Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.     

Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls

The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [³H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half-maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene-treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [³H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K_d) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [³H]NPA binding.     

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     

Polar Transport of Auxin across Gravistimulated Roots of Maize and Its Enhancement by Calcium

The effect of Ca on the polar movement of [³H]indoleacetic acid ([³H] IAA) in gravistimulated roots was examined using 3-day-old seedlings of maize (Zea mays L.). Transport of label was measured by placing an agar donor block containing [³H]IAA on one side of the elongation zone and measuring movement of label across the root into an agar receiver block on the opposite side. In vertically oriented roots, movement of label across the elongation zone into the receiver was slight and was not enhanced by incorporating 10 millimolar CaCl₂ into the receiver block. In horizontally oriented roots, movement of label across the root was readily detectable and movement to a receiver on the bottom was about 3-fold greater than movement in the opposite direction. This polarity was abolished in roots from which the caps were removed prior to gravistimulation. When CaCl₂ was incorporated into the receivers, movement of label across horizontally oriented intact roots was increased about 3-fold in both the downward and upward direction. The ability of Ca to enhance the movement of label from [³H]IAA increased with increasing Ca concentration in the receiver up to 5 to 10 millimolar CaCl₂. With the inclusion of CaCl₂ in the receiver blocks, gravity-induced polar movement of label into receiver blocks from applied [³H]IAA was detectable within 30 minutes, and asymmetric distribution of label within the tissue was detectable within 20 minutes. The results indicate that gravistimulation induces a physiological asymmetry in the auxin transport system of maize roots and that Ca increases the total transport of auxin across the root.     

Effect of Photoperiod on Metabolism of [H] Gibberellins A(1), 3-epi-A(1), and A(20) in Agrostemma githago L

To determine whether daylength influences the rate of metabolism of gibberellins (GAs) in the long-day (LD) rosette plant Agrostemma githago L., [³H]GA₂₀ and [³H]GA₁ were applied under short day (SD) and LD. Both were metabolized faster under LD than under SD. [³H]GA₂₀ was metabolized to a compound chromatographically identical to 3-epi-GA₁. [³H]GA₁ was metabolized to two acidic compounds, the major metabolite having chromatographic properties similar to, but not identical with GA₈. [³H]3-epi-GA₁ applied to plants under LD was metabolized much more slowly than was [³H]GA₁, and formed a very polar metabolite which did not partition into ethyl acetate at pH 2.5. Very polar metabolites were also formed after the feeds of [³H]GA₂₀ and [³H]GA₁. It was not possible to characterize these very polar compounds further because of their apparent instability. The results obtained suggest that in Agrostemma GA₂₀ is the precursor of 3-epi-GA₁, but there is at present no evidence indicating the precursor of GA₁.     

A saturable site responsible for polar transport of indole-3-acetic acid in sections of maize coleoptiles

The velocity of transport and shape of a pulse of radioactive indole-3-acetic acid (IAA) applied to a section of maize (Zea mays L.) coleoptile depends strongly on the concentration of nonradioactive auxin in which the section has been incubated before, during, and after the radioactive pulse. A pulse of [³H]IAA disperses slowly in sections incubated in buffer (pH 6) alone; but when 0.5–5 M IAA is included, the pulse achieves its maximum velocity of about 2 cm h^-1. At still higher IAA concentrations in the medium, a transition occurs from a discrete, downwardly migrating pulse to a slowly advancing profile. Specificity of IAA in the latter effect is indicated by the observation that benzoic acid, which is taken up to an even greater extent than IAA, does not inhibit movement of [³H]IAA. These results fully substantiate the hypothesis that auxin transport consists of a saturable flux of auxin anions (A^-) in parallel with a nonsaturable flux of undissociated IAA (HA), with both fluxes operating down their respective concentration gradients. When the anion site saturates, the movement of [³H]IAA is nonpolar and dominated by the diffusion of HA. Saturating polar transport also results in greater cellular accumulation of auxin, indicating that the same site mediates the cellular efflux of A^-. The transport inhibitors napthylphthalamic acid and 2,3,5-triiodobenzoic acid specifically block the polar A^- component of auxin transport without affecting the nonsaturable component. The transport can be saturated at any point during its passage through the section, indicating that the carriers are distributed throughout the tissue, most likely in the plasmalemma of each cell.Abbreviations A^- auxin anion - HA undissociated auxin - IAA indole-3-acetic acid - NPA N-1-napthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

