Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol-3yl-acetic acid

Phenylacetic acid (PAA), a naturally-occurring acidic plant growth substance, was readily taken up by pea (Pisum sativum L. cv. Alderman) stem segments from buffered external solutions by a pH-dependent, non-mediated diffusion. Net uptake from a 0.2 M solution at pH 4.5 proceeded at a constant rate for at least 60 min and, up to approx. 100 M, the rate of uptake was directly proportional to the external concentration of the compound. The net rate of uptake of PAA was not affected by the inclusion of indol-3yl-acetic acid (IAA) in the uptake medium (up to approx. 30 M) and, unlike the net uptake of IAA, was not stimulated by N-1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid. At an external concentration of 0.2 M and pH 4.5, the net rate of uptake of PAA was about twice that of IAA. It was concluded that the uptake of PAA did not involve the participation of carriers and that PAA was not a transported substrate for the carriers involved in the uptake and polar transport of IAA. Nevertheless, the inclusion of 3–100 M unlabelled PAA in the external medium greatly stimulated the uptake by pea stem segments of [1-¹⁴C]IAA (external concentration 0.2 M). It was concluded that whilst PAA was not a transported substrate for the NPA-sensitive IAA efflux carrier, it interacted with this carrier to inhibit IAA efflux from cells. Over the concentration range 3–100 M, PAA progressively reduced the stimulatory effect of NPA on IAA uptake, indicating that PAA also inhibited carrier-mediated uptake of IAA. The consequences of these observations for the regulation of polar auxin transport are discussed.Abbreviations IAA indol-3yl-acetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid     

Auxin carriers in Cucurbita vesicles

Carrier-mediated uptake of indole-3-acetic acid (IAA) by microsomal vesicles from Cucurbita pepo L. hypocotyls was strongly inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D; i ₅₀= 0.3 M) but only weakly by 1-naphthylacetic acid (NAA). The fully ionised auxin indol-3-yl methanesulphonic acid also inhibited (i ₅₀=3 M). The same affinity ranking of these auxins for the uptake carrier, an electroimpelled auxin anion-H⁺ symport, is demonstrable in hypocotyl segments. The specificity of the auxin-anion eflux carrier was tested by the ability of different nonradioactive auxins to compete with [³H]IAA and reduce the stimulation of net radioactive uptake by N-1-naphthylphthalamic acid (NPA), a noncompetitive inhibitor of this carrier. By this criterion, NAA and IAA had comparable affinities, with 2,4-D interaction more weakly. Stimulation of [³H]IAA uptake by NAA, as a result of competition for the efflux carrier, could also be demonstrated when a suitable concentration of 2,4-D was used selectively to inhibit the uptake carrier. However, when [³H]NAA was used, no stimulation of its association with vesicles by NPA, 2,3,5-triiodobenzoic acid, or nonradioactive NAA was found. In hypocotyl segments, [³H]NAA net uptake was much less sensitive to NPA stimulation than was [¹⁴C]IAA uptake. The apparent contradictions concerning NAA could be explained by carrier-mediated auxin efflux making a smaller relative contribution to the overall transport of NAA than of IAA. The relationship between carrier specificity as manifested in vitro and the specificity of polar auxin transport is discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - IMS indol-3-yl methanesulphonic acid - NAA 1-naphthylacetic aci - NPA N-1-naphthylphthalamic acid     

The action of specific inhibitors of auxin transport on uptake of auxin and binding of N-1-naphthylphthalamic acid to a membrane site in maize coleoptiles

Using both 1-mm segments of corn (Zea mays L.) coleoptiles and a preparation of membranes isolated from the same source, we have compared the effectiveness of several inhibitors of geotropism and polar transport in stimulating uptake of auxin (indole-3-acetic acid, IAA) into the tissue and in competing with N-1-naphthylphthalamic acid (NPA) for a membrane-bound site. Low concentrations of 2,3,5-triiodobenzoic acid (TIBA), NPA, 2-chloro-9-hydroxyfluorene-9-carboxylic acid (morphactin), and fluorescein, eosin, and mercurochrome all stimulated net uptake of [³H]IAA by corn coleoptile tissues while higher concentrations reduced the uptake of both [³H]IAA and another lipophilic weak acid, [¹⁴C]benzoic acid. Since low concentrations of fluorescein and its derivatives competed for the same membrane-bound site in vitro as did morphactin and NPA, the basis for both the specific stimulation of auxin accumulation and the inhibition of polar auxin transport by all these compounds may be their ability to interfere with the carrier-mediated efflux of auxin anions from cells. At higher concentrations, the decrease in accumulation of weak acids was nonspecific and thus may be the result of acidification of the cytoplasm and a general decrease in the driving force for uptake of the weak acids. Triiodobenzoic acid was an exception. Low concentration of TIBA (0.1–1 M) were much less effective than NPA in competing for the NPA receptor in vitro, but little different from NPA in ability to stimulate auxin uptake. One possibility is that TIBA, a substance which is polarly transported, may compete with auxin for the polar transport site while NPA, morphactin, and the fluorescein derivatives may render this site inactive.Abbreviations C1-NPA 2,3,4,5-tetrachloro-N-1-naphthylphthalamic acid - IAA indole-3-acetic acid - -NAA -naphthaleneacetic acid - -NAA -naphthalenacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Targeting of auxin carriers to the plasma membrane: effects of monensin on transmembrane auxin transport in Cucurbita pepo L. tissue

