Ascorbic acid as negative effector of the peroxidase-catalyzed degradation of indole-3-acetic acid

Ascorbic acid is a strong inhibitor of indole-3-acetic oxidation catalyzed by commercial horse-radish peroxidase. In the presence of excess ascorbic acid, the indole-acetic acid oxidation catalysis is apparently blocked. The activity of peroxidase for indoleacetic acid at pH 3.7 and 33°C, in the presence of 2,4-dichlorophenol and MnCl₂ as promotors was measured by polarographic technique. The K_m was 0.27 m M and the maximum velocity was 1.02 mmol O₂ (mg protein)⁻¹ min⁻¹. Dixon plots lead to an apparent K_i of 1.25 (μ M for ascorbic acid and the inhibition was apparently competitive. Ascorbic acid, besides appearing to be a strong inhibitor of the IAA oxidase activity of peroxidase, seemed to protect IAA from total degradation. Addition of more than 5 μ M ascorbic acid produced both an exponential increase in the lag time before the onset of reaction and, at the end, an oxidation protection of 26 μ M IAA when 111 μ M IAA was present at the stawrt. The possibility of ascorbic acid-IAA auxin from endogenous oxidation in plants, is proposed.     

Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid,naphthalene-1-acetic acid,and indole-3-acetic acid in suspension-cultured tobacco cells

Accumulation of radiolabelled naphthalene-1-acetic acid (1-NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-acetic acid (IAA) has been measured in suspension-cultured tobacco (Nicotiana tabacum) cells. In this paper is presented a simple methodology allowing activities of the auxin influx and efflux carriers to be monitored independently by measuring the cellular accumulation of [³H]NAA and [¹⁴C]2,4-D. We have shown that 1-NAA enters cells by passive diffusion and has its accumulation level controlled by the efflux carrier. By contrast, 2,4-D uptake is mostly ensured by the influx carrier and this auxin is not secreted by the efflux carrier. Both auxin carriers contribute to IAA accumulation. The kinetic parameters and specificity of each carrier have been determined and new information concerning interactions with naphthylphthalamic acid, pyrenoylbenzoic acid, and naphthalene-2-acetic acid are provided. The relative contributions of diffusion and carrier-mediated influx and efflux to the membrane transport of 2,4-D, 1-NAA, and IAA have been quantified, and the data indicate that plant cells are able to modulate over a large range their auxin content by modifying the activity of each carrier.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 1-NAA naphthalene-1-acetic acid - 2-NAA naphthalene-2-acetic acid - NPA N-1-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - V_m maximum transport capacity of the carrier In honour of Professor Dieter Klämbt's 65th birthdayThe authors thank Drs. A.E. Geissler and G.F. Katekar (CSIRO, Canberra City, Australia) for providing auxin efflux carrier inhibitors CPD, CPP, and PBA, and Dr. H. Barbier-Brygoo (Institut des Sciences Végétales, CNRS, Gif-sur-Yvette, France) for helpful discussions. This work was supported by funds from the Centre National de la Recherche Scientifique (UPR0040).     

Expression of an auxin-inducible promoter of tobacco in Arabidopsis thaliana

The expression of the auxin-inducible Nt103-1 gene of tobacco was studied in Arabidopsis thaliana. For this purpose we introduced a gene fusion between the promoter of the gene and the -glucuronidase reporter gene (GUS) into Arabidopsis thaliana. The expression and location of GUS activity were studied histochemically in time and after incubation of seedlings on medium containing auxins or other compounds. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and 1-naphthylacetic acid (1-NAA) were able to induce GUS activity in the root tips of transgenic seedlings. The auxin transport inhibitor 2,3,5-triiodobenzoic acid was able to induce GUS activity not only in the root tip, but also in other parts of the root. Induction by the inactive auxin analog 3,5-dichlorophenoxyacetic acid was much weaker. Compounds like glutathione and the heavy metal CuSO₄ were weak inducers. GUS activity observed after induction by glutathione was located in the transition zone. Salicylic acid and compounds increasing the concentration of hydrogen peroxide in the cell were also very well able to induce GUS activity in the roots. The possible involvement of hydrogen peroxide as a second messenger in the pathway leading to the induction of the Nt103-1 promoter is discussed.     

