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.     

Auxin Concentrations in Nodes and Internodes ofImpatiens sultani

Greater concentrations of auxin at nodes than in internodes,resulting from some nodal barrier to basipetal transport, havelong been postulated as the cause of early differentiation ofinitially isolated xylem and cambium at the nodes. However,this study, using [¹⁴C] indole-3-acetic acid (IAA) applied apicallyand gas chromatography-mass spectrometry, found that in stemsofImpatiens sultanithe IAA concentrations (per unit f. wt) atnodes were similar to those in adjacent internodes, though alittle greater at nodes if expressed per unit length of stemand a little less per unit d. wt. By contrast, in decapitatedshoots and in stem explants of dicotyledons, loss of the apicalsource of basipetally flowing auxin can result in auxin drainagewith some auxin retention in the uppermost remaining nodes.When [¹⁴C]IAA was applied apically to shoots for 4 h and stemexplants were excised, the explants had no nodal accumulationinitially whereas comparable explants incubated for 20 h revealedsignificant nodal accumulation. If decapitation leads both tonodal auxin accumulation and to adventitious abscission justabove the node, this fits the hypothesis that abscission sitesare positioned where auxin concentration decreases locally inthe apical direction. Difficulties in quantifying nodal auxindynamics are discussed, and some crude estimates of metabolicrates and locations of the auxin are presented.Copyright 1999Annals of Botany Company Abscission, auxin,Impatiens sultani, indole-3-acetic acid, node.     

Auxin-Growth Relationships in Maize Coleoptiles and Pea Internodes and Control by Auxin of the Tissue Sensitivity to Auxin

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied ³H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.     

The role of cytokinin in sieve tube regeneration and callose production in wounded coleus internodes

Cytokinin proved to be a controlling factor in sieve tube regeneration around wounded collateral bundles in an in vivo system in which the endogenous cytokinin level had been minimized. Both kinetin and zeatin were applied in aqueous solution to the bases of excised, mature internodes of Coleus blumei Benth. that had an active vascular cambium. Each internode also received indoleacetic acid (IAA) in lanolin at its apical end. Under either low (0.1% w/w) or high (1.0% w/w) auxin concentrations, the control internodes (without exogenous cytokinin) exhibited small amounts of sieve tube regeneration. At appropriate concentrations, both kinetin and zeatin induced a significant increase in sieve tube regeneration around the wound. However, the highest concentration of kinetin tested (50 μg/mL) completely inhibited this process. Kinetin was the most effective with high auxin (1.0% IAA), while zeatin was the most effective with low auxin level (0.1% IAA). Kinetin and zeatin showed the strongest promotive effect at 10 μg/mL and 20 μg/mL, respectively. Both cytokinins also induced supplementary phloem regeneration further from the wound surface. In addition to their effects on vascular tissue regeneration, both cytokinins promoted callose production. This was most evident on the sieve plates of the regenerated sieve tube members and on the walls of the parenchyma cells around the wound. The largest deposits of callose were found in both regenerated sieve tube members and parenchyma cells at the highest cytokinin concentration tested (50 μg/mL). The possible role of cytokinin in controlling callose accumulation in the sieve tubes during autumn is discussed.     

Polarity of IAA Effect on Sieve-Tube and Xylem Regeneration in Coleus and Tomato Stems

A technique is described for the processing of regenerated xylem and sieve tubes from the same wound area for microscopic and quantitative comparison.

Regeneration was examined in internodes of 2 developmental stages in Coleus: internode 2, elongating, characteristic of primary growth; and internode 5, non-elongating, characteristic of secondary growth.

Transport of indoleacetic acid (IAA) in excised number 5 internodes of Coleus is strictly polar, in a basipetal direction, judging by a regeneration bioassay involving both sieve tube strands and xylem cells. Similar results were obtained with tomato.

If isolated number 5 Coleus internodes are not treated with hormone, they regenerate no xylem cells and a small number of sieve tube strands. With increasing concentrations of IAA added apically, the number of regenerated sieve tube strands (and, with higher concentrations, of xylem cells) increases progressively up to 1% IAA, the highest concentration applied.

Internode 2 of Coleus regenerates fewer xylem cells or sieve tube strands than internode 5, whether on the otherwise intact plant or with a given concentration of IAA added apically. The amount of regenerated xylem increases with added apical IAA, except that the highest concentration gives no further increase. The number of xylem cells regenerated in intact plants occurs at the same interpolated IAA concentration as in number 5 internodes. No concentration of IAA tried provided replacement of intact number of sieve tube strands in internode 2.

