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.     

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.     

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.     

Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport in Cucurbita pepo L. Hypocotyl Segments

Pretreatment of 2?0 mm segments of etiolated zucchini (Cucurbitapepo L.) hypocotyl with cycloheximide (CH) or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide(MDMP) eliminated the stimulation by N-1-naphthylphthalamicacid (NPA) of net uptake of [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA),but had relatively little effect on the net uptake of IAA inthe absence of NPA. The efflux of [1-¹⁴C]IAA from preloadedsegments was not substantially affected by inhibitor pretreatmentin the absence of NPA, but CH pretreatment significantly inhibitedthe reduction of efflux caused by NPA. Pretreatment with CHor MDMP did not affect net uptake by segments of the pH probe[2-¹⁴C]5,5-dimethyl-oxazolidine-2,4-dione ([2-¹⁴C]DMO), or thenet uptake of [¹⁴C]-labelled 3-O-methylglucose ([¹⁴C]3-0-MeGlu),suggesting that neither inhibitor affected intracellular pHor the general function of proton symporters in the plasma membrane.Both compounds reduced the incorporation of label from [³⁵S]methionineinto trichloroacetic acid (TCA)-insoluble fractions of zucchinitissue, confirming their inhibitory effect on protein synthesis. The steady-state association of [³H]IAA with microsomal vesiclesprepared from zucchini hypocotyl tissue was enhanced by theinclusion of NPA in the uptake medium. The stimulation by NPAof [³H]IAA association with microsomes was substantially reducedwhen the tissue was pretreated with CH. However, CH pretreatmentdid not affect the level of high affinity NPA binding to themembranes indicating that treatments did not result in lossof NPA receptors. It is suggested that the auxin transport site on the effluxcarrier system and the receptor site for NPA may reside on separateproteins linked by a third, rapidly turned-over, transducingprotein. Key words: Auxin carriers, auxin efflux, Cucurbita pepo, phytotropin receptors     

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.     

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

 The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsisthaliana (L.) Heynh. seedlings. Moreover, amino acyl-β-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [³H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [¹⁴C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux. Received: 2 December 1999 / Accepted: 16 January 2000     

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     

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.     

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 membranes of lupin hypocotyls

The pH-driven accumulation of [³H]indolyl-3-acetic acid (IAA) has been found to occur in membrane vesicles of lupin (Lupinus albus L.) hypocotyls. Most of this association of auxin with membranes is very sensitive to osmotic shock, high concentrations of permeable weak acids, incubation at 20° C for 20 min and to some ionophores. Long incubation times also depress the ability to accumulate radioactive IAA but this ability can be partially restored by a treatment that presumably reconstitutes the pH gradient across the membranes. Two specific inhibitors of auxin transport, N-1-naphtylphthalamic acid and 2,3,5-triiodobenzoic acid, stimulate net IAA uptake with an optimum at about 10^-6 M (pH 5.0). At least two auxin carriers appear to be present in the lupin membrane vesicles. An uptake carrier seems to be saturated at 10^-7 M IAA in the presence of N-1-naphtylphthalamic acid, but higher IAA concentrations are needed to saturate an efflux carrier. The uptake carrier also shows a high affinity for IAA and 2,4-dichlorophenoxyacetic acid and a low affinity for 1-naphthylacetic acid.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indolyl-3-acetic acid - NAA naphthalene-1-acetic acid - NIG nigeriein - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid - VAL valinomycin     

Evidence supporting a model of voltage-dependent uptake of auxin into Cucurbita vesicles

The accumulation of [¹⁴C]indole-3-acetic acid (IAA), of [³H]tetra-phenyl phosphonium ion as a membrane potential probe, and of [¹⁴C]butyric acid as probe for pH gradients was studied with membrane vesicles from etiolated hypocotyls of Cucurbita pepo. Ion gradients (K⁺, H⁺) were applied in the presence and absence of specific ionophores e.g. valinomycin or carbonylcyanide m-chlorophenylhydrazone. In all cases tested, the accumulation of [¹⁴C]IAA equals neither potential probe nor pH-probe accumulation, but represents. an intermediate between the two. Auxin molecules seem to be taken up as positively charged ions and a pH gradient is required for accumulation. The uptake mechanism thus appears to be a specific, carrier-mediated cotransport of the anion of IAA and no less than two protons. The initial rates of auxin uptake by the saturable influx carrier, of permeation through the membrane, and of efflux by the phytotropin-affected efflux carrier were analysed.Abbreviations BA butyric acid - CCCP carbonylcyanid-3-chlorophenylhydrazone - CPD 2-carboxylphenyl-3-phenylpropan-1,3-dion - IAA indole-3-acetic acid - IAA anion of IAA - IAAH undissociated form of IAA - 2-NAA 2-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - TPP⁺ tetra-phenyl phosphonium ion     

