Ionic Current Changes Associated with the Gravity-Induced Bending Response in Roots of Zea mays L

A vibrating probe was used to measure the changes in ionic currents around gravistimulated roots of Zea mays L. in an effort to determine whether these currents are involved in stimulus transduction from the root cap to the elongation zone. We did not observe a migration of the previously reported auxin-insensitive current efflux associated with gravity sensing (T. Björkman, A.C. Leopold [1987] Plant Physiol 84:841-846) back from the root cap. Instead, beginning 10 to 15 min after gravistimulation, an asymmetry in current developed simultaneously along the root around the meristem and apical regions of the elongation zone. This asymmetry comprised a proton efflux from the upper surface, which was superimposed on the symmetrical pattern around the vertical root. The gravity-induced proton efflux was inhibited by the application of the auxin transport inhibitor, 2,3,5-triiodobenzoic acid, whereas the calcium channel blocker, lanthanum, had little effect. Because the onset of the gravity-induced current asymmetry coincided both spatially and temporally with the onset of the differential growth response, we suggest that this current efflux may result from auxin-requiring acid-growth phenomena in the upper root tissue. The implications of this simultaneous onset of both proton efflux and elongation for theories about gravity stimulus transduction are discussed.     

Actin Turnover-Mediated Gravity Response in Maize Root Apices: Gravitropism of Decapped Roots Implicates Gravisensing Outside of the Root Cap

The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extents suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth process. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap.Key Words: actin cytoskeleton, gravisensing, graviresponding, root cap     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

Positional effect of cell inactivation on root gravitropism using heavy-ion microbeams

When primary root apical tissues of Arabidopsis thaliana were irradiated by heavy-ion microbeams with 120 microm diameter, strong inhibition of root elongation and curvature were observed at the root tip. Irradiation of the cells that become the lower part of the root cap after gravistimulation showed strong inhibition of root curvature, whereas irradiation of the cells that become the upper part of the root cap after gravistimulation did not show severe damage in either root curvature or root growth. Further analysis using smaller area microbeams with 40 microm diameter indicated that the greatest inhibition of curvature occurred at the root tip and the next greatest inhibition occurred in the cells in the lower part of the root cap. These results indicate not only that the root tip and columella cells are the most sensitive sites for root gravity, but also that signalling of root gravity would go through the lower part of the cap cells after perception.     

Changes in IAA responsiveness in the elongation region of graviresponding mung bean roots

IAA responsiveness of sections of root tissue taken from the top and bottom of mung bean roots was assessed prior to and at varying times following gravistimulation. Prior to gravistimulation, root tissue sections from the sides of the elongation zone responded similarly to IAA. After gravistimulation (within 5 min), root sections from the bottom of the elongation zone became more responsive to IAA than sections collected from the upper side of the elongation zone. The change in IAA responsiveness of these tissue sections was transient with root sections from both the top and bottom of the elongation zone again exhibiting similar responsiveness to IAA following 15 minutes of gravistimulation.These studies also examined if the root tip is required for the gravity-induced shift in IAA responsiveness in the tissues of the elongation zone. The IAA responsiveness of top and bottom sections of the elongation zone from decapped mung bean roots was assessed at varying times following gravistimulation. The responsiveness to IAA of top and bottom sections changed rapidly in decapped roots, just as had been previously found for intact roots. Although the alteration in responsiveness was transient in decapped roots (just as intact roots), the time it took for the sections to recover previous responsiveness to IAA was extended.These results suggest that the initial growth response of graviresponding roots may be due to a change in the IAA responsiveness of tissues in the elongation zone and not an asymmetric accumulation of IAA on the lower side of the elongation zone. The results also indicate that the gravity-induced shift in IAA responsiveness in the elongation zone occurs independently of the root cap, suggesting that the cells in the elongation region can perceive and respond to gravity independently of the root cap during the intial phases of the gravity response.     

A Role for the TOC Complex in Arabidopsis Root Gravitropism

Arabidopsis (Arabidopsis thaliana) roots perceive gravity and reorient their growth accordingly. Starch-dense amyloplasts within the columella cells of the root cap are important for gravitropism, and starchless mutants such as pgm1 display an attenuated response to gravistimulation. The altered response to gravity1 (arg1) mutant is known to be involved with the early phases of gravity signal transduction. arg1 responds slowly to gravistimulation and is in a genetically distinct pathway from pgm1, as pgm1 mutants enhance the gravitropic defect of arg1. arg1 seeds were mutagenized with ethylmethane sulfonate to identify new mutants that enhance the gravitropic defect of arg1. Two modifier of arg1 mutants (mar1 and mar2) grow in random directions only when arg1 is present, do not affect phototropism, and respond like the wild type to application of phytohormones. Both have mutations affecting different components of the Translocon of Outer Membrane of Chloroplasts (TOC) complex. mar1 possesses a mutation in the TOC75-III gene; mar2 possesses a mutation in the TOC132 gene. Overexpression of TOC132 rescues the random growth phenotype of mar2 arg1 roots. Root cap amyloplasts in mar2 arg1 appear ultrastructurally normal. They saltate like the wild type and sediment at wild-type rates upon gravistimulation. These data point to a role for the plastidic TOC complex in gravity signal transduction within the statocytes.     