Treatment of etiolated zucchini (Cucurbita pepo L.) hypocotyl tissue with sub-micromolar concentrations of the cationophore monensin rapidly (<20 min) inhibited the transport catalytic activity of the specific auxin-anion efflux carrier and reduced the inhibition of this carrier by the phytotropin N-1-naphthylphthalamic acid (NPA). Monensin inhibited the basipetal polar transport of indol-3yl-acetic acid (IAA) in long (30 mm) zucchini segments. At concentrations lower than 10^–5 mol·dm^–3 monensin did not affect uptake of the pH probe [2-¹⁴C]5,5-dimethyloxazolidine-2,4-dione (DMO) or that of the membrane-potential probe tetra[¹⁴C-phenyl]phosphonium bromide (TPP⁺), did not affect the response of IAA net uptake to external Ca²⁺ concentration and did not alter the metabolism of IAA. It was concluded that low concentrations of monensin inhibit transport through the Golgi apparatus of auxin efflux carrier protein and that the efflux carriers turn over very rapidly in the plasma membrane. Monensin pretreatment did not affect the saturable binding of [³H]NPA to microsomal membranes, indicating that the auxin-efflux catalytic sites and the NPA-binding sites are located on separate proteins. At higher concentrations (10^–5 mol·dm^–3) monensin inhibited both mediated uptake and mediated efflux components of IAA transport. This effect was at least in part attributable to perturbation by monensin of the driving forces for mediated uptake since high concentrations of monensin also reduced the uptake of DMO and TPP⁺.Abbreviations CH cycloheximide - DMO 5,5-dimethyloxazolidine-2,4-dione - MDMP 2-(4-methyl-2,6-dinitroanlilino)N-methyl-propionamide - NPA N-1-naphthylphthalamic acid - TPP⁺ tetraphenylphosphonium ion We thank Mrs. R.P. Bell for technical assistance and Drs. G.F. Katekar and M.A. Venis for generous gifts of NPA. S.W. was supported by the U.K. Science and Engineering Research Council.     

Components of auxin transport in stem segments of Pisum sativum L.

1. The uptake of indol-3-yl acetic acid ([1-¹⁴C]IAA, 0–2.0 M) into light-grown pea stem segments was measured under various conditions to investigate the extent to which mechanisms of auxin transport in crown gall suspension culture cells (Rubery and Sheldrake, Planta 118, 101–121, 1974) are also found in a tissue capable of polar auxin transport. — 2. IAA uptake increased as the external pH was lowered. IAA uptake was less than that of benzoic acid (BA), naphthylacetic acid (NAA) or 2,4 dichlorophenoxyacetic acid (2,4D) under equivalent conditions. TIBA enhanced net IAA uptake through inhibition of efflux, and to a lesser extent, also increased uptake of NAA and 2,4D while it had no effect on BA uptake. — 3. Both DNP and, at higher concentrations, BA, reduced IAA uptake probably because of a reduction of cytoplasmic pH. However, low concentrations of both BA and DNP caused a slight enhancement of IAA net uptake, possibly through a reduction of carrier-mediated IAA efflux. In the presence of TIBA, the inhibitory effects of DNP and BA were more severe and there was no enhancement of uptake at low concentrations. — 4. Non-radioactive IAA (10 M) reduced uptake of labelled IAA but further increases in concentration up to 1.0 mM produced first an inhibition (0–10 min) of labelled IAA uptake, followed by a stimulation at later times. Non-radioactive 2,4 D decreased, but was not observed to stimulate, uptake of labelled IAA. In the presence of TIBA labelled IAA uptake was inhibited by non-radioactive IAA regardless of its concentration. — 5. Sulphydryl reagents PCMB and PCMBS promoted or inhibited IAA uptake depending, respectively, on whether they penetrated or were excluded from the cells. The penetrant PCMB also reduced the promotion of labelled IAA uptake by TIBA or by high concentrations of added non-labelled IAA. — 6. Our findings are interpreted as being consistent with the diffusive entry of unionised IAA into cells together with some carrier-mediated uptake. Auxin efflux from the cells also appears to have a carrier-mediated contribution, at least part of which is inhibited by TIBA, and which has a capacity at least as great as that of the uptake carrier. The data indicate that pea stem segments contain cells whose mechanisms of trans-membrane auxin transport fit the model of polar auxin transport proposed from experiments with crown gall suspension cells, although differences, particularly of carrier specificity, are apparent between the two systems.Abbreviations IAA indol-3-yl acetic acid - BA benzoic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - DNP 2,4-dinitrophenol - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzene sulphonic acid This work was performed in Cambridge during the tenure of a sabbatical leave by P.J.D. Supported by a grant for supplies from the American Philosophical Society to P.J.D.     

