The kinetics of abscisic acid action on root growth and gravitropism

Using an auxanometer and time-lapse cinematography we have studied the timing of abscisic acid (ABA) effects on elongation, gravitropic curvature, and hydrogen-ion efflux in several cultivars of maize (Zea mays L.). The effect of high concentrations (e.g. 0.1 mM) of ABA on root elongation is triphasic, including 1) a period of promotion lasting approximately 12 h, 2) a subsequent period of increasing inhibition lasting approximately 12h, and 3) gradual recovery to a rate within approximately 80% of the control rate. With lower concentrations of ABA (e.g. 0.1 μM) only the transient promotive phase is seen. Abscisic acid enhances ethylene biosynthesis in roots of maize but suppression of ethylene biosynthesis does not prevent the long-term inhibitory action of ABA on growth. Application of ABA (0.1 mM) to the upper surface of horizontally placed roots accelerates positive gravitropism. Application of ABA to the lower surface retards gravitropism and in some cases causes the roots to curve upward against the direction of gravity. These observations are consistent with our finding that the initial effect of ABA on root elongation is stimulatory. Since root gravitropism is rapid enough to be completed within the stimulatory phase of ABA action, the data argue against hypotheses of gravitropism based upon accumulation of ABA to inhibitory levels on the lower side of a hirizontal root.     

Complex physiological and molecular processes underlying root gravitropism

Gravitropism allows plant organs to guide their growth in relation to the gravity vector. For most roots, this response to gravity allows downward growth into soil where water and nutrients are available for plant growth and development. The primary site for gravity sensing in roots includes the root cap and appears to involve the sedimentation of amyloplasts within the columella cells. This process triggers a signal transduction pathway that promotes both an acidification of the wall around the columella cells, an alkalinization of the columella cytoplasm, and the development of a lateral polarity across the root cap that allows for the establishment of a lateral auxin gradient. This gradient is then transmitted to the elongation zones where it triggers a differential cellular elongation on opposite flanks of the central elongation zone, responsible for part of the gravitropic curvature. Recent findings also suggest the involvement of a secondary site/mechanism of gravity sensing for gravitropism in roots, and the possibility that the early phases of graviresponse, which involve differential elongation on opposite flanks of the distal elongation zone, might be independent of this auxin gradient. This review discusses our current understanding of the molecular and physiological mechanisms underlying these various phases of the gravitropic response in roots.     

Hormonal interactions during root tropic growth: hydrotropism versus gravitropism

Terrestrial plants have evolved remarkable morphological plasticity that enables them to adapt to their surroundings. One of the most important traits that plants have acquired is the ability to sense environmental cues and use them as a basis for governing their growth orientation. The directional growth of plant organs relative to the direction of environmental stimuli is a tropism. The Cholodny–Went theory proposes that auxin plays a key role in several tropisms. Recent molecular genetic studies have strongly supported this hypothesis for gravitropism. However, the molecular mechanisms of other tropisms are far less clear. Hydrotropism is the response of roots to a moisture gradient. Since its re-discovery in 1985, root hydrotropism has been shown to be common among higher plant species. Additionally, in some species, gravitropism interferes with hydrotropism, suggesting that both shared and divergent mechanisms mediating the two tropisms exist. This hypothesis has been supported by recent studies, which provide an understanding of how roots sense multiple environmental cues and exhibit different tropic responses. In this review, we focus on the overlapping and unique mechanisms of the hormonal regulation underlying gravitropism and hydrotropism in roots.     

Auxin fluxes in the root apex co-regulate gravitropism and lateral root initiation

Root architecture plays an important role in water and nutrient acquisition and in the ability of the plant to adapt to the soil. Lateral root development is the main determinant of the shape of the root system and is controlled by external factors such as nutrient concentration. Here it is shown that lateral root initiation and root gravitropism, two processes that are regulated by auxin, are co-regulated in Arabidopsis. A mathematical model was generated that can predict the effects of gravistimulations on lateral root initiation density and suggests that lateral root initiation is controlled by an inhibitory fields mechanism. Moreover, gene transactivation experiments suggest a mechanism involving a single auxin transport route for both responses. Finally, co-regulation may offer a selective advantage by optimizing soil exploration as supported by a simple quantitative analysis.     

PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism

Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.     

Circumnutation and gravitropism cause root waving in Arabidopsis thaliana

Arabidopsis thaliana roots grow in a wavy pattern on inclinedagar plates. This waving behaviour has been interpreted as representinga gravitropism-dependent thigmotropic response. We argue insteadthat this root waving represents primarily a flattened spiralgrowth pattern resulting from circumnutation and gravitropism. Key words: Arabidopsis, circumnutation, gravitropism, roots, thigmotropism     

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     

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.     

