Intracellular localization of the active process in polar transport of auxin

Summary The cytoplasm of maize coleoptile cells was displaced to either the apical or basal ends of the cells by centrifuging (1750xg for 10 min) segments in which protoplasmic streaming had been stopped by pretreatment with cytochalasin B. Centrifugation toward the base of the segment promotes the subsequent basipetal transport of indole-3-acetic acid, whereas apical centrifugation dramatically inhibits this transport. Apical centrifugation neither promotes acropetal transport nor reverses the polarity of auxin transport. Experiments in which the amyloplasts were separated from the bulk of the cytoplasm indicate that the basipetal transport is independent of both the position and pressure exerted by the amyloplasts but is strongly dependent on the amount of cytoplasm at the basal end of the cells. These effects of centrifugation on auxin transport lead to the conclusion that the metabolic component of the transport is a polar secretion of auxin localized in the basal plasma membrane of each cell.     

Transport of 14C-lableled indoleacetic acid in Vicia root segments

IAA transport in Vicia root segments was investigated for comparisonwith that in intact roots. Lanolin paste (1-mm-wide ring) oragar blocks (3?3?1.5mm), both containing IAA-2-¹⁴C were appliedto the surface or a cut end of the root segments, respectively;transported ¹⁴C was collected in receiver agar blocks placedon the cut end of the segments. When lanolin paste was appliedto 5-mm segments, basipetal transport of IAA predominated overacropetal transport. When agar blocks were applied to 1- and2-mm segments, the same was true; in longer segments (3 and5 mm long), however, basipetal movement occurred predominantlyat first but was surpassed by acropetal movement after 2–3hr. Among the segments tested (regions 2–4, 4–6and 8–10 mm from the tip), the most apical one showedthe distinctest predominancy of basipetal movement. The velocitiesof the acropetal and basipetal movement of the ¹⁴C were estimatedat 3–3.8 and 8–12 mm/hr, respectively. Autoradiographicstudy and the experiment in which wire was inserted longitudinallythrough the central part of the segments showed that basipetalmovement occurred mainly through the outer part of the rootsand acropetal movement mainly through the central cylinder.The present results were compatible with those obtained previouslywith intact roots. Some properties of polar movement, such asits specificity, inhibition by TIBA, and dependency on terneprature are described. (Received March 22, 1978; )     

Auxin Transport in Roots: V. EFFECTS OF TEMPERATURE ON THE MOVEMENT OF IAA IN ZEA ROOTS

The effects of temperature on the polar movement of IAA through6-mm and 12-mm segments of Zea mays roots have been investigatedover the range from 1 to 50°C. At all temperatures an acropetal polar movement of IAA predominated,although at low temperatures and at 50°C the 6-mm segmentsshowed a transient basipetal polarity, before the persistentacropetal polarity developed. At 1°C the differences betweenacropetal and basipetal movement of IAA were less distinct thanat the other temperatures. There is, however, a marked metabolically-dependentacropetal movement of IAA through the tissues at 1°C, becausewhen the segments were deprived of oxygen the acropetal movementwas severely reduced while the basipetal movement was reducedto a smaller extent. At 1°C and at 5°C there was alwaysa persistent basipetal polarity of IAA movement through 6-mmand 12-mm segments under anaerobic conditions. The velocity of acropetal movement (mm h^–1) was the samethrough the 6-mm and the 12-mm segments and was markedly affectedby temperature. It increased from 1°C to a maximum valueof 8 mm h^–1 at 31°C and then decreased again at 40and 50°C. The velocity of basipetal movement could be assessedonly at 1 and 5°C at which temperatures it was greater thanthe velocity of acropetal movement, and virtually independentof segment length. The acropetal flux of IAA (cpm h^–1) was much less through12-mm segments than through 6-mm segments. For both lengthsof segment, however, the flux showed a complex relationshipwith ambient temperature, increasing from 1°C to a maximumat 10–15°C, declining to a minimum value at 31°Cand then rising again at 40 and 50°C. The basipetal fluxof IAA could be astimated only at 1 and 5°C at which itwas very much smaller than the acropetal flux. The amount of IAA in the receiver blocks increased linearlywith time at the lower temperatures. At temperatures withinthe range 15°C to about 31°C, however, the amount ofIAA in the receiver blocks began to decline if the transportperiods exceeded a certain length. The time at which this declinein the IAA in the receiver block began was related to the ambienttemperature. Chromatographic analysis indicated one radioactive substancein receiver blocks at the apical end of segments supplied withIAA-1-¹⁴C at the basal end after transport periods of 6 h at25°C, and 72 h at 5°C. The Rf of this substance wasclosely similar to that of the radioactive IAA supplied in thedonor blocks.     

