GROWTH INHIBITION

Water stress can be estimated by measuring water potential. Water moves according to a water potential gradient within a plant and between the plant and its environment. Water is taken up from the environment and then absorbed into cells, which results in increased cell volume. This increase in cell volume is also driven by a water potential gradient between the outside and inside of the cell. Changes to cell volume are thought to be determined by several factors, namely differences between osmotic potential and turgor pressure, water permeability across the plasma membrane and cellular surface area. The growth responses of terrestrial plants are particularly sensitive to water shortages (Mullet and Whitsitt, 1996), as these decrease the environmental water potential and reduce water movement into plants. However, water shortage does not result in a large difference between osmotic potential and turgor pressure (
0.1–0.2 MPa). Thus, sensitivity to changes in cell volume may explain drought-induced inhibition of the growth response. Following a decrease in environmental water potential, cell growth is inhibited via adjustments to the osmotic potential, that is, solute concentration. Sugars and amino acids are thought to be instrumental in osmotic adjustment, as their distribution is unlikely to affect cellular components such as enzymes and membranes. Sugars and amino acids can be taken up by cells or obtained via the degradation of osmotically inactive compounds. High molecular mass solutes such as proteins and starch do not contribute to osmotic potential. Until recently, ions have not been considered suitable osmotic

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components because alterations to their distribution could cause charge effects on enzymatic processes. However, comprehensive analyses of plant growth, metabolite composition, enzyme activity and gene expression in Arabidopsis exposed to different drought conditions have identified potassium and organic acids as the primary components for osmotic adjustment (Hummel et al., 2010). In addition, Arabidopsis cells use enzymes to perform these osmotic adjustments without altering the primary metabolic system. This lack of metabolic change is in sharp contrast to the drought-induced alterations to expression of genes involved in metabolism, transport, signalling and transcription, as well as genes encoding hydrophilic proteins (Bray, 2004; Kawaguchi et al., 2004; Kreps et al., 2002; Seki et al., 2002). Water permeability can be important for cell volume increases. Water molecules may pass passively through the lipid bilayers of the plasma membrane or they may be transported via water channels. Aquaporins are the most well-known water channels. These integral membrane proteins contain six transmembrane domains, with both the C- and N-termini found on the cytoplasmic side of the membrane. In plants, important water uptake roles are performed by aquaporin family members that localize to the plasma membrane and vacuole (Kjellbom et al., 1999). While overexpression of a plasma membrane-localized aquaporin improves the growth of transgenic tobacco plants under well-watered conditions, these plants wilt easily under drought conditions (Aharon et al., 2003). Recently, drought conditions were shown to decrease aquaporin levels in the plasma membrane of Arabidopsis (Lee et al., 2009). This decrease resulted from aquaporin degradation in the endoplasmic reticulum via a ubiquitin-mediated protein degradation pathway. The observations described above suggest that cells may control water permeability to regulate water uptake during times of drought. B. SEVERE DROUGHT CONDITIONS DAMAGE PLANT CELLS

Under severe drought conditions, the water potential outside of a plant may be lower than inside, resulting in the dehydration of cells and wilting from loss of turgor pressure. Excessive water loss can irreversibly damage plant cells, and at the macro level, increased ion leakage can be detected (Blum and Ebercon, 1981; Whitlow et al., 1992). Electron microscopy of barley leaf cells under drought conditions shows that volume reduction is achieved by deformation and folding of the cell surface (Pearce and Beckett, 1987). Although ultrastructural particles are usually present in biomembranes such as the plasma membrane, tonoplast and chloroplast envelopes, freezefracture observations reveal patches lacking intramembranous particles (free IMP) in well-watered wheat plants that are rapidly exposed to drought

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conditions (Pearce, 1985). The presence of IMP-free patches or patches with few IMPs is associated with the formation of lamellae between membranes. These patches appear similar to the plasma membrane lesions that occur following exposure to subzero temperatures, which freeze extracellular water (Steponkus et al., 1993). Lamellar and hexagonal II structures are wellknown lipid formations. For example, the lipid bilayer of the plasma membrane represents a unit of lamellar structure, whereas lipids form tube-like units in the hexagonal II structure, and these tubes are packed into a regular pattern. On the plasma membrane, freeze-induced lesions result during the transition from a lamellar to a hexagonal II structure. Lamellae are thought to appear during the loss of cytoplasmic water, when the plasma membrane closes to other membranes in the endomembrane system. Under drought conditions, free-IMP patches are thought to be induced by ion leakage from the cytoplasm. Loss of these ions results in irreversible lesions on the plasma membrane, and accumulation of such lesions will eventually kill the plants. Severe drought conditions also cause loss of available water from cellular macromolecules such as enzymes, the lipid membrane and polysaccharide components of the cell wall (Hoekstra et al., 2001; Moore et al., 2008). These macromolecules are conjugated to water molecules via hydrogen bonds, and this association is necessary for basic molecular function. The loss of water molecules results in significant structural and functional changes in cellular macromolecules. For instance, the loss of water molecules from polysaccharides (e.g. cellulose) causes the cell wall to tighten, reducing elasticity (Moore et al., 2008). Irreversible structural changes result from the complete loss of water molecules from macromolecules, a process referred to as desiccation. C. AVOIDANCE AND TOLERANCE OF SEVERE DROUGHT CONDITIONS

