Dependence of Basipetal Polar Transport of Auxin upon Aerobic Metabolism

The movement of IAA-¹⁴C through coleoptile segments of Avena and Zea has been investigated under aerobic and anaerobic conditions. The results are as follows: Zea. Using a 5-mm segment and a 2-hour transport period anaerobic conditions reduced the total uptake of ¹⁴C from an apical donor by 74% and the proportion of the total found in the receiving block by at least 45%. Anaerobic conditions reduced total uptake from a basal donor by 58% but no ¹⁴C reached the apical receiving block in either air or N₂. Uptake from apical and basal donor blocks in N₂ is closely similar.

The presence of ¹⁴C in the basal receiving blocks, and its absence in the apical receiving blocks, in N₂ suggests that even in anaerobic conditions movement of IAA is polarized basipetally, although the movement occurs at only a fraction of the rate found in air.

Anaerobic conditions induced a similar reduction in basipetal movement of IAA in upper and lower 5-mm segments taken from the apical 10 mm of a Zea coleoptile.

Using 10-mm Zea segments no ¹⁴C was recovered in the receiving blocks at the basal end of the segment after 2 and 4 hours in N₂ whereas large amounts were recovered in air.

Avena: Using 5-mm segments and a 2-hour transport period the total uptake of ¹⁴C from an apical donor is reduced by 83%. Movement of ¹⁴C into the basal donor is totally inhibited in N₂. Total uptake of ¹⁴C from a basal donor is reduced by 61% in nitrogen and no ¹⁴C reached the apical receiving blocks regardless of the atmospheric conditions.

A time course for the movement of ¹⁴C into the basal and apical receiving blocks through 5-mm segments showed that in air the amount in the basal receivers increased for 4 hours and then remained approximately uniform. In N₂ no significant ¹⁴C reached the receivers until 6 to 8 hours after the application of donors but even then the amounts were about 12 to 14% of that in aerobic receivers. Movement of ¹⁴C into apical receivers was similar in air and in nitrogen and even after 6 to 8 hours the amount of radioactivity barely reached significant levels.

     

THE TIME-COURSE OF POLAR MOVEMENT OF GIBBERELLIN THROUGH ZEA ROOTS

The polarity of movement of gibberellin through sections cut from near the root tips of Zea mays L. was studied, using methods like those we previously used in roots for auxin and in petioles for auxins, cytokinins, and gibberellic acid (GA-3). One μg GA-3 was added in a donor agar block and gibberellin activity in the receiver agar at the opposite end of the section was measured directly with a modified barley endosperm bioassay. The movement of gibberellin was away from the root tip (basipetal) and thus opposite in direction to the polarity of auxin through such root sections. The time-course of basipetal movement was dissimilar to that for gibberellin or auxin movement through petiole sections. It took 14-18 hr for gibberellin activity equivalent to 6 ng GA-3 to collect in the basal receivers on roots. Apical receivers showed activity equivalent to 1.6 ng GA-3 at 14-18 hr. Less than 0.01 ng equivalent GA-3 was collected from sections to which GA-3 was not added, so the 6 and 1.6 ng were almost entirely due to the added GA-3. These general conclusions were confirmed with an experiment using ¹⁴C-GA-3. A decline in activity in receivers was found in some experiments at 18 hr, paralleling earlier results with GA-3, IAA, and adenine in petioles and IAA in roots.     

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.     

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.     

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.     

The transport of radiolabeled indoleacetic acid and its conjugates in nodal stem segments of Phaseolus vulgaris L.