A protein from maize labeled with azido-IAA has novel β-glucosidase activity

A biologically active and photolabile auxin analog, 5-azido-[7-³H]indole-3-acetic acid ([³H]N₃IAA), was used to search for auxin-binding proteins in cytosolic extracts from maize coleoptiles (Zea mays L.) and identified a protein with a molecular mass of 60 kDa (p60). Binding of [³H]N₃IAA is highly specific as demonstrated by competition analysis with functionally relevant auxin analogs. p60 is found in coleoptiles and roots of etiolated maize seedlings and was detected in cytosolic as well as in microsomal fractions. The protein binds to 1-naphthylacetic acid (1-NAA) sepharose and is eluted with auxins. A purification scheme resulting in homogenous p60 protein was devised and it was shown that p60 has β-d -glucoside glucohydrolase activity (E.C.3.2.1.21). The hydrolytic activity of p60 for the synthetic substrate p-nitro-phenyl-β-d -glucopyranoside is diminished by 1-NAA. p60 shows high substrate specificity since it hydrolyzes indoxyl-O-glucoside, but not β-(1,4)-cellobiose, IAA-inositol or IAA-amino acid conjugates. The present data suggest that p60 might be involved in the hydrolysis of auxin conjugates.     

Translocation and Metabolism of Endosperm-Applied [2-C] Indoleacetic Acid in Etiolated Avena sativa L. Seedlings

The role of free indole-3-acetic acid (IAA) in the endosperm of Avena sativa L. seedlings was investigated to determine its contribution to free IAA in the shoot. [2-¹⁴C]IAA was injected into the endosperm of darkgrown seedlings and the transport and metabolism of the [¹⁴C]-labeled compounds determined. It was concluded that translocation of free IAA directly from the endosperm is probably not a significant source of free IAA in the shoot, mainly because even small amounts of [¹⁴C]IAA introduced into the endosperm were rapidly metabolized. This suggested that, in Avena, free IAA does not normally exist in the liquid endosperm.     

Inhibition of conjugation of indole-3-acetic acid with amino acids by 2,6-dihydroxyacetophenone in Teucrium canadense

2,6-Dihydroxyacetophenone and five structurally related compounds were tested for their effects on metabolism of[2-¹⁴C]IAA in stem segments of 3-week-old American germander (Teucrium canadense). Pre-treatment of the plants with 2 mM 2,6-dihydroxyacetophenone for 12 hr significantly reduced the formation of two radioactive metabolites, which were tentatively identified as N-(indole-3-acetyl)-L-aspartic acid and N-(indole-3-acetyl)-L-glutamic acid. The chemical pre-treatment also decreased the level of a less polar metabolite chromatographically indistinguishable from oxindole-3-acetic acid, an oxidative product of IAA, and other unidentified metabolites of IAA. Concomitantly, the level of free [2-¹⁴C]IAA increased significantly in the treated tissue. 2,4-, 2,5- and 3,4-Dihydroxyacetophenones, as well as 3-bromo-2,6-dihydroxyacetophenone and 2-hydroxy-6-methoxyacetophenone, did not show a similar effect.     

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.     