Effects of G-protein probes on interactions between auxin efflux carriers and phytotropin receptors in zucchini (Cucurbita pepo L.) microsomal membranes

Microsomal vesicles prepared from etiolated hypocotyl tissue of zucchini (Cucurbita pepo L. cv. All Green Bush) exhibited saturable N-1-naphthylphthalamic acid ([³H]NPA) binding, NPA-stimulated association of indol-3yl-acetic acid ([³H]IAA), and saturable binding of guanosine 5-O-[3-thiotriphosphate] (GTP--[³⁵S]). These vesicles were used to test the possibility that NPA receptors might interact with IAA-anion efflux carriers by coupling through a GTP-binding protein (G-protein). Unlabelled GTP--S or guanosine 5-O-[2-thiodiphosphate] (GDP--S) had no effect on saturable NPA binding or on the NPA-stimulated association of IAA with microsomes. NPA did not affect saturable binding of GTP--[³⁵S] to microsomes, either in the presence or absence of saturating concentrations of unlabelled GTP--S or GDP. It is concluded that the occupancy of phytotropin receptors is not transduced to auxin efflux carriers by a GTP-binding protein.     

Flavonoids and flavonoid sulphates as probes of auxin-transport regulation in Cucurbita pepo hypocotyl segments and vesicles

The accumulation of IAA by sealed microsomal vesicles prepared from hypocotyls of dark-grown Cucurbita pepo L. (zucchini) seedlings was stimulated by N-1-naphthylphthalamic acid (NPA: an inhibitor of carrier-mediated auxin efflux and hence of polar auxin transport) as well as by quercetin and certain other flavonoids with a specificity pattern similar to that previously shown for their NPA-like effects on auxin transport and inhibition of NPA binding to saturable sites. In contrast, putatively nonpenetrant negatively charged quercetinsulphate esters did not stimulate such auxin accumulation although they were able to oppose stimulation by NPA or quercetin itself. However, the binding of NPA to hypocotyl microsomes was 30- to 80-fold more strongly inhibited by the quercetin sulphates than by unsubstituted quercetin. As with vesicles, net IAA uptake by hypocotyl segments (2 mm) from dark-grown zucchini was stimulated less effectively by quercetin-sulphate esters than by quercetin itself. We discuss the implications of these observations for the accessibility of the NPA receptor from cell wall or cytoplasm and for the coupling of its occupancy to inhibition of the auxin efflux carrier.Abbreviations ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - NPA N-1-naphthylphthalamic acid - PMSF phenylmethylsulphonyl fluoride This work was supported by a Studentship (I.J.F.) from the Science and Engineering Research Council and by the Gatsby Charitable Foundation. We are particularly grateful to Dr. W. Michalke for a preprint and permission to use his method of microsome preparation in advance of publication.     