The transport of radiolabeled indoleacetic acid (IAA), and some of its conjugates, was investigated in nodal stem segments of Phaseolus vulgaris L. Donor agar blocks containing either [2-acetyl-¹⁴C]-IAA; [2-acetyl-¹⁴C]-indole-3-acetyl-L-aspartate (IAAsp); [2-acetyl-¹⁴C]-indole-3-acetyl-L-glycine (IAGly); or [2-acetyl-¹⁴C]-indole-3-acetyl-L-alanine (IAAla) were placed on either the apical or basal cut surface of stem segments each bearing an axillary bud at the midline. In some experiments, a receiver block was placed on the end opposite to the donor. After transport was terminated, the segments were divided into five equal sections plus the bud, and the radioactivity of donors, receivers and each part of the stem segment was counted.For all four substances tested, the amount of ¹⁴C transported to the axillary bud from the base was the same or greater than that from the apical end. After basipetal transport, the distribution of ¹⁴C in the segment declined sharply from apex to base. The inverse was true for acropetal transport. Transport for the three IAA conjugates did not differ substantially from each other.The IAA transport inhibitor, N-1-naphthylphthalamic acid (NPA), inhibited basipetal ¹⁴C-IAA transport to the base of the stem segment but did not alter substantially the amount of ¹⁴C-IAA recovered from the bud. Transport of ¹⁴C-IAA from the apical end to all parts of the stem segment declined when the base of the section was treated with nonradioactive IAA. Taken together with data presented in the accompanying article [Tamas et al. (1989) Plant Growth Regul 8: 165–183], these results suggest that the transport of IAA plays a role in axillary bud growth regulation, but its effect does not depend on the accumulation of IAA in the axillary bud itself.     

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.     

Polar transport and accumulation of indole-3-acetic acid during root regeneration by Pinus lambertiana embryos

Summary The relation of indoleacetic acid (IAA) transport to accumulation of auxin at the base of cuttings and to polar root formation was investigated with small cuttings from germinating embryos of Pinus lambertiana.The transport of endogenous auxin participates in regeneration of roots. This is shown by the facts that (1) more than 40% of the cuttings rooted without addition of exogenous indoleacetic acid; (2) the first regeneration always occurred at the basal tip of a slanting cut; and (3) 2,3,5-triiodobenzoic acid (TIBA), a specific inhibitor of auxin transport, totally inhibited rooting. Addition of IAA to the medium increased the number of roots formed per rooting hypocotyl.Sections of hypocotyls excised from dormant embryos and tested immediately after 2 h hydration were capable of polar transport of IAA. This polarity increased during the first 3 days of culture because of a marked increase in basipetal transport. Culturing the cuttings in 1 M IAA for 3–5 days doubled both the basipetal transport of 1-¹⁴C-IAA by hypocotyl segments and the accumulation of radioactivity at the base of cuttings.The extent of the accumulation at the base of cuttings was similar at early (2 days, first mitoses) and late stages (5 days, organized meristem) of regeneration and was not affected by removal of the regenerating region immediately prior to uptake and transport of ¹⁴C-IAA. The accumulation was inhibited by TIBA. In terms of increase in wet and dry weight and mitotic activity, the cotyledons rather than the regenerating root meristems were the most actively growing region of the cuttings. The upper part of the hypocotyl elongated more than the region of the slanting cut where regeneration was occurring.These results provide no support for the idea that the regenerating root controls the direction of polar transport by acting as a sink. The results are consistent with the view that polar auxin transport delivers auxin to the base of the cutting and raises the local concentration to levels sufficient to promote root formation.     

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.     

The Lateral Transport of 2, 4-Dichlorophenoxyacetic Acid in Horizontal Hypocotyl Segments of Helianthus annuus

The longitudinal and lateral transport of 2, 4-D-[1-¹⁴C] hasbeen studied in 6-mm horizontally disposed segments of the hypocotylof Helianthun annuus. The technique involved the asymmetricapplication of the auxin in agar donor blocks to either theupper or lower halves of the cut surface of the segments andthe measurement of ¹⁴C accumulating with time in split receiverblocks on the upper and lower halves of the cut surfaces atthe opposite ends. A highly significant effect of gravity inducinga polar migration of 2, 4-D to the lower side has been establishedand represents, under optimal conditions, about 10 per centof the total 2, 4-D in transit. This lateral polar movementshows a tendency to saturate at higher donor concentrations(5 mg/1) and is affected by temperature in precisely the sameway as the basipetal longitudinal transport. The optimal flux(intensity of transport) occurs at about 35 °C or slightlyabove, and above 40 °C both transport systems are seriouslyimpaired. There is some evidence that gravity increases thevelocity of 2, 4-D transport in the lowermost tissues of thehorizontal hypocotyl but does not affect the transport intensity.     