IAA can exert a regenerative stimulus on both xylem and sieve tubes in the area immediately adjacent to the site of its application.

     

A role for polar auxin transport in rhizogenesis

Internodal shoot sections of the easy-to-root Forsythia×intermedia cv. Lynwood, and the difficult-to-root Syringa vulgaris cv. Madame Lemoine were used in vitro to investigate the role of polar auxin transport (PAT) in rhizogenesis. Syringa internodes required the distal application of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or naphthaleneacetic acid to induce rooting, while 2,4-dichlorophenoxyacetic acid was ineffective. In contrast, Forsythia internodes rooted equally well when IBA was applied at either end of the internode. Using [³H]IAA showed transport of exogenous auxin was basipetal, and that despite similar transport velocities, the intensity of auxin transport in Syringa was greater than in Forsythia. Basipetal transport of exogenous auxin was blocked using the PAT inhibitors 2,3,5-triiodobenzoic acid (TIBA) and naringenin (Nar); where Forsythia proved more sensitive to TIBA, but less so to Nar, in comparison with Syringa. In both species, percentage rooting and the number of roots formed were greater in 5-mm-long internodes than in shorter internodes. The results demonstrate the importance of PAT for root initiation in Syringa, whereas Forsythia tissue appears to be more sensitive to the direct application of auxin.     

Transport of exogenous auxin in two-branched dwarf pea seedlings (Pisum sativum L.)

Dwarf pea plants bearing two cotyledonary shoots were obtained by removing the epicotyl shortly after germination, and the patterns of distribution of ¹⁴C in these plants was investigated following the application of [¹⁴C]IAA to the apex of one shoot. Basipetal transport to the root system occurred, but in none of the experiments was ¹⁴C ever detected in the unlabelled shoot even after transport periods of up to 48 h. This was true both of plants with two equal growing shoots and of plants in which one shoot had become correlatively inhibited by the other, and in the latter case applied whether the dominant or subordinate shoot was labelled. In contrast, when [¹⁴C]IAA was applied to a mature foliage leaf of one shoot transfer of ¹⁴C to the other shoot took place, although the amount transported was always low. Transport of ¹⁴C from the apex of a subordinate shoot on plants bearing one growing and one inhibited shoot was severely restricted compared with the transport from the dominant shoot apex, and in some individual plants no transport at all was detected. Removal of the dominant shoot apex rapidly restored the capacity of the subordinate shoot to transport apically-applied [¹⁴C]IAA, and at the same time led to rapid cambial development and secondary vascular differentiation in the previously inhibited shoot. Applications of 1% unlabelled IAA in lanolin to the decapitated dominant shoot maintained the inhibition of cambial development in the subordinate shoot and its reduced capacity for auxin transport. These results are discussed in relation to the polarity of auxin transport in intact plants and the mechanism of correlative inhibition.Abbreviations IAA Indol-3-yl-acetic acid - TIBA 2,3,5-triiodobenzoic acid - 2,4D 2,4-dichlorophenoxyacetic acid - IAAsp Indol-3-yl-acetyl aspartic 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     

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.     

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.     

Role of Cytokinin in Vessel Regeneration in Wounded Coleus Internodes

Cytokinin was found to be a controlling or limiting factor invessel regeneration around a wound in internodes of Coleus blumeiBenth. in which the endogenous cytokinin level was minimized.The cytokinin was applied in aqueous solution to the base ofexcised, mature internodes that had an active vascular cambium.Each internode also received IAA in lanolin at its apical end.Under low (0.1 %, w/w) or high (10%, w/w) auxin concentrations,the control internodes (without exogenous cytokinin) exhibitedsmall amounts of vessel regeneration. At appropriate concentrationszeatin, kinetin and 6-benzylamino-purine (BAP) induced a significantincrease in vessel regeneration around the wound. The threecytokinins also induced novel patterns of supplementary regenerationfurther from the wound surface. Kinetin and BAP showed the strongestpromoting effect at 5 and 10 µg ml^–1, while zeatinwas most effective at 20 µg ml^–1. At a low (0.1%) auxin level zeatin was the most effective cytokinin, whereaskinetin was the most effective one at high (1 %) auxin. An inhibitoryeffect on vessel regeneration was observed at the highest kinetinconcentration tested (50 µg ml^–1). The regenerationof vessels induced by cytokinin was very polar. Many more regeneratedvessel members differentiated below the wound than above it,and the regeneration process proceeded acropetally from thebase of the internode to its upper parts. Our results implya basipetal polar increase in cambium responsiveness along thestem axis from internode 5 to 7. The possible significance ofsuch a basipetal increase in cambium sensitivity in wood formationin trees is discussed. Auxin, Coleus blumei, cytokinin, vascular differentiation, vessel regeneration, wound xylem     