Effects of Cations on Hormone Transport in Primary Roots of Zea mays

We examined the influence of aluminum and calcium (and certain other cations) on hormone transport in corn roots. When aluminum was applied unilaterally to the caps of 15 mm apical root sections the roots curved strongly away from the aluminum. When aluminum was applied unilaterally to the cap and ³H-indole-3-acetic acid was applied to the basal cut surface twice as much radioactivity (assumed to be IAA) accumulated on the concave side of the curved root as on the convex side. Auxin transport in the apical region of intact roots was preferentially basipetal, with a polarity (basipetal transport divided by acropetal transport) of 6.3. In decapped 5 mm apical root segments, auxin transport was acropetally polar (polarity = 0.63). Application of aluminum to the root cap strongly promoted acropetal transport of auxin reducing polarity from 6.3 to 2.1. Application of calcium to the root cap enhanced basipetal movement of auxin, increasing polarity from 6.3 to 7.6. Application of the calcium chelator, ethylene-glycol-bis-(β-aminoethylether)-N,N,N′, N′-tetraacetic acid, greatly decreased basipetal auxin movement, reducing polarity from 6.3 to 3.7. Transport of label after application of tritiated abscisic acid showed no polarity and was not affected by calcium or aluminum. The results indicate that the root cap is particularly important in maintaining basipetal polarity of auxin transport in primary roots of corn. The induction of root curvature by unilateral application of aluminum or calcium to root caps is likely to result from localized effects of these ions on auxin transport. The findings are discussed relative to the possible role of calcium redistribution in the gravitropic curvature of roots and the possibility of calmodulin involvement in the action of calcium and aluminum on auxin transport.     

Polar transport of kinetin in tissues of radish

Polar transport of kinetin-8-¹⁴C occurred in segments of petioles, hypocotyls, and roots of radish (Raphanus sativus L.). The polarity was basipetal in petioles and hypocotyls and acropetal in roots. In segments excised from seedlings with fully expanded cotyledons, indole-3-acetic acid was required for polarity to develop. In hypocotyl segments isolated at this stage, basipetal and acropetal movements were equal during the first 12 hours of auxin treatment after which time acropetal movement declined. Pretreatment with auxin eliminated this delay in the appearance of polarity. In hypocotyl segments excised from seedlings with expanding cotyledons, exogenous auxin was unnecessary for polarity. Potassium cyanide abolished polarity at both stages of growth by allowing increased acropetal movement. The rate of accumulation of kinetin in receiver blocks was greater than the in vivo increase in cytokinin content of developing radish roots.     