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.     

Transport of the Auxin 2,4-Dichlorophenoxyacetic Acid Through Absiccion Zones, Pulvini, and Petioles of Phaseolus vulgaris

Measurements were made of the transport of 2,4-dichlorophenoxyacetic acid-¹⁴C (2,4-D) through segments cut from the region of the distal abscission zone in young and old primary leaves of Phaseolus vulgaris L. When old leaves were used basipetal transport of 2,4-D in segments including pulvinar tissue, abscission zone, and petiolar tissue was much less than in wholly petiolar segments. In both young and old plants, segments consisting entirely of pulvinar tissue transported 2,4-D basipetally at a velocity about half that in petiolar tissue. At both ages the flux of 2,4-D through pulvinar tissue was less than that through petiolar tissue. In segments from old leaves the flux through pulvinar tissue was much less than in young plants; the flux through petiolar tissue changed little with age. There was no change with age in the velocity of basipetal transport. The distribution of ¹⁴C along segments including the abscission zone showed no marked discontinuity. It was concluded that the pulvinus limited the basipetal movement of 2,4-D through segments from old leaves which included both pulvinar and petiolar tissue, but there was no evidence that the abscission zone itself was a barrier to auxin transport.     

Movement and metabolism of kinetin-C and of adenine-C in coleus petiole segments of increasing age

To see if polar movement was typical of growth-regulators other than auxins, the movement of adenine-8-¹⁴C and of kinetin-8-¹⁴C was studied in segments cut from petioles of increasing age. No polarity was found. In time-course experiments lasting 24 hr, kinetin showed a progressive increase of radioactivity in receiver blocks, while adenine showed a maximum at 8 hr with a decline thereafter. More kinetin moved through older segments than through younger ones. There was no difference in net loss as far as the position of the donor block is concerned. However, the loss of radioactivity from adenine donor blocks was much higher than the loss of radioactivity from kinetin donor blocks.     

Strongly acidic auxin indole-3-methanesulfonic Acid: synthesis of [C]indole-3-methanesulfonic Acid and studies of its chromatographic, spectral, and biological properties

A radiochemical synthesis is described for [¹⁴C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [¹⁴C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles.     

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.     

Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport in Cucurbita pepo L. Hypocotyl Segments

Pretreatment of 2?0 mm segments of etiolated zucchini (Cucurbitapepo L.) hypocotyl with cycloheximide (CH) or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide(MDMP) eliminated the stimulation by N-1-naphthylphthalamicacid (NPA) of net uptake of [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA),but had relatively little effect on the net uptake of IAA inthe absence of NPA. The efflux of [1-¹⁴C]IAA from preloadedsegments was not substantially affected by inhibitor pretreatmentin the absence of NPA, but CH pretreatment significantly inhibitedthe reduction of efflux caused by NPA. Pretreatment with CHor MDMP did not affect net uptake by segments of the pH probe[2-¹⁴C]5,5-dimethyl-oxazolidine-2,4-dione ([2-¹⁴C]DMO), or thenet uptake of [¹⁴C]-labelled 3-O-methylglucose ([¹⁴C]3-0-MeGlu),suggesting that neither inhibitor affected intracellular pHor the general function of proton symporters in the plasma membrane.Both compounds reduced the incorporation of label from [³⁵S]methionineinto trichloroacetic acid (TCA)-insoluble fractions of zucchinitissue, confirming their inhibitory effect on protein synthesis. The steady-state association of [³H]IAA with microsomal vesiclesprepared from zucchini hypocotyl tissue was enhanced by theinclusion of NPA in the uptake medium. The stimulation by NPAof [³H]IAA association with microsomes was substantially reducedwhen the tissue was pretreated with CH. However, CH pretreatmentdid not affect the level of high affinity NPA binding to themembranes indicating that treatments did not result in lossof NPA receptors. It is suggested that the auxin transport site on the effluxcarrier system and the receptor site for NPA may reside on separateproteins linked by a third, rapidly turned-over, transducingprotein. Key words: Auxin carriers, auxin efflux, Cucurbita pepo, phytotropin receptors     

