Transport of indoleacetic acid in intact roots of Phaseolus coccineus

Summary Indoleacetic acid (IAA)-5-³H (2×10^-9) was applied to intact roots of Phaseolus coccineus seedlings at the apex or 2 cm above the apex, and the movement of IAA-³H and its metabolites traced by sectioning and chromatography. Basipetal movement of label occurred for 2 cm or less, declining exponentially, and the amount increased with time. Acropetal transport from above the apex showed quantitatively less movement of radioactivity. After a 6h treatment period a decline of label occurred in the first 0.5cm, below which there was a long distance movement of small amounts of label, mainly in IAA, towards the apex where the label concentrated by a factor of approximately 2. Short-distance basipetal movement consisted of about equal amounts of IAA and metabolites, and only metabolites were found in areas more basipetal than 2cm. Label from solutions of sucrose-¹⁴C and ³H₂O followed the same general pattern of movement as label from IAA-³H, except that acropetal movement of water showed a steady decrease in the amount of label as the distance from the area of application increased. The short distance basipetal transport of label with the breakdown of IAA-³H indicates that the extent of basipetal movement was limited by catabolic processes. The acropetal pattern of IAA-³H movement with the concentration of the transported material close to the apex, is possibly the result of transport in the phloem.     

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.     

Patterns of auxin and abscisic acid movement in the tips of gravistimulated primary roots of maize

Because both abscisic acid (ABA) and auxin (IAA) have been suggested as possible chemical mediators of differential growth during root gravitropism, we compared with redistribution of label from applied ³H-IAA and ³H-ABA during maize root gravitropism and examined the relative basipetal movement of ³H-IAA and ³H-ABA applied to the caps of vertical roots. Lateral movement of ³H-ABA across the tips of vertical roots was non-polar and about 2-fold greater than lateral movement of ³H-IAA (also non-polar). The greater movement of ABA was not due to enhanced uptake since the uptake of ³H-IAA was greater than that of ³H-ABA. Basipetal movement of label from ³H-IAA or ³H-ABA applied to the root cap was determined by measuring radioactivity in successive 1 mm sections behind the tip 90 minutes after application. ABA remained largely in the first mm (point of application) whereas IAA was concentrated in the region 2–4 mm from the tip with substantial levels found 7–8 mm from the tip. Pretreatment with inhibitors of polar auxin transport decreased both gravicurvature and the basipetal movement of IAA. When roots were placed horizontally, the movement of ³H-IAA from top to bottom across the cap was enhanced relative to movement from bottom to top whereas the pattern of movement of label from ³H-ABA was unaffected. These results are consistent with the hypothesis that IAA plays a role in root gravitropism but contrary to the idea that gravi-induced asymmetric distribution of ABA contributes to the response.     

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.     

Effect of auxin on acropetal auxin transport in roots of corn

Acropetal [¹⁴C]indoleacetic acid (IAA) transport was investigated in roots of corn. At least 40 to 50% of this movement is dependent on activities in the root apex. Selective excision of various populations of cells comprising the root apex, e.g. the root cap, quiescent center, or proximal meristem show that the proximal meristem is the critical region in the apex with regard to influencing IAA movement. The quiescent center has no influence and the root cap has only a minor effect. Excision and replacement of the proximal meristem with an exogenous supply of 10⁻⁸ to 10⁻⁹ molar IAA prevents the reduction in acropetal IAA transport which would normally occur in the absence of this meristem. Substituting 10⁻⁹ molar IAA for the excised root cap brings about a significant increase in the amount of IAA moved acropetally, as compared to intact roots with the root cap still in place. From this and previous work, it is concluded that IAA synthesis occurring in the proximal meristem stimulates the movement of IAA from the basal to apical end of the root.     

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.     

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

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.     

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.     

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.     

Effect of Inversion on Growth and Movement of Indole-3-acetic Acid in Coleoptiles

The effect of a 180° displacement from the normal vertical orientation on longitudinal growth and on the acropetal and basipetal movement of ¹⁴C-IAA was investigated in Avena sativa L. and Zea mays L. coleoptile sections. Inversion inhibits growth in intact sections (apex not removed) and in decapitated sections supplied apically with donor blocks containing auxin. Under aerobic conditions, inversion inhibits basipetal auxin movement and promotes acropetal auxin movement, whereas under anaerobic conditions, it does not influence the movement of auxin in either direction. Inversion retards the basipetal movement of the peak of a 30-minute pulse of auxin in corn.

The inversion-induced inhibition of basipetal auxin movement is not explained by an effect of gravity on production, uptake, destruction, exit from sections, retention in tissue, or purely physical movement of auxin. It is concluded that inversion (a) inhibits basipetal transport, the component of auxin movement that is metabolically dependent, and as a result (b) inhibits growth and (c) promotes acropetal auxin movement.

     

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.     

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.     