Relation Between Simultaneous Ca and Sr Transport Rates in Isolated Segments of Vetch, Barley, and Pine Roots

Root segments of vetch, barley, and pine were exposed to a nutrient solution containing (85)Sr and (45)Ca tracers. Translocation was measured from solutions containing stable ions at concentrations of 2.5 mm Ca, and at either 0.5 mm or 2.5 mm Sr. Polar transport was established between 12 and 18 hr in barley, and between 16 and 22 hr in vetch. Acropetal transport remained below 5% of basipetal transport of tracer during these intervals. Transport in both vetch and barley usually declined before an elapsed time of 24 hr unlike corn, which maintained its steady state beyond 24 hr. Pine was radically different in that it showed no difference between acropetal and basipetal transport rates and had very low rates. Sr transport in all plants studied to date paralleled that of Ca and the ratio Sr:Ca transported was equal to the ratio Sr:Ca in the nutrient. In vetch, stable Ca transport was reduced to one-fifth when Sr concentration was increased from 0.5 mm to 2.5 mm. Yet stable Sr transport did not change, indicating that the effect on transport was not due to competitive inhibition. A similar effect was less pronounced in barley, but could not be detected in pine. The magnitude of the transport rates varied considerably among the various species, corn having the greatest followed by barley, vetch, and pine in decreasing order. Transport did not correlate with root weight or surface area; it amounted to from 0.03 to 0.60 nanomoles per hr in these experiments as compared to 7 nanomoles per hr previously established in corn (in all cases, 55 mm segments, sectioned 10 mm from apex).     

Genetic and Chemical Reductions in Protein Phosphatase Activity Alter Auxin Transport, Gravity Response, and Lateral Root Growth

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.     

Auxin-Promoted Transport of Metabolites in Stems of Phaseolus vulgaris L.: EFFECTS REMOTE FROM THE SITE OF HORMONE APPLICATION

Phloem transport in stems of Phaseolus vulgaris was found tobe sensitive to treatment with the auxin transport inhibitor,2,3,5-triidobenzoic acid (TIBA). The response was dependenton the concentration of TIBA applied. A concentration of TIBA(0?5% in lanolin) which did not interfere with normal phloemtransport proved inhibitory to both basipetal transport of IAAand the acropetal component of IAA-promoted metabolite transport.In contrast, both acropetal IAA transport and basipetal IAA-promotedmetabolite transport were unaffected by TIBA treatment. Theinhibitory effect of TIBA on acropetal IAA-promoted transportwas overcome by providing IAA below the point of TIBA application.Both acropetal and basipetal IAA-promoted transport in stemsegments were unaccompanied by any corresponding changes inthe accumulation of [¹⁴C]sucrose by the segments.     

Basipetally polarised transport of [3H]gibberellin A1 and [14C]gibberellin A3, and acropetal polarity of [14C]indole-3-acetic acid transport,in stelar tissues of Phaseolus coccineus roots

Summary Movement of both [³H]GA₁ and [¹⁴C]GA₃ through root segments from P. coccineus seedlings was basipetally polarised. The basipetal/acropetal ratio of radioactivity from [³H]GA₁ in agar receiver blocks was 9.2 for apical, elongating segments, and 4.0 for more basal, non-elongating segments. Polarity of gibberellin transport was restricted to the stele, and absent from cortical tissues. Transport of [¹⁴C]IAA through root segments to agar receivers was preferentially acropetal, particularly so in the stele. Despite the existence of basipetal polarity of gibberellin transport in the root, [³H]GA₁ injected into cotyledons moved into and acropetally along the seedling root.     

Basipetal auxin transport is required for gravitropism in roots of Arabidopsis

Auxin transport has been reported to occur in two distinct polarities, acropetally and basipetally, in two different root tissues. The goals of this study were to determine whether both polarities of indole-3-acetic acid (IAA) transport occur in roots of Arabidopsis and to determine which polarity controls the gravity response. Global application of the auxin transport inhibitor naphthylphthalamic acid (NPA) to roots blocked the gravity response, root waving, and root elongation. Immediately after the application of NPA, the root gravity response was completely blocked, as measured by an automated video digitizer. Basipetal [(3)H]IAA transport in Arabidopsis roots was inhibited by NPA, whereas the movement of [(14)C]benzoic acid was not affected. Inhibition of basipetal IAA transport by local application of NPA blocked the gravity response. Inhibition of acropetal IAA transport by application of NPA at the root-shoot junction only partially reduced the gravity response at high NPA concentrations. Excised root tips, which do not receive auxin from the shoot, exhibited a normal response to gravity. The Arabidopsis mutant eir1, which has agravitropic roots, exhibited reduced basipetal IAA transport but wild-type levels of acropetal IAA transport. These results support the hypothesis that basipetally transported IAA controls root gravitropism in Arabidopsis.     

