Auxin metabolism and transport during gravitropism

Auxins are endogenous, growth-regulating compounds in plants: for decades investigators have hypothesized that plants change their growth rates and patterns in response to environmental signals by changing their transport of, metabolism of, or sensitivity to their endogenous auxins. The Cholodny-Went hypothesis, for example, postulates that plants respond to tropic signals by changing the distribution of free indoleacetic acid within their tissues. This hypothesis was based on data from experiments investigating phototropism and gravitropism in oat ( Avena sativa L.) and maize ( Zea mays L.) coleoptiles. The results of recent experiments support the Cholodny-Went hypothesis for maize coleoptile gravitropism. Recent experiments conducted on the gravitropisms of other developmental stages of grasses, and other species of plants, however, indicate that the Cholodny-Went hypothesis may not adequately describe how all plants respond to gravity.     

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.     

Phototropism in Higher Plants — Controversies and Caveats1

Recent literature on light-induced changes in the distribution of growth and of endogenous growth regulators in the development of phototropic curvature is reviewed. It is concluded that in a great many cases the Cholodny-Went hypothesis is sufficient to account for the growth changes bringing about curvature, though in certain dicotyledonous seedlings differential effects of light on the synthesis of growth inhibitors across the tissue from the lighted to the shaded side are implicated. The problems in the interpretation of results from experiments in which more than one photoresponse may be simultaneously induced are discussed and methods of circumventing them considered. Action spectroscopy suggests that dicotyledonous seedlings may have the same phototropic photoreceptor as monocotyledonous seedlings.     

Root gravitropism

When a plant root is reoriented within the gravity field, it responds by initiating a curvature which eventually results in vertical growth. Gravity sensing occurs primarily in the root tip. It may involve amyloplast sedimentation in the columella cells of the root cap, or the detection of forces exerted by the mass of the protoplast on opposite sides of its cell wall. Gravisensing activates a signal transduction cascade which results in the asymmetric redistribution of auxin and apoplastic Ca²⁺ across the root tip, with accumulation at the bottom side. The resulting lateral asymmetry in Ca²⁺ and auxin concentration is probably transmitted to the elongation zone where differential cellular elongation occurs until the tip resumes vertical growth. The Cholodny-Went theory proposes that gravity-induced auxin redistribution across a gravistimulated plant organ is responsible for the gravitropic response. However, recent data indicate that the gravity-induced reorientation is more complex, involving both auxin gradient-dependent and auxin gradient-independent events.     

Evidence for Two Indoleacetic Acid-Induced Growth Responses in the Avena Straight-Growth Indoleacetic Acid Assay

Floating Avena sativa L. cv Victory coleoptile segments were used to determine whether the straight-growth indoleacetic acid (IAA) assay can be reconciled with the Avena curvature assay and the Cholodny-Went theory of photo- and gravitropism. Measurements of segment length after 5 h yield sigmoid-shaped IAA dose-response curves with the growth rate leveling off at 1 [mu]M. However, measurements made at 24 h generate bell-shaped curves with maximal growth being induced by 10 [mu]M IAA. The difference between short- and long-term IAA dose-response curves is not due to IAA degradation; instead, it is the result of two growth responses to IAA. The initial one is rapid, responds to low concentrations of IAA, and lasts for 12 h. The second response is less sensitive to IAA than the first one. It appears after 6 h but is not obvious until the last 12 h of a 24-h incubation. The profile of short-term IAA dose-response curves reflects the initial growth response, whereas that of the 24-h curve is the sum of both growth responses. Linear-linear plots of 5- and 24-h dose-response curves show that coleoptile segment growth rate is proportional to IAA concentration up to 0.3 [mu]M. When the efficiency of IAA action is taken into account, it is found that the most effective IAA concentration for short and long incubations is 0.4 [mu]M. It is concluded that the Avena straight-growth IAA assay is as sensitive as the Avena coleoptile curvature assay, and that it is consistent with the Cholodny-Went theory.     