THE EFFECTS OF TEN PHENOLIC COMPOUNDS ON HYPOCOTYL GROWTH AND MITOCHONDRIAL METABOLISM OF MUNG BEAN

Ten phenolic compounds were examined for their effect on mung bean (Phaseolus aureus L.) hypocotyl growth and on respiration and coupling parameters of isolated mung bean hypocotyl mitochondria. Three compounds—tannic, gentisic, and p-coumaric acids—inhibited hypocotyl growth and when incubated with isolated hypocotyl mitochondria released respiratory control, inhibited respiration, and prevented substrate-supported Ca²⁺ and PO₄ transport. Vanillic acid also inhibited hypocotyl growth and reduced mitochondrial Ca²⁺ uptake but did not affect respiration or respiratory control of isolated mitochondria. This is the first compound reported to selectively inhibit Ca²⁺ uptake in plant mitochondria. Two other phenolic compounds—α, 3,5-resorcylic and protocatechuic acids—showed no significant effect on hypocotyl growth and did not affect mitochondrial oxidative phosphorylation either separately or in various combinations. Four phenolic compounds—ferulic, caffeic, p-hydroxybenzoic, and syringic acids—showed a significant reduction in mung bean hypocotyl growth but did not inhibit any of the mitochondrial processes examined. The results show that phenolic compounds which alter respiration or coupling responses in isolated mitochondria also inhibit hypocotyl growth and may reflect a mechanism of action for these natural growth inhibitors.     

Biochemical Bases for the Loss of Basipetal IAA Transport with Advancing Physiological Age in Etiolated Helianthus Hypocotyls: Changes in IAA Movement, Net IAA Uptake, and Phytotropin Binding

Basipetal transport of [¹⁴C]IAA in hypocotyl segments isolated from various regions of etiolated Helianthus annuus L. cv NK 265 seedlings declines with increasing physiological age. This decline was the result of a reduction in both transport capacity and apparent velocity. Net IAA uptake was greater and the abilities of auxin transport inhibitors to stimulate net IAA uptake were reduced in older tissues. Net IAA accumulation by microsomal vesicles exhibited a similar behavior with respect to age. Specific binding of [³H]N-1-naphthylphthalamic acid (NPA) to microsomes prepared from young and older hypocotyl regions was saturable and consistent with a single class of binding sites. The apparent affinity constants for NPA binding in microsomes prepared from young versus older tissues were 6.4 and 10.8 nanomolar, respectively, and the binding site densities for young versus old tissues were 7.44 and 3.29 picomoles/milligram protein, respectively. Specific binding of [³H]NPA in microsomes prepared from both tissues displayed similar sensitivities toward unlabeled flurenol and exhibited only slight differences in sensitivity toward 2,3,5-triiodobenzoic acid. These results demonstrate that the progressive loss of basipetal IAA transport capacity in etiolated Helianthus hypocotyls with advancing age is associated with substantial alterations in the phytotropin-sensitive, IAA efflux system and they suggest that these changes are, at least partially, responsible for the observed reduction of polar IAA transport with advancing tissue age.     

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     

Developmental aspects ofl-valine uptake by mouse intestine

Summary ¹⁴C-l-Valine uptake by intestinal segments of mice of various ages, ranging between 20-day fetuses and adults, was studied in vitro. 1 mMl-Valine was accumulated against a concentration gradient by processes which showed saturation kinetics. There appeared to be a two-fold increase ofl-valine accumulation after the 2nd postnatal day and a three-fold increase in adult mice. Fetal transport of valine only occurred at pH 7.4 but was not Na⁺ dependent. In contrast, valine transport became increasingly Na⁺ dependent and the pH optimum widened, ranging between 5–8. A series of amino acids, including representatives of the imino acid and dibasic groups, failed to inhibit valine uptake while leucine and isoleucine manifested mutual inhibition with valine. It is speculated that in the mouse intestine,l-valine is transported by at least two mechanisms, one functioning in the fetus, not requiring Na⁺, but pH dependent and another which developes postnatally, is Na-dependent and functions over a wide pH optimum.