In arid regions, terrestrial plants have evolved several strategies for resisting water stress under severe drought conditions. In angiosperms, the most common mechanism for avoiding a water shortage is the control of germination timing. Succulents such as cacti have tissue modifications for water storage, and Eucalyptus roots penetrate deeply into soil to reach the available water. These examples are part of a plant’s water loss avoidance strategy, which also includes modifications to vegetative organs such as the leaf and stem. The other type of strategy is to generate increased tolerance to the water deficit by protection of cellular components. The seed is a major developmental phase that can be used to withstand water deficit. By sensing the presence of water, seeds are able to time the onset of germination appropriately. An extreme example of desiccation tolerance is found in the resurrection plant Ramonda serbica, which can

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recover from almost complete water loss (Farrant, 2000). When exposed to moderate drought conditions, many plants, including Arabidopsis, can acclimate by increasing drought tolerance (Mullet and Whitsitt, 1996; Whitlow et al., 1992). When rapid exposure to severe drought conditions follows a period of moderate drought conditions, plants will show reduced ion leakage (Blum and Ebercon, 1981; Mullet and Whitsitt, 1996; Whitlow et al., 1992). In barley, fewer IMP-free patches are formed in the leaves of plants that have experienced mild drought than in well-watered plants (Pearce, 1985). During drying, unsaturated fatty acids increase in the plasma membranes of Arabidopsis leaf cells (Gigon et al., 2004). A similar increase in unsaturated fatty acid species occurs under non-freezing cold stress and represents the acquisition of freezing tolerance (Uemura et al., 1995). The relationship between changes in fatty acid composition and freezing tolerance is also observed in other plants such as rye and oat (Steponkus et al., 1993). Interestingly, resurrection plants can survive more than 90% water loss by increasing the unsaturated fatty acids in the plasma membrane during drying (Quartacci et al., 2002). Freezing and drought cause similar stresses, as both induce dehydration of cells. Therefore, the physiological responses observed in the membrane under dehydrating conditions may relate to the molecular mechanisms for tolerance to dehydration.

III. MECHANISMS FOR ROOT HYDROTROPISM AND ITS POSSIBLE FUNCTIONS IN DROUGHT AVOIDANCE As far as we are aware, root hydrotropism was first described by Knight (1811). Knight wrote as follows: ‘‘When a tree, which requires much moisture, has sprung up or been planted, in a dry soil, in the vicinity of water, it has been observed, that much the largest portion of its roots has been directed towards the water.’’ Since then, root hydrotropism has fascinated many plant physiologists. Water acquisition is critical for plant survival on land and it is easy enough to imagine the importance of root hydrotropism. However, root hydrotropism was poorly understood until its ‘‘rediscovery’’. Its obscurity occurred because roots also display gravitropism (directed growth towards the centre of gravity; see next section), and researchers were unable to differentiate between the two tropisms. In addition, it was difficult to establish an experimental system that could maintain a stable moisture gradient. For these reasons, the existence of root hydrotropism remained in doubt for many years. It was in 1985 that Jaffe et al. rediscovered root hydrotropism using an agravitropic mutant of pea (Jaffe et al., 1985). When this pea mutant, ageotropum, was grown under moisture-saturated

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conditions, its roots emerged from the soil and grew into the humid air; however, when the atmosphere was dried, its roots would bend towards the moistened soil. This unequivocal demonstration of the existence of hydrotropism prompted a few researchers to begin to elucidate the nature of hydrotropism. More importantly, this demonstration showed that gravitropism often interferes with hydrotropism. Hence, in the early days, physiological studies were performed using the roots of agravitropic mutants or clinorotation (continuous rotation of samples to nullify the effects of gravity vector) of the seedlings. Following development of these experimental systems, root hydrotropism has been observed in pea, cucumber, maize, wheat and Arabidopsis (Mizuno et al., 2002; Oyanagi et al., 1995; Takahashi and Scott. 1991, 1993; Takahashi et al., 2002). From these experiments, many researchers have concluded that the ability to express root hydrotropism might be universal among monocots and dicots; however, arguments for and against the existence of root hydrotropism continue (Coutts and Nicoll, 1993; Plaut et al., 1996). Very recently, our group identified MIZUKUSSEI1, a gene responsible for root hydrotropism (Kobayashi et al., 2007). Since homologues of MIZU-KUSSEI1 are found across terrestrial plant species, it is likely that the capability to express root hydrotropism has been strongly conserved. In this section, we will describe physiological and genetic studies of root hydrotropism, as understanding the molecular mechanisms underlying this phenomenon could assist with the improvement of plant growth in arid areas. A. MECHANISM FOR SENSING HYDROSTIMULATION

