Effects of temperature and sink activity on the transport of 14C-labelled indol-3yl-acetic acid in the intact pea plant (Pisum sativum L.)

The velocity and intensity of basipetal transport of ¹⁴C-labelled indol-3yl-acetic acid (IAA) applied to the apical bud of the intact pea plant were influenced by the temperature to which the stem was exposed and were not influenced by changes in the temperature of the root system when this was controlled independently between 5°C and 35°C. The velocity of transport increased steadily with temperature to a maximum in excess of 35°C and then fell sharply with further increase in temperature. The Q₁₀ for velocity, determined from Arrhenius plots, was low (ca. 1.3). Transport intensity increased to a maximum at about 25°C (Q₁₀=2.2) and then declined gradually with further increase in temperature. It is suggested that transport velocity and transport intensity are controlled independently.The characteristics of auxin transport through the stem were not affected by removal of the root system, or by the withdrawl of root aeration. Labelled IAA did not pass a region of the stem cooled to about 1.0°C, or through a narrow zone of stem tissue killed by heat treatment. In the latter case the heat treatment was shown not to interfere with the upward transport of water in the xylem. Labelled IAA continued to move into, and to accumulate in, the tissues immediately above a cooled or heat-killed region of the stem. It was concluded that the long-distance basipetal transport of auxin through the stem of the intact plant is driven by the transporting cells themselves and is independent of the activity of sinks for the transported auxin.The fronts of the observed tracer profiles in the stem were closely fitted by error function diffusion analogue curves. However, diffusion of IAA alone could not account for the observed characteristics of the transport and it is suggested that the curvilinear fronts of the profiles resulted from a diffusive mixing of exogenous IAA (or IAA-carrier complexes) with endogenous IAA already in the transport pathway.Abbreviations IAA indol-3yl-acetic acid - IAAsp indol-3yl-acetyl aspartic acid - CFM methyl 2-chloro-9-hydroxyfluorene-9-carboxylate (morphactin) - TIBA 2,3,5-triiodobenzoic acid - ABA abscisic acid     

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

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

The Effect of IAA on Sugar Accumulation and Basipetal Transport of 14C-labelled Assimilates in Relation to Root Formation in Phaseolus vulgaris Cuttings

The effect of indol-3yl-acetic acid on root formation, accumulation of 80% ethanol-soluble sugars and basipetal transport of ¹⁴C-labelled assimilates has been investigated in Phaseolus vulgaris (cv. Canadian Wonder) hypocotyl cuttings. The removal of leaves reduced root formation in the hypocotyl, while excision of the apical bud was less detrimental. The expression of the IAA effect in inducing more roots was dependent on the area of leaves, and was found to be better when all leaves were present. Sugars accumulated slowly at the base of cuttings during a four-day period after excision, and IAA greatly enhanced this accumulation. By comparing sugar content at the base of green and starved cuttings it was established that IAA greatly increased it concurrently with root formation. IAA applied in solution to the hypocotyl greatly enhanced the basipetal transport of ¹⁴C-labelled assimilates and their accumulation at the hypocotyl during a 24-h period. The IAA-induced accumulation was found to be connected with a greater mobilization of labelled assimilates from upper parts of the cutting. Experiments involving pretreatment with IAA and transport in cuttings already possessing root primordia, suggest a dual effect of IAA: (I) a direct effect on transport, and (2) an increase in the root-“sink”. It is concluded that both may be operating in inducing basipetal accumulation of labelled assimilates. It is suggested that one of the roles of IAA in promoting rooting of cuttings is to increase sugar availability at the site of root formation.     

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.     

The specificity of auxin transport in intact pea seedlings (Pisum sativum L.)

Summary When eight ¹⁴C-labelled auxin and non-auxin compounds were applied to the apical buds of intact dwarf pea seedlings (Pisum sativum L.), only [1-¹⁴C]indoleacetic acid ([¹⁴C]IAA) and -[1-¹⁴C] naphthaleneacetic acid ([¹⁴C]NAA) underwent appreciable basipetal transport during the first 24 h; over a longer period (72 h) considerable basipetal transport of the auxin [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([¹⁴C]2,4-D) also occurred, but at a very much lower velocity (ca. 1.4–2.2 mm·h^-1). The movement of 2,4-D possessed many of the characteristics of a typical auxin transport. During uptake and transport IAA and NAA were extensively metabolised to the corresponding aspartates, and to ethanol-insoluble/NaOH-soluble compounds; little metabolism of 2,4-D was observed. None of the non-auxin compounds applied (sorbose, sucrose, leucine, adenine and kinetin) underwent appreciable basipetal transport from the apical bud. All but sorbose were extensively metabolised by the apical tissues. Little metabolism of sorbose itself was detected.The results suggest that the long-distance basipetal auxin transport system from the apical bud of intact plants is specific for auxins; the specificity may result from the affinity of auxins for specific transport sites.     