Adventitious rooting: examining the role of auxin in an easy- and a difficult-to-root plant

Therooting responses of cuttings of difficult-to-root lilac (Syringavulgaris) and easy-to-root forsythia(Forsythia×intermedia)were compared. The rooting ability of lilac cuttings declined over the growingseason (May–June). There was also a decline in the initial concentrationof free IAA at the base of the cuttings, but there was not a tight relationshipbetween basal IAA concentration and rooting ability. Polar auxin transportability was measured in lilac and forsythia during the period of maximum growthby [³H]IAA application to stem internodal tissue. Transport abilitydeclined in lilac over this time period, particularly in terms of transportintensity and percentage of [³H]IAA transported. In contrast thechanges in polar auxin transport ability in forsythia were less marked. Thisdifference between species was maintained in winter hardwood cuttings, withforsythia tissue showing greater polar auxin transport ability than lilac. Theimportance of polar auxin transport for adventitious rooting was demonstratedinboth lilac and forsythia softwood cuttings by use of the polar transportinhibitor 2,3,5-triiodobenzoic acid (TIBA). Overall the results indicate thatdifferences in polar auxin transport ability between lilac and forsythiacontribute to differences in rooting ability.     

Simultaneous gas chromatography-mass spectrometry quantification of endogenous [C]- and applied [C]indole-3yl-acetic Acid levels in growing maize roots

The use of stable indole-3yl-acetic acid (IAA) labeled by 6 atoms of ¹³C allowed, after [¹³C]IAA treatment, simultaneous gas chromatography-mass spectrometry quantifications of both endogenous [¹²C]IAA and applied [¹³C]IAA levels in Zea mays L. roots. Root material was immersed for 1 hour in a buffered (pH 6.0) solution without or with [¹³C]IAA at 10⁻⁷ molar. Both applied and endogenous IAA were thus measured for three zones of the roots (apical, elongating, differentiating) directly after treatment and also 2 hours later. Growth was followed over a 4 hour period. Roots not immersed elongated more than control roots (immersed in buffer), which grew more than IAA-treated roots. Immersion in buffer induced a large decrease (−68%) of [¹²C]IAA in the apical part of control roots, whereas immersion in [¹³C]IAA prevented most of it. No significant difference between control and treated roots occurred in the two other zones. Two hours after treatment, [¹³C]IAA had completely disappeared from the elongating zone even though [¹²C]IAA level was essentially stable. A direct relationship occurred between the level of IAA in the elongating zone and the growth of the root. This relationship was strongly disturbed if unmetabolized [¹³C]IAA was present. However, the relationship returned to its initial state when significant amounts of free [¹³C]IAA were no longer detectable. These results are discussed in terms of the stability of both types of compounds and the utility of the method of using stable isotopes of hormones, for the understanding of hormonal regulation of plant growth.     

Transfer of exogenous auxin from the phloem to the polar auxin transport pathway in pea (Pisum sativum L.)

When [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA) was applied to the upper surface of a mature foliage leaf of garden pea (Pisum sativum L. cv. Alderman), ¹⁴C effluxed basipetally but not acropetally from 30-mm-long internode segments excised 4 h after the application of [1-¹⁴C]IAA. This basipetal efflux was strongly inhibited by the inclusion of 3.10^–6 mol· dm³ N-1-naphthylphthalamic acid (NPA) in the efflux buffer. In contrast, when [¹⁴C] sucrose was applied to the leaf, the efflux of label from stem segments excised subsequently was neither polar nor sensitive to NPA. The [1-¹⁴C]IAA was initially exported from mature leaves in the phloem — transport was rapid and apolar; label was recovered from aphids feeding on the stem; and label was recovered in exudates collected from severed petioles in 20 mM ethylenediaminetetraacetic acid. No ¹⁴C was detected in aphids feeding on the stems of plants to which [1-¹⁴C]IAA had been applied apically, even though the internode on which they were feeding transported considerable quantities of label. Localised applications of NPA to the stem strongly inhibited the basipetal transport of apically applied [1-¹⁴C]IAA, but did not affect transport of [1-¹⁴C]IAA in the phloem. These results demonstrate for the first time that IAA exported from leaves in the phloem can be transferred into the extravascular polar auxin transport pathway but that reciprocal transfer probably does not occur. In intact plants, transfer of foliar-applied [1-¹⁴C]IAA from the phloem to the polar auxin transport pathway was confined to immature tissues at the shoot apex. In plants in which all tissues above the fed leaf were removed before labelling, a limited transfer of IAA occurred in more mature regions of the stem.Abbreviations IAA indol-3yl-acetic acid - EDTA ethylenediaminetetraacetic acid - NPA N-1-naphthylphthalamic acid We are grateful to the Nuffield Foundation for supporting this research under the NUF-URB95 scheme and for the provision of a bursary to A.J.C. We thank Professor Dennis A. Baker for constructive comments on a draft of this paper and Mrs. Rosemary Bell for her able technical assistance.     