Auxin uptake and action of N-1-naphthylphthalamic acid in corn coleoptiles

The validity of a chemiosmotic hypothesis for uptake of weak acids as an explanation for the accumulation of auxin by cells has been explored further by comparing the uptake of indole-3-acetic acid (IAA) by 1-mm segments of corn (Zea mays L.) coleoptiles with that of benzoic acid and two neutral indoles, indoleethanol and indoleacetonitrile, which do not ionize. These substances, while structurally related to IAA lack both auxin activity and polar transport. Uptake of IAA and benzoic acid increase with decreasing external pH, whereas the uptake of the two neutral indoles is independent of external pH.Although metabolism of IAA, during 90 min or less, is minimal and without significant effect on its uptake, metabolism of benzoic acid appears responsible for the apparent saturation of benzoic acid uptake at high concentrations. An inhibitor of auxin transport, N-1-naphthylphathalamic acid (NPA), stimulates uptake of IAA but has no effect on uptake of either benzoic acid or the two neutral indoles. Thus, NPA does not affect the driving forces for accumulation of weak acids but probably specifically decreases the flux of the auxin anions relative to undissociated auxin. Since the electrochemical potential of auxin anions is usually higher in than outside cells, blocking the anion flux with NPA would enhance auxin uptake. Azide, which abolishes accumulation of both IAA and benzoic acid, may simply collapse the pH gradient across the plasma membrane.In the absence of NPA, increasing concentrations of auxins or the analogoue -naphthaleneacetic acid (-NAA) exert two opposing effects on the uptake of IAA-depression and stimulation. Stimulation results from saturating the anion flux. With uptake fully stimulated by NPA, however, increasing concentrations of auxins or analogues only depress uptake of [³H]IAA. These results are consistent with more than one path for auxin transport each with a different dependence on concentration. In depressing NPA-stimulated IAA uptake, the effectiveness of -NAAIAA-NAA benzoic acid, a specificity similar to that of an auxin binding site in vitro that has been implicated by others in auxin transport. The results support the general hypothesis that cellular auxin uptake and polar transport through tissues are chemiosmotically coupled to the electrochemical potential of auxin and protons.Abbreviations IAA indole-3-acetic acid - -NAA -naphthaleneacetic acid - -NAA -naphthaleneacetic acid - NPA N-1-naphthylphthalamic acid     

The effects of 2,4-dinitrophenol and chemical modifying reagents on auxin transport by suspension-cultured crown gall cells

1. The effects of 2,4-dinitrophenol (DNP) and chemical modifying reagents on the transport of indol-3-yl acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4 D) by suspension-cultured crown gall cells of Parthenocissus tricuspidata Planch. were investigated. 2. DNP smoothly reduced uptakes of both benzoic acid and 2,4 D but IAA uptake at pH 6.0 was not inhibited by concentrations below 20 mol/l except in the presence of 2,3,5-triiodobenzoic acid (TIBA) whose stimulatory effect was thereby abolished. DNP stimulated the efflux of 2,4 D and of IAA in the presence of TIBA. Without TIBA, DNP first inhibited but later stimulated IAA efflux. —3. Low concentrations of N-ethylmaleimide (NEM) (<5 mol/l) abolished TIBA-stimulation of net IAA uptake while not affecting (or slightly promoting) net uptake of IAA alone, whose inhibition needs greater NEM concentrations. Diethylpyrocarbonate behaved similarly. The poorly-penetrant p-chloromercuriben-zenesulphonic acid did not cause a marked differential inhibition of the TIBA stimulation. — 4. Together with earlier data, the results support a two-carrier model comprising a common carrier for IAA and 2,4 D, previously suggested to be an auxin anion/proton symport, and also an electrogenic carrier, specific for IAA anions, and inhibited by TIBA. The role of such carriers in polar auxin transport is discussed.Abbreviations IAA Indol-3-yl acetic acid - 2,4 D 2,4-Dichlorophenoxyacetic acid - BA Benzoic acid - DNP 2,4-Dinitrophenol - NEM N-ethylmaleimide - PCMBS p-Chloromercuribenzenesulphonic acid - TIBA 2,3,5-Triiodobenzoic acid     

A saturable site responsible for polar transport of indole-3-acetic acid in sections of maize coleoptiles

The velocity of transport and shape of a pulse of radioactive indole-3-acetic acid (IAA) applied to a section of maize (Zea mays L.) coleoptile depends strongly on the concentration of nonradioactive auxin in which the section has been incubated before, during, and after the radioactive pulse. A pulse of [³H]IAA disperses slowly in sections incubated in buffer (pH 6) alone; but when 0.5–5 M IAA is included, the pulse achieves its maximum velocity of about 2 cm h^-1. At still higher IAA concentrations in the medium, a transition occurs from a discrete, downwardly migrating pulse to a slowly advancing profile. Specificity of IAA in the latter effect is indicated by the observation that benzoic acid, which is taken up to an even greater extent than IAA, does not inhibit movement of [³H]IAA. These results fully substantiate the hypothesis that auxin transport consists of a saturable flux of auxin anions (A^-) in parallel with a nonsaturable flux of undissociated IAA (HA), with both fluxes operating down their respective concentration gradients. When the anion site saturates, the movement of [³H]IAA is nonpolar and dominated by the diffusion of HA. Saturating polar transport also results in greater cellular accumulation of auxin, indicating that the same site mediates the cellular efflux of A^-. The transport inhibitors napthylphthalamic acid and 2,3,5-triiodobenzoic acid specifically block the polar A^- component of auxin transport without affecting the nonsaturable component. The transport can be saturated at any point during its passage through the section, indicating that the carriers are distributed throughout the tissue, most likely in the plasmalemma of each cell.Abbreviations A^- auxin anion - HA undissociated auxin - IAA indole-3-acetic acid - NPA N-1-napthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