Maintenance of polar auxin transport in Zea coleoptiles by anaerobic metabolism

Summary The basipetal movement of IAA in 5-mm Zea coleoptile segments is drastically reduced under anaerobic conditions, but it remains greater than acropetal movement which is closely similar in the presence and absence of oxygen. The polarity of IAA movement has thus been confirmed in Zea coleoptile segments which have been deprived of oxygen. This net polar flux is dependent upon anaerobic metabolism since it is abolished in the presence of the metabolic inhibitiors sodium fluoride and iodoacetic acid.Acropetal movement of IAA is unaffected by the presence of sodium fluoride in air or anaerobic conditions. Uptake of IAA from a basal donor is not affected by sodium fluoride in air, but under anaerobic conditions the inhibitor decreased uptake by approximately 13%.Under anaerobic conditions both inhibitors reduce basipetal movement of IAA to the level of acropetal movement, and both decrease the total uptake of IAA from an apical donor by up to 30–45%. Under aerobic conditions sodium fluoride has no marked effect upon either the uptake of IAA from an apical donor or the basipetal movement of IAA by the segments. On the other hand, iodoacetic acid greatly decreased the uptake of IAA by the segments in air, but the same fraction of the total IAA taken up was recovered in the receiving block in the presence and absence of the inhibitor.This research was supported by Grant Number 83/6 to Professor M. B. Wilkins from the U. K. Agricultural Research Council.     

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.     

THE MOVEMENT OF IAA-C14 IN THE HYPOCOTYL OF PHASEOLUS VULGARIS

The amount of IAA-C¹⁴ transported basipetally through excised hypocotyl sections was strongly affected by the pH of the donor blocks, less so by the pH of the receivers. The effect of donor pH was mostly on uptake. The small amount of acropetal movement was not noticeably affected by pH. Sucrose added to the donor resulted in increased basipetal transport. The time-course of C¹⁴ movement into basal receivers followed a linear course from 1.5 to 3 hr as expected, but there was no net loss from the donors until after 30-45 min. The usual type of velocity calculation, which assumes uptake starting from zero time, would therefore be lower than the true value. Basipetal transport through segments cut from various positions in the hypocotyl and from seedlings of various ages was maximal in 6-8-day-old hypocotyl segments cut 25-30 mm below the cotyledons. Acropetal movement was minimal at all positions of all ages tested.     

Decarboxylation and transport of auxin in segments of sunflower and cabbage roots

Summary The movement of ¹⁴C from indole-3-acetic acid (IAA) ¹⁴C has been examined in 5 mm root segments of dark-grown seedlings of Helianthus annuus and Brassica oleracea. Contaminants from distilled water, phosphate buffer and the razor-blade cutter increase the decarboxylation of IAA-¹⁴C, and cutting of root segments results in an activation of IAA-destroying enzymes at the cut surfaces. When these sources of errors were eliminated the following was shown: a) Both in sunflower and cabbage there is a slight acropetal flux of ¹⁴C through the root segments into the agar receiver blocks. The amount of ¹⁴C found in the receiver blocks increases with the lenght of the transport period. b) When the root segments, after the transport period, are cut in two equal parts and these assayed separately, the amounts of ¹⁴C in the two parts indicate a greater acropetal than basipetal transport. c) The total radioactivity of the receiver blocks is in part due to IAA-¹⁴C and in part to ¹⁴CO₂, the latter being a result of enzymatic destruction of auxin. d) Addition of ferulic acid, an inhibitor of IAA oxidases, to the receiver blocks markedly inhibits the decarboxylation of IAA-¹⁴C and thus increases the amount transported. This effect is more pronounced after a 20 hr than after a 6 hr transport period.     

Auxin biosynthesis and metabolism in isolated roots of Zea mays

The synthesis and metabolism of indole-3-acetic acid (IAA) was investigated in isolated roots of corn, Zea mays L. Roots were cultured aseptically in media supplemented with either ¹⁴C-tryptophan or ¹⁴C-IAA. Exogenously supplied IAA is rapidly and completely metabolized by root tissues. The main site in the root for the synthesis of IAA is in the apex. Removal of either the root cap or the quiescent center, or the root cap and the quiescent center from the apex has no effect on the IAA-synthesizing ability of the apex. Subdividing the terminal 2.1 cm of the root into various segments and culturing them separately stimulates IAA synthesis in these isolated root tissues. Roots in culture maintain relatively constant IAA levels, reflecting the precise controls of the level of this hormone.     

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.     

Polar Movement of Indole-3-acetic Acid-C in Roots of Lens and Phaseolus

A critical review of the few papers on IAA-¹⁴C movement in roots revealed apparent contradictions, as well as flaws in experimental design that would be apt to cause artifacts. The movement of ¹⁴C from IAA-¹⁴C was studied in sections of Lens and Phaseolus roots, using a system 20 or more times as sensitive as any previously used. To make sure that our results with roots could be compared validly with published work on petioles and stems, we used the same techniques as we had earlier used for shoot structures. The results with Lens were similar in many ways to those for shoots: net movement into receiver blocks was very strongly polar, followed a linear course for several hours, and showed a velocity of the same order of magnitude as in shoots (and, in fact, very close in absolute value to that found in Coleus stem cylinders). Also, as with shoots, all the radioactivity in receiver blocks ran to the R_F of IAA. The time-course of loss of counts from donor blocks was similar to that found in shoots. The 2 most striking differences from shoots were 1) the very low percentage of added ¹⁴C that was moved into the receivers (about one-tenth of the values for bean petioles), and 2) the fact that the polar movement was acropetal in roots, rather than basipetal as in shoots. Results with Phaseolus roots were similar to those for Lens, although an additional complication with Phaseolus roots was the indication of a transitory stage of weak basipetal polarity in the first few hours after excising the section. This stage was followed in a few hours by a stronger acropetal polarity.