Abscisic acid and apical dominance in Phaseolus coccineus L.

Abscisic acid (ABA) in lanolin, applied to the internode of decapitated runner bean plants enhances the outgrowth of lateral buds. The optimum concentration of the paste is 10^-5 M. The effect of ABA is counteracted by indoleacetic acid (IAA) but not by gibberellic acid (GA₃). There is no effect when ABA is applied to the apical bud or lateral buds of intact plants. However, 13.2 ng given to the lateral buds of decapitated plants stimulate their growth, whereas higher concentrations are inhibitory. Consequently, ABA enhances growth of lateral buds directly, but only when apical dominance is already weakened. The growth of the decapitated 2^nd internode was not affected by ABA. Radioactivity from [2-¹⁴C] ABA, applied to nonelongating 2^nd internode stumps of decapitated runner bean plants moves to the lateral buds, whereas [1-¹⁴C]IAA-and [³H]GA₁-translocation is much weaker. ABA transport is inhibited if IAA or [³H]GA₁ is applied simultaneously. In elongating internodes [¹⁴C]ABA is almost completely immobile. [¹⁴C]IAA-and [³H]GA₁-translocation is not affected by ABA. The amount of radioactivity from labelled ABA, translocated to the lateral buds, is highest during the early stages of bud outgrowth.Abbreviations ABA 2,4-cis, trans-(+)-abscisic acid - GA gibberellic acid - IAA indoleacetic acid - p.l. plain lanolin     

Relationship of indole-3-butyric acid and adventitious rooting in M.26 apple (Malus Pumila mill.) shoots cultured in vitro

The role of indole-3-butyric acid (IBA) in adventitious root formation was studied by analyzing the uptake and subsequent metabolism of IBA in shoots of M.26 apple (Malus pumila Mill.) rootstock grown in vitro. Roots were induced by exposing shoots to 4 M IBA and [³H]IBA for 5 days in the dark and then transferring them to plant growth regulator (PGR)-free medium in the light until roots formed. Approximately 50% of the total radioactivity applied was taken up from the agar medium by the shoots during the 5-day incubation period in IBA. Indole-3-butyric acid metabolism was studied by extraction and high-performance liquid chromatographic (HPLC) separation of [³H]IBA and metabolites from the basal sections of treated shoots. The major [³H]IBA metabolite co-eluted with authentic [¹⁴C]indole-3-acetic acid (IAA) suggesting that IBA was converted to IAA in the shoots. The proportion of newly synthesized IAA present as conjugates was higher at the end of the 5-day IBA treatment period than after 13 days in PGR-free medium. There appeared to be no conjugation of IBA at any time.     

Myo-Inositol Esters of Indole-3-acetic Acid as Seed Auxin Precursors of Zea mays L

Indole-3-acetyl-myo-inositol esters constitute 30% of the low molecular weight derivatives of indole-3-acetic acid (IAA) in seeds of Zea mays. [¹⁴C]Indole-3-acetyl-myo-inositol was applied to a cut in the endosperm of the seed and found to be transported from endosperm to shoot at 400 times the rate of transport of free IAA. The rate of transport of indole-3-acetyl-myo-inositol from endosperm to shoot was 6.3 picomoles per shoot per hour and thus adequate to serve as the seed auxin precursor for the free IAA diffusing downward from the shoot tip. Indole-3-acetyl-myo-inositol is the first seed auxin precursor to be identified.     

