Influence of 2,3,5-triiodobenzoic acid on the transport and metabolism of IAA in lupin hypocotyls

The influence of 2,3,5-triiodobenzoic acid (TIBA) on the transport and metabolism of indolyl-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L) hypocotyls. Double isotope-labeled IAA [(5-³H)-IAA plus (1-¹⁴C)-IAA] was applied to the cut surface of decapitated seedlings. This confirmed that the species mobilized was unaltered IAA and permitted us to measure the in vivo decarboxylation of applied IAA. A pretreatment with TIBA applied to the cut surface produced a partial or drastic inhibition in the basipetal IAA movement at 0.5 or 100 M, respectively. Since TIBA inhibits auxin polar transport by interfering with the efflux carrier, the above results suggest that 100 M TIBA is sufficient to saturate the binding sites in the transporting cells. Compared to the control plants, in vivo decarboxylation of IAA was enhanced in 0.5 M TIBA-treated plants, while no decarboxylation was detected after treatment with 100 M TIBA. The in vitro decarboxylation of (1-¹⁴C)-IAA catalyzed by purified peroxidase was moderately activated by 100 M and unaffected by 0.5 M TIBA. The paradoxical effect of TIBA in vivo vs in vitro assays suggests that the in vivo effect of TIBA on IAA oxidation might be the consequence of the action of TIBA on the auxin transport system. Thus, transport reduction by 0.5 M TIBA caused a temporary accumulation of IAA in that apical region of the hypocotyl which has the highest capacity to decarboxylate IAA. In the presence of 100 M TIBA, a concentration which presumably saturates the efflux carriers, most of the added IAA can be expected to be located in the transporting cells where, according to the present data, IAA decarboxylation cannot take place.     

The effects of 3,5-diiodo-4-hydroxybenzoic acid on the oxidation of IAA and auxin-induced ethylene production by cress root segments

Summary In previous research here, 3,5-diiodo-4-hydroxybenzoic acid (DIHB) was shown to promote the elongation of roots of cress (Lepidium sativum) seedlings growing in light, and to inhibit the auxin-induced production of ethylene in this tissue. Although DIHB is a cofactor for the oxidation of indole-3-acetic acid (IAA) by horse-radish peroxidase, it inhibits the decarboxylation of [1-¹⁴C]IAA by segments excised from cress roots. The inhibition by DIHB of ethylene production by this tissue does not, therefore, arise from a reduction of IAA levels. These findings are discussed in relation to the effects of DIHB on cress root growth.Abbreviations IAA indole-3-acetic acid - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DCP 2,4-dichlorophenol - 2,4-D 2,4-dichlorophenoxyacetic acid This study forms part of a research project to be submitted by M.L.R. for PhD degree and supported by a grant from Consejo Nacional de Ciencia y Tecnología (México).     

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.     

Identification of the metabolites of Indole-3-acetic acid in growing hypocotyls of Lupinus albus

The products of indole-3-acetic acid (IAA) metabolism by incubating hypocotyl sections and decapitated seedlings of Lupinus albus were investigated. Single treatments using [1-¹⁴C]-IAA, [2-¹⁴C]-IAA or [5-³H]-IAA and double treatments using [1-¹⁴C]-IAA+[5-³H]-IAA were carried out. Extracts from treated plant material were analyzed by paper chromatography (PC), Thin layer chromatography (TLC), and high performance liquid chromatography (HPLC). When hypocotyl sections were incubated in [2-¹⁴C]-IAA, several IAA decarboxylation products including indole-3-aldehyde (IA1), indole-3-methanol (IM), 3-hydroxymethyloxindole (HMOx), methyleneoxindole (MOx) and 3,3-bisindolylmethane (BIM) were detected in the 95% ethanol extract; a latter extraction with 1M NaOH rendered IAA, IM and BIM, suggesting that conjugated auxins were formed in addition to conjugated IM. In sections incubated with [1-¹⁴C]-IAA, the 1M NaOH extraction also produced IAA so confirming the formation of conjugated auxins. The same decarboxylation products and two conjugated auxins, indole-3-acetylaspartic acid (IAAsp) and 1-O-(indole-3-acetyl)--D-glucose (IAGlu), were detected in the acetonitrile extracts from decapitated seedlings treated with [5-³H]-IAA. After a double isotope treatment ([1-¹⁴C]-IAA+[5-³H]-IAA) of decapitated seedlings, the ratio ¹⁴C/³H measured in the HPLC fractions of the acetonitrile extracts confirmed the presence of decarboxylation products as well as conjugated auxins.     

