Decarboxylation and Transport of Auxin in Segments of Sunflower and Cabbage Roots. II. A Chromatographic Study Using IAA-1-14C and IAA-5-3H

The polar movement of IAA has been examined in 5-mm root segments of Brassica oleracea and Helianthus annum. The movement was studied partly with IAA-1-¹⁴C and partly with IAA-5-³H. In both plants a slight acropetal flux of ¹⁴C and IAA-³H was found through the segments. The recovered radioactivity in the agar receiver blocks and in the receiver end of the segments increased as a function of time. A large portion of the applied IAA was converted on the cut surfaces and in the tissues of the segments. Chromatographic analysis indicated different destruction products when estimated by scintillation counting and by spraying with in-dole reagent (DMCA). Chromatograms run in isopropanol: ammonia: water, 8:1:1, yielded three different substances, one spot near the starting line and one near the front, neither of which has been identified. Finally there was a spot with R_f 0.4–0.6, probably representing IAA.     

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; )     

Effects of Morphactin on Indol-3yl-acetic Acid Transport, Growth, and Geotropic Response in Cereal Coleoptiles

The effects of the morphactin 2-ehloro-9-hydroxyfluorene-9-carboxylicacid methyl ester [CFM] on growth, geotropic curvature and transportand metabolism of indol-3yl-acetic acid [IAA-5-³H] in the coleoptilesof Zea mays and A vena saliva have been investigated. A strongcorrelation has been found to exist between the inhibition ofthe geotropic response and the inhibition of auxin transport.CFM supplied at concentrations sufficient to abolish auxin transporthas been shown to promote the elongation of Zea, but not ofAvena, coleoptile segments. CFM does not change the patternof metabolism of IAA in Zea coleoptile segments. In these segmentsIAA is metabolized when its concentration is high, but the radioactivitytransported basipetally, or laterally in geotropically stimulatedcoleoptiles, is virtually confined to the IAA molecule. Radioactivityexported into the basal receiver blocks is wholly confined toIAA. It is concluded that CFM inhibits the geotropic responsein coleoptiles by suppression of the longitudinal and lateralauxin transport mechanisms. The growth-promoting propertiesof this substance cannot be linked with its effects on eitherauxin metabolism or transport.     

The movement of 2,4-dichlorophenoxy acetic acid in root segments of Pisum sativum L.

Summary An acropetal polarisation of the movement of 2,4-dichlorophenoxy acetic acid (2,4-D) through subapical segments of Pisum seedling primary roots has been monitored throughout a 60 h transport period in darkness at 25° C using [1-¹⁴C]2,4-D and [2-¹⁴C]2,4-D. Uptake of 2,4-D does not proceed at a constant rate; periods in which the amount of ¹⁴C in the root segments and receiver blocks increases rapidly are followed by periods in which the amount of radioactivity remains relatively constant or declines slightly. These oscillations do not appear to be related to the time of day at which the experiments are begun or ended. Immobilisation and degradation of 2,4-D during transport in the segments seems to be low. Replacement of [1-¹⁴C]2,4-D donor blocks after 25 h by blocks containing unlabelled 2,4-D results in continued transport of the compound into receiver blocks, with only small amounts of ¹⁴C remaining in the root tissues. Radioactivity is also exported from the segments into the blocks used to replace the donor blocks, with larger amounts being exported into the blocks applied to the apical ends than into those applied to the basal ends of the segments. This radioactivity may be taken-up again by the segments but more ¹⁴C is exported into these blocks towards the end of the experiments. The possibility of regular oscillations in uptake and movement of 2,4-D in Pisum root segments is discussed.     