A 2,4-D-inducible glutathione S-transferase from soybean (Glycine max). Purification, characterisation and induction

Glutathione S-transferases (GSTs; EC 2.5.1.18) have recently been proposed to form one large group among the auxin-induced proteins. However. the properties and regulation of such auxin-responsive GSTs in the plant still await detailed investigation. In this study, a 2,4-dichloro-phenoxyacetic acid (2,4-D)-inducible GST isozyme from soybean ( Glycine max [L.] Merr. cv. Williams) was purified to near homogeneity by anion-exchange and affinity chromatography on S-hexylglutathione agarose. The native enzyme had a molecular mass of 49 kDa, as determined by gel filtration, and consisted of 26-kDa subunits. The purified GST conjugated glutathione to 1-chloro-2,4-dinitrobenzene and to the herbicide metolachlor, but not to the other GST substrates atrazine. fluorodifen or trans-cinnamic acid. The N-termmal amino acid sequence shared significant homology with the deduced polypeptide sequences of two 2,4-D-inducible genes from tobacco, par A and CNT107 . The levels of the 26-kDa GST subunit protein in soybean hypocotyls were analysed by immunoblotting. At micromolar concentrations, 2,4-D induced a transient increase in net accumulation of GST, whereas indole-3-acetic acid or I-naphthaleneacetic acid did not increase the GST levels. Known inhibitors of polar auxin transport, including 2.3.5-tri-iodobenzoic acid. N-I-naphthylphthalamic acid and analogues thereof, differed widely in their ability to elicit GST protein accumulation. It is concluded that the induction of soybean GST by 2,4-D and by some of the auxin transport inhibitors is not related to auxin activity or to changes in the endogenous auxin levels.     

Invertase and auxin-induced elongation in internodal segments of Phaseolus vulgaris

A close positive correlation was observed between segment elongation and the specific activity of soluble acid invertase in stem segments of P. vulgaris incubated for 21 hr in the presence of IAA or of several synthetic auxins and auxin analogues. Optimum concentrations for the stimulation of growth and invertase activity were similar and varied from 10^?6 M (2,4-D) through 10^?5 M (IAA, IBA, α-NAA, β-NAA) to greater than 10^?4 (IPA, PoAA, trans-cinnamic acid). The weak activity of trans-cinnamic acid, a competitive inhibitor of auxin action, may have resulted from cis-trans isomerization during incubation. The concentration of hexose sugars in the segments fell during incubation in the presence of auxin, the greatest decline in hexose concentration occurring in the presence of compounds exhibiting the greatest stimulation of growth.     

Regulation of genes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxyacetic acid in Arabidopsis