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     

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.     

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     

Metabolism of [14C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots

Reverse-phase high-performance liquid chromatography was used to analyse ¹⁴C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [¹⁴C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [¹⁴C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [¹⁴C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid     

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.     

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     

Kainic acid,AMPA, and dihydrokainic acid effect on uptake and efflux ofd-[3H]aspartic acid in cerebellar slices

In this study we show that the glutamate ionotropic agonist kainate (KA) stimulates the efflux of preloadedd-[³H]aspartate (D-[³H]Asp) and inhibits the uptake of this amino acid in cerebellar slices. The effect of this agonist on the efflux of D-[³H]Asp is sensitive to(i) 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2-3-dione (NBQX), indicating the involvement of KA/(RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and is(ii) partially tetrodotoxin (TTX)-sensitive, indicating that pre-(TTX-insensitive) and post-synaptic (TTX-sensitive) KA/AMPA receptors are involved. In contrast, the effect on uptake is NBQX- and TTX-insensitive indicating a direct interaction with glutamate transporters. AMPA inhibited D-[³H]Asp uptake and had no effect on D-[³H]Asp efflux. In the same system, the uptake but not the efflux of D-[³H]Asp was affected by dihydrokainate (DHK). The DHK-induced uptake inhibition occurred in the presence of TTX. NBQX inhibited DHK-induced effect at 5 mM but not at 1 mM DHK concentrations.     

Uptake and Growth-promoting Activity of Indoleacetic Acid in Segments of Cold-hardened Wheat Coleoptiles

Seedings of winter wheat (Triticum aestivum L. cv. Kharkov MC 22) were grown at 24 C (unhardened) and 4 C (hardened). Indoleacetic acid (IAA) was added to excised coleoptile segments after lengthy incubation and their responses were determined by photometric auxanometry at both 25 C and 5 C. The segments' rates of uptake of ¹⁴CIAA were also compared at both temperatures. Cold hardening had no significant effect on the rates of elongation and uptake in a saturating concentration of IAA (2 to 10 μM) at either temperature. Elongation was more sensitive to temperature of measurement than was uptake. At suboptimal concentrations of IAA and 25 C, hardened coleoptiles took up [2-¹⁴C]-IAA twice as fast but elongated half as fast as unhardened coleoptiles. This and the lack of effect of cold hardening on apparent uptake of [1-¹⁴C]-IAA raised the possibility that a higher rate of IAA-decarboxylation was coupled with the higher rate of uptake of IAA by hardened coleoptiles. Homeostatic hormonal regulation was also evident in the same endogenous rates of elongation of segments of cold-hardened and unhardened coleoptiles.     

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     

A comparison between 3,5-diiodo-4-hydroxybenzoic acid and 2,3,5-triiodobenzoic acid. I. Effects on growth and gravireaction of maize roots

A comparison between the effects of DIHB and TIBA on growth and gravireaction of 15 mm primary maize ( Zea mays L. cv. LG 11) roots is presented. Intact roots were pretreated in the dark for 1 h with buffered solutions (pH 5.0 or 6.0) containing DIHB (10, 50, 100 μ M ). The plantlets were then maintained either vertically or horizontally in the dark or the light, and growth and gravireaction were recorded using a macrophotographic technique. Pretreatment with DIHB slightly inhibited growth and delayed gravireaction. These effects were most marked with DIHB at 100 μ M and were enhanced when DIHB was applied at pH 5.0. Similar effects were observed in roots pretreated with TIBA, but at a lower concentration (1 μ M ). The similarities between DIHB and TIBA as regards both chemical structure and the inhibition of gravireaction and growth, lead us to suggest that a major mode of action of DIHB, like TIBA, is the inhibition of indol-3yl-acetic acid transport.     

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     

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.     

Forskolin and 3-Isobutyl-l-Methylxanthine Increase Basal and Sodium Nitroprusside-Elevated Cyclic GMP Levels in Adult Guinea-Pig Cerebellar Slices