Auxin transport in roots

Summary The net uptake and movement of radioactivity by 12-mm root segments of Zea mays have been studied as a function of time at 5, 15 and 25° C. Segments were supplied with an agar donor block containing 1 M IAA-1-¹⁴C or IAA-2-¹⁴C continuously or for a limited period of time (pulse-labelling). In the latter case the original donor block was replaced either by a plain agar block or by one containing 1 M unlabelled IAA. Receiver blocks were placed at the other end of the segments.The net uptake of radioactivity from the donor block at 15° C was greater at the basal end than at the apical end of the segment. At 5 and 15° C, the net uptake from a basal donor was virtually linear with time but at 25° C the rate of net accumulation decreased after about 10 h. Decarboxylation of IAA undoubtedly occurred at 15 and 25° C when the concentration in the tissue attained a high value.An acropetally polarised movement of radioactivity into the receiver blocks occurred regardless of whether the results were based on the actual amounts of radioactivity in the receiver block, or on the amounts in the receiver block expressed as a percentage of the net total radioactivity accumulated from the donor block. Only one radioactive substance was present in the receiver block and it ran to the same R_f as IAA in the isopropanol: ammonium: water solvent system.The amounts of radioactivity moving into that part of the root segment at least 6 mm distant from the end in contact with either an apical or a basal donor block were assessed. An acropetal polarity in the movement of radioactivity was observed on the basis of the actual amounts of radioactivity in these distal parts of the segments, but no such polarity was evident when the amounts of radioactivity were expressed as a percentage of the net total accumulated from the donor block. At least 3 radioactive substances were present in the tissue in addition to the substance running to the same R_f as IAA. The distribution of radioactivity in the segment cannot therefore be used to assess the distribution of IAA.Acropetal movement of radioactivity into an apical receiver block is not dependent upon the continued uptake of IAA at the basal end of the segment. No distinct pulses of radioactivity were detected moving through the root segments.Only a small part of the radioactivity in the root segment appears to be located in the polar transport system, while the bulk is not. The polarity found in the movement of the bulk radioactivity within the segment seems to be related to the polarity in IAA uptake from the donor blocks.     

Metabolism of [14C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots

Reverse-phase high-performance liquid chromatography was used to analyse ¹⁴C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [¹⁴C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [¹⁴C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [¹⁴C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid     

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.     

Localization and identification of auxin in roots of Zea mays

Summary Roots of 3.5-day-old seedlings of Zea mays cv. Giant White Horsetooth contain an extractable auxin which has chromatographic properties and reactions to chromogenic sprays identical with those of indole-3-acetic acid (IAA). By separating stele from cortex (and root tips) before extraction it was shown that the auxin is localized predominantly in the stele, with little being found in the cortex. Whole roots, isolated cortices and isolated steles accumulate and metabolize exogenously applied IAA-1-¹⁴C. The stelar tissue is distinguished from whole roots and cortical tissue in having a different pattern of IAA metabolism.     

Relationship Between Metabolism and the Lateral Transport of IAA in Corn Coleoptiles

The lateral movement of IAA in coleoptiles of Zea mays has been investigated under aerobic and anaerobic conditions. The IAA-1-¹⁴C was supplied asymmetrically to the apical end of the segment. The results were as follows: A) In air more ¹⁴C was found in the lower half of horizontal segments supplied with an upper donor than in the half opposite the donor in vertical segments. The enhanced lateral movement of ¹⁴C in geotropically stimulated segments of corn coleoptiles under aerobic conditions has thus been confirmed. B) This increased lateral movement of ¹⁴C in geotropically stimulated segments is greatly reduced, but is not completely abolished, under anaerobic conditions. C) The lateral movement of ¹⁴C in vertical segments is significantly less under anaerobic conditions than in air. D) Under anaerobic conditions, the lateral movement of ¹⁴C in horizontal segments can be reduced to the level found in vertical segments by pre-soaking the tissue in a 1 mm solution of the metabolic inhibitor sodium fluoride for 2 hours. The inhibitor has no effect on lateral movement of ¹⁴C in vertical anaerobic segments. E) In air, sodium fluoride has no effect on the lateral movement of ¹⁴C in either vertical or horizontal segments.     