5′-Isothiocyanato-N-1-naphthylphthalamic acid: a chemically reactive site-directed irreversible ligand for phytotropin receptors

A chemically reactive analog of the phytotropin N-1-naphthylphthalamic acid (NPA) was synthesized and evaluated as a site-directed irreversible ligand for the NPA receptor. The NPA analog (5-isothiocyanato-N-1-naphthylphthalamic acid; NCS-NPA) was synthesized in two steps. Pretreatment of etiolated Helianthus hypocotyl segments with NCS-NPA at concentrations in excess of 1 M resulted in a dose-dependent inhibition of basipetal [¹⁴C]IAA transport. Net uptake of IAA by hypocotyl segments was stimulated by NCS-NPA at concentrations of 1 M or greater. NCS-NPA inhibited the saturable binding of [³H]NPA in Helianthus microsomes in a dose-dependent fashion with 50% inhibition occurring at NCS-NPA concentrations of 3 to 10 nM. The binding affinity of [³H]NPA in microsomes pretreated with NCS-NPA followed by extensive washing was substantially reduced. These results demonstrate that NCS-NPA is a site-directed irreversible ligand for the NPA receptor and suggest that it may be of use in the purification and characterization of this biologically important receptor.Abbreviations ANPA 5-amino-naphthylphthalamic acid - IAA indole-3-acetic acid - NCS-NPA 5-isothiocyanato-N-1-naphthylphthalamic acid - NPA N-1-naphthylphthalamic acid - TLC thin-layer chromatography     

Biochemical Bases for the Loss of Basipetal IAA Transport with Advancing Physiological Age in Etiolated Helianthus Hypocotyls: Changes in IAA Movement, Net IAA Uptake, and Phytotropin Binding

Basipetal transport of [¹⁴C]IAA in hypocotyl segments isolated from various regions of etiolated Helianthus annuus L. cv NK 265 seedlings declines with increasing physiological age. This decline was the result of a reduction in both transport capacity and apparent velocity. Net IAA uptake was greater and the abilities of auxin transport inhibitors to stimulate net IAA uptake were reduced in older tissues. Net IAA accumulation by microsomal vesicles exhibited a similar behavior with respect to age. Specific binding of [³H]N-1-naphthylphthalamic acid (NPA) to microsomes prepared from young and older hypocotyl regions was saturable and consistent with a single class of binding sites. The apparent affinity constants for NPA binding in microsomes prepared from young versus older tissues were 6.4 and 10.8 nanomolar, respectively, and the binding site densities for young versus old tissues were 7.44 and 3.29 picomoles/milligram protein, respectively. Specific binding of [³H]NPA in microsomes prepared from both tissues displayed similar sensitivities toward unlabeled flurenol and exhibited only slight differences in sensitivity toward 2,3,5-triiodobenzoic acid. These results demonstrate that the progressive loss of basipetal IAA transport capacity in etiolated Helianthus hypocotyls with advancing age is associated with substantial alterations in the phytotropin-sensitive, IAA efflux system and they suggest that these changes are, at least partially, responsible for the observed reduction of polar IAA transport with advancing tissue age.     

Targeting of auxin carriers to the plasma membrane: differential effects of brefeldin A on the traffic of auxin uptake and efflux carriers

Monensin and brefeldin A (BFA), inhibitors of Golgi-mediated protein secretion, rapidly perturb the transport catalytic activity of specific plasma membrane-associated efflux carriers for indole-3-acetic acid (IAA) and inhibit polar transport of IAA. To determine if these responses result solely from perturbation of the efflux carrier or whether specific auxin uptake carrier function is also affected, the influence of BFA on the cellular transport of a range of auxins with contrasting affinities for specific auxin uptake and efflux carriers was investigated in zucchini (Cucurbita pepo L.) hypocotyl tissue. In-flight addition of BFA (3 · 10⁻⁵ mol · dm⁻³) caused a rapid (lag < 10 min) and substantial (fourfold) increase in the rate of [1-¹⁴C]IAA net uptake by zucchini hypocotyl tissue. In the presence of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA; 3 · 10⁻⁶ mol · dm⁻³), BFA slightly reduced the rate of [1-¹⁴C]IAA net uptake. Stimulation of [1-¹⁴C]IAA net uptake by BFA was concentration-dependent. In the absence of BFA, net uptake of [1-¹⁴C]IAA exhibited the characteristic biphasic response to increasing concentrations of competing cold IAA but in the presence of BFA, [1-¹⁴C]IAA uptake decreased smoothly with increase in concentration of competing unlabelled IAA, indicating a loss of auxin efflux carrier activity but retention of functional uptake carriers. The half-time for mediated efflux of [1-¹⁴C]IAA from preloaded zucchini tissue was substantially increased by BFA (t_1/2 = 51 min, controls; 107 min, BFA-treated). Treatment with BFA and/or NPA did not significantly affect the net uptake by, or efflux from, zucchini tissue of [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([1-¹⁴C]2,4-D), a substrate for the auxin uptake carrier but not the auxin efflux carrier. Uptake of [1-¹⁴C]2,4-D declined smoothly with increasing concentrations of competing unlabelled IAA whether or not BFA was included in the uptake medium, confirming the failure of BFA to perturb auxin uptake carrier function. Transport of 1-[4-³H]naphthaleneacetic acid (1-NAA) exhibited little response to BFA or NPA, confirming that it is only a weakly transported substrate for the efflux carrier in zucchini cells. Received: 12 November 1997 / Accepted: 27 January 1998     