Abstract: In this study, the interaction between 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) in [³H]adenine-or [³H]-guanine-prelabelled adult guinea-pig cerebellar slices was investigated. Basal levels of [³H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1-100 μM). However, forskolin elicited a concentration-dependent, saturable potentiation of sodium nitroprusside (SNP)-stimulated [³H]cGMP accumulation (forskolin EC₅₀ value of 0.98 β 0.23 μM; 10 μM forskolin produced a 1.8 β 0.3-fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001-10 mM). forskolin also elicited a large stimulation of [³H]-cAMP in [³H]adenine-prelabelled guinea-pig cerebellar slices; however, 1,9-dideoxyforskolin failed to elicit either a [³H]cAMP response or a potentiation of the SNP-induced [³H]cGMP response at concentrations up to 100 μM. Pretreatment with oxyhaemoglobin (50 μM) inhibited the response to SNP (1 mM) and forskolin (10 μM), as well as the response evoked by the combination of SNP and forskolih. A^G-Nitro-l -arginine (100 μM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP-induced [³H]cGMP levels. The protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 100 μM), staurosporine (10 μM), polymyxin B (100 μM), and Ro 31-8220 (10 μM) had no effect on the [³H]cGMP response to either SNP or the combination of SNP plus forskolin. N⁶,2′-Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [³H]cGMP levels induced by SNP. 3-lso-butyl-1-methylxanthine reproduced the effect of forskolin on SNP-induced [³H]cGMP levels, but a less-than-additive effect was observed when the response to SNP was studied in the presence of forskolin and 3-isobutyl-1-methylxanthine. Taken together, these results infer that crosstalk between cyclic nucleotides takes place in guinea-pig cerebellar slices, and that cAMP may regulate cGMP-mediated responses in this tissue.     

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     

The transport of radiolabeled indoleacetic acid and its conjugates in nodal stem segments of Phaseolus vulgaris L.

The transport of radiolabeled indoleacetic acid (IAA), and some of its conjugates, was investigated in nodal stem segments of Phaseolus vulgaris L. Donor agar blocks containing either [2-acetyl-¹⁴C]-IAA; [2-acetyl-¹⁴C]-indole-3-acetyl-L-aspartate (IAAsp); [2-acetyl-¹⁴C]-indole-3-acetyl-L-glycine (IAGly); or [2-acetyl-¹⁴C]-indole-3-acetyl-L-alanine (IAAla) were placed on either the apical or basal cut surface of stem segments each bearing an axillary bud at the midline. In some experiments, a receiver block was placed on the end opposite to the donor. After transport was terminated, the segments were divided into five equal sections plus the bud, and the radioactivity of donors, receivers and each part of the stem segment was counted.For all four substances tested, the amount of ¹⁴C transported to the axillary bud from the base was the same or greater than that from the apical end. After basipetal transport, the distribution of ¹⁴C in the segment declined sharply from apex to base. The inverse was true for acropetal transport. Transport for the three IAA conjugates did not differ substantially from each other.The IAA transport inhibitor, N-1-naphthylphthalamic acid (NPA), inhibited basipetal ¹⁴C-IAA transport to the base of the stem segment but did not alter substantially the amount of ¹⁴C-IAA recovered from the bud. Transport of ¹⁴C-IAA from the apical end to all parts of the stem segment declined when the base of the section was treated with nonradioactive IAA. Taken together with data presented in the accompanying article [Tamas et al. (1989) Plant Growth Regul 8: 165–183], these results suggest that the transport of IAA plays a role in axillary bud growth regulation, but its effect does not depend on the accumulation of IAA in the axillary bud itself.     

Characteristics of adventitious root formation in cotyledon segments of mango (Mangifera indica L. cv. Zihua): two induction patterns, histological origins and the relationship with polar auxin transport

Cotyledon segments derived from zygote embryos of mango (Mangifera indica L. cv. Zihua) were cultured on agar medium for 28 days. Depending on different pre-treatments with plant growth regulators, two distinct patterns of adventitious roots were observed. A first pattern of adventitious roots was seen at the proximal cut surface, whereas no roots were formed on the opposite, distal cut surface. The rooting ability depended on the segment length and was significantly promoted by pre-treatment of embryos with indol-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for 1 h. A pre-treatment with the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) completely inhibited adventitious root formation on proximal cut surfaces. A second pattern of roots was observed on abaxial surfaces of cotyledon segments when embryos were pre-treated with 2,700 μM 1-naphthalenacetic acid (NAA) for 1 h. Histological observations indicated that both patterns of adventitious roots originated from parenchymal cells, but developmental directions of the root primordia were different. A polar auxin transport assay was used to demonstrate transport of [³H] indole-3-acetic acid (IAA) in cotyledon segments from the distal to the proximal cut surface. In conclusion, we suggest that polar auxin transport plays a role in adventitious root formation at the proximal cut surface, whereas NAA levels (influx by diffusion; carrier mediated efflux) seem to control development of adventitious roots on the abaxial surface of cotyledon segments.