Auxin transport in roots

Summary The reasons underlying the initial increase and subsequent decrease in the amount of radioactivity in the receiver block at the apical end of a Zea root segment supplied with a basal donor block containing labelled IAA have been investigated.The phenomenon was observed in segments supplied with IAA-1-¹⁴C, IAA-2-¹⁴C and IAA-5-³H. An acropetal polarity in the movement of radioactivity into the receiver blocks was observed using donor blocks containing IAA-5-³H at concentrations as low as 10^-10M.The decrease in the amount of radioactivity in the receiver block begins after 6–8 h of transport at 25° C, and is unaffected by renewal of the donor block every 2 h, or the presence of 2% sucrose in the donor and receiver blocks.The net export of radioactivity into the receiver block at the apical end of the segment virtually ceases after 6–8 h of transport at 25° C, and is not prolonged by the presence of 2% sucrose in the donor and receiver blocks. At 10° C, net export of radioactivity continues for at least the first 50 h of transport, and the amount of radioactivity in a continuously applied receiver block continues to increase over this period.Receiver blocks removed from the apical end of segments after 8 h of transport and placed on planchettes show little or no decrease in the amount of radioactivity they contain as a function of time, in marked contrast to those left in contact with the segment.There is a marked, and metabolically dependent, resorption of radioactivity from the receiver block at the apical end of the segment after about 8 h of transport at 25° C; most of the resorbed radioactivity remains in the apical 2–4 mm of the segment.There is a loss of radioactive CO₂ from segments supplied with a basal donor block containing 10^-6M IAA-1-¹⁴C at 25° C, the emission beginning after 6–8 h of transport. Segments similarly supplied with 10^-6M IAA-2-¹⁴C did not begin to lose radioactive CO₂ until after about 10–12 h of transport.The ability of the segments to transport radioactivity in a polar manner declines with time after they are excised from the root, regardless of whether their cut ends are kept in the intervening period in contact with plain agar blocks, or ones containing unlabelled IAA at 10^-6M. By the 6th h after excision at 25° C no transport of radioactivity through the segments and into the receiver blocks could be detected in either the aropetal or basipetal direction.The decrease in radioactivity in the receiver block after transport periods of 6–8 h at 25° C is therefore due to (1) a cessation of net export of radioactivity into the block, and (2) the onset of a metabolically-dependent, net resorption of radioactivity. At this time substantial amounts of radioactive CO₂ begin to be evolved from segments supplied with IAA-1-¹⁴C, whereas with IAA-2-¹⁴C radioactive CO₂ is not evolved for a further 4–6 h.     

Auxin Transport in Tendril Segments of Passiflora caerulea

The movement of auxin through tendril segments of Passiflora caerulca L. has been investigated using IAA-2-¹⁴C. It has been shown that (1) flux of IAA through the segments is strongly polarized basipetally: (2) the amount of ¹⁴C recovered in the basal receiver blocks increases linearly within a transport period of 6 h; (3) velocity of basipetal transport is 14.5 mm h^?1; (4) at least 70% of the radioactivity in the receiver blocks is confined to the IAA molecule: approximately 55% of ¹⁴C from methanolic extracts of the segments is IAA: (5) at low temperatures (2–4°C) the basipetal transport is abolished; (6) white light promotes basipetal transport, and this effect is abolished in a CO₂-free atmosphere; (7) no difference could be detected in ¹⁴C content between dorsal and ventral halves of tendril segments nor among individual dorsal and ventral receiver blocks.     

The lateral transport of IAA in intact coleoptiles of Avena sativa L. and Zea mays L. during geotropic stimulation

Summary Movement of IAA was studied in excised coleoptile apices and whole seedlings of Zea mays L. and Avena sativa L. during geotropic stimulation. A micropipette technique permitted the application of [5-³H]IAA at predetermined points on the coleoptiles with minimal tissue damage.When [5-³H]IAA was applied to the upper side of a horizontal excised Zea coleoptile, about 60% of the recoverable radioactivity had moved into the lower half after 2 h. In contrast, when application was made to the lower side of a horizontal excised coleoptile, only 4% of the radioactivity migrated to the upper half. There was, thus, a net downward movement of 56%. Similar patterns of distribution were found for radioactivity in both the tissue and the basal receiver blocks. In horizontal shoot tissues of intact Zea seedlings a net downward movement of about 30% of the recoverable radioactivity occurred after 1 h of geotropic stimulation. Comparable experiments with Avena indicated a net downward movement of 6–12% in excised apices of coleoptiles and in the intact shoot. In both Zea and Avena chromatographic analyses of tissue and receiver blocks indicated that the movement of radioactivity reflected that of IAA.In Zea coleoptiles, the lateral migration of radioactivity after 2 h was 3 to 4 times greater in the apical tissues than in the basal tissues. A significant net downward movement of radioactivity was detected after 10 min of geotropic stimulation in the extreme apex of Zea coleoptiles but not in the more basal regions.These experiments show that downward lateral transport of IAA occurs in intact shoots of Zea and Avena seedlings upon geotropic stimulation. Lateral transport of IAA had previously been demonstrated only in sub-apical segments of Zea coleoptiles.     

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.     

