Asymmetric Auxin Distribution Research Articles

Inositol 1,4,5-trisphosphate and Ran expression during simulated and real microgravity

In order to gain further insight into the signal transduction pathway concerning gravitropism, we studied the expression profiles of mRNA in etiolated sunflower (Helianthus annuus L.) seedlings. Differential-display reverse transcriptase PCR product assayed by capillary electrophoresis revealed the small GTPase Ran, regulating nuclear import and export of proteins. Parallel analysis of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) release by a highly advanced system of metal-dye detection combined with high-performance liquid chromatography provided evidence that the second messenger Ins(1,4,5)P3 is modulated by changes of the gravity vector. Investigations by fast clinorotation and sounding rockets established a positive correlation between the Ins(1,4,5)P3 level and the expression rate of Ran mRNA during simulated and real microgravity. Since an asymmetric distribution of auxin during graviresponse is suggested to induce differential cell elongation, additional information on the perception and transduction pathways was achieved by auxin stimulation experiments. While we were able to demonstrate an auxin-dependent production of Ins(1,4,5)P3, the expression of Ran mRNA was not affected by auxin. Finally, besides the phosphoinositide system as one element of the signal transduction chain linking graviperception to graviresponse, a Ran-mediated interaction model of extracellular microgravity signal perception and intercellular transduction pathway is proposed.

Spatiotemporal asymmetric auxin distribution: a means to coordinate plant development.

The plant hormone auxin plays crucial roles in regulating plant growth development, including embryo and root patterning, organ formation, vascular tissue differentiation and growth responses to environmental stimuli. Asymmetric auxin distribution patterns have been observed within tissues, and these so-called auxin gradients change dynamically during different developmental processes. Most auxin is synthesized in the shoot and distributed directionally throughout the plant. This polar auxin transport is mediated by auxin influx and efflux facilitators, whose subcellular polar localizations guide the direction of auxin flow. The polar localization of PIN auxin efflux carriers changes in response to developmental and external cues in order to channel auxin flow in a regulated manner for organized growth. Auxin itself modulates the expression and subcellular localization of PIN proteins, contributing to a complex pattern of feedback regulation. Here we review the available information mainly from studies of a model plant, Arabidopsis thaliana, on the generation of auxin gradients, the regulation of polar auxin transport and further downstream cellular events.

PIN2 Polarity Requires Vesicular Cycling and Proteasomal Degradation

PIN2 is part of the auxin efflux carrier that controls the asymmetric distribution of auxin, which is a key regulator of root gravitropism. Abas et al. show that root meristems reorient to point their tips down in response to being positioned horizontally in the gravitational field (gravistimulation) by generating an asymmetric distribution of PIN2 such that PIN2 was more abundant on the lower side of the root tip. Before positioning the root horizontally, PIN2 abundance was equal in the lateral sides of the root. Gravistimulation initially caused PIN2 to be removed from the upper root cells' plasma membrane and appear in intracellular vesicles. The decrease in PIN2 abundance (measured in isolated membrane fractions or from the upper cells by microscopic techniques) and gravitropic root bending was blocked if the root meristems were treated with an inhibitor of the proteasome (MG132) or brefeldin A (BFA), which inhibits vesicular trafficking. Plants expressing a mutant PIN2 (wav6-52) do not respond to gravity, and Abas et al. showed that wav6-52 root meristems had increased abundance of PIN2, which accumulated in intracellular vesicles and the plasma membrane in unstimulated roots and did not redistribute in response to gravistimulation. Furthermore, wav6-52 roots exhibited aberrant auxin activity in the lateral root cap cells and elongation zone, which is not seen in wild-type roots. Auxin also exhibited feedback onto PIN2, with application of an auxin decreasing PIN2 abundance. Uniform pretreatment of the roots with an auxin analog followed by gravistimulation caused the internalization of PIN2 from both the upper and lower root cells. Auxin would only accumulate in the lower cells and thus may have served as a signal to reset the system once the root bent and was oriented vertically again. The constitutive cycling of PIN2 provides an opportunity for the cells of the upper root to selectively trigger degradation of PIN2, thereby decreasing its abundance and decreasing auxin flow to the upper side of the root to mediate bending in response to gravity. L. Abas, R. Benjamins, N. Malenica, T. Paciorek, J. Wiřniewska, J. C. Moulinier-Anzola, T. Sieberer, J. Friml, C. Luschnig, Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat. Cell Biol. 8 , 249-256 (2006). [PubMed]

Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism

Root gravitropism describes the orientation of root growth along the gravity vector and is mediated by differential cell elongation in the root meristem. This response requires the coordinated, asymmetric distribution of the phytohormone auxin within the root meristem, and depends on the concerted activities of PIN proteins and AUX1 - members of the auxin transport pathway. Here, we show that intracellular trafficking and proteasome activity combine to control PIN2 degradation during root gravitropism. Following gravi-stimulation, proteasome-dependent variations in PIN2 localization and degradation at the upper and lower sides of the root result in asymmetric distribution of PIN2. Ubiquitination of PIN2 occurs in a proteasome-dependent manner, indicating that the proteasome is involved in the control of PIN2 turnover. Stabilization of PIN2 affects its abundance and distribution, and leads to defects in auxin distribution and gravitropic responses. We describe the effects of auxin on PIN2 localization and protein levels, indicating that redistribution of auxin during the gravitropic response may be involved in the regulation of PIN2 protein.

Asymmetric distribution of auxin correlates with gravitropism and phototropism but not with autostraightening (autotropism) in pea epicotyls.

The relationships between the distribution of the native auxin indole-3-acetic acid (IAA) and tropisms in the epicotyl of red light-grown pea (Pisum sativum L.) seedlings have been investigated. The distribution measurement was made in a defined zone of the third internode, using (3)H-IAA applied from the plumule as a tracer. The tropisms investigated were gravitropism, pulse-induced phototropism, and time-dependent phototropism. The investigation was extended to the phase of autostraightening (autotropism) that followed gravitropic curvature. It was found that IAA is asymmetrically distributed between the two halves of the zone, with a greater IAA level occurring on the convex side, at early stages of gravitropic and phototropic curvatures. This asymmetry was found in epidermal peels and, except for one case (pulse-induced phototropism), no asymmetry was detected in whole tissues. It was concluded, in support of earlier results, that auxin asymmetry mediates gravitropism and phototropism and that the epidermis or peripheral cell layers play an important role in the establishment of auxin asymmetry in pea epicotyls. During autostraightening, which results from a reversal of growth asymmetry, the extent of IAA asymmetry was reduced, but its direction was not reversed. This result demonstrated that autostraightening is not regulated through auxin distribution. In this study, the growth on either side of the investigated zone was also measured. In some cases, the measured IAA distribution could not adequately explain the local growth rate, necessitating further detailed investigation.

Functional redundancy of PIN proteins is accompanied by auxin-dependent cross-regulation of PIN expression

Plant development displays an exceptional plasticity and adaptability that involves the dynamic, asymmetric distribution of the phytohormone auxin. Polar auxin flow, which requires polarly localized transport facilitators of the PIN family, largely contributes to the establishment and maintenance of the auxin gradients. Functionally overlapping action of PIN proteins mediates multiple developmental processes, including embryo formation, organ development and tropisms. Here we show that PIN proteins exhibit synergistic interactions, which involve cross-regulation of PIN gene expression in pin mutants or plants with inhibited auxin transport. Auxin itself positively feeds back on PIN gene expression in a tissue-specific manner through an AUX/IAA-dependent signalling pathway. This regulatory switch is indicative of a mechanism by which the loss of a specific PIN protein is compensated for by auxin-dependent ectopic expression of its homologues. The compensatory properties of the PIN-dependent transport network might enable the stabilization of auxin gradients and potentially contribute to the robustness of plant adaptive development.