Generally, tropism comprises three steps: sensing, signal transmission and response. The Cholodny-Went hypothesis suggests that the detection of environmental stimuli leads to lateral auxin redistribution (Went and Thimann, 1937), and recent molecular genetic studies have demonstrated that, for the most part, this hypothesis holds for the gravitropic response in roots. The Cholodny-Went hypothesis will be described in detail in the Section IV.A. As root hydrotropism is often masked by gravitropism, early investigations focused primarily on interactions between these two tropisms in various plant species. Several lines of evidence have suggested that the sensing apparatus for hydrostimulation resides in the root tip. In pea and maize, microsurgical removal of the root tip caused diminishment of root hydrotropism (Takahashi and Scott, 1993; Takahashi and Suge, 1991), and this tropism was lost completely following laser ablation of columella cells in the roots of Arabidopsis seedling (Miyazawa et al., 2008). Further, when agar blocks

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containing different concentrations of sorbitol are applied bilaterally to the root cap of an agravitropic pea mutant, the roots respond to gradients in water potential of 0.5–1.5 MPa by bending away from the sorbitol agar block (Takano et al., 1995). It is likely that plants respond to hydrostimulation at the cellular level, reacting to differences or gradients in water potential. As mentioned previously, a water potential gradient is formed across the root cap between the dry and wet sides. Although the molecules that form the sensory apparatus remain unknown, a mechano-sensitive ion channel responsive to osmotic stresses has been reported (Kung, 2005). Such a channel could be responsible for inducing differences in water potential between extracellular and intracellular regions. Recent studies using homologues of such genes have led to the hypothesis that these channels might be involved in the perception of hydrotropic stimuli (Haswell and Meyerowitz, 2006; Nakagawa et al., 2007). However, no experimental evidence on the involvement of these channels is demonstrated, rather it has been shown that a range of single and multiple knockout mutants for genes encoding such proteins showed no alteration in hydrotropism (Fujii et al., unpublished). In yeast, osmotic stress leads to the activation of a mitogen-activated protein kinase, Hog1p, via Sln1p and Sho1p (Reiser et al., 2003). Similarly, the Arabidopsis histidine kinase AHK1 has been implicated as a positive regulator in the stress response (Tran et al., 2007). Future studies will verify whether such proteins are involved in sensing hydrostimulation. B. MECHANISM FOR HYDROSTIMULATION SIGNAL TRANSMISSION

The gravistimulation sensing apparatus resides in the columella cells of the root tip. Interactions between hydrotropism and gravitropism may occur in roots, as both stimuli are sensed by the same or nearby cells. The hydrotropism of maize is affected by different intensities of gravistimulation (Takahashi and Scott, 1991), and the starch granules that play a role in Arabidopsis graviperception are degraded upon hydrostimulation (Takahashi et al., 2003). Indeed, a starchless mutant of Arabidopsis shows not only decreased gravitropism but also enhanced hydrotropism (Takahashi et al., 2003). However, the commonality of this phenomenon among plant species awaits further study. Calcium ions may be involved in the interaction between hydrotropism and gravitropism. Several studies have identified calcium ions as important signal transducers for gravitropism (Plieth and Trewavas, 2002; Sedbrook et al., 1996; Toyota et al., 2008), and treatment with a calcium ion chelator leads to the inhibition of both hydrotropism and gravitropism in pea and Arabidopsis roots (Takahashi and Suge, 1991; Kaneyasu et al., unpublished

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results). However, the exact role that calcium ions play in such an interaction remains unclear. Phospholipase D proteins may also be involved in the interaction between these tropisms. Phospholipase Ds are known to be involved in cellular responses to biotic and abiotic stresses (Bargmann and Munnik, 2006; Li et al., 2009). A recent report showed that the mutation of a gene encoding phospholipase D2 produces defects in both gravitropism and hydrotropism in Arabidopsis roots (Taniguchi et al., 2010). As the gene encoding phospholipase D2 is expressed in root tip cells, it is proposed that this enzyme functions as a signal transducer for drought stress (Taniguchi et al., 2010). However, the phenotypes of these ahydrotropic mutants are quite subtle and, thus, comprehensive studies of multiple phospholipase D mutants will be needed to fully understand the role that these enzymes play in hydrotropism. Although these findings represent important clues towards a future understanding of the mechanisms underlying hydrotropic signalling, the contributions of calcium and/or phospholipase Ds to hydrotropic signalling remain no more than speculation at this time. C. MECHANISM FOR HYDROTROPIC ROOT BENDING

Auxin plays a central role in many aspects of plant morphogenesis, including gravitropism. In the gravitropic response of roots, auxin plays an important role by transmitting the gravity signal from sensing cells, that is, columella cells, to the root elongation zone. When gravity is sensed, auxin is transported preferentially to the lower side of the root, which leads to the accumulation of auxin in the elongation zone (Muday, 2001). Auxin influx and efflux carriers play critical roles in this transport system. When seedlings are treated with inhibitors of either auxin influx or efflux, gravitropism is severely inhibited. Recent molecular genetic analyses of Arabidopsis have identified the auxin-related molecules that function in the gravitropic response in roots (Abas et al., 2006; Friml et al., 2002; Swarup et al., 2005). Classical studies on root hydrotropism have shown that the auxin efflux inhibitor TIBA inhibits the hydrotropic response in pea (Takahashi and Suge, 1991). In cucumber roots, the auxin efflux inhibitor also caused a reduction in the hydrotropic response (Morohashi et al., unpublished). Moreover, hydrotropic bending of pea seedling roots occurs when auxin is redistributed and accumulated at the concave side of the elongation zone (Takano, 1999). In cucumber, hydrotropic bending is associated with differential accumulation of mRNA for the auxin-inducible gene CsIAA1, with the higher mRNA levels being detected on the concave side than convex side of the elongation zone (Mizuno et al., 2002). These results strongly suggest that differential auxin transport from the root tip to the elongation zone is crucial for the hydrotropic response, at