Mutations in Arabidopsis multidrug resistance-like ABC transporters separate the roles of acropetal and basipetal auxin transport in lateral root development

Auxin affects the shape of root systems by influencing elongation and branching. Because multidrug resistance (MDR)-like ABC transporters participate in auxin transport, they may be expected to contribute to root system development. This reverse genetic study of Arabidopsis thaliana roots shows that MDR4-mediated basipetal auxin transport did not affect root elongation or branching. However, impaired acropetal auxin transport due to mutation of the MDR1 gene caused 21% of nascent lateral roots to arrest their growth and the remainder to elongate 50% more slowly than the wild type. Reporter gene analyses indicated a severe auxin deficit in the apex of mdr1 but not mdr4 lateral roots. The mdr1 deficit was explained by 40% less acropetal auxin transport within the mdr1 lateral roots. The slow elongation of mdr1 lateral roots was rescued by auxin and phenocopied in the wild type by an inhibitor of polar auxin transport. Confocal microscopy analysis of a functional green fluorescent protein-MDR1 translational fusion showed the protein to be auxin inducible and present in the tissues responsible for acropetal transport in the primary root. The protein also accumulated in lateral root primordia and later in the tissues responsible for acropetal transport within the lateral root, fully supporting the conclusion that auxin levels established by MDR1-dependent acropetal transport control lateral root growth rate to influence root system architecture.     

Linear velocity of cyclic adenosine 3',5'-monophosphate transport in corn coleoptiles

The transport of cyclic adenosine 3′, 5′-monophosphate in corn coleoptile segments is very rapid. The linear velocity of basipetal transport is 183 millimeters per hour, while the velocity of acropetal transport is 79 millimeters per hour. Transport velocity as well as intensity thus appear to be polar in the corn coleoptile. Application of metabolic inhibitors such as cyanide, ouabain, and 2,4-dinitrophenol increase rather than decrease the velocity and intensity of transport. The mechanism of transport in light of these data is discussed.     

Auxin Transport in Roots: VI. MOVEMENT OF IAA THROUGH DIFFERENT ZONES OF ZEA ROOTS

The movement of IAA through 6-mm segments excised 1 mm, 7 mm,and 13 mm behind the apex of the primary root of Zea mays seedlingshas been investigated at temperatures between 10 and 25°C. In all segments, and at all temperatures, the movement of IAAwas polarized acropetally, more IAA being found in apical receiverblocks than in basal ones after transport periods of up to 24h. The amounts of IAA which moved acropetally through a segmentdecreased as the segment was taken at an increasing distancebehind the root apex. Similarly, at least after transport periodsof 8 h, more IAA moved basipetally through the apical segmentthan through the basal ones. At 10°C the velocity of acropetal movement was similar inall three segments, but the acropetbut the acropetal flux wasgreatest in the apical segment and smallest in the most basalone. The same situation appears to exist at the other temperatures. The flux and velocity of the acropetal movement of IAA througha 6-mm segment taken 7 mm behind the apex of the root were similarto those previously reported for the acropetal movement througha 12-mm segment excised 1 mm behind the apex. The smaller amountsof IAA which move acropetally through longer root segments aretherefore attributable to a limitation of the flux in the mostbasal regions of the segment.     