POLAR TRANSPORT OF GROWTH-REGULATORS IN PITH AND VASCULAR TISSUES OF COLEUS STEMS

Movement of IAA-C¹⁴ and 2,4-D-C¹⁴ through cylinders of known size and histology was compared using liquid scintillation counting. Both auxins showed strongly polar movement, even through pith parenchyma cut from Coleus internode #5, the youngest internode to have ceased elongation. The polar movement was correlated with sizable elongation of the excised cylinders. Velocities of basipetal movement for a given auxin, as determined by the intercept method, showed small or negligible differences between pith and “corner” cylinders. (Corner cylinders comprised mostly vascular tissue, plus some cortical, pith, and epidermal cells.) For IAA, basipetal velocities ranged from 2.1 to 3.3 mm per hr; for 2,4-D, they were 0.6–0.8. For both auxins there was much more net loss into corner than into pith cylinders, a difference associated with the fact that corner cylinders showed 10 times as many cells in transection. More 2,4-D moved basipetally through corner than through pith cylinders and the reverse was true of IAA. By chromatographic evidence, all the radioactivity in the basal receiving blocks was still associated with the auxin molecules.     

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     

Control of Internode Length in Pisum sativum (Further Evidence for the Involvement of Indole-3-Acetic Acid)

The effects of altered endogenous indole-3-acetic (IAA) levels on elongation in garden pea (Pisum sativum L.) plants were investigated. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) were applied to elongating internodes of wild-type and mutant lkb plants. The lkb mutant was included because elongating lkb internodes contained 2- to 3-fold less free IAA than those of the wild type. In the wild type, TIBA reduced both the IAA level and internode elongation below the site of application. Both TIBA and HFCA strongly promoted the elongation of lkb internodes and also raised IAA levels above the application site. The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) also markedly increased internode elongation in lkb plants and virtually restored petioles and tendrils to their wild-type length. In contrast, treatment of wild-type plants with TIBA, HFCA, or 2,4-D caused little or no increase in elongation above the application site. The ethylene synthesis inhibitor aminoethoxyvinylglycine also increased stem elongation in lkb plants, and combined application of HFCA and aminoethoxy-vinylglycine restored lkb internodes to the wild-type length. It is concluded that the level of IAA in wild-type internodes is necessary for normal elongation, and that the reduced stature of lkb plants is at least partially attributable to a reduction in free IAA level in this mutant.     

Effects of Applied IAA on the Position of Abscission Sites Induced in Wounded Explants from Impatiens sultani Internodes

Previous work established that if segments of Impatiens sultaniinternodes are explanted and incubated on a suitable medium,they tend to undergo abscission by a transverse separation layerthat differentiates a short distance above the explant base.The present study has shown that the position of the abscissionsite can be modified experimentally. When an explant was splitdown to midlength and auxin (IAA) was applied to the top ofone of the two arms, abscission often occurred at or near thebase of the other arm. Again, when IAA was applied to the explantlaterally midway along its length, abscission often occurredjust above the application point. These two modifications ofabscission sites had been predicted by a hypothesis statingthat separation layers tend to be positioned where auxin concentrationdecreases in the morphologically upward direction. Studies with[¹⁴C]IAA confirmed that the separation layers above the explantbase, and in the two experimentally modified sites, did indeedarise where the concentration decreased upwards. Also, woundingaltered the position of abscission in these explants in waysthat can be interpreted in terms of the above hypothesis coupledwith the destruction of auxin that occurs at wound surfaces.In this system, auxin is acting as a morphogen: its concentrationgradients provide positional information. Impatiens sullani Hook., abscission, auxin, IAA, morphogen, positional control, separation layer, wounding     