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least in the plant species examined thus far. However, the hydrotropic responses of some auxin influx- or efflux-associated gravitropic mutants of Arabidopsis are comparable to the wild type (Takahashi et al., 2002). Moreover, this hydrotropic response was not reduced by inhibitors of auxin influx or efflux (Kaneyasu et al., 2007). While polar auxin transport is unnecessary for the hydrotropic response in Arabidopsis, an inhibitor of the auxin response substantially reduces both gravitropism and hydrotropism (Kaneyasu et al., 2007). These findings imply that the auxin response is necessary for both gravitropism and hydrotropism, but that the regulatory mechanisms for auxin dynamics may differ between them. At present, we do not know why only some plant species require polar auxin transport for hydrotropism. There might be an as-yet unidentified mechanism that regulates auxin redistribution or activity in a species-specific manner. To understand root hydrotropism from both an ecological and evolutionary point of view, we should make further efforts to determine this species specificity. Thus, experimental systems for hydrotropism must be established in multiple species for the requisite physiological studies to be performed. D. MOLECULAR IDENTIFICATION OF GENES RESPONSIBLE FOR HYDROTROPISM IN ARABIDOPSIS ROOTS

As described above, Arabidopsis uses a mechanism for hydrotropism that differs from that for gravitropism. Two strategies were adopted to clarify this molecular mechanism, transcriptomic identification of genes responsible for hydrotropism and isolation and analyses of mutants showing abnormal hydrotropism. Recent developments in DNA microarray technology have enabled comprehensive profiling of genes that are up- or down-regulated in response to environmental stresses, gravity and light (Killan et al., 2007; Kimbrough et al., 2004; Ma et al., 2003). Public release of the Arabidopsis transcriptome database has allowed researchers to determine the uniqueness or commonalities of different signalling pathways. To investigate the transcriptional changes associated with root hydrotropism, a microarray analysis was performed on hydrostimulated roots of Arabidopsis seedlings (Moriwaki et al., 2010). Among the 22,810 genes identified, 322 were up-regulated and 468 were down-regulated under hydrostimulated condition. Despite the intimate relationship between hydrotropism and gravitropism, there was little overlap between the genes responsible for these two tropisms. Thus, the transcriptional regulation of hydrotropism differs from that of gravitropism. However, a significant overlap was observed between the genes responsible for hydrostimulation, ABA and water stress, including drought, salt and osmotic stresses (Moriwaki et al., 2010). Thus, it was concluded that ABA and water-stress

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responses contribute to the regulation of hydrotropism at the transcriptional level. In addition, Ponce et al. (2008) reported that the application of ABA disrupted the hydrotropic response in the roots of wild-type Arabidopsis seedlings. Although these results have furthered our knowledge of hydrotropism at the molecular level, they raise two questions: (1) How are ABA and water-stress signals integrated into the signalling pathway of root hydrotropism? (2) Is there a similar molecular system in other plant species? Comparative genetic analyses will need to be performed in other plant species to answer these questions. The establishment of an experimental system to examine hydrotropism in the roots of Arabidopsis seedling has led to genetic screening for defects in root hydrotropism (Eapen et al., 2003; Kobayashi et al., 2007). Cassab and her colleagues reported two mutants named no hydrotropic response1 and 2 (nhr1, 2; Eapen et al., 2003). Although the genes responsible for these mutants have not yet been identified, physiological studies of the semidominant mutant, nhr1, showed abnormal root tip cells and enhanced gravitropism (Eapen et al., 2003). Subsequent reports have suggested that nhr1 exhibits pleiotropic phenotypes, most of which can be explained by enhanced accumulation of the phytohormone ABA (Ponce et al., 2008; QuirozFigueroa et al., 2010). In addition, these authors showed that ABA treatment of wild-type roots not only reduces the starch granules in the root tip but also produces enhanced gravitropism. Although these findings have led to the hypothesis that ABA negatively regulates root hydrotropism (Ponce et al., 2008; Quiroz-Figueroa et al., 2010), the roots of some ABA-deficient and insensitive mutants have shown reductions in both hydrotropism and gravitropism (Takahashi et al., 2002). As hydrostimulation is related to mechanisms for combating water stress, it is possible that NHR1 might play a role in the drought response and/or drought-triggered ABA signalling. Clearly, ABA synthesis and signalling are important for root hydrotropism (Moriwaki et al., 2010; Takahashi et al., 2002); however, more detailed studies of the nhr1 mutant will be necessary to determine the molecular mechanism underlying root hydrotropism in its pleiotropic phenotypes, as well as the identification of the gene responsible for the phenotype. At the same time as work was being undertaken on nhr1, our group isolated two ahydrotropic mutants, mizu-kussei1 (miz1) and miz2 (Kobayashi et al., 2007; Miyazawa et al., 2009a). Although these mutants were completely ahydrotropic, they retained normal gravitropism, which enabled the dissection of molecular mechanisms unique to root hydrotropism. These are the only ahydrotropic mutants in which the responsible genes have been identified. MIZ1 encodes a protein of 297 amino acids that contains a domain of unknown function (DUF617), and MIZ2 encodes a guanine exchange factor for ADP-ribosylation factor (ARF-GEF), which is known as GNOM