Changes in root cap pH are required for the gravity response of the Arabidopsis root

Although the columella cells of the root cap have been identified as the site of gravity perception, the cellular events that mediate gravity signaling remain poorly understood. To determine if cytoplasmic and/or wall pH mediates the initial stages of root gravitropism, we combined a novel cell wall pH sensor (a cellulose binding domain peptide-Oregon green conjugate) and a cytoplasmic pH sensor (plants expressing pH-sensitive green fluorescent protein) to monitor pH dynamics throughout the graviresponding Arabidopsis root. The root cap apoplast acidified from pH 5.5 to 4.5 within 2 min of gravistimulation. Concomitantly, cytoplasmic pH increased in columella cells from 7.2 to 7.6 but was unchanged elsewhere in the root. These changes in cap pH preceded detectable tropic growth or growth-related pH changes in the elongation zone cell wall by 10 min. Altering the gravity-related columella cytoplasmic pH shift with caged protons delayed the gravitropic response. Together, these results suggest that alterations in root cap pH likely are involved in the initial events that mediate root gravity perception or signal transduction.     

Ethylene-dependent adjustment of metabolite profiles in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seedlings during gravitropic response

Metabolite profile adjustments under impact of ethylene synthesis inhibitors were studied in Arabidopsis thaliana (L.) Heynh. seedlings during reorientation of plants relative to the gravity vector (gravistimulation). Metabolite profiles were compared with Principal Component Analysis. We have shown that significant changes in metabolite profiles developed within 60 min of gravistimulation and were most pronounced in 2 mm root tips including the root cap, apical meristem and elongation zone. Gravistimulation resulted in the increased levels of valine, leucine, serine, γ-aminobutyric acid, nicotinic acid, and decreased levels of several monosaccharides, malate and oxalate. Treatment with ethylene synthesis inhibitor, aminoethoxyvinylglycine (10 μM), escaped the effect of gravistimulation on root tip metabolite profile. Metabolite profile adjustments revealed in this study suggest that ethylene may be involved into the regulation of Arabidopsis metabolome during the gravitropic response.     

Mapping the Functional Roles of Cap Cells in the Response of Arabidopsis Primary Roots to Gravity

The cap is widely accepted to be the site of gravity sensing in roots because removal of the cap abolishes root curvature. Circumstantial evidence favors the columella cells as the gravisensory cells because amyloplasts (and often other cellular components) are polarized with respect to the gravity vector. However, there has been no functional confirmation of their role. To address this problem, we used laser ablation to remove defined cells in the cap of Arabidopsis primary roots and quantified the response of the roots to gravity using three parameters: time course of curvature, presentation time, and deviation from vertical growth. Ablation of the peripheral cap cells and tip cells did not alter root curvature. Ablation of the innermost columella cells caused the strongest inhibitory effect on root curvature without affecting growth rates. Many of these roots deviated significantly from vertical growth and had a presentation time 6-fold longer than the controls. Among the two inner columella stories, the central cells of story 2 contributed the most to root gravitropism. These cells also exhibited the largest amyloplast sedimentation velocities. Therefore, these results are consistent with the starch-statolith sedimentation hypothesis for gravity sensing.     

Gravitropic curvature of maize roots is not preceded by rootcap asymmetry

We tested whether the first response to gravistimulation is an asymmetry in the root tip that results from differential growth of the rootcap itself. The displacement of markers on the rootcap surface of maize (Zea mays L. cv. Merit) roots was quantified from videotaped images using customized software. The method was sensitive enough to detect marker displacements down to 15 microns and root curvature as early as 8 min after gravistimulation. No differential growth of the upper and lower sides of the cap occurred before or during root curvature. Fewer than a third of all gravistimulated roots developed an asymmetrical outline of the root tip after curvature had started, and this asymmetry did not occur in the rootcap itself. Our data support the view that the regions of gravitropic sensing and curvature are spatially separate during all phases of gravitropism in maize roots.     