Gibberellin-regulated XET is differentially induced by auxin in rice leaf sheath bases during gravitropic bending

The asymmetric distribution of auxin plays a fundamental role in plant gravitropism, yet little is understood about how its lateral distribution stimulates growth. In the present work, the asymmetric distribution not only of auxin, but also that of gibberellins (GAs), was observed in rice leaf sheath bases following gravistimulation. Gravistimulation induced the transient accumulation of greater amounts of both IAA and GA in the lower halves of the leaf sheath bases of rice seedlings. OsGA3ox1, a gene of active GA synthesis, was differentially induced by gravistimulation. Furthermore, 2,3,5-tri-iodobenzoic acid (TIBA), an inhibitor of auxin transport, substantially decreased the asymmetric distribution of IAA and the gradient of OsGA3ox1 expression. Externally applied GA(3) restored the gravitropic curvature of rice leaf sheaths inhibited by either TIBA or by ancymidol, a GA synthesis inhibitor. The expression of XET (encoding xyloglucan endotransglycosylase) was differentially induced in the lower halves of gravistimulated leaf sheath bases and was also up-regulated by exogenous IAA and GA(3). Both ancymidol and TIBA decreased the gradient of XET expression. These data suggest that the asymmetric distribution of auxin effected by gravistimulation induced a gradient of GAs via asymmetric expression of OsGA3ox1 in rice leaf sheath bases, and hence caused the asymmetric expression of XET. Cell wall loosening in the curvature site of the leaf sheath triggered by the expression of XET would contribute to gravitropic growth.

Asymmetric Auxin Response Precedes Asymmetric Growth and Differentiation of asymmetric leaf1 and asymmetric leaf2 Arabidopsis Leaves

We have analyzed the development of leaf shape and vascular pattern in leaves mutant for ASYMMETRIC LEAVES1 (AS1) or AS2 and compared the timing of developmental landmarks to cellular response to auxin, as measured by expression of the DR5:beta-glucuronidase (GUS) transgene and to cell division, as measured by expression of the cycB1:GUS transgene. We found that the earliest visible defect in both as1 and as2 first leaves is the asymmetric placement of auxin response at the distal leaf tip. This precedes visible changes in leaf morphology, asymmetric placement of the distal margin gap, formation of margin gaps along the leaf border, asymmetric distribution of marginal auxin, and asymmetry in cell division patterns. Moreover, treatment of developing leaves with either exogenous auxin or an auxin transport inhibitor eliminates asymmetric auxin response and subsequent asymmetric leaf development. We propose that the initial asymmetric placement of auxin at the leaf tip gives rise to later asymmetries in the internal auxin sources, which subsequently result in asymmetrical cell differentiation and division patterns.

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.

Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis.

Long-standing models propose that plant growth responses to light or gravity are mediated by asymmetric distribution of the phytohormone auxin. Physiological studies implicated a specific transport system that relocates auxin laterally, thereby effecting differential growth; however, neither the molecular components of this system nor the cellular mechanism of auxin redistribution on light or gravity perception have been identified. Here, we show that auxin accumulates asymmetrically during differential growth in an efflux-dependent manner. Mutations in the Arabidopsis gene PIN3, a regulator of auxin efflux, alter differential growth. PIN3 is expressed in gravity-sensing tissues, with PIN3 protein accumulating predominantly at the lateral cell surface. PIN3 localizes to the plasma membrane and to vesicles that cycle in an actin-dependent manner. In the root columella, PIN3 is positioned symmetrically at the plasma membrane but rapidly relocalizes laterally on gravity stimulation. Our data indicate that PIN3 is a component of the lateral auxin transport system regulating tropic growth. In addition, actin-dependent relocalization of PIN3 in response to gravity provides a mechanism for redirecting auxin flux to trigger asymmetric growth.

Post-transcriptional control of the Arabidopsis auxin efflux carrier EIR1 requires AXR1.

