Rainer Hertel

In-vitro auxin binding to particulate cell fractions from corn coleoptiles.

SummaryWhen low concentrations (e.g. 10-6 M) of labelled 3-indoleacetic acid (14C-IAA) or α-naphthaleneacetic acid (14C-NAA) are added in vitro to homogenates of corn coleoptiles, radioactivity is reversibly bound to pelletable particles. From the saturation kinetics of the binding it is possible to estimate an apparent KM between 10-6 M and 10-5 M and a concentration of specific sites of 10-7–10-6 M per tissue volume.The binding is auxin-specific. Among many compounds tested, only auxins and such auxin analogues that are known to interact directly with auxin in transport and/or growth were found to interfere with this binding. For instance, the growth-active d-dichlorophenoxyisopropionic acid at 10-4 M inhibits 14C-NAA binding more than the less active l-isomer.The auxin-binding fractions are practically free of DNA and cytochrome-C oxidase and contain binding sites for 1-naphthylphthalamic acid. The results are discussed in context with the hyothesis—derived mainly from physiological data—that auxin receptors are localized at the plasma membrane.

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.

1-N-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid : In-vitro binding to particulate cell fractions and action on auxin transport in corn coleoptiles.

SummaryAuxin transport in corn coleoptile sections was inhibited by 2,3,5-triiodobenzoic acid (TIBA) as well as by 1-N-naphthylphthalamic acid (NPA); this inhibition was effected within 1 min of application.A particulate cell fraction-presumably plasma-membrane vesicles-specifically binds NPA and properties of these binding sites were studied using 3H-NPA and a pelletting technique. The saturation kinetics of the physiological NPA effect, i.e. the inhibition of auxin transport, is similar to that of the specific in-vitro NPA binding. Half saturation of the inhibitory effect was found with about 5×10-7 M TIBA and with 10-7 M NPA. Both substances also decreased the speed of movement of auxin pulses within coleoptile sections.NPA dissociates from its binding site when the particulate cell material is centrifuged through an NPA-free cushion. The NPA that is washed from its binding site can be used in another binding test without any apparent change and is chromatographically unaltered. Therefore, the NPA binding is probably reversible and non-covalent. Inhibition of auxin transport by TIBA or NPA could also be reversed when the coleoptile sections were washed in buffer.The movement of 131I-TIBA in corn coleoptiles appears to be polar in a basipetal direction. Higher concentrations of indoleacetic acid or TIBA inhibited this polar movement, suggesting that TIBA moves in the same channels as auxin. With 3H-NPA, however, no polar transport could be detected. Together with the in-vitro binding results, these data indicate that TIBA acts directly at the auxin receptor while NPA has a different receptor site.The effect of TIBA and NPA on elongation, with or without auxin, is neglegible in comparison to their effects on auxin transport.

Properties of auxin binding sites in different subcellular fractions from maize coleoptiles.

In-vitro binding of labeled auxins to sedimentable particles was tested in subcellular fractions from homogenates of maize (Zea mays L.) coleoptiles. The material was fractionated by differential centrifugation or on sucrose density gradients. It was confirmed that the major saturable binding activity (site I) for 1-naphthyl[1-14C]acetic acid is associated with vesicles derived from the endoplasmatic reticulum. A second type of specific auxin binding (site II) could be distinguished by several criteria, e.g. by the low affinity towards phenylacetic acid. The particles carrying site II could be clearly separated from markers of the endoplasmatic reticulum, the plasmalemma, the mitochondria and the nuclei, while their density as well as sedimentation velocity correlated with particle-bound acid phosphatase, indicating a localization at the tonoplast. In contrast to site I, binding at site II was hardly affected by a supernatant factor and by sulfhydryl groups. However, the specificity pattern of site II towards auxins and auxin analogs was very similar to that of site I tested in the presence of supernatant factor. The existence of a third auxin receptor localized in plasma membrane-rich gradient fractions was indicated by a preferential in-vitro binding of 2,4-dichlorophenoxyacetic acid.

Auxin transport in membrane vesicles from Cucurbita pepo L.

Association of 14C-labelled indole-3-acetic acid (IAA) with membrane particles from zucchini (Cucurbita pepo L.) hypocotyls — previously described as “site III binding” (M. Jacobs and R. Hertel, 1978, Planta 142, 1–10) — is reinterpreted as a carrier-mediated uptake into closed and sealed vesicles driven by a pH gradient. Accumulation of the radioactive auxin is saturable, sensitive to the protonophore, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), and to nigericin, and requires a pH gradient across the membranes with proton concentration greater outside than inside. The pH gradient decays within 1–2 h at 4°C and can be restored by re-equilibration of the particle preparation at more alkaline pH followed by return to more acidic medium. Osmotic shock and sonication release the IAA from the vesicles. 1-N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both inhibitors of auxin transport in vivo, increase the amount of net IAA accumulation in the vesicles, presumably by blocking efflux. Analogs of NPA less active or inactive in vivo are respectively less active or inactive in vitro. It is proposed that these membrane particles are outside-out plasma membrane vesicles, and that they perform the essential functions of auxin transport according to the chemiosmotic theory, with a specific, saturable proton symport uptake and an export anion carrier which is inhibited by NPA and TIBA.