Role of cytokinin and auxin in shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism

BACKGROUND AND AIMS: Development and architecture of plant roots are regulated by phytohormones. Cytokinin (CK), synthesized in the root cap, promotes cytokinesis, vascular cambium sensitivity, vascular differentiation and root apical dominance. Auxin (indole-3-acetic acid, IAA), produced in young shoot organs, promotes root development and induces vascular differentiation. Both IAA and CK regulate root gravitropism. The aims of this study were to analyse the hormonal mechanisms that induce the root's primary vascular system, explain how differentiating-protoxylem vessels promote lateral root initiation, propose the concept of CK-dependent root apical dominance, and visualize the CK and IAA regulation of root gravitropiosm. KEY ISSUES: The hormonal analysis and proposed mechanisms yield new insights and extend previous concepts: how the radial pattern of the root protoxylem vs. protophloem strands is induced by alternating polar streams of high IAA vs. low IAA concentrations, respectively; how differentiating-protoxylem vessel elements stimulate lateral root initiation by auxin-ethylene-auxin signalling; and how root apical dominance is regulated by the root-cap-synthesized CK, which gives priority to the primary root in competition with its own lateral roots. CONCLUSIONS: CK and IAA are key hormones that regulate root development, its vascular differentiation and root gravitropism; these two hormones, together with ethylene, regulate lateral root initiation.     

The axr4 auxin-resistant mutants of Arabidopsis thaliana define a gene important for root gravitropism and lateral root initiation

To understand the molecular mechanism of auxin action, mutants of Arabidopsis thaliana with altered responses to auxin have been identified and characterized. Here the isolation of two auxin-resistant mutants that define a new locus involved in auxin response, named AXR4, is reported. The axr4 mutations are recessive and map near the ch1 mutation on chromosome 1. Mutant plants are specifically resistant to auxin and defective in root gravitropism. Double mutants between axr4 and the recessive auxin-resistant mutants axr1-3 and aux1-7 were characterized to ascertain possible genetic interactions between the mutations. The roots of the axr4 axr1-3 double mutant plants are less sensitive to auxin, respond more slowly to gravity, and form fewer lateral roots than either parental single mutant. These results suggest that the two mutations have additive or even synergistic effects. The AXR1 and AXR4 gene products may therefore act in separate pathways of auxin response or perhaps perform partially redundant functions in a single pathway. The axr4 aux1-7 double mutant has the same sensitivity to auxin as the aux1-7 mutant but forms far fewer lateral roots than either parental single mutant. The aux1-7 mutation thus appears to be epistatic to axr4 with respect to auxin-resistant root elongation, whereas in lateral root formation, the effects of the two mutations are additive. The complexity of the genetic interactions indicated by these results may reflect differences in the mechanism of auxin action during root elongation and the formation of lateral roots. The AXR4 gene product, along with those of the AXR1 and AUX1 genes, is important for normal auxin sensitivity, gravitropic response in roots and lateral root formation.     

Interaction of root gravitropism and phototropism in Arabidopsis wild-type and starchless mutants

Root gravitropism in wild-type Arabidopsis and in two starchless mutants, pgm1-1 and adg1-1, was evaluated as a function of light position to determine the relative strengths of negative phototropism and of gravitropism and how much phototropism affects gravitropic measurements. Gravitropism was stronger than phototropism in some but not all light positions in wild-type roots grown for an extended period, indicating that the relationship between the two tropisms is more complex than previously reported. Root phototropism significantly influenced the time course of gravitropic curvature and the two measures of sensitivity. Light from above during horizontal exposure overestimated all three parameters for all three genotypes except the wild-type perception time. At the irradiance used (80 micromol m(-2) s(-1)), the shortest periods of illumination found to exaggerate gravitropism were 45 min of continuous illumination and 2-min doses of intermittent illumination. By growing roots in circumlateral light or by gravistimulating in the dark, corrected values were obtained for each gravitropic parameter. Roots of both starchless mutants were determined to be about three times less sensitive than prior estimates. This study demonstrates the importance of accounting for phototropism in the design of root gravitropism experiments in Arabidopsis.     

The kinetics of analyte capture on nanoscale sensors

This article presents a number of kinetic analyses related to binding processes relevant to capture of target analyte species in nanoscale cantilever-type devices designed to detect small concentrations of biomolecules. The overall analyte capture efficiency is a crucial measure of the ultimate sensitivity of such devices, and a detailed kinetic analysis tells us how rapidly such measurements may be made. We have analyzed the capture kinetics under a variety of conditions, including the possibility of so-called surface-enhanced ligand capture. One of the modalities studied requires ligand capture through a cross-linking mechanism, and it was found that this mode may provide a robust and sensitive approach to biomolecular detection. For the two modalities studied, we find that detection of specific biomolecules down to concentration levels of 1 nM or less appear to be quite feasible for the device configurations studied.     