Hormones andCuscuta development: IAA uptake transport and metabolism in relation to growth in the absence and presence of applied cytokinin

Transport of 1-¹⁴C-IAA in successive stem segments ofCuscuta was strictly basipetal in growing and non growing regions of the vine with a flux velocity of 10–12 mm/h (intercept method). This transport showed a distinct peaked profile, increasing from a low value at 10 mm from the apex to a maximum between 50 and 90 mm before declining to a low value again around 160 mm at which elongation growth ceased. The IAA transport profile paralleled the in vivo growth rate profile, though the latter peaked ahead of transport. A better correlation was observed between the profile of growth responsiveness of the vine to exogenous IAA application and the profile of IAA transport. Growth responsiveness was determined as the differential in growth rate of stem segments in vitro in the absence and presence of growth optimal concentration of IAA (10 m). Retention of exogenous IAA in the stem was maximal where transport decreased, and this coincided with the region of maximal conjugation of applied 1-¹⁴C-IAA to aspartic acid to form indoleacetylaspartate (IAAsp). In addition to aspartate, IAA was conjugated to a small extent to an unidentified compound. IAA destruction by decarboxylation was greatest where transport was low, particularly in the nongrowing region, where lignification occurred (i.e., beyond 180 mm). At concentrations up to 20 M, a pulse of 1-¹⁴C-IAA chased by cold IAA moved as a peak (with a peak displacement velocity of 12–18 mm/h) in the growth region of the vine, but became diffusionlike where growth either fell off steeply or ceased. At a higher (50 M) IAA concentration, though uptake was not saturated, transport in the growth region became diffusionlike, indicating saturation of the system. Reduced IAA flux in the region where growth responsiveness to IAA declined coincided with the region of increased IAA conjugation. However, it cannot be concluded whether increased IAA conjugation was the cause or effect of decreased IAA flux. Application of benzyladenine to the vines in vivo, a treatment that elicited haustoria formation by 72 h, resulted in the inhibition of both IAA transport and elongation growth rate in the subapical region. In vitro treatment of vine segments with BA similarly increased IAA retention and decreased IAA transport. IAA loss was suppressed, and conjugation to IAAsp was enhanced.     

Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in pea cuttings

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea ( Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.     

The effect of cycloheximide on IAA-stimulated transport of 14C-ABA and 14C-sucrose in long pea epicotyl segments

The effect of cycloheximide (CH) on the indol-3yl-acetic acid (IAA)-stimulated transport of ¹⁴C-labelled abscisic acid (ABA) and ¹⁴C-labelled sucrose was studied in 110 mm long pea epicotyl segments. IAA application resulted in elongation growth of the segments. This effect was decreased by CH treatment which also reduced [¹⁴C] ABA and [¹⁴C] sucrose accumulation in the growing apical part of the segments. A reduction in [¹⁴C] IAA uptake and in protein synthesis in this part of the segments was also observed. The simultaneous inhibition of protein synthesis and reduction of [¹⁴C] ABA and [¹⁴C] sucrose transport suggests that IAA can stimulate the transport of ABA and sucrose through a protein synthesis-based elongation growth.     

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     

Auxin transport and the regulation of petiole abscission in cotton (Gossypium hirsutum L.): A comparison of indol-3yl-acetic acid and phenylacetic acid

Distal applications of indol-3yl-acetic acid (IAA) to debladed cotyledonary petioles of cotton (Gossypium hirsutum L.) seedlings greatly delayed petiole abscission, but similar applications of phenylacetic acid (PAA) slightly accelerated abscission compared with untreated controls. Both compounds prevented abscission for at least 91 h when applied directly to the abscission zone at the base of the petiole. The contrasting effects of distal IAA and PAA on abscission were correlated with their polar transport behaviour-[1-¹⁴C]IAA underwent typical polar (basipetal) transport through isolated 30 mm petiole segments, but only a weak diffusive movement of [1-¹⁴C]PAA occurred.Removal of the shoot tip substantially delayed abscission of subtending debladed cotyledonary petioles. The promotive effect of the shoot tip on petiole abscission could be replaced in decapitated shoots by applications of either IAA or PAA to the cut surface of the stem. Following the application of [1-¹⁴C]IAA or [1-¹⁴C]PAA to the cut surface of decapitated shoots, only IAA was transported basipetally through the stem. Proximal applications of either compound stimulated the acropetal transport of [¹⁴C]sucrose applied to a subtending intact cotyledonary leaf and caused label to accumulate at the shoot tip. However, PAA was considerably less active than IAA in this response.It is concluded that whilst the inhibition of petiole abscission by distal auxin is mediated by effects of auxin in cells of the abscission zone itself, the promotion of abscission by the shoot tip (or by proximal exogenous auxin) is a remote effect which does not require basipetal auxin transport to the abscission zone. Possible mechanisms to explain this indirect effect of proximal auxin on abscission are discussed.     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