Auxin transport in roots

Summary The movement of IAA has been investigated in roots of dark-grown seedlings of Zea mays using IAA-I-¹⁴C.With 6-mm segments excised 1 mm below the apex of the root it has been shown that: (a) There is a strictly acropetal flux of IAA through the tissues, the amount of IAA found in an apical receiving block increasing almost linearly with increasing transport period up to about 6–7 hours, but thereafter declining for at least a further 18 hours. The onset of this decline appears to be dependent upon the concentration of IAA in the donor block. (b) The amount of IAA recovered in the apical receiving block increases with increasing concentration of IAA in the donor block over the range from 0.1–10 M, with transport periods of both 4 and 9 hours. (c) The radioactivity in the receiving block is confined to the IAA molecule. (d) The orientation of the segment with respect to gravity did not significantly affect the acropetal polar flux of IAA in the tissue.With non-decapitated 7-mm root apices it has been found that the presence of the apex has no effect on the strictly acropetal flux of IAA in the tissues, but that it entirely prevented the emergence of IAA into an apical receiving block.     

Relationship between polar basipetal auxin transport and acropetal Ca2+ transport into tomato fruits

The dependence of acropetal Ca²⁺ transport on polar basipetal indoleacetic acid (IAA) transport was investigated in excised tomato fruits ( Lycopersicon esculentum L. Mill.) using an in vitro fruit system. Auxin transport inhibitors like triiodobenzoic acid (TIBA), chlorofluorenolmethyl ester (CME) and naphthylphthalamic acid (NPA) were used in order to investigate the effect of restricted polar basipetal auxin transport on the acropetal transport of ⁴⁵Ca²⁺, ⁸⁶Rb⁺ and ⁹⁸Sr²⁺ into the same fruits. TIBA and CME inhibited basipetal transport of IAA. particularly in 10- to 12-day-old tomato fruits, and simultaneously restricted the acropetal transport of ⁴⁵Ca²⁺. The auxin transport inhibitors failed to significantly reduce the upward transport of ⁸⁶Rb⁺ and the transport of ⁹⁶Sr²⁺ was less inhibited than that of ⁴⁵Ca²⁺. TIBA and CME did not significantly affect the acropetal transport of labelled water into the fruit, nor the cation-exchange capacity or K⁺ and Mg²⁺ concentrations in the tomato fruit. These results support the view that a part of the Ca²⁺-specific acropetal transport into tomato fruits is associated with the polar basipetal IAA transport. This Ca²⁺ transport is independent of the transpiration stream into the fruit and the cation exchange capacity of the fruit tissue.     

Morphogenesis in Selaginella: II. Auxin Transport in the Root (Rhizophore)

The rhizophore of Selaginella willdenovii Baker develops from the ventral angle meristem. The morphological nature of this organ has been in dispute. The purpose of this investigation was to obtain physiological evidence to support the contention that the rhizophore is a root and not a shoot. This was accomplished by studying the movement of ³H-indoleacetic acid and ¹⁴C-indoleacetic acid in Selaginella rhizophores. In 6-millimeter tissue segments, twice as much radioactivity accumulated in acropetal receivers as in basipetal. During 1 hour of transport in intact roots auxin traveled twice as far in the acropetal direction as basipetal. A significant amount of radioactivity transported in the tissue was found to co-chromatograph with cold indoleacetic acid. Decarboxylation accounted for 10% loss of activity from donors. The data provide sufficient physiological evidence that this organ is morphogenetically a root.     

Basipetal and Acropetal Auxin Transport in Relation with Temperature

In stem sections of lentil seedlings, there is a typical polar movement of IAA labelled with ¹⁴C. The degree of polarity, expressed as the ratio of basipetal to acropetal transport, was (25°C) 7.6. A decrease (from 25° to 15°C) and an increase (from 25° to 30°C) of temperature cause a reduction of the IAA uptake by the sections and a decrease of both the basipetal and the acropetal translocation of IAA. Results suggest that the basipetal as well as the acropetal movement of auxin, are dependent of a metabolical component which is discussed.     