The Mechanism of Auxin Transport as a Model for Auxin Action

Summary Auxin is transported by two elements in the plasma membrane: a symport and an exit carrier. Driven by a pH difference and an electric gradient, auxin uptake into the cytoplasm may lead to a 10- to 100 fold accumulation. For the exit carrier, a gating cycle is proposed which transiently opens a gate for Ca 2+ -influx. The cycle also accounts for the optimum type responses to external stimuli such as gravity, and to auxin itself. Furthermore, the model describes the decrease in sensitivity (= adaptation) of the auxin transport system following a step-up in stimulus. Adaptation can be inhibited by ethylene. With respect to optimum curve, to adaptation and also to analog specificity, the auxin response system, e.g. of cell elongation, resembles the transport mechanism; the two processes, however, are not identical. It is proposed that in evolution, from one auxin-transporting/responding system two such mechanisms - and perhaps a third more sensitive mechanism for the root — arose by gene duplication and diversification. Beyond the auxin carrier in the plasmalemma, in higher plants a homologous system operates on an inner membrane system, e.g. on the tonoplast of shoots and coleoptiles. This tonoplast-located carrier catalyzing net auxin influx into the vacuole, is proposed to act as a Ca 2+ gate. As a result of carrier cycling, Ca 2+ leaks into the cytoplasm where, in turn, Ca 2+ enhances the flow of Golgi vesicles and thus cell elongation.

N-1-napthylphthalamic-acid-binding activity of a plasma membrane-rich fraction from maize coleoptiles

SummaryPlasma membrane-rich fractions were prepared from maize coleoptiles by low-shear homogenization and differential and sucrose-gradient centrifugation. Plasma membrane fragments were identified using a specific cytochemical stain based on phosphotungstic acid prepared in chromic acid. In a comparison of 10 different cell fractions of varying plasma membrane content, the N-1-napthylphthalamic-acid (NPA)-binding activity of the fractions was directly proportional to the content of plasma membrane. The NPA binding appears to be strong KM between 10-8 and 10-7 M) but non-covalent. NPA is known to inhibit auxin transport efficiently and quickly. Thus, the results are consistent with the localization of auxin transport sites at the plasma membrane of plant cells.

Auxin binding to subcellular fractions from Cucurbita hypocotyls: In vitro evidence for an auxin transport carrier

An auxin binding sive, with characteristics different from the previously described auxin binding sites I and II in maize coleoptiles, is reported in homogenates of zucchini (Cucurbita pepo L. cv. Black Beauty) hypocotyls. Evidence from differential centrifugation and sucrose and metrizamide density gradients indicates that the site is localized on the plasma membrane. The site has a KD of 1–2×10−6 M for indole acetic acid and has a pH optimum of 5.0. Binding specificity measured with several auxins, weak auxins, and anti-auxins generally parallels the activities of the same compounds as inhibitors of auxin transport. 1-N-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid (2,3,5-TIBA), both auxin transport inhibitors in vivo, increase specific auxin binding to this site. 3,4,5-TIBA, which can partially reverse 2,3,5-TIBAs transport inhibition when the two substances are added together in vivo, partially reverses 2,3,5-TIBAs increase in specific auxin binding to the plasma membrane site when added with 2,3,5-TIBA in vitro. Preliminary investigations indicate that a similar plasma membrane site exists in maize (Zea mays L.) coleoptiles. It is suggested that different conformations of this site may function during active auxin transport.

Localization of “in vitro” binding of the fungal toxin fusicoccin to plasma-membrane-rich fractions from corn coleoptiles

Abstract Radioactive fusicoccin ([3H]FC) associates in vitro with subcellular, post-mitochondrial particles from corn coleoptiles, as shown by the appearance of radioactivity in high speed pellets. This binding is specific, not readily chaseable, and saturable by 10−4M unlabelled FC. Auxins do not cross-compete for these sites. The binding is heat-labile at 60°C and has a pH optimum around 6. Differential centrifugation and isopycnic sucrose density gradients with parallel determination of markers indicate the localization of the FC binding sites at the plasmalemma. This is in agreement with the known in vivo effects of this fungal toxin.

Initial phases of gravity-induced lateral auxin transport and geotropic curvature in corn coleoptiles.

SummaryWild-type corn coleoptiles showed an initial downward bending upon transfer from the vertical to the horizontal position. Strong upward curvature started only 15–30 min after the begin of horizontal exposure.Little, if any at all, initial downward geotropic bending was found with amylomaize coleoptiles at 1 X g. With stronger stimuli (10 or 20 X g) the amylomaize mutant reacted initially strongly in the “wrong” direction, i.e. opposite to the later response.When wild-type coleoptiles had been symmetrically prestimulated for 60 min with alternating 2-min horizontal exposures from opposite sides, no initial downward bending occurred if the plane of horizontal exposure was maintained from pretreatment to the continuous horizontal stimulation of the test. If, however, the coleoptiles were rotated 90° around their long axis between pretreatment and test, the initial downward bending reaction developed as in the non-prestimulated controls. Thus changes in reactivity remained localized to the site of stimulation.Following the same pretreatments used for the curvature measurements, lateral 3H-IAA transport was measured in coleoptile segments for 10 or 12.5 min. The auxin distribution found was strikingly parallel to the bending for all pretreatments.The dependence of reaction pattern on the duration of prestimulation in the same plane was tested. The function indicates a “half life” of 10–20 min for the change in sensitivity. The findings are discussed in view of a model of overstimulation and adaptation.