The role of auxin efflux carriers in the reversible loss of polar auxin transport in the pea (Pisum sativum L.) stem

Correlatively inhibited pea shoots (Pisum sativum L.) did not transport apically applied ¹⁴C-labelled indol-3yl-acetic acid ([¹⁴C]IAA), and polar IAA transport did not occur in internodal segments cut from these shoots. Polar transport in shoots and segments recovered within 24 h of removing the dominant shoot apex. Decapitation of growing shoots also resulted in the loss of polar transport in segments from internodes subtending the apex. This loss was prevented by apical applications of unlabelled IAA, or by low temperatures (approx. 2° C) after decapitation. Rates of net uptake of [¹⁴C]IAA by 2-mm segments cut from subordinate or decapitated shoots were the same as those in segments cut from dominant or growing shoots. In both cases net uptake was stimulated to the same extent by competing unlabelled IAA and by N-1-naphthylphthalamic acid. Uptake of the pH probe [¹⁴C]-5,5-dimethyloxazolidine-2,4-dione from unbuffered solutions was the same in segments from both types of shoot. Patterns of [¹⁴C]IAA metabolism in shoots in which polar transport had ceased were the same as those in shoots capable of polar transport. The reversible loss of polar IAA transport in these systems, therefore, was not the result of loss or inactivation of specific IAA efflux carriers, loss of ability of cells to maintain transmembrane pH gradients, or the result of a change in IAA metabolism. Furthermore, in tissues incapable of polar transport, no evidence was found for the occurrence of inhibitors of IAA uptake or efflux. Evidence is cited to support the possibility that the reversible loss of polar auxin transport is the result of a gradual randomization of effluxcarrier distribution in the plasma membrane following withdrawal of an apical auxin supply and that the recovery of polar transport involves reestablishment of effluxcarrier asymmetry under the influence of vectorial gradients in auxin concentration.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid This work was supported by grant no. GR/D/08760 from the U.K. Science and Engineering Research Council. We thank Mrs. R.P. Bell for technical assistance.     

Studies on the evolution of auxin carriers and phytotropin receptors: Transmembrane auxin transport in unicellular and multicellular Chlorophyta

The characteristics of transmembrane transport of ¹⁴C-labelled indol-3yl-acetic acid ([1-¹⁴C]IAA) were compared in Chlorella vulgaris Beij., a simple unicellular green alga, and in Chara vulgaris L., a branched, multicellular green alga exhibiting axial polarity and a high degree of cell and organ specialization. In Chara thallus cells, three distinguishable trans-plasmamembrane fluxes contributed to the net uptake of [1-¹⁴C]-IAA from an external solution, viz.: a non-mediated, pH-sensitive influx of undissociated IAA (IAAH); a saturable influx of IAA; and a saturable efflux of IAA. Both saturable fluxes were competitively inhibited by unlabelled IAA. Association of [³H]IAA with microsomal preparations from Chara thallus tissue was competitively inhibited by unlabelled IAA. Results indicated that up-take carriers occurred in the membranes at a much higher density than efflux carriers. The efflux component of IAA net uptake by Chara was not affected by several phytotropins (N-1-naphthylphthalmic acid, NPA; 2-(1-pyrenoyl)benzoic acid; and 5-(2-carboxyphenyl)-3-phenylpyrazole), which are potent non-competitive inhibitors of specific auxin-efflux carriers in more advanced plant groups, and no evidence was found for a specific association of [³H]NPA with Chara microsomal preparations. It was concluded that Chara lacked phytotropin receptors. Net uptake of [1-¹⁴C]IAA also was unaffected by 2,3,5-triiodobenzoic acid except at concentrations ( 10^–1 mol · m^–3) high enough to depress cytoplasmic pH (determined by uptake of 5,5-dimethyloxazolidine-2,4-dione). Chlorella cells accumulated [1-¹⁴C]IAA from an external solution by pH-sensitive diffusion of IAA across the plasma membrane and anion (IAA^–) trapping, but no evidence was found in Chlorella for the occurrence of IAA carriers. These results indicate that carrier systems capable of mediating the transmembrane transport of auxins appeared at a very early stage in the evolution of green plants, possibly in association with the origin of a differentiated, multicellular plant body. Phytotropin receptors evolved independently of the carriers.Abbreviations CPP 5-(2-carboxyphenyl)-3-phenylpyrazole - DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - TIBA 2,3,5-triiodobenzoic acid We thank the Nuffield Foundation for the award of an Undergraduate Research Bursary to J.E.D.-F., Dr. G.F. Katekar, C.S.I.R.O., Canberra, Australia for generous gifts of phytotropins, and Mrs. R.P. Bell for technical support.     