Influence of 2,3,5-Triiodobenzoic Acid and 1-N-Naphthylphthalamic Acid on Indoleacetic Acid Transport in Carnation Cuttings: Relationship with Rooting

³H-IAA transport in excised sections of carnation cuttings was studied by using two receiver systems for recovery of transported radioactivity: agar blocks (A) and wells containing a buffer solution (B). When receivers were periodically renewed, transport continued for up to 8 h and ceased before 24 h. If receivers were not renewed, IAA transport decreased drastically due to immobilization in the base of the sections. TIBA was as effective as NPA in inhibiting the basipetal transport irrespective of the application site (the basal or the apical side of sections). The polarity of IAA transport was determined by measuring the polar ratio (basipetal/acropetal) and the inhibition caused by TIBA or NPA. The polar ratio varied with receiver, whereas the inhibition by TIBA or NPA was similar. Distribution of immobilized radioactivity along the sections after a transport period of 24 h showed that the application of TIBA to the apical side or NPA to the basal side of sections, increased the radioactivity in zones further from the application site, which agrees with a basipetal and acropetal movement of TIBA and NPA, respectively. The existence of a slow acropetal movement of the inhibitor was confirmed by using ³H-NPA. From the results obtained, a methodological approach is proposed to measure the variations in polar auxin transport. This method was used to investigate whether the variations in rooting observed during the cold storage of cuttings might be related to changes in polar auxin transport. As the storage period increased, a decrease in intensity and polarity of auxin transport occurred, which was accompanied by a delay in the formation and growth of adventitious roots, confirming the involvement of polar auxin transport in supplying the auxin for rooting. Received April 19, 1999; accepted December 2, 1999     

Auxin transport in roots

Summary Light promotes the net acropetal movement of ¹⁴C through 6-mm subapical segments of dark-grown roots of Zea mays supplied at their basal ends with 1 M IAA-1-¹⁴C in agar blocks. This promotion occurs only when the segments are irradiated during the transport period, and both red and blue light appear to be as effective as white light at the radiant flux densities used in this investigation. The promotion is not found if the segments are pretreated with light and then returned to darkness before the trasport of IAA-1-¹⁴C is determined. The very slight basipetal movement of ¹⁴C through the segments supplied with an apical source of IAA-1-¹⁴C is unaffected by light.Only one radioactive substance is found in the apical receiver blocks. This substance has an R_f virtually identical to those of the stock solution of IAA incorporated into the donor block and of unlabelled IAA. The movement of radioactivity into the receiver blocks through, the illuminated segments therefore appears to reflect the movement of IAA. Light thus increases the acropetal movement of IAA through the Zea root segment.The primary roots of Zea mays var. Giant Horse Tooth seedlings grown in total darkness do not exhibit a positive geotropic response. When the seed is orientated with the embryo uppermost the radicle grows out horizontally. On exposure to light, however, the roots bend down. This reaction appears about 3–9 hours after the onset of illumination, and white, red and blue light appear to be equally effective at the flux densities employed in this study. Green light in the spectral band between 510–530 nm did not appear to induce this positive geotropic responsiveness.     

Endogenous and exogenous auxin in the control of root growth

The endogenous indol-3yl-acetic acid (IAA) of detipped apical segments from roots of maize (cv ORLA) was greatly reduced by an exodiffusion technique which depended upon the preferential acropetal transport of the phytohormone into buffered agar. When IAA was applied to the basal cut ends of freshly prepared root segments only growth inhibitions were demonstrable but after the endogenous auxin concentration had been reduced by the exodiffusion technique it became possible to stimulate growth by IAA application. The implications of the interaction between exogenous and endogenous IAA in the control of root segment growth are discussed with special reference to the role of endogenous IAA in the regulation of root growth and geotropism.Abbreviations IAA indol-3yl-acetic acid - GC-MS gas chromatography-mass spectrometry