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(Kobayashi et al., 2007; Miyazawa et al., 2009a). Thus far, our investigations have indicated that MIZ1 expression is restricted to the root tip, the mature region of primary roots and leaf hydathodes (Kobayashi et al., 2007). As hydrostimulation sensing is thought to occur at the root tip, it is likely that MIZ1 functions during an early stage of the root hydrotropic response. As stated above, MIZ1 contains a domain of unknown function, which we termed the MIZ domain. Genes encoding this domain are found in terrestrial plants but not in algae, fungi, bacteria or animals. Searches of the Arabidopsis genome revealed 12 genes encoding the MIZ domain; however, as yet, there are no reports describing its function. The missense mutation in miz1-1 is found in a conserved amino acid within the MIZ domain, which suggests that this domain plays an important role in root hydrotropism (Takahashi et al., 2009). Moreover, amino acid sequences outside the MIZ domain are not conserved among the gene family. With the exception of slightly reduced root phototropism, the miz1 mutant shows no obvious morphological defects other than the ahydrotropic root phenotype (Kobayashi et al., 2007). Phototropism of the hypocotyl appears normal in the miz1 mutant. Moreover, normal root hydrotropism is observed in nph1 mutants, which lack phototropin1. Although much of the relationship between root phototropism and hydrotropism remains to be elucidated, these findings suggest that MIZ1 may function in a common signalling pathway (Takahashi et al., 2002). Further investigations of MIZ1 will shed new light on the as-yet unidentified relationships between hydrotropism and phototropism in roots. Unlike MIZ1, MIZ2 encodes a well-described protein, GNOM (Miyazawa et al., 2009a). GNOM plays important roles in membrane trafficking and it is responsible for the dissociation of GDP from ARFs (Anders and Ju¨rgens, 2008). The guanine exchange activity of ARF-GEFs resides within the Sec7 domain, which exhibits strong homology to the yeast protein Sec7p. Embryonic lethality is observed in most severe gnom alleles that contain mutations inside the Sec7 domain. The conserved domains, DCB and HUS, are located upstream of the Sec7 domain, while the conserved domains, HDS 1–3, are located downstream. Interestingly, miz2 is a missense mutation that causes a single amino acid change in the less conserved region flanking the HDS1 domain. The phenotype of this mutant was surprising, as nearly all the defects associated with gnom alleles involve altered auxin transport. Proper localization of the auxin efflux facilitator requires GNOM function, and defects in localization cause agravitropic root growth, as well as abnormal organ patterning and vein formation (Geldner et al., 2003, 2004). The miz2 mutants were not defective in organ patterning or vein formation and showed normal localization of the mutant GNOM (gnommiz2), as well as the auxin efflux

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facilitator, PIN1 (Miyazawa et al., 2009b). Pharmacological and genetic studies support the suggestion that polar auxin transport is unnecessary for hydrotropism in Arabidopsis roots; however, these findings imply that GNOM performs an as-yet undiscovered role in root hydrotropism, as well as being instrumental for polar auxin transport. When a primary root of Arabidopsis is grown on an inclined agar plate, it exhibits an oscillatory pattern called waving (Okada and Shimura, 1990). Although we do not understand the exact reason why this root movement is induced, it is likely that the graviresponse and/or touch response is involved in the phenomenon (Oliva and Dunand, 2007). This root movement can be interpreted as reflecting the obstacle-avoidance response. It would be interesting to know whether or not there is any integration between the signalling pathways for root hydrotropism and wavy root growth. Some mutants with a short wavelength phenotype also demonstrate enhanced hydrotropism (Takahashi et al., 2002). Moreover, all reported ahydrotropic mutants also exhibit abnormal wavy growth. However, the waving phenotypes of the ahydrotropic mutants vary. For example, nhr1 shows enhanced wavy growth, while miz1 and miz2 exhibit decreased wavy growth (Eapen et al., 2003; Kobayashi et al., 2007; Miyazawa et al., unpublished result). Currently, we do not know why these opposite phenotypes are found in ahydrotropic mutants. However, it is clear that the signalling pathway for root hydrotropism interacts with many other root navigation signalling pathways.