The auxin efflux carrier EIR1 (also known as AGR and AtPIN2) is a key mediator of the response of Arabidopsis roots to gravity [1,2]. This response is thought to require the establishment of a transient auxin gradient in the root meristem, resulting in differential cell elongation [3]. Recent reports suggest that EIR1 is essential for the asymmetric distribution of auxin in the root meristem [4-7], but the regulatory aspects of this process are still not fully understood. Here, we studied the regulation of EIR1 in Arabidopsis using two reporters: one was a translational fusion that contained the entire EIR1 coding sequence, and the other a transcriptional fusion that had no EIR1 coding sequence. We found that EIR1 is controlled at the post-transcriptional level. The translational fusion was unstable in response to changes in auxin homeostasis, and was destabilized by cycloheximide. In contrast, the protein was stabilized in the axr1-3 mutant, which is auxin resistant and defective in auxin responses such as root gravitropism [8,9]. AXR1 is thought to participate in ubiquitin-mediated control of protein stability [10-12]. The dependence of EIR1 reporter expression on auxin concentrations and AXR1 suggests that auxin transport is regulated through a feedback regulatory loop that affects protein stability in response to auxin.

Time course and auxin sensitivity of cortical microtubule reorientation in maize roots.

The kinetics of MT [microtubule] reorientation in primary roots of Zea mays cv. Merit, were examined 15, 30, 45, and 60 min after horizontal positioning. Confocal microscopy of longitudinal tissue sections showed no change in MT orientation 15 and 30 min after horizontal placement. However, after 45 and 60 min, MTs of the outer 4-5 cortical cell layers along the lower side were reoriented. In order to test whether MT reorientation during graviresponse is caused by an auxin gradient, we examined the organization of MTs in roots that were incubated for 1 h in solutions containing 10(-9) to 10(-6) M IAA. IAA treatment at 10(-8) M or less showed no major or consistent changes but 10(-7) M IAA resulted in MT reorientation in the cortex. The auxin effect does not appear to be acid-induced since benzoic acid (10(-5) M) did not cause MT reorientation. The region closest to the maturation zone was most sensitive to IAA. The data indicate that early stages of gravity induced curvature occur in the absence of MT reorientation but sustained curvature leads to reoriented MTs in the outer cortex. Growth inhibition along the lower side of graviresponding roots appears to result from asymmetric distribution of auxin following gravistimulation.

An Auxin-Responsive Promoter Is Differentially Induced by Auxin Gradients during Tropisms.

We constructed a chimeric gene consisting of a soybean small auxin up RNA (SAUR) promoter and leader sequence fused to an Escherichia coli [beta]-glucuronidase (GUS) open reading frame and a 3[prime] untranslated nopaline synthase sequence from Agrobacterium tumefaciens. This chimeric gene was used to transform tobacco by Agrobacterium-mediated transformation. In R2 etiolated transgenic tobacco seedlings, GUS expression occurred primarily in elongation regions of hypocotyls and roots. In green plants, GUS was expressed primarily in the epidermis and cortex of stems and petioles, as well as in elongation regions of anther filaments in developing flowers. GUS expression was responsive to exogenous auxin in the range of 10-8 to 10-3 M. During gravitropism and phototropism, the GUS activity became greater on the more rapidly elongating side of tobacco stems. Auxin transport inhibitors and other manipulations that blocked gravitropism also blocked the asymmetric distribution of GUS activity in gravistimulated stems. Light treatment of dark-grown seedlings resulted in a rapid decrease in GUS activity. Light-induced decay in GUS activity was fully reversed by application of auxin. Taken together, our results add support for the formation of an asymmetric distribution of auxin at sites of action during tropism.

The ?acid growth effect? and geotropism

The curvature developed by segments of sunflower hypocotyl exposed to gravitational stimulus was enhanced in buffer solutions between pH 3.4 and 4.0 in the absence of added auxin. This effect was observed both when the segments were submerged during the stimulus and when they floated near the surface of the solution. 5-10 min in a horizontal position was sufficient to induce subsequent curvature.Straight growth of the segments was also promoted in buffers of this pH range.The acid effect on curvature was insensitive to KAsO2, HgCl2 and cycloheximide, inhibitors which drastically reduced auxin-induced curvature. Furthermore, acid buffer, but not auxin, restored the ability of segments taken from etiolated and "starved" plants to respond to gravity. These results suggest that the polarisation following gravistimulus may not be resticted to the asymmetric distribution of auxin and auxin co-factors but may involve a general physiological asymmetry.