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     

Quantitative predictions for the chemiosmotic uptake of auxin

1. The predictions of a general kinetic model for the chemiosmotic uptake of auxin and other weak acids are compared with experimental results for the auxin indoleacetic acid. The proposed mechanism involves diffusional flux of undissociated acid, a saturable, voltage-sensitive flux of anion (A^-), and a carrier-mediated symport of H⁺ and A^-, all operating in parallel. During much of uptake, the electrochemical gradients are such that the net symport and the net anion flux are in opposition: the symport contributes more to influx; the anion path, to efflux. The voltage-sensitive flux of A^- therefore constitutes a leak. 2. The presence of a symport, whose carrier can distribute across the membrane in response to the internal and external concentrations of auxin, can speed the rate of uptake, but does not by itself alter the accumulation of auxin at equilibrium. 3. The accumulation ratio at equilibrium is less at low concentrations of auxin than at higher concentrations, indicating the presence of a saturable anion path. The concentration dependence of the transition depends on several factors, and is not a reliable indicator of the A^--carrier binding constant. 4. Observed uptake near neutral pH appears larger than is consistent with a voltage-sensitive anion flux being the only carrier-mediated path across the membrane. This observation provides indirect evidence for the presence of an auxin-proton symport in addition to a saturable A^- carrier. 5. The change in kinetics of uptake of [³H]indole-3-acetic acid (IAA), observed as the total concentration of IAA is raised from 0.1 to 100 M, is consistent with either (i) a symport that saturates at low concentrations, or (ii) activation of an A^- efflux by intermediate concentrations of auxin. 6. The data on the concentration dependence of uptake of auxin are not consistent with a multi-proton symport.Abbreviations A^- auxin anion - HA weak acid, particularly IAA - HXA carrier in electroneutral complex with a proton and the auxin anion - H₂XA carrier in electroneutral complex with two protons and the auxin anion - IAA indole-3-acetic acid - X auxin carrier - XA carrier-auxin anion complex     

Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites

Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3-yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in double labelling experiments using [2,3,4,5-³H]1-N-naphthylphthalamic acid and 3-indolyl-[2-¹⁴C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (K_D at 10^-8mol·l^-1) for one site and low affinity (K_D at 10^-6 mol·l^-1) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (K_D below 10^-8 mol·l^-1) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles.Abbreviations IAA Indol-3-yl acetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - TIBA 2,3,5-triiodobenzoic acid W.M. was supported in part by an allowance from CSIRO and in part by a fellowship of the Deutsche Forschungsgemeinschaft; he acknowledges the friendly hospitality of the CSIRO Division of Plant Industry. The authors thank R. Hertel (Freiburg) for valuable discussion.     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: inhibition of polar auxin transport in intact plants and stem segments

The transport of [¹⁴C]phenylacetic acid (PAA) in intact plants and stem segments of light-grown pea (Pisum sativum L. cv. Alderman) plants was investigated and compared with the transport of [¹⁴C]indiol-3yl-acetic acid (IAA). Although PAA was readily taken up by apical tissues, unlike IAA it did not undergo long-distance transport in the stem. The absence of PAA export from the apex was shown not to be the consequence of its failure to be taken up or of its metabolism. Only a weak diffusive movement of PAA was observed in isolated stem segments which readily transported IAA. When [1-¹⁴C]PAA was applied to a mature foliage leaf in light, only 5.4% of the ¹⁴C recovered in ethanol extracts (89.6% of applied ¹⁴C) had been exported from the leaf after 6.0 h. When applied to the corresponding leaf, [¹⁴C]sucrose was readily exported (46.4% of the total recovered ethanol-soluble ¹⁴C after 6.0 h). [1-¹⁴C]phenylacetic acid applied to the root system was readily taken up but, after 5.0 h, 99.3% of the recovered ¹⁴C was still in the root system.When applied to the stem of intact plants (either in lanolin at 10 mg·g^-1, or as a 10^-4 M solution), unlabelled PAA blocked the transport through the stem of [1-¹⁴C]IAA applied to the apical bud, and caused IAA to accumulate in the PAA-treated region of the stem. Applications of PAA to the stem also inhibited the basipetal polar transport of [1-¹⁴C]IAA in isolated stem segments. These results are consistent with recent observations (C.F. Johnson and D.A. Morris, 1987, Planta 172, 400–407) that no carriers for PAA occur in the plasma membrane of the light-grown pea stem, but that PAA can inhibit the carrier-mediated efflux of IAA from cells. The possible functions of endogenous PAA are discussed and its is suggested that an important role of the compound may be to modulate the polar transport and-or accumulation by cells of IAA.Abbreviations IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - IIBA 2,3,5-triiodobenzoic 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     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

In-vitro auxin transport in membrane vesicles from maize coleoptiles

When membrane vesicles from maize (Zea mays L.) coleoptiles are extracted at high buffer strength, a pH-driven, saturable association of [¹⁴C] indole-3-acetic acid is found, similar to the in-vitro auxin-transport system previously described for Cucurbita hypocotyls. The phytotropins naphthylphthalamic acid and pyrenoylbenzoic acid increase net uptake, pressumably by inhibiting the auxin-efflux carrier.Abbreviations IAA indole-3-acetic acid - ION3 ionophore mixture of carbonylcyanide-3-chlorophenylhydrazone, nigericin and valinomycin - 1-NAA, 2-NAA 1-, 2-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid     

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.