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.     

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     

A comparison between 3,5-diiodo-4-hydroxybenzoic acid and 2,3,5-triiodobenzoic acid. II. Effects on uptake and efflux of IAA in maize roots

An explanation is sought for the inhibition of maize root growth and gravireaction brought about by treatment with 3,5-diiodo-4-hydroxybenzoic acid (DIHB). The effects of DIHB and 2,3,5-triiodobenzoic acid (TIBA) on the uptake and efflux of [³H]-indol-3yl-acetic acid (IAA) were tested using segments prepared from the elongation zone (2 to 7 mm region) of maize (Zea mays L. cv. LG11) roots. The uptake of [³H]-IAA (21 nM) by root segments incubated in buffered solutions (pH 5.0) was measured over a 5-min time-course. No significant effect of DIHB at 100 μM was observed, whereas TIBA at 10 μM slightly stimulated the uptake of [³H]-IAA. This experiment was repeated with the addition of non-radioactive IAA (total IAA concentration 1.0 μM). Up to 3 min DIHB (100 μM) had no significant effect, but thereafter a slight stimulation of IAA net uptake was observed. Treatment with TIBA (10 μM) stimulated the accumulation of IAA in the segments. The effects of DIHB (10, 50, 100 μM) and TIBA (10 and 50 μM) on the efflux of [³H]-IAA from segments that had been pretreated in [³H]-IAA (22 nM) were then tested. Treatment with DIHB or TIBA at pH 5.0 inhibited IAA efflux; the inhibition by TIBA was more marked than that produced by DIHB. This experiment was repeated using DIHB (10, 50, 100 μM) buffered at pH 6.0, and an inhibition of IAA efflux was again observed. Both DIHB (10 μM) and TIBA (10 μM) inhibited the binding of [³H]-NPA to a 5000–48000 g membrane fraction prepared from whole maize roots. The effects of the two substances were similar: 40% inhibition of specific binding by DIHB and 41% inhibition by TIBA. This indicates that DIHB, like TIBA, binds to the N-1-naphthyl-phthalamic acid-sensitive carrier for IAA efflux. It is concluded that DIHB, like TIBA, inhibits IAA transport at the level of efflux. The similarity between DIHB and TIBA as regards chemical structure and their inhibitory effects on IAA efflux and NPA binding strongly suggest that they act on the same carrier for IAA efflux across the plasmalemma.     

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.     

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     

Characterization of the growth and auxin physiology of roots of the tomato mutant,diageotropica

Roots of the tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([³H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [³H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.Abbreviations BCA bicinchoninic acid - IAA indole 3-acetic acid - dgt diageotropica - IC₅₀ concentration for 50% inhibition of growth - NPA N-1-naphthylphthalamic acid - SCB-1 semicarbazone 1 This research was supported by grants from Sandoz Agro, Inc. (G.K.M), the National Aeronautics and Space Administration (NASA) and the National Science Foundation (T.L.L), and NASA (D.L.R.).     

Induction of 33-kD and 60-kD Peroxidases during Ethylene-Induced Senescence of Cucumber Cotyledons

Ethylene enhanced the senescence of cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. The effect of 10 microliters per liter ethylene was inhibited by 1 millimolar silver thiosulfate, an inhibitor of ethylene action. An increase in proteins with molecular weights of 33 to 30 kilodaltons and lower molecular weights (25, 23, 20, 16, 12, and 10 kilodaltons) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after ethylene enhanced senescence. The measurement of DNase and RNase activity in gels indicated that these new proteins were not nucleases. Two proteins from ethylene-treated cotyledons were purified on the basis of their association with a red chromaphore and subsequently were identified as peroxidases. The molecular weights and isoelectric points (pI) of two of these peroxidases were 33 kilodaltons (cationic, pI = 8.9) and 60 kilodaltons (anionic, pI = 4.0). The observation that [³⁵S]Na₂SO₄ was incorporated into these proteins during ethylene-enhanced senescence suggests that these peroxidases represent newly synthesized proteins. Antibodies to the 33-kilodalton peroxidase precipitated two in vitro translation products from RNA isolated from ethylene-treated but not from control cucumber seedlings. This indicates that the increase in 33-kilodalton peroxidase activity represents de novo protein synthesis. Both forms of peroxidase degraded chlorophyll in vitro, which is consistent with the hypothesis that peroxidases have catabolic or scavenging functions in senescent tissues.     

Effect of pH on IAA Uptake by Maize Root Segments

The uptake of [5-³H]indoleacetic acid (IAA) by Zea mays L. root segments involves nonsaturable and saturable processes. The pH optimum of the saturable component was found to be 5.0. The proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone inhibited at 100 micromolar the saturable component of IAA uptake but had no effect on non-saturable uptake. This indicates that the saturable component of IAA uptake is dependent on the proton gradient across the plasmalemma. The high level of proton extrusion in the elongation zone of the root will stimulate nonsaturable and saturable uptake of IAA in that zone.     