Applicability of the chemiosmotic polar diffusion theory to the transport of indol-3yl-acetic acid in the intact pea (Pisum sativum L.)

The transport of exogenous indol-3yl-acetic acid (IAA) from the apical tissues of intact, light-grown pea (Pisum sativum L. cv. Alderman) shoots exhibited properties identical to those associated with polar transport in isolated shoot segments. Transport in the stem of apically applied [1-¹⁴C]-or [5-³H]IAA occurred at velocities (approx. 8–15 mm·h^-1) characteristic of polar transport. Following pulse-labelling, IAA drained from distal tissues after passage of a pulse and the rate characteristics of a pulse were not affected by chases of unlabelled IAA. However, transport of [1-¹⁴C]IAA was inhibited through a localised region of the stem pretreated with a high concentration of unlabelled IAA or with the synthetic auxins 1-napthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, and label accumulated in more distal tissues. Transport of [1-¹⁴C]IAA was also completely prevented through regions of the intact stem treated with N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid.Export of IAA from the apical bud into the stem increased with total concentration of IAA applied (labelled+unlabelled) but approached saturation at high concentrations (834 mmol·m^-3). Transport velocity increased with concentration up to 83 mmol·m^-3 IAA but fell again with further increase in concentration.Stem segments (2 mm) cut from intact plants transporting apically applied [1-¹⁴C]IAA effluxed 93% of their initial radioactivity into buffer (pH 7.0) in 90 min. The half-time for efflux increased from 32.5 to 103.9 min when 3 mmol·m^-3 NPA was included in the efflux medium. Long (30 mm) stem sections cut from immediately below an apical bud 3.0 h after the apical application of [1-¹⁴C]IAA effluxed IAA when their basal ends, but not their apical ends, were immersed in buffer (pH 7.0). Addition of 3 mmol·m^-3 NPA to the external medium completely prevented this basal efflux.These results support the view that the slow long-distance transport of IAA from the intact shoot apex occurs by polar cell-to-cell transport and that it is mediated by the components of IAA transmembrane transport predicted by the chemiosmotic polar diffusion theory.Abbreviations IAA indol-3yl-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

The specificity of carrier-mediated auxin transport by suspension-cultured crown gall cells

1. The specificity of the auxin transport system of suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch- is examined with regard to 2,4-Dichlorophenoxyacetic acid (2,4 D), l-Naphthylacetic acid (NAA) and Benzoic acid (BA) as well as for indole-3-acetic acid (IAA). — 2. All four weak acids can be accumulated by the cells from a medium more acidic than the cytoplasm. This is by virtue of non-specific passive diffusion of their lipid-soluble protonated forms down a concentration gradient. The corresponding anionic species are much less permeant. The extent of the accumulation is dependent on the pH difference that is maintained by the cells between their cytoplasm and the incubation medium. Studies of the concentration dependence of BA and NAA net uptake at a series of external pHs suggest that an acidification of the cytoplasm can be eventually brought about by the entry of weak acid into the cells. — 3. The uptake of 2,4 D, as well as that of IAA, has a saturable carrier-mediated component in addition to the passive diffusion of the undissociated acid. These saturable components probably represent anion uptake and appear to be mediated by a common carrier. The kinetic studies provided no evidence for the participation of carriers in the transport of BA or NAA. — 4. It was shown that the efflux of 2,4 D also has a carrier-mediated component and it is suggested that both the influx and efflux of IAA and 2,4 D occur on a common carrier. — 5. The inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA), stimulates the net uptake of IAA by inhibiting carrier-mediated efflux of IAA from the cells. However, TIBA could not be demonstrated to have a significant effect on 2,4 D transport and any perturbation that occurs is very small in comparison with its effect on IAA movement. To account for this, the proposed common carrier could exhibit some difference in its internal binding characteristics betweend 2,4 D and IAA. An alternative explanation is that a second carrier is present, which mediates IAA efflux only, and which is inhibited by TIBA. — 6. TIBA has no significant effect on the transport of either BA or NAA, except to bring about an inhibition of net uptake, and a corresponding stimulation of efflux, when it is present at concentrations sufficient to acidify the cytoplasm. —7. The crown gall cells are compared to intact plant tissues capable of polar auxin transport with regard to the specificities exhibited for the transport of the auxins IAA, 2,4 D and NAA and the non-auxin BA.Abbreviations IAA indol-3-yl acetic acid - 2,4 D 2,4-Dichlorophenoxyacetic acid - NAA 1-Naphthylacetic acid - BA Benzoic acid - TIBA 2,3,5-triiodobenzoic acid     

Transfer of exogenous auxin from the phloem to the polar auxin transport pathway in pea (Pisum sativum L.)