Cross-talk in plant hormone signalling: what Arabidopsis mutants are telling us

Genetic screens have been extremely useful in identifying genes involved in hormone signal transduction. However, although these screens were originally designed to identify specific components involved in early hormone signalling, mutations in these genes often confer changes in sensitivity to more than one hormone at the whole-plant level. Moreover, a variety of hormone response genes has been identified through screens that were originally designed to uncover regulators of sugar metabolism. Together, these facts indicate that the linear representation of the hormone signalling pathways controlling a specific aspect of plant growth and development is not sufficient, and that hormones interact with each other and with a variety of developmental and metabolic signals. Following the advent of arabidopsis molecular genetics we are beginning to understand some of the mechanisms by which a hormone is transduced into a cellular response. In this Botanical Briefing we review a subset of genes in arabidopsis that influence hormonal cross-talk, with emphasis on the gibberellin, abscisic acid and ethylene pathways. In some cases it appears that modulation of hormone sensitivity can cause changes in the synthesis of an unrelated hormone, while in other cases a hormone response gene defines a node of interaction between two response pathways. It also appears that a variety of hormones may converge to regulate the turnover of important regulators involved in growth and development. Examples are cited of the recent use of suppressor and enhancer analysis to identify new nodes of interaction between these pathways.     

Novel software for analysis of root gravitropism: comparative response patterns of Arabidopsis wild-type and axr1 seedlings

In an earlier study (Evans, Ishikawa & Estelle 1994, Planta 194, 215-222) we used a video digitizer system to compare the kinetics of auxin action on root elongation in wild-type seedlings and seedlings of auxin response mutants of Arabidopsis thaliana (L.) Heynh. We have since modified the system software to allow determination of elongation on opposite sides of vertical or gravistimulated roots and to allow continuous measurement of the angle of orientation of sequential subsections of the root during the response. We used this technology to compare the patterns of differential growth that generate curvature in roots of the Columbia ecotype and in the mutants axr1-3, axr1-12 and axr2, which show reduced gravitropic responsiveness and reduced sensitivity to inhibition by auxin. The pattern of differential growth during gravitropism differed in roots of wild-type and axr1 seedlings. In wild-type roots, initial curvature resulted from differential inhibition of elongation in the distal elongation zone (DEZ). This was followed by an acceleration of elongation along the top side of the DEZ. In roots of axr1-3, curvature resulted from differential stimulation of elongation whereas in roots of axr1-12 the response was variable. Roots of axr2 did not exhibit gravitropic curvature. The observation that the pattern of differential growth causing curvature is dramatically altered by a change in sensitivity to auxin is consistent with the classical Cholodny-Went theory of gravitropism which maintains that differential growth patterns induced by gravistimulation are mediated primarily by gravi-induced shifts in auxin distribution. The new technology introduced with this report allows automated determination of stimulus response patterns in the small but experimentally popular roots of Arabidopsis.     

Severely Reduced Gravitropism in Dark-Grown Hypocotyls of a Starch-Deficient Mutant of Nicotiana sylvestris

Gravitropism in dark-grown hypocotyls of the wild type was compared with a starch-deficient Nicotiana sylvestris mutant (NS 458) to test the effects of starch deficiency on gravity sensing. In a time course of curvature measured using infrared video, the response of the mutant was greatly reduced compared to the wild type; 72 hours after reorientation, curvature was about 10° for NS 458 and about 70° for wild type. In dishes maintained in a vertical orientation, wild-type hypocotyls were predominantly vertical, whereas NS 458 hypocotyls were severely disoriented with about 5 times more orientational variability than wild type. Since the growth rates were equal for both genotypes and phototropic curvature was only slightly inhibited in NS 458, the mutation probably affects gravity perception rather than differential growth. Our data suggest that starch deficiency reduces gravitropic sensitivity more in dark-grown hypocotyls than in dark- or light-grown roots in this mutant and support the hypothesis that amyloplasts function as statoliths in shoots as well as roots.     

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type     

Photoregulation of hypocotyl growth: geotropic evidence for the operation of two photosystems

Abstract. The rate of curvature of etiolated cress ( Lepi-dium sativum L. ) hypocotyls in response to gravity (negative geotropism) was retarded by red or blue light; far-red irradiation was without effect. The timing of the irradiation period in relation to the presentation for geostimulus markedly affected the response. When seedlings were irradiated during the 1–2 h period of geostimulus, blue light was more effective than red at retarding curvature; when seedlings were irradiated prior to geostimulus, only red light affected geocurvature. These results are interpreted as a further example of the kinetically distinct effects of red and blue light on hypocotyl development. Blue light elicited a rapid, immediate response effective only during the period of irradiation; red light induced a response characterized by a lag period and persistence in subsequent darkness. Etiolated mustard seedlings showed similar responses to light and gravity. The results are discussed in relation to the possibility that two photosystems operate in hypocotyl growth.     