The kinetics of root gravitropism: dual motors and sensors

The Cholodny-Went theory of tropisms has served as a framework for investigation of root gravitropism for nearly three quarters of a century. Recent investigations using modern techniques have generated findings consistent with the classical theory, including confirmation of asymmetrical distribution of polar auxin transport carriers, molecular evidence for auxin asymmetry following gravistimulation, and generation of auxin response mutants with predictable lesions in gravitropism. Other results indicate that the classical model is inadequate to account for key features of root gravitropism. Initiation of curvature, for example, occurs outside the region of most rapid elongation and is driven by differential acceleration rather than differential inhibition of elongation. The evidence indicates that there are two motors driving root gravitropism, one of which appears not to be auxin regulated. We have recently developed technology that is capable of maintaining a constant angle of gravistimulation at any selected target region of a root while continuously monitoring growth and curvature kinetics. This review elaborates on the advantages of this new technology for analyzing gravitropism and describes applications of the technology that reveal (1) the existence of at least two phases to gravitropic motor output, even under conditions of constant stimulus input and (2) the existence of gravity sensing outside of the root cap. We propose a revised model of root gravitropism including dual sensors and dual motors interacting to accomplish root gravitropism, with only one of the systems linked to the classical Cholodny-Went theory.     

Role of cytokinin in the regulation of root gravitropism

The models explaining root gravitropism propose that the growth response of plants to gravity is regulated by asymmetric distribution of auxin (indole-3-acetic acid, IAA). Since cytokinin has a negative regulatory role in root growth, we suspected that it might function as an inhibitor of tropic root elongation during gravity response. Therefore, we examined the free-bioactive-cytokinin-dependent ARR5::GUS expression pattern in root tips of transformants of Arabidopsis thaliana (L.) Heynh., visualized high cytokinin concentrations in the root cap with specific monoclonal antibodies, and complemented the analyses by external application of cytokinin. Our findings show that mainly the statocytes of the cap produce cytokinin, which may contribute to the regulation of root gravitropism. The homogenous symmetric expression of the cytokinin-responsive promoter in vertical root caps rapidly changed within less than 30 min of gravistimulation into an asymmetrical activation pattern, visualized as a lateral, distinctly stained, concentrated spot on the new lower root side of the cap cells. This asymmetric cytokinin distribution obviously caused initiation of a downward curvature near the root apex during the early rapid phase of gravity response, by inhibiting elongation at the lower side and promoting growth at the upper side of the distal elongation zone closely behind the root cap. Exogenous cytokinin applied to vertical roots induced root bending towards the application site, confirming the suspected inhibitory effect of cytokinin in root gravitropism. Our results suggest that the early root graviresponse is controlled by cytokinin. We conclude that both cytokinin and auxin are key hormones that regulate root gravitropism.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-004-1381-8     

Rapid Changes in the Pattern of Electric Current around the Root Tip of Lepidium sativum L. following Gravistimulation

Using a highly sensitive vibrating electrode, the pattern of naturally occurring electric currents around 1-day-old primary roots of Lepidium sativum L. growing vertically downward and the current pattern following gravistimulation of the root has been examined. A more or less symmetrical pattern of current was found around vertically oriented, downward growing roots. Current entered the root at the root cap, the meristem, and the beginning of the elongation zone and left the root along most of the elongation zone and in the root hair zone. After the root was tilted to a horizontal position, we observed current flowing acropetally at the upper side of the root cap and basipetally at the lower side within about 30 seconds in most cases. After a delay of several minutes, acropetally oriented current was also found flowing along the upper side of the meristematic zone. The apparent density of the acropetal current in the root cap region increased and then decreased with time. Gravitropic curvature was first visible approximately 10 minutes after tilting of the root to the horizontal position. Since the change in the pattern of current in the root cap region precedes bending of the root and is different for the upper and lower side, a close connection is suggested between the current and the transduction of information from the root cap to the elongation zone following graviperception in the cap.     

Hydrotropism: root growth responses to water

The survival of terrestrial plants depends upon the capacity of roots to obtain water and nutrients from the soil. Directed growth of roots in relation to a gradient in moisture is called hydrotropism and begins in the root cap with the sensing of the moisture gradient. Even though the lack of sufficient water is the single-most important factor affecting world agriculture, there are surprisingly few studies on hydrotropism. Recent genetic analysis of hydrotropism in Arabidopsis has provided new insights about the mechanisms that the root cap uses to perceive and respond simultaneously to moisture and gravity signals. This knowledge might enable us to understand how the root cap processes environmental signals that are capable of regulating whole plant growth.     

Basipetal propagation of gravity-induced surface pH changes along primary roots of <Emphasis Type="Italic">Lepidium sativum</Emphasis> L.