Auxin and Ethylene-stimulated Adventitious Rooting in Relation to Tissue 9

We have previously shown that both endogenous auxin and ethylenepromote adventitious root formation in the hypocotyls of derootedsunflower (Helianthus annuus) seedlings. Experiments here showedthat promotive effects on rooting of the ethylene precursor,1-aminocyclopropane-l-carboxylic acid (ACC) and the ethylene-releasingcompound, ethephon (2-chloro-ethylphosphonic acid), dependedon the existence of cotyledons and apical bud (major sourcesof auxin) or the presence of exogenously applied indole-3-aceticacid (IAA). Ethephon, ACC, aminoethoxyvinylglycine (an inhibitorof ethylene biosynthesis), and silver thiosulphate (STS, aninhibitor of ethylene action), applied for a length of timethat significantly influenced adventitious rooting, showed noinhibitory effect on the basipetal transport of [³H]IAA. Theseregulators also had no effect on the metabolism of [³H]IAA andendogenous IAA levels measured by gas chromatography-mass spectrometry.ACC enhanced the rooting response of hypocotyls to exogenousIAA and decreased the inhibition of rooting by IAA transportinhibitor, N-1-naphthylphthalamic acid (NPA). STS reduced therooting response of hypocotyls to exogenous IAA and increasedthe inhibition of rooting by NPA. Exogenous auxins promotedethylene production in the rooting zone of the hypocotyls. Decapitationof the cuttings or application of NPA to the hypocotyl belowthe cotyledons did not alter ethylene production in the rootingzone, but greatly reduced the number of root primordia. We concludethat auxin is a primary controller of adventitious root formationin sunflower hypocotyls, while the effect of ethylene is mediatedby auxin. Key words: Auxin, ethylene, adventitious rooting, sunflower     

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     

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     

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     

Effect of ethylene on [C]indole-3-acetic Acid metabolism in leaf tissues of woody plants

The effect of ethylene on [¹⁴C]indole-3-acetic acid (IAA) metabolism was investigated in defoliation sensitive leaf tissues of citrus (Citrus sinensis) and resistant leaf tissues of eucalyptus (Eucalyptus camaldulensis). IAA metabolites were fractionated into 80% ethanol-soluble, H₂O-soluble, NaOH-soluble, and insoluble components. In citrus, pretreatment with 25 microliters per liter ethylene for 24 hours significantly increased the amount of ethanol- and H₂O-extractable conjugates during the first hour of incubation in [¹⁴C]IAA and increased 3- to 4-fold the formation of NaOH-extractable conjugates during the entire 6-hour incubation period. However, induction of the IAA-aspartate conjugation system was inhibited by ethylene. In eucalyptus, ethylene pretreatment only slightly stimulated the formation of IAA metabolites. Increased formation of ethanol-extractable conjugates in ethylene-pretreated eucalyptus tissues was observed only after 6 hours of incubation. Chromatographic analysis indicated that the ethanol and H₂O extracts of both species contained various low molecular weight conjugates, whereas in citrus leaf tissues high molecular weight conjugates accounted for most of the greater radioactivity detected in the NaOH extracts as a result of ethylene-pretreatment. It is suggested that ethylene may reduce the level of endogenous IAA in citrus leaf tissues by stimulating IAA conjugation.     

Ontogenetic Changes in the Transport of Indol-3yl-acetic Acid into Maize Roots from the Shoot and Caryopsis

The quantities of endogenous indol-3yl-acetic acid (IAA) in endosperms and scutella of 6-day-old maize seedlings (Zea mays L. cv Giant White Horsetooth) were determined by a fluorimetric method. Endosperms were found to contain 33.4 nanograms IAA per plant, and scutella 7.5 nanograms IAA per plant. [5-³H]IAA applied to endosperms of 6-day-old seedlings moved into the roots and radioactivity accumulated at the apex of the primary root within 8 hours. Two to 7-day-old seedlings were treated simultaneously with [5-³H]IAA in the endosperm and [2-¹⁴C] IAA on the shoot apex. The patterns of transport into the root were found to change during ontogeny: in successively older plants, transport from the shoot into the roots increased relative to transport from the endosperm into the roots. The auxin required for the growth of maize roots could, therefore, partially be contributed by the shoot and endosperm. Ontogenetic changes in the relative importance of these two supplies could be of significance for the integration of growth and development between shoot and root.     

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     

5'-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1-Naphthylphthalamic Acid : Synthesis and Binding Properties in Curcurbita pepo L

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5′-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [³H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K_0.5 = 2.8 × 10⁻⁷ molar. The K_0.5 for NPA under these conditions is 2 × 10⁻⁸ molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [³H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [³H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.     

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.     