Nitric oxide mediates gravitropic bending in soybean roots

Plant roots are gravitropic, detecting and responding to changes in orientation via differential growth that results in bending and reestablishment of downward growth. Recent data support the basics of the Cholodny-Went hypothesis, indicating that differential growth is due to redistribution of auxin to the lower sides of gravistimulated roots, but little is known regarding the molecular details of such effects. Here, we investigate auxin and gravity signal transduction by demonstrating that the endogenous signaling molecules nitric oxide (NO) and cGMP mediate responses to gravistimulation in primary roots of soybean (Glycine max). Horizontal orientation of soybean roots caused the accumulation of both NO and cGMP in the primary root tip. Fluorescence confocal microcopy revealed that the accumulation of NO was asymmetric, with NO concentrating in the lower side of the root. Removal of NO with an NO scavenger or inhibition of NO synthesis via NO synthase inhibitors or an inhibitor of nitrate reductase reduced both NO accumulation and gravitropic bending, indicating that NO synthesis was required for the gravitropic responses and that both NO synthase and nitrate reductase may contribute to the synthesis of the NO required. Auxin induced NO accumulation in root protoplasts and asymmetric NO accumulation in root tips. Gravistimulation, NO, and auxin also induced the accumulation of cGMP, a response inhibited by removal of NO or by inhibitors of guanylyl cyclase, compounds that also reduced gravitropic bending. Asymmetric NO accumulation and gravitropic bending were both inhibited by an auxin transport inhibitor, and the inhibition of bending was overcome by treatment with NO or 8-bromo-cGMP, a cell-permeable analog of cGMP. These data indicate that auxin-induced NO and cGMP mediate gravitropic curvature in soybean roots.     

Small bending,big curvature

<正>The classical "Cholodny-Went theory" predicted that directional stimuli trigger the redistribution of auxin, which governs the differential growth of plant organs through potent effects on cell expansion, thereby establishing an"auxin-then-growth" paradigm; this theory has been validated for both gravitropism and phototropism in plants(reviewed in Muthert et al., 2020).     

Elongation and gravitropic responses of Arabidopsis roots are regulated by brassinolide and IAA

Exogenously applied brassinolide (BL) increased both gravitropic curvature and length of primary roots of Arabidopsis at low concentration (10(-10) M), whereas at higher concentration, BL further increased gravitropic curvature while it inhibited primary root growth. BRI1-GFP plants possessing a high steady-state expression level of a brassinosteroid (BR) receptor kinase rendered the plant's responses to gravity and root growth more sensitive, while BR-insensitive mutants, bri1-301 and bak1, delayed root growth and reduced their response to the gravitropic stimulus. The stimulatory effect of BL on the root gravitropic curvature was also enhanced in auxin transport mutants, aux1-7 and pin2, relative to wild-type plants, and increasing concentration of auxin attenuated BL-induced root sensitivity to gravity. Interestingly, IAA treatment to the roots of bri1-301 and bak1 plants or of plants pretreated with a BL biosynthetic inhibitor, brassinazole, increased their sensitivity to gravity, while these treatments for the BL-hypersensitive transgenic plants, BRI1-GFP and 35S-BAK1, were less effective. Expression of a CYP79B2 gene, encoding an IAA biosynthetic enzyme, was suppressed in BL-hypersensitive plant types and enhanced in BL-insensitive or -deficient plants. In conclusion, our results indicate that BL interacts negatively with IAA in the regulation of plant gravitropic response and root growth, and its regulation is achieved partly by modulating biosynthetic pathways of the counterpart hormone.     