Transport Gradient of Indoleacetic Acid in Pine Seedlings

Movement of indoleacetic acid (IAA-1-¹⁴C) through 5-min segments from stems and roots of Pinus taeda L., P. jeffreyi Gtrev. and Balf., and P. ponderosa Laws, seedlings was measured. All three species showed the same general pattern of IAA-1-¹⁴C transport. Predominantly downward movement occurred at all points along the stem and root; least IAA-1-¹⁴C transport occurred in segments obtained near the shoot apex; and most occurred in segments obtained near or just below the cotyledons. This pattern of transport gradient is different from that reported in other plants. These results could be interpreted in either of two ways: (1) that transport limitations may control movement of the growth-regulator, or (2) that the transport gradient may be an indication of endogenous growth-regulator levels.     

Abscisic Acid Action on Basipetal Auxin Transport

Several experiments have been performed to analyse the ABA effects on the basipetal transport of IAA-2-¹⁴C, using sections of epicotyls prepared from etiolated Lens seedlings. The sections were incubated in an ABA solution or ABA was applied in the donor blocks containing IAA. For each type of assay, the uptake (analyses of the donor blocks) and the movement of IAA-C¹⁴ (analyses of the receiver blocks) were inhibited by ABA. The distribution of continuous decrease of the radioactivity, along the sections' axis, showed a ¹⁴C level from the apical towards the basal segments. ABA caused a decrease in the ¹⁴C concentration for the total sections, but a relative increase for the basal segment. When ABA was applied simultaneously with IAA in the donor blocks, the transport velocity of IAA, through the sections, was not changed significantly, while an ABA pretreatment caused a significant decrease.     

Die Wirkung von Licht auf den Transport von 2-[14C]Abscisinsäure in Bohnenwurzelsegmenten

Summary Light promotes the acropetal movement of 2-[¹⁴C]ABA through root segments of Phaseolus coccineus L. The promotion occurs only when the segments are irradited during the transport period. Red light and blue light are as effective as white light. There is no significant promotion when the segments are irradiated with green light or when they are kept in darkness during the transport period after preceding light treatment. Light has no effect on basipetal ABA-transport.

---

Abkürzungen ABA Abscisinsäure - D Dunkel - GA Gibberellinsäure - IAA Indolessigsäure - L Licht - PPO Diphenyloxazol     

Effects of Light on the Georeaction and Growth Inhibitor Content of Roots

The positive geotropic response of the apical segments prepared from the primary roots of Zea mays depends upon at least one growth inhibitor, produced by the root cap, moving basipetally into the extending zone of the root in which it accumulates in the lower part. Anjou maize reacts in both darkness and light while Kelvedon maize is, for the first few hours, geotropic only in light. The production (or activity) of the growth-inhibiting substance — tested by using vertical half-decapitated root segments — is quite similar to the georeaction. This finding provides strong evidence that, in the case of Kelvedon maize roots, the inhibitory substance may depend on light. Observations related to the root segment of Anjou and Kelvedon maizes of which the tips are exchanged, are in agreement with the above results.     

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.     

Kinetics of the light-induced georeactivity of maize roots

Apical root segments of Zea mays L. cv. Orla 264 undergo some geotropic curvature in complete darkness but the curvature increases considerably if prior to geostimulation the segments are given a light pretreatment. If the light treatment is follwed by a dark treatment before the root is geostimulated the light-induced response is not changed by dark periods up to 2 h but declines with longer ones, and disappears completely after 5 h of darkness.     

Transport and metabolism of indole-3-acetic Acid in coleus petiole segments of increasing age

Transport and metabolism of IAA-1-¹⁴C in Coleus blumei Benth. was studied by means of a combination of liquid scintillation counting, autoradiography and thin-layer chromatography. Transport of IAA in petiole segments of increasing age (No. 2-8) was strictly polar in a basipetal direction. No acropetal movement occurred in either young or old tissues. The greatest amount, expressed as a percentage of the radioactivity lost from the donor block, was found in basal receivers on petiole number 2. There was gradually less transport in older segments. The recovery as a percentage of the radioactivity not accounted for by donor and receiver blocks, measured by counting the radioactivity in an acetonitrile-extract of petiole segments, was low: 25 to 50%. In this acetonitrile-soluble fraction evidence for different radioactive compounds was found, depending on the age of the tissue. A possible relationship between the amounts of auxin transported in the tissue and its corresponding metabolism is discussed.