活性氧对外源IAA诱导的ACC合酶活性的影响

本文试图从活性氧的角度阐明外源IAA诱导ACC合酶活性的机制.绿豆(Phaseolus radiatus L.)幼苗的乙烯产生及ACC合酶活性从萌发的第5天开始上升,到第10天达到高峰,接着下降.10 μmol/L的外源IAA能明显促进绿豆幼苗乙烯的产生及ACC合酶的活性,同时也促进了超氧阴离子自由基(O(-)/(*)2)、过氧化氢(H2O2)的产生.显示外源IAA诱导的ACC合酶的活性与其诱导的活性氧的产生具有某种相关性.外源O(-)/(*)2处理能明显提高绿豆幼苗的乙烯产生速率及ACC合酶的活性,而外源H2O2无论对乙烯产生或ACC合酶的活性均没有明显的作用.外加O(-)/(*)2的清除剂SOD对绿豆幼苗乙烯的产生及ACC合酶活性的提高有一定的抑制作用,而外源过氧化氢酶却没有明显的作用.为此我们可以得出结论:外源IAA诱导的绿豆幼苗ACC合酶活性的提高可能是由于其诱导的O(-)/(*)2产生的升高引起的,这可能也是高等植物中调控乙烯生物合成的机制之一;而IAA诱导的H2O2产率的升高并不是其诱导ACC合酶活性升高的原因.     

Disruption of the Polar Auxin Transport System in Cotton Seedlings following Treatment with the Defoliant Thidiazuron

The effect of the defoliant thidiazuron (TDZ) on basipetal auxin transport in petiole segments isolated from cotton (Gossypium hirsutum L. cv LG102) seedlings was examined using the donor/receiver agar block technique. Treatment of intact seedlings with TDZ at concentrations of 1 micromolar or greater resulted in a dose-dependent inhibition of ¹⁴C-IAA transport in petiole segments isolated 1 or 2 days after treatment. Using 100 micromolar TDZ, the inhibition was detectable 19 hours after treatment and was complete by 27 hours. Both leaves and petiole segments exhibited a marked increase in ethylene production following treatment with TDZ at concentrations of 0.1 micromolar or greater. The involvement of ethylene in this TDZ response was evaluated by examining the effects of two inhibitors of ethylene action: silver thiosulfate, 2,5-norbornadiene. One day after treatment, both inhibitors effectively antagonized the TDZ-induced inhibition of auxin transport. Two days after TDZ treatment both inhibitors were ineffective. The decrease in IAA transport in TDZ treated tissues was associated with increased metabolism of IAA. The transport of ¹⁴C-2,4-dichlorophenoxyacetic acid was also inhibited by TDZ treatment. This inhibition was not accompanied by increased metabolism. Incorporation of TDZ into the receiver blocks had no effect on auxin transport. The ability of the phytotropin N-1-naphthylphthalamic acid to stimulate IAA uptake from a bathing medium was reduced in TDZ-treated tissues. This reduction is thought to reflect a decline in the auxin efflux system following TDZ treatment.     

IAA transport during the phototropic responses of intact Zea and Avena coleoptiles

Summary Transport of indolyl-3-acetic acid (IAA) was studied during the phototropic responses of intact shoots and detached coleoptiles of Zea mays L. and Avena sativa L. The use of a high specific activity [5-³H]IAA and glass micropipettes enabled asymmetric application of the IAA to be made to individual coleoptiles with minimal tissue damage.A unilateral stimulus of 2.59×10^-11 einstein cm^-2 of blue light, probably in the dose range of the first positive phototropic response, caused significant net lateral movement of radioactivity from [5-³H]IAA away from the illuminated side of intact shoots and detached coleoptile apices of both Avena and Zea. The magnitude of the net lateral movement was 15.3% in Zea seedlings and 12.3% in Avena seedlings. Chromatographic analyses indicated that the movement of radioactivity reflected that of IAA. A phototropic stimulus of 1.24×10^-7 einstein cm^-2, which was probably in the second positive dose range, caused significant lateral movement of radioactivity in intact shoots and detached coleoptiles of Zea but not of Avena.In intact Zea seedlings, neither phototropic dosage affected the longitudinal transport of IAA. In intact Avena seedlings, first positive stimulation inhibited longitudinal transport only when the IAA was applied to the illuminated side of the coleoptile, but second positive stimulation inhibited basipetal movement of IAA regardless of the side of application.Exposing the intact seedlings to red light before phototropic stimulation abolished lateral transport after a first positive stimulus in Zea and in Avena.Phototropic stimulation can thus induce a lateral transport of IAA towards the shaded side of the coleoptiles of both Zea and Avena seedlings, and can affect longitudinal movement of IAA in the coleoptile of Avena. However, since phototropic curvature was observed under certain conditions in the absence of either of these effects, the extent to which they are involved in the induction of asymmetric growth in a stimulated coleoptile has yet to be resolved.