The auxin-resistant diageotropica mutant of tomato responds to gravity via an auxin-mediated pathway

 Hypocotyls of the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) do not elongate in response to exogenous auxin, but can respond to gravity. This appears paradoxical in light of the Cholodny-Went hypothesis, which states that shoot gravicurvature results from asymmetric stimulation of elongation by auxin. While light-grown dgt seedlings can achieve correct gravitropic reorientation, the response is slow compared to wild-type seedlings. The sensitivity of dgt seedlings to inhibition of gravicurvature by immersion in auxin or auxin-transport inhibitors is similar to that of wild-type plants, indicating that both an auxin gradient and auxin transport are required for the gravitropic response and that auxin uptake, efflux, and at least one auxin receptor are functional in dgt. Furthermore, dgt gravicurvature is the result of asymmetrically increased elongation as would be expected for an auxin-mediated response. Our results suggest differences between elongation in response to exogenous auxin (absent in dgt) and elongation in response to gravistimulation (present but attenuated in dgt) and confirm the presence of two phases during the gravitropic response, both of which are dependent on functional auxin transport. Received: 16 July 1999 / Accepted: 24 September 1999     

Happy end in sight after 70 years of controversy

Differential auxin transport from the columella to lateral root cap cells as a result of root gravitropism has recently been shown using a GFP-based auxin biosensor. Together with the recent finding of gravity-dependent localization of an auxin efflux carrier, these results strongly support the Cholodny-Went hypothesis for the tropic response, which has been disputed for 70 years.     

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.     

Gravitropism in roots of intermediate-starch mutants of Arabidopsis

Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated, by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as. determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the slarchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to that of the starchless mutant, it appears that 51–60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.     

Growth distribution during first positive phototropic curvature of maize coleoptiles*

Abastract Measurements of growth increments on the shaded and the irradiated sides of phototropically stimulated maize (Zea mays L.) coleoptiles, obtained over the entire fluence range of the first positive curvature, indicate that the curvature is induced by growth stimulation on the shaded side and compensating inhibition on the irradiated side (length increments on the coleoptile flanks were determined 100 min after 30 s phototropic induction with blue light). At high fluences of blue light, overall stimulation of growth takes place, but this tendency is largely eliminated when only the tip of the coleoptile is irradiated. Time courses for growth increments obtained for the maximum first positive response show that the growth stimulation on the shaded side and the growth inhibition on the irradiated side commence almost simultaneously 20-30 min after the phototropic induction. The growth on the irradiated side almost ceases, but the growth rate on the shaded side is doubled, relative to the control rate. The onset of differential growth migrates basipetally from the tip at a velocity similar to that for polar auxin transport. The first positive phototropic response of the coleoptile is concluded to be the consequence of lateral redistribution of growth, which is not necessarily accompanied by changes in the net growth. The results are consonant with the Cholodny-Went theory of tropisms, in which lateral redistribution of auxin is considered to be the cause of tropic responses.     

What remains of the Cholodny-Went theory? Introduction

The Cholodny-Went theory, which states that plant tropisms in roots and shoots are due to the unequal distribution of the growth regulator auxin in reponse to light or gravity, presented a simple and direct explanation of these phenomena. This was significant in the 1930s when simple explanations were lacking, and much research into this theory has been performed since that time. From the 1980s forward, there has been much questioning and controversy surrounding the theory. In this introduction to 15 brief articles, the author describes how leading scientists in the field were asked to air their opinions of the theory and formulate questions for each other. The resulting articles are presented in an attempt to stimulate discussion and give insights into the state of the Cholodny-Went theory in the 1990s.     

Auxin,gibberellins and the gravitropic response of grass leaf sheath pulvini

The role of hormones in mediating tropic responses has been a central question in plant biology. Another key issue concerns how interactions between hormones regulate plant responses. In the September 2007 issue of Physiologia Plantarum, we published a paper relevant to both these questions.¹ This paper focuses on gravitropism in the barley leaf sheath pulvinus. The results support the Cholodny-Went theory on hormones and tropic responses, and highlight how an environmental factor (gravity) appears to first affect auxin content and consequently that of bioactive gibberellins (GAs). It appears that while GAs do not actually trigger the gravitropic bending of barley pulvini, they do act to magnify the bending response.Key words: auxin, gibberellin, Cholodny-Went theory, barley, pulvinus, gravitropism, mutant