While there is ample evidence for a role of auxin in root gravitropism, the seeming rapidity of gravi-induced changes in electrical parameters has so far been an argument against auxin being a primary signal in gravitropic signal transmission. To address this problem, we re-investigated the effect of gravistimulation on membrane voltages of Lepidium sativum L. and Vigna mungo L. root cells. In our hands, gravistimulation did not induce changes in membrane voltage in cells of the root cap statenchyma, root meristem or apical elongation zone that can be correlated with the orientation of the cells relative to the gravity vector. While these results challenge a model of rapid electrically based signal transmission, there is evidence for a slower signal propagation along gravistimulated L. sativum roots. Using multiple proton-selective microelectrodes to simultaneously measure surface pH on opposite root flanks at different distances from the root tip, we observed gravi-induced asymmetric pH changes at the surface of all investigated root zones. Upon gravistimulation, the surface pH decreased on the physically upper root flank and increased on the lower flank. The pH asymmetry appeared first [2.1+/-0.4 min (mean +/- SD) after tilting] at the root cap and then - with incrementing lag times - at the meristem (after 2.5+/-0.3 min at 300 micro m from root tip; after 3.7+/-0.4 min at 700 micro m) and apical elongation zone (4.8+/-0.5 min at 1,000 micro m), suggesting a basipetal progression of differential surface acidification at a rate of 250-350 micro m min(-1), consistent with reported auxin transport rates.     

Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.     

Correlations between Gravitropic Curvature and Auxin Movement across Gravistimulated Roots of Zea mays

We compared the kinetics of auxin redistribution across the caps of primary roots of 2-day-old maize (Zea mays, cv Merit) seedlings with the time course of gravitropic curvature. [³H] indoleacetic acid was applied to one side of the cap in an agar donor and radioactivity moving across the cap was collected in an agar receiver applied to the opposite side. Upon gravistimulation the roots first curved upward slightly, then returned to the horizontal and began curving downward, reaching a final angle of about 67°. Movement of label across the caps of gravistimulated roots was asymmetric with preferential downward movement (ratio downward/upward = ca. 1.6, radioactivity collected during the 90 min following beginning of gravistimulation). There was a close correlation between the development of asymmetric auxin movement across the root cap and the rate of curvature, with both values increasing to a maximum and then declining as the roots approached the final angle of curvature. In roots preadapted to gravity (alternate brief stimulation on opposite flanks over a period of 1 hour) the initial phase of upward curvature was eliminated and downward bending began earlier than for controls. The correlation between asymmetric auxin movement and the kinetics of curvature also held in comparisons between control and preadapted roots. Both downward auxin transport asymmetry and downward curvature occurred earlier in preadapted roots than in controls. These findings are consistent with suggestions that the root cap is not only the site of perception but also the location of the initial redistribution of effectors that ultimately leads to curvature.     

Negative gravitropic response of roots directs auxin flow to control root gravitropism

Root tip is capable of sensing and adjusting its growth direction in response to gravity, a phenomenon known as root gravitropism. Previously, we have shown that negative gravitropic response of roots (NGR) is essential for the positive gravitropic response of roots. Here, we show that NGR, a plasma membrane protein specifically expressed in root columella and lateral root cap cells, controls the positive root gravitropic response by regulating auxin efflux carrier localization in columella cells and the direction of lateral auxin flow in response to gravity. Pharmacological and genetic studies show that the negative root gravitropic response of the ngr mutants depends on polar auxin transport in the root elongation zone. Cell biology studies further demonstrate that polar localization of the auxin efflux carrier PIN3 in root columella cells and asymmetric lateral auxin flow in the root tip in response to gravistimulation is reversed in the atngr1;2;3 triple mutant. Furthermore, simultaneous mutations of three PIN genes expressed in root columella cells impaired the negative root gravitropic response of the atngr1;2;3 triple mutant. Our work revealed a critical role of NGR in root gravitropic response and provided an insight of the early events and molecular basis of the positive root gravitropism.     

Effect of Inhibitors of Auxin Transport and of Calmodulin on a Gravisensing-Dependent Current in Maize Roots

Some characteristics of the gravity sensing mechanism in maize root caps were investigated using a bioelectric current as an indicator of gravity sensing. This technique involves the measurement of a change in the current density which arises at the columella region coincidently with the presentation time. Two inhibitors of auxin transport, triiodobenzoic acid and naphthylphthalamic acid, blocked gravitropic curvature but not the change in current density. Two inhibitors of calmodulin activity, compound 48/80 and calmidazolium, blocked both curvature and gravity-induced current. The results suggest that auxin transport is not a component of gravity sensing in the root cap. By contrast, the results suggest that calmodulin plays an intrinsic role in gravity sensing.