Effects of Auxin Transport Inhibitors on Gibberellins in Pea

The effects of the auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA), 9-hydroxyfluorene-9-carboxylic acid (HFCA), and 1-N-naphthylphthalamic acid (NPA) on gibberellins (GAs) in the garden pea (Pisum sativum L.) were studied. Application of these compounds to elongating internodes of intact wild type plants reduced markedly the endogenous level of the bioactive gibberellin A₁ (GA₁) below the application site. Indole-3-acetic acid (IAA) levels were also reduced, as was internode elongation. The auxin transport inhibitors did not affect the level of endogenous GA₁ above the application site markedly, nor that of GA₁ precursors above or below it. When plants were treated with [¹³C,³H]GA₂₀, TIBA reduced dramatically the level of [¹³C,³H]GA₁ recovered below the TIBA application site. The internodes treated with auxin transport inhibitors appeared to be still in the phase where endogenous GA₁ affects elongation, as indicated by the strong response to applied GA₁ by internodes of a GA₁-deficient line at the same stage of expansion. On the basis of the present results it is suggested that caution be exercised when attributing the developmental effects of auxin transport inhibitors to changes in IAA level alone. Received April 13, 1998; accepted April 14, 1998     

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     

RECOVERY OF NATIVE AND APPLIED AUXIN FROM THE LIGHT-GROWN ‘ALASKA’ PEA SEEDLING

Scott , Tom K., and Winslow R. Briggs . (Stanford U., Stanford, Calif.) Recovery of native and applied auxin from the light-grown ‘Alaska’ pea seedling. Amer. Jour. Bot. 49(10): 1056–1063. Illus. 1962.—The physiological status of both endogenous and exogenously applied auxin was compared in the epicotyl of the 9-day-old light-grown ‘Alaska’ pea (Pisum sativum L.) by means of agar-diffusion and short-term ether extraction. A detailed analysis of endogenous auxin revealed a linear basipetal decrease in diffusible auxin within the growing region. A decrease in extractable auxin occurred only within the most mature region. The capacity for uptake of indole-3-acetic acid (IAA), applied in lanolin paste, was compared in different regions of the epicotyl. The fifth and most apical internode had the greatest capacity for uptake as measured by extraction. A reduced capacity was found in more basal internodes. The transport rate of applied IAA, under conditions of optimal uptake, was 10–12 mm/hr. An application of IAA for 24 hr resulted in a dramatic increase in auxin content throughout the length of the epicotyl compared to that found in the normal control. There was no apparent gradation in content from apex to base. An increase of diffusible auxin was also found, but only in the fourth and third internodes. That no such increase was detected in the basal 3 internodes suggested that the auxin transport system within this region had special properties related to a transition between shoot and root vascular patterns.     

Growth and anthocyanin synthesis in excised Sorghum internodes

Summary Ligh-induced anthocyanin synthesis in excised dark-grown internodes of Sorghum was depressed by the addition of auxin to the incubating medium at physiological concentrations. Both IAA and the synthetic auxin, 2,4-D, reduced anthocyanin yield. Similar results were obtained with isolated internode segments and in internodes incubated with coleoptiles (the major source of endogenous auxins) attached. Auxin increased the duration of the lag phase before anthocyanin synthesis began and reduced the rate during the subsequent linear phase. Elongation continued longer with IAA than without it and anthocyanin formation did not begin until extension growth had ceased or was slowing down in both cases; the rate of anthocyanin synthesis in the IAA solution remained depressed compared with that in buffer even after extension growth had ceased in both.At low concentrations IAA stimulated elongation growth without reducing anthocyanin yield and it is unlikely that the effect of IAA on anthocyanin synthesis results from the increased utilisation in growth of substrates needed for anthocyanin formation. The results of reciprocal transfer experiments from dark to light, and vice versa, showed that the action of IAA was associated with its presence in the incubating medium during the irradiation period. If present only in darkness, before or after transfer to light, IAA did not reduce anthocyanin formation; in the former case total yield was increased by IAA as a result of the stimulation of elongation growth, the concentration of anthocyanin remaining unchanged.GA₃ also decreased anthocyanin content; the effect was greater in sections incubated with coleoptiles attached and it is possible that GA₃ acts by increasing the concentration of endogenous auxins. However, CCC, which has been reported to decrease endogenous auxin levels, also reduced anthocyanin yield.The effect of IAA was not influenced by the presence of ascorbate in the incubating medium, nor did added ascorbate result in the formation of any acylated cyanidin derivative in internodes maintained in darkness.Possible relationships between light-induced anthocyanin formation and the photo-inhibition of elongation are discussed.     

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.