IV. MECHANISMS FOR OTHER ROOT TROPISMS RELATED TO DROUGHT AVOIDANCE Plant roots show positive gravitropism and negative phototropism. Under normal conditions, soil water conditions reflect the effects of gravity on precipitation and the soil surface is assumed to be arid when exposed to sunshine. Thus, roots show positive gravitropism and negative phototropism to orient growth appropriately. In this section, we will give a brief summary of the molecular mechanisms underlying these two tropisms. As positive gravitropism and negative phototropism are particularly important for avoiding drought conditions, we will also discuss their regulation under water-stressed conditions. A. ROOT GRAVITROPISM

Physiological analyses of roots in which the root cap was removed surgically indicated that the gravity sensor for root gravitropism resides within the root cap. The root cap is organized by specialized cells, namely columella and lateral

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root cap cells. Laser ablation experiments have indicated that columella cells are essential for gravitropism (Blancaflor et al., 1998). Columella cells contain starch-filled plastids, termed amyloplasts. Due to the dense nature of starch, amyloplasts sediment under the force of gravity and accumulate at the lower side of the cell. As this sedimentation is observed immediately after gravistimulation, it is thought to be the trigger for gravi-sensing (Sack, 1991). This hypothesis is supported by genetic analyses of starchless Arabidopsis mutants and Kiss et al. (1989, 1996) showed that the roots of starchless mutants exhibit a weaker response to gravity than the wild type. Amyloplast sedimentation generates a physical signal that is converted into a biochemical signal, which initiates the graviresponse. However, the mechanisms underlying these processes are not fully understood. One possibility is that the sedimented amyloplasts activate mechano-sensitive ion channels in intercellular membranes or the plasma membrane. The activated channels lead to changes in Hþ and Ca2þ flow, and these ions have been postulated to play important roles in gravity signal transduction within root cap statocytes (Fasano et al., 2001; Perera et al., 2001). The starchless mutant pgm1 does not show pH changes after gravistimulation, which suggests that changes in root cap pH depend upon amyloplast sedimentation (Fasano et al., 2001). ALTERED RESPONSE TO GRAVITY1 (ARG1) and its homologue ARL2 play key roles in gravity signal transduction (Boonsirichai et al., 2003). These genes encode DnaJ-like proteins. Following gravistimulation, no changes in cytosolic pH were detected in the roots of an arg1 mutant; however, the mutant did exhibit reduced gravitropism. Moreover, the arg1 mutant phenotype was recovered by the expression of ARG1 in the root cap, which suggests that ARG1 functions during early gravisignal transduction (Boonsirichai et al., 2003). According to the Cholodny-Went hypothesis, gravistimulation induces an asymmetrical auxin gradient within gravistimulated organs, and auxin-dependent growth inhibition results in a downward curvature of roots. Indeed, an asymmetric pattern of auxin-responsive gene expression occurs rapidly in columella and lateral root cap cells following gravistimulation (Ottenschla¨ger et al., 2003). Pharmacological and genetic disruption of auxin transport inhibits the asymmetrical auxin redistribution and tropic responses in the root (Fujita and Syono, 1996; Li et al., 1991; Luschnig et al., 1998; Marchant et al., 1999). A combination of auxin influx and efflux transporters mediates auxin transport through the root cell files. In Arabidopsis roots, AUX1/LAX family proteins function as auxin influx carriers. As the mutation of AUX1 completely disrupts root gravitropism, it is likely that the AUX1-mediated influx of auxin is essential for gravitropism (Marchant et al., 1999). Localization analyses have shown that AUX1 is expressed in lateral root cap and stele cells, and that the expression of AUX1 in lateral root cap cells alone can rescue

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gravitropism in an aux1 mutant (Swarup et al., 2001, 2005). These results indicate that gravitropism requires auxin redistribution via the lateral root cap. The PINFORMED (PIN) auxin transporters have been shown to play critical roles in auxin efflux. PIN3 localizes symmetrically in the plasma membrane of columella cells but will relocalize to the lower side of the cell upon gravistimulation (Friml et al., 2002). This gravity-induced relocalization was not observed in mutants of ARG1 or ARL2, which suggests that ARG1/ARL2-dependent signalling is required for lateral relocalization of PIN3 (Harrison and Masson, 2007). Surprisingly, a pin3 mutant only exhibited a weak reduction in the root gravitropic response (Friml et al., 2002); however, this finding may be due to the redundancy of function among the PIN protein family (Vieten et al., 2005). In roots, PIN2 localizes to the apical membrane of epidermal cells and the basal membrane of cortical cells, and pin2 mutants completely lack root gravitropism (Luschnig et al., 1998; Muller et al., 1998). Following gravistimulation, epidermal PIN2 proteins present on the upper side of the cell were rapidly internalized and degraded (Abas et al., 2006). This degradation process results in an asymmetric distribution of PIN2, which is consistent with the lateral auxin distribution in the root. It is well known that the subcellular localization of PIN proteins is strictly determined by regulatory proteins. For instance, basal localization is regulated by GNOM, and apical localization is regulated by PINOID protein kinase (Friml et al., 2004; Geldner et al., 2003). Indeed, root gravitropic responses are disrupted by mutations in either GNOM or PINOID (Geldner et al., 2004; Sukumar et al., 2009). Later phases of gravitropism depend upon auxin-regulated transcription, which is controlled by a combination of two transcriptional regulators, AUXIN RESPONSE FACTOR (ARF) and AUX/IAA protein. The Arabidopsis genome contains 23 genes that encode ARF proteins, but two closely related genes, ARF7 and ARF19, are required for gravitropism (Okushima et al., 2005). Under low auxin concentrations, AUX/IAA binds ARF and represses its activity. In the presence of high auxin concentrations, auxin binds and activates the SCFTIR1 complex, which degrades AUX/IAA proteins. As expected, a mutation that stabilizes AUX/IAA proteins will also disrupt gravitropism (Fukaki et al., 2002; Weijers et al., 2005). B. REGULATION OF GRAVITROPISM BY WATER STRESS