Ontogenetic Changes in the Transport of Indol-3yl-acetic Acid into Maize Roots from the Shoot and Caryopsis

The quantities of endogenous indol-3yl-acetic acid (IAA) in endosperms and scutella of 6-day-old maize seedlings (Zea mays L. cv Giant White Horsetooth) were determined by a fluorimetric method. Endosperms were found to contain 33.4 nanograms IAA per plant, and scutella 7.5 nanograms IAA per plant. [5-³H]IAA applied to endosperms of 6-day-old seedlings moved into the roots and radioactivity accumulated at the apex of the primary root within 8 hours. Two to 7-day-old seedlings were treated simultaneously with [5-³H]IAA in the endosperm and [2-¹⁴C] IAA on the shoot apex. The patterns of transport into the root were found to change during ontogeny: in successively older plants, transport from the shoot into the roots increased relative to transport from the endosperm into the roots. The auxin required for the growth of maize roots could, therefore, partially be contributed by the shoot and endosperm. Ontogenetic changes in the relative importance of these two supplies could be of significance for the integration of growth and development between shoot and root.     

C(6)-[benzene ring]-indole-3-acetic Acid: a new internal standard for quantitative mass spectral analysis of indole-3-acetic Acid in plants

Indole-3-acetic acid (IAA) labeled with ¹³C in the six carbons of the benzene ring is described for use as an internal standard for quantitative mass spectral analysis of IAA by gas chromatography/selected ion monitoring. [¹³C₆]IAA was compared to the available deuterium labeled compounds and shown to offer the advantages of nonexchangeability of the isotope label, high isotopic enrichment, and chromatographic properties identical to that of the unlabeled compound. The utility of [¹³C₆]IAA for measurement of endogenous IAA levels was demonstrated by analysis of IAA in Lemna gibba G-3.     

Auxin carriers in Cucurbita vesicles

Carrier-mediated uptake of indole-3-acetic acid (IAA) by microsomal vesicles from Cucurbita pepo L. hypocotyls was strongly inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D; i ₅₀= 0.3 M) but only weakly by 1-naphthylacetic acid (NAA). The fully ionised auxin indol-3-yl methanesulphonic acid also inhibited (i ₅₀=3 M). The same affinity ranking of these auxins for the uptake carrier, an electroimpelled auxin anion-H⁺ symport, is demonstrable in hypocotyl segments. The specificity of the auxin-anion eflux carrier was tested by the ability of different nonradioactive auxins to compete with [³H]IAA and reduce the stimulation of net radioactive uptake by N-1-naphthylphthalamic acid (NPA), a noncompetitive inhibitor of this carrier. By this criterion, NAA and IAA had comparable affinities, with 2,4-D interaction more weakly. Stimulation of [³H]IAA uptake by NAA, as a result of competition for the efflux carrier, could also be demonstrated when a suitable concentration of 2,4-D was used selectively to inhibit the uptake carrier. However, when [³H]NAA was used, no stimulation of its association with vesicles by NPA, 2,3,5-triiodobenzoic acid, or nonradioactive NAA was found. In hypocotyl segments, [³H]NAA net uptake was much less sensitive to NPA stimulation than was [¹⁴C]IAA uptake. The apparent contradictions concerning NAA could be explained by carrier-mediated auxin efflux making a smaller relative contribution to the overall transport of NAA than of IAA. The relationship between carrier specificity as manifested in vitro and the specificity of polar auxin transport is discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - IMS indol-3-yl methanesulphonic acid - NAA 1-naphthylacetic aci - NPA N-1-naphthylphthalamic acid