The importance of root gravitropism for inter-root competition and phosphorus acquisition efficiency: results from a geometric simulation model

We have observed that low soil phosphorus availability alters the gravitropic response of basal roots in common bean (Phaseolus vulgaris L.), resulting in a shallower root system. In this study we use a geometric model to test the hypotheses that a shallower root system is a positive adaptive response to low soil P availability by (1) concentrating root foraging in surface soil horizons, which generally have the highest P availability, and (2) reducing spatial competition for P among roots of the same plant. The growth of nine root systems contrasting in gravitropic response over 320 h was simulated in SimRoot, a dynamic three-dimensional geometric model of root growth and architecture. Phosphorus acquisition and inter-root competition were estimated with Depzone, a program that dynamically models nutrient diffusion to roots. Shallower root systems had greater P acquisition per unit carbon cost than deeper root systems, especially in older root systems. This was due to greater inter-root competition in deeper root systems, as measured by the volume of overlapping P depletion zones. Inter-root competition for P was a significant fraction of total soil P depletion, and increased with increasing values of the P diffusion coefficient (De), with root age, and with increasing root gravitropism. In heterogenous soil having greater P availability in surface horizons, shallower root systems had greater P acquisition than deeper root systems, because of less inter-root competition as well as increased root foraging in the topsoil. Root P acquisition predicted by SimRoot was validated against values for bean P uptake in the field, with an r ² between observed and predicted values of 0.75. Our results support the hypothesis that altered gravitropic sensitivity in P-stressed roots, resulting in a shallower root system, is a positive adaptive response to low P availability by reducing inter-root competition within the same plant and by concentrating root activity in soil domains with the greatest P availability. This revised version was published online in August 2006 with corrections to the Cover Date.     

Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement

The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with CI-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units.     

The endodermis and shoot gravitropism

Shoots and roots of higher plants exhibit negative and positive gravitropism, respectively. A variety of gravitropic mutants have recently been isolated from Arabidopsis, the characterization of which demonstrates that the molecular mechanisms of the gravitropic responses in roots, hypocotyls and inflorescence stems are different. The cytological and molecular analysis of two mutants, shoot gravitropism 1 (sgrl), which is allelic to scarecrow (scr), and sgr7, which is allelic to short-root(shr), indicate that the endodermis is the site of gravity perception in shoots. These data suggest a new model for shoot gravitropism.     

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.     

Model for kinetics of myosin-V molecular motors

A hand-over-hand model is presented for the processive movement of myosin-V based on previous biochemical experimental results and structural observations of nucleotide-dependent conformational changes of single-headed myosins. The model shows that the ADP-release rate of the trailing head is much higher than that of the leading head, thus giving a 1 : 1 mechanochemical coupling for the processive movement of the motor. It explains well the previous finding that some 36-nm steps consist of two substeps, while other 36-nm steps consist of no substeps. Using the model, the calculated kinetic behaviors of myosin-V such as the main and intermediate dwell time distributions, the load dependence of the average main and intermediate dwell time and the load dependence of occurrence frequency of the intermediate state under various nucleotide conditions show good quantitative agreement with previous experimental results.     

Tomato root growth, gravitropism, and lateral development: correlation with auxin transport.

Tomato (Lycopersicon esculentum, Mill.) roots were analyzed during growth on agar plates. Growth of these roots was inhibited by the auxin transport inhibitors naphthylphthalamic acid (NPA) and semicarbazone derivative I (SCB-1). The effect of auxin transport inhibitors on root gravitropism was analyzed by measurement of the angle of gravitropic curvature after the roots were reoriented 90 degrees from the vertical. NPA and SCB-1 abolished both the response of these roots to gravity and the formation of lateral roots, with SCB-1 being the more effective at inhibition. Auxins also inhibited root growth. Both auxins tested has a slight effect on the gravity response, but this effect is probably indirect, since auxins reduced the growth rate. Auxins also stimulated lateral root growth at concentration where primary root growth was inhibited. When roots were treated with both IAA and NPA simultaneously, a cumulative inhibition of root growth was found. When both compounds were applied together, analysis of gravitropism and lateral root formation indicated that the dominant effect was exerted by auxin transport inhibitors. Together, these data suggest a model for the role of auxin transport in controlling both primary and lateral root growth.     

Development of extracellular electric pattern aroundLepidium roots: its possible role in root growth and gravitropism

Summary Using a vibrating probe technique, four distinct electric patterns around growing cress roots were observed. The growth rate of the root with a particular one of them was apparently faster than that with the others. No direct correlation between the intensity of electric field and the root growth rate could be found. When gravistimulation was applied to the root, the electric pattern changed to be suitable for elongation of the gravitropic curvature. It is probable that change in electric pattern is related to growth of the root under a given environment.