Many researchers have demonstrated that the gravitropic response is modulated by water conditions. For instance, Sharp and Davies (1985) showed that water depletion accelerates root distribution in deeper soils. Osmotic stress also

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enhances the gravitropic response of maize roots (Leopold and LaFavre, 1989). These observations led to the hypothesis that water stress enhances the gravitropic response. Drought stress is known to induce ABA biosynthesis in roots (Zhang and Davies, 1987). ABA functions not only as a signal molecule for different types of drought response, but also as a regulator of root gravitropism. ABA treatment increases the gravitropic curvature of maize roots in a dose-dependent manner (Wilkins and Wain, 1976). In Arabidopsis, the ABA synthesis and signalling mutants, aba1 and abi2, show slightly reduced gravitropic responses (Takahashi et al., 2002). In contrast, Moore (1990) found a normal graviresponse in ABA-deficient mutants of maize, which suggests that while changes in ABA homeostasis may enhance the gravitropic response, ABA is not essential for gravitropism. Although numerous findings have described relationships between ABA and gravitropism, we have little understanding of how ABA functions in gravitropism under drought conditions. Ethylene is another important stress response hormone in plants. Usually, high concentrations of ethylene up-regulate auxin biosynthesis and, thus, inhibit the elongation of root cells (Swarup et al., 2007). To maintain root elongation under drought conditions, ABA represses ethylene synthesis (Sharp and LeNoble, 2002; Spollen et al., 2000). However, the mutation of EIN2, which is a positive regulator of ethylene signalling, causes a reduction of root growth under osmotic stress conditions (Wang et al., 2007). This finding suggests that ethylene signalling is an important factor in root growth under drought conditions. In addition, there is increasing evidence to indicate that ethylene also regulates the gravitropic response. Studies have shown decreased gravitropic responses following early phase application of ethylene gas or the ethylene precursor ACC in maize or Arabidopsis roots, respectively (Buer et al., 2006; Lee et al., 1990). Pharmacological inhibition of ethylene synthesis also reduced the gravitropism of maize roots (Chang et al., 2004; Lee et al., 1990). Inhibition of ethylene synthesis by drug treatments also reduced the gravitropism of maize roots (Chang et al., 2004; Lee et al., 1990). Thus, it appears that ethylene can both positively and negatively affect gravitropism. As the auxin transport inhibitor NPA abolishes the effect of ethylene on gravitropism (Lee et al., 1990), it is likely that ethylene affects the gravityinduced asymmetrical distribution of auxin. Recent work has demonstrated that flavonoids may function as ethylene-dependent regulators of gravitropism (Buer et al., 2006). In Arabidopsis roots, ethylene treatment inhibits the gravitropic response; however, this gravitropic inhibition is lost in chalcone synthase-deficient tt4 mutants (Buer and Muday, 2004). Flavonoid synthesis is modulated by environmental changes such as gravistimulation, and ACC treatment delays gravity-induced flavonoid synthesis (Buer and Muday, 2004; Buer et al., 2006). In addition, PIN mRNA expression patterns are

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altered in the tt4 mutant, which shows disruption to the polar localization of some PIN proteins (Peer et al., 2004). These results indicate that flavonoids are important for maintaining the auxin flow required for gravitropism, and that ethylene inhibits root gravitropism in Arabidopsis via modulation of flavonoid synthesis. Osmotic stress is also known to induce flavonoid accumulation (Chalker-Scott, 1999), which suggests that under water-stress conditions, flavonoid regulation of the gravitropic response occurs downstream of ethylene. Although many researchers have pointed out that drought signals modulate the gravitropic response, the obvious ecological benefits of root gravitropism under drought conditions are not fully understood. Mutants in signalling or synthesis of the stress signal hormones ABA and ethylene show poor tolerance to water deficits or salinity (Cao et al., 2007). However, ABA and ethylene play multiple roles in stress tolerance and, thus, a reduced gravitropic response would not be the sole factor contributing to the phenotypes observed with ABA and ethylene signalling or synthesis mutants under stress conditions. In addition, the roots of aux1 mutants exhibit a completely agravitropic phenotype, but no difference was detected in the survival ratio of aux1 and wild-type seedlings, even under drought conditions (Vartanian, 1996). As this field progresses, it is anticipated that novel approaches will assist in the elucidation of the ecological functions of the root gravitropic response. C. ROOT PHOTOTROPISM

Typically, roots show negative phototropism in response to blue or white light and this assists in the development of the root system in soil. In contrast, Arabidopsis roots also exhibit positive phototropism in response to red light; however, the ecological function of this response is not known (Ruppel et al., 2001). Arabidopsis has three major classes of photoreceptors, that is, the phytochromes, cryptochromes and phototropins. Genetic analyses of phototropic mutants have revealed that the phototropin family is essential for blue light perception and that these proteins are required for negative phototropism in roots (Christie et al., 1998; Sakai et al., 2001). It has been shown that two phytochromes, namely PHYA and PHYB, are required for positive phototropism in roots (Kiss et al., 2003). The mechanisms for signal transduction in root phototropism remain largely unknown. Isolation of the phototropism mutant nph3 led to the identification of NPH3, which encodes a plant-specific protein containing a BTB domain and a coiled-coil domain (Motchoulski and Liscum, 1999). NPH3 interacts with a phototropin, PHOT1, via the coiled-coil domain