When [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA) was applied to the upper surface of a mature foliage leaf of garden pea (Pisum sativum L. cv. Alderman), ¹⁴C effluxed basipetally but not acropetally from 30-mm-long internode segments excised 4 h after the application of [1-¹⁴C]IAA. This basipetal efflux was strongly inhibited by the inclusion of 3.10^–6 mol· dm³ N-1-naphthylphthalamic acid (NPA) in the efflux buffer. In contrast, when [¹⁴C] sucrose was applied to the leaf, the efflux of label from stem segments excised subsequently was neither polar nor sensitive to NPA. The [1-¹⁴C]IAA was initially exported from mature leaves in the phloem — transport was rapid and apolar; label was recovered from aphids feeding on the stem; and label was recovered in exudates collected from severed petioles in 20 mM ethylenediaminetetraacetic acid. No ¹⁴C was detected in aphids feeding on the stems of plants to which [1-¹⁴C]IAA had been applied apically, even though the internode on which they were feeding transported considerable quantities of label. Localised applications of NPA to the stem strongly inhibited the basipetal transport of apically applied [1-¹⁴C]IAA, but did not affect transport of [1-¹⁴C]IAA in the phloem. These results demonstrate for the first time that IAA exported from leaves in the phloem can be transferred into the extravascular polar auxin transport pathway but that reciprocal transfer probably does not occur. In intact plants, transfer of foliar-applied [1-¹⁴C]IAA from the phloem to the polar auxin transport pathway was confined to immature tissues at the shoot apex. In plants in which all tissues above the fed leaf were removed before labelling, a limited transfer of IAA occurred in more mature regions of the stem.Abbreviations IAA indol-3yl-acetic acid - EDTA ethylenediaminetetraacetic acid - NPA N-1-naphthylphthalamic acid We are grateful to the Nuffield Foundation for supporting this research under the NUF-URB95 scheme and for the provision of a bursary to A.J.C. We thank Professor Dennis A. Baker for constructive comments on a draft of this paper and Mrs. Rosemary Bell for her able technical assistance.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Effect of Cyclanilide on Auxin Activity

Cyclanilide is a plant growth regulator that is registered for use in cotton at different stages of growth, to either suppress vegetative growth (in combination with mepiquat chloride) or accelerate senescence (enhance defoliation and boll opening, used in combination with ethephon). This research was conducted to study the mechanism of action of cyclanilide: its potential interaction with auxin (IAA) transport and signaling in plants. The activity of cyclanilide was compared with the activity of the auxin transport inhibitors NPA and TIBA. Movement of [³H]IAA was inhibited in etiolated corn coleoptiles by 10 μM cyclanilide, NPA, and TIBA, which demonstrated that cyclanilide affected polar auxin transport. Although NPA inhibited [³H]IAA efflux from cells in etiolated zucchini hypocotyls, cyclanilide had no effect. NPA did not inhibit the influx of IAA into cells in etiolated zucchini hypocotyls, whereas cyclanilide inhibited uptake 25 and 31% at 10 and 100 μM, respectively. Also, NPA inhibited the gravitropic response in tomato roots (85% at 1 μM) more than cyclanilide (30% at 1 μM). Although NPA inhibited tomato root growth (30% at 1 μM), cyclanilide stimulated root growth (165% of control at 5 μM). To further characterize cyclanilide action, plasma membrane fractions from etiolated zucchini hypocotyls were obtained and the binding of NPA, IAA, and cyclanilide studied. Cyclanilide inhibited the binding of [³H]NPA and [³H]IAA with an IC₅₀ of 50 μM for both. NPA did not affect the binding of IAA, nor did IAA affect the binding of NPA. Kinetic analysis indicated that cyclanilide is a noncompetitive inhibitor of both NPA and IAA binding, with inhibition constants (K _i) of 40 and 2.3 μM, respectively. These data demonstrated that cyclanilide interacts with auxin-regulated processes via a mechanism that is distinct from other auxin transport inhibitors. This research identifies a possible mechanism of action for cyclanilide when used as a plant growth regulator.