Transport & Distribution of Auxin during Tropistic Response. I. The Lateral Migration of Auxin in Geotropism

A central tenet of the theory of tropisms is that shoots are enabled to bend toward light and away froml the earth by a migration of auxin across the stimiulated plant. Following Cholodny's proposal of such a lateral migration (12), Went in 1928 (31) shoX-ed directly that when the tips of Avena coleoptiles are illuminated from one side, more auxin diffuses out of the darker than out of the lighter side. The evidence was almost simultaneously extended to geotropism by Dolk, whose doctoral thesis (15) provi(lel a careful demonstration that when Avena coleoptile tips are held horizontal more auxin diffuses out of the lower than out of the upper side. Dolk's experiments further showed that when auxin is externally applied to horizontal sections cut from decapitated coleoptiles, it undergoes a similar asymmetric distribution. For both phototropism and geotropism the asymmetric distribution of endogenous auxin was soon confirmed by other workers, and the observations were extended both by diffusion and by extraction to dicotyledonous seedlings as well (see Went & Thimann (33) and Thimann & Curry (27) for reviews). Subsequently a large aggregate of concepts, though actually not many additional data, have crystallized around this simple notion of lateral auxin transport. In recent years, however, the lateral transport hypothesis has been seriously questioned, primarily because a number of workers failed to find any asymmetric distribution within the tissue when radioactive indoleacetic acid was applied to stimulated plant parts, and radioactivity, instead of growthpromoting activity, was the criterion. Negative results with plants placed horizontally or illuminated unilaterally were reported for more than one species by several groups of workers, notably Bunning et al. (10), Gordlon and Eib (19), Reisener (21, 22), Reisener and Simon (23), and Ching and Fang (11). On the other hand the original measurements, based on lioassay of endogenouts auxin diffusing from the basal surface of stimulated plant sections into agar blocks, have been confirmed and extended; Briggs et al. (9) and Gillespie and Briggs (16) have found clear-cut asymmetric distribution. Briggs (8) presents still more evidence for the reproducibility of these results. Much of this work has recently been reviewed by Anker (2). Such striking disagreement between two methods of measurement calls for explanation. If a plant indeed translocates its native indoleacetic acid, it must without discrimination move exogenous indoleacetic acid in the same way, providing that the applied auxin is made accessible to the transport system. It is of fundamental importance that when IAA-C14 is applied to oat coleoptile sections, the radioactivity transported out into agar blocks is indeed biologically active as auxin (18). On the other hand, several interpretations of the discrepancy may be considered. In the first place, the endogenous auxin which becomes asymmetrically distributed might not be indoleacetic acid (IAA). This is, however, made improbable by the repeated demonstrations that IAA is the auxin produced by Avena coleoptile tips (34, 20, 25) and by the fact that it does restore geotropic sensitivity to decapitated Avena coleoptile sections placed horizontally in solutions (1). As a second alternative, the material which becomes asymmetrically distributed might not be the auxin itself, but an auxin precursor or some other factor controlling the synthesis of auxin. Thirdly, it is important to note that the measurements of radioactivity have invariably been made on the tissue itself or on tissue extracts, while most of the measurements of biological activity have been made on agar blocks receiving the transported auxin. Thus gravity or light might conceivably cause the lateral migration of a factor controlling polar transport, so that although the amounts of auxin on the two sides of the plant remained the same, the amounts transported through the tissue into receiver blocks might be different. It would, indeed, be surprising if careful determinations of radioactivity in the tissue failed to reveal some change, but it is not impossible. Mechanisms of this and otlher kinds involving changes in auxin production. release, transport, or utilization on one side (without change in the anmounts present at any moment) have been postulated 1 Received Nov. 5, 1962. 2 This work was supported in part by a grant from the National Science Foundation, No. G9084, to Professor K. V. Thimann, and by predoctoral fellowships to B. Gillespie from the National Science Foundation (1959-61) and from the Public Health Service, Division of General Medical Sciences (No. CPM-8662-C1, 1961-63).