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and with CULLIN3, which is a subunit of E3 ubiquitin ligase, via its BTB domain (Inada et al., 2004; Motchoulski and Liscum, 1999). In another phototropic mutant, rpt2, the NPH3-like gene RPT2 is disrupted. Inada et al. (2004) demonstrated that RPT2 interacts with both PHOT1 and NPH3. These results suggest that RPT2 functions as an adaptor between PHOT1 and NPH3. PHYTOCHROME KINASE SUBSTRATE1 (PKS1) was isolated originally from a screen using PHYA-interacting factor (Fankhauser et al., 1999). PKS1 also interacts with PHOT1 (Lariguet et al., 2006), and PKS1 mutants show reductions in both positive and negative root phototropism (Boccalandro et al., 2008; Molas and Kiss, 2008). Further, enhanced gravitropism was observed in a pks1 mutant, which indicates that PKS1 is involved in the transduction of both gravity and light signals (Boccalandro et al., 2008). In addition to gravitropism, pharmacological analysis has demonstrated that auxin-dependent asymmetric growth is required for root phototropism (Fujita and Syono, 1997). However, the molecular machinery required to establish an auxin gradient in response to light perception remains unclear. Recently, Ruzicka et al. (2010) demonstrated that PIS1 encodes an ABC transporter that can transport the IAA precursor, IBA. As the roots of PIS1 mutants exhibit slightly reduced phototropism and gravitropism (Fujita and Syono, 1997), it is possible that PIS1-mediated auxin transport may contribute to the establishment of a phototropic auxin gradient. D. ECOLOGICAL FUNCTION OF PHOTOTROPISM

In contrast to the research on gravitropism, there is little evidence from which to determine whether or not root phototropism is enhanced by drought or its related signals, such as salt, ABA and ethylene. Experiments on the phototropic mutant phot1 have indicated that root phototropism could contribute to the orientation of the root system, directing growth away from the soil surface to avoid drought conditions (Galen et al., 2007). Thus, root phototropism represents an important strategy for drought adaptation in plants.

V. CONCLUSIONS AND PERSPECTIVES Considering their sessile nature and the necessity for water acquisition, plants have evolved unique mechanisms to avoid or tolerate drought and to utilize the resources available at the site of germination. To improve plant survival under water-deficient conditions, it is necessary to understand and utilize drought tolerance and/or avoidance mechanisms. Intense study has led to many

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discoveries regarding the mechanisms underlying drought tolerance in plants, and these are described in the other chapters. However, it is important to develop plants with enhanced drought avoidance capabilities, as these seedlings would have the ability to acquire water for continued survival and to sustain growth. Roots exhibit tropisms in response to many environmental cues, including gravity, light and moisture gradients. Among these, the root hydrotropism response to moisture gradients is thought to function in water-stress avoidance as well as in the efficient uptake of water and nutrients from the soil (Takahashi et al., 2009). As described in this chapter, clarification of molecular mechanisms underlying root hydrotropism is just beginning, and our knowledge is too fragmented to determine the entire process. Nevertheless, we now have some clues to add to the puzzle, at least in Arabidopsis. Detailed studies that connect the physiological, genetic and molecular biological information of root hydrotropism will help develop a broader understanding of this phenomenon. In addition, it is important to clarify the mechanisms that integrate the different environmental cues that lead to the directional growth of roots. The ecological significance of root hydrotropism must also be elucidated. Thus far, contradictory results have been reported (e.g. Cole and Mahall, 2006; Tsuda et al., 2003) and these problems will need to be addressed using sophisticated experimental systems and a wide variety of plant species and/or ecotypes. Along with such studies, different types of hydrotropism mutants will be useful for developing a more detailed understanding of this phenomenon. We believe that future investigations will lead to the elucidation of all root tropism mechanisms, and especially hydrotropism. Once we understand root tropisms at the molecular level, these processes may be manipulated to develop plants with enhanced survival capabilities under water-stress conditions, improvements that will be beneficial for overcoming current climate changes.

ACKNOWLEDGEMENTS This work is supported by Grants-in-Aid for Scientific Research (B: 20370017) from JSPS, Grants-in-Aid for Scientific Research on Priority Areas (No. 22120004) from the MEXT, the Global COE Program J03 (Ecosystem Management Adapting to Global Change) of the MEXT to H. T., Special Research Grant of Global COE Program J03 for T. Y. (No. 27220004) and JSPS Research Fellowship for Young Scientists to T. M. (No. 09J06705). Y. M. is supported by the Funding Program for Next-Generation WorldLeading Researchers (GS002). This work was carried out as a part of the ‘‘Ground-based Research Announcement for Space Utilization’’, promoted by the Japan Space Forum.

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