Auxin transport in intact pea seedlings (Pisum sativum L.): The inhibition of transport by 2,3,5-triiodobenzoic acid

Summary The application of 2,3,5-triiodobenzoic acid (TIBA, 10 mg·g^-1 in lanolin) to the stem of intact pea seedlings (Pisum sativum L.) inhibited the basipetal transport of ¹⁴C from indoleacetic acid-1-¹⁴C (IAA-1-¹⁴C) applied to the apical bud, but not the transport of ¹⁴C in the phloem following the application of IAA-1-¹⁴C or sucrose-¹⁴C to mature foliage leaves. It was concluded that fundamentally different mechanisms of auxin transport operate in these two pathways.When TIBA was applied at the same time as, or 3.0 h after, the application of IAA-1-¹⁴C to the apical bud, ¹⁴C accumulated in the TIBA-treated and higher internodes; when TIBA was applied 24.0 h before the IAA-1-¹⁴C, transport in the stem above the TIBA-treated internode was considerably reduced. TIBA treatments did not consistently influence the total recovery of ¹⁴C, or the conversion of free IAA to indoleaspartic acid (IAAsp). These results are discussed in relation to the possible mechanism by which TIBA inhibits auxin transport,.Attention is drawn to the need for more detailed studies of the role of the phloem in the transport of endogenous auxin in the intact plant.Abbreviations TIBA 2,3,5-triiodobenzoic acid - IAAsp indoleaspartic 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     

Cell length,light and14C-labelled indol-3yl-acetic acid transport inPisum satisum L. andPhaseolus vulgaris L

The putative auxin-transporting cells of the intact herbaceous dicotyledon are the young, differentiating vascular elements. The length of these cells was found to be considerably greater in dwarf (Meteor) than in tall (Alderman) varieties ofPisum sativum L., and to be greater in etiolated than in light-grown plants ofP. sativum cv Meteor andPhaseolus vulgaris L. cv Mexican Black. Under given light conditions during transport these large differences in cell length did not influence the shapes of the transport profiles or the velocity of transport of¹⁴C-labelled indol-3yl-acetic acid (IAA) applied to the apical bud. However, in both etiolated and light-grown bean and dwarf pea plants the velocity of transport in darkness was ca. 25% lower than that in light. Under the same conditions of transport velocities in bean were about twice those observed in the dwarf pea. Exposure to light during transport increased the rate of export of¹⁴C from the labelled shoot apex in green dwarf pea plants but not in etiolated plants. The light conditions to which the plants were exposed during growth and transport had little effect on the rates of uptake of IAA from the applied solutions. The results indicate that the velocity of auxin transport is independent of the frequency of cell-to-cell interfaces along the transport pathway and it is suggested that in intact plants auxin transport is entirely symplastic.     

Occurrence of Endogenous Indol-3yI-Aspartic Acid in Light and Dark-Grown Bean Seedlings (Phaseolus vulgaris)

An acid ether-soluble, strongly growth-stimulating substance revealed by the Avena coleoptile straight-growth test in methanol extracts from bean seedlings (Phaseolus vulgaris L.) was identified as indol-3yl-aspartic acid (IAAsp). Points of agreement between synthetic IAAsp and the investigated growth stimulator were indicated by chromatographic behavior, elution volume in gel filtration, mobility in paper electrophoresis, “colour reaction” with DMCA reagent, ability to form indol-3yl-acetic acid (IAA) and aspartic acid after hydrolysis and, finally, biological activity in the Avena test. Furthermore, some experiments demonstrated the occurrence of an inhibitor in extracts from light-grown tissue. This masked the stimulation of IAAsp in the Avena test when the extracts had been chromatographed in isopropanol: NH₃:H₂ O. A comparison of the levels of IAAsp between green and etiolated tissue did not reveal any distinct difference, demonstrating that the IAA conjugate IAAsp does not participate in the regulation of the photomorphogenesis.     

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     

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.     

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.     

Effects of osmotic stress on polar auxin transport in Avena mesocotyl sections

Segments of mesocotyls of Avena sativa L. transported [1-¹⁴C]indol-3yl-acetic acid (IAA) with strictly basipetal polarity. Treatment of the segments with solutions of sorbitol caused a striking increase in basipetal auxin transport, which was greatest at concentrations around 0.5 M. Similar effects were observed with mannitol or quebrachitol as osmotica, but with glucose or sucrose the increases were smaller. Polar transport was still detectable in segments treated with 1.2 M sorbitol. The effects of osmotic stress on the polar transport of auxin were reversible, but treatment with sorbital solutions more concentrated than 0.5 M reduced the subsequent ability of mesocotyl segments to grow in response to IAA. The increased transport of auxin in the osmotically stressed segments could not be explained in terms of an increased uptake from donor blocks. The velocity of transport declined with higher concentrations of osmoticum. The reasons for the enhancement of auxin transport by osmotic stress are not known.     

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.