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) regulates plant growth and development and mimics auxins in exhibiting a biphasic mode of action. Although gene regulation in response to the natural auxin indole acetic acid (IAA) has been examined, the molecular mode of action of 2,4-D is poorly understood. Data from biochemical studies, (Grossmann (2000) Mode of action of auxin herbicides: a new ending to a long, drawn out story. Trends Plant Sci 5:506–508) proposed that at high concentrations, auxins and auxinic herbicides induced the plant hormones ethylene and abscisic acid (ABA), leading to inhibited plant growth and senescence. Further, in a recent gene expression study (Raghavan et al. (2005) Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis. Funct Integr Genomics 5:4–17), we have confirmed that at high concentrations, 2,4-D induced the expression of the gene NCED1, which encodes 9-cis-epoxycarotenoid dioxygenase, a key regulatory enzyme of ABA biosynthesis. To understand the concentration-dependent mode of action of 2,4-D, we further examined the regulation of whole genome of Arabidopsis in response to a range of 2,4-D concentrations from 0.001 to 1.0 mM, using the ATH1-121501 Arabidopsis whole genome microarray developed by Affymetrix. Results of this study indicated that 2,4-D induced the expression of auxin-response genes (IAA1, IAA13, IAA19) at both auxinic and herbicidal levels of application, whereas the TIR1 and ASK1 genes, which are associated with ubiquitin-mediated auxin signalling, were down-regulated in response to low concentrations of 2,4-D application. It was also observed that in response to low concentrations of 2,4-D, ethylene biosynthesis was induced, as suggested by the up-regulation of genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Although genes involved in ethylene biosynthesis were not regulated in response to 0.1 and 1.0 mM 2,4-D, ethylene signalling was induced as indicated by the down-regulation of CTR1 and ERS, both of which play a key role in the ethylene signalling pathway. In response to 1.0 mM 2,4-D, both ABA biosynthesis and signalling were induced, in contrast to the response to lower concentrations of 2,4-D where ABA biosynthesis was suppressed. We present a comprehensive model indicating a molecular mode of action for 2,4-D in Arabidopsis and the effects of this growth regulator on the auxin, ethylene and abscisic acid pathways. Experiment station: Plant Biotechnology Centre, Primary Industries Research Victoria, Department of Primary Industries, La Trobe University, Bundoora, Victoria 3086, and the Victorian Microarray Technology Consortium (VMTC).     

The effect of auxin (indole-3-acetic acid) on the growth rate and tropism of the sporangiophore of <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis> and identification of auxin-related genes

The roles of fungal auxins in the regulation of elongation growth, photo-, and gravitropism are completely unknown. We analyzed the effects of exogenous IAA (indole-3-acetic acid), various synthetic auxins including 1-NAA (1-naphthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), and the auxin transport inhibitor NPA (N-1-naphtylphtalamic acid) on the growth rate and bending of the unicellular sporangiophore of the zygomycete fungus, Phycomyces blakesleeanus. Sporangiophores that were submerged in an aqueous buffer responded to IAA with a sustained enhancement of the growth rate, while 1-NAA, 2,4-D, and NPA elicited an inhibition. In contrast, sporangiophores kept in air responded to IAA with a 20 to 40% decrease of the growth rate, while 1-NAA and NPA elicited an enhancement. The unilateral and local application of IAA in the growing zone of the sporangiophore elicited in 30 min a moderate negative tropic bending in wild type C2 and mutant C148madC, which was, however, partially masked by a concomitant avoidance response caused by the aqueous buffer. Auxin transport-related genes ubiquitous in plants were found in a BLAST search of the Phycomyces genome. They included members of the AUX1 (auxin influx carrier protein 1), PILS (PIN-LIKES, auxin transport facilitator protein), and ABCB (plant ATP-binding cassette transporter B) families while members of the PIN family were absent. Our observations imply that IAA represents an intrinsic element of the sensory transduction of Phycomyces and that its mode of action must very likely differ in several respects from that operating in plants.     

Oxidation of NADH by hypocotyl segments from soybean is stimulated by 2,4-D

Sections cut from regions of cell elongation of hypocotyls of dark-grown soybean seedlings oxidized externally supplied NADH as estimated from the decrease in A₃₄₀ measured spectrophotometrically. The oxidation of NADH by 1-cm sections was stimulated 1.5- to 2-fold by 1 μM of the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D-Stimulated oxidation of NADH was resistant to cyanide. Stimulations were also given by the naturally occurring auxin, indole-3-acetic acid (IAA) but not by the growth inactive 2,4-D analog 2,3-dichlorophenoxyacetic acid (2,3-D) and the growth inactive β-naphthaleneacetic acid (β-NAA). Since NADH is a membrane impermeant substrate, the findings confirm studies with inside-out and right-side-out vesicles that show the 2,4-D-stimulated NADH oxidase to be located at the external cell surface. Cut surfaces are not responsible for the activity as shown by experiments with lanolin-sealed sections. The external NADH oxidase measurements do not require special equipment and exhibit characteristics normally associated with enzyme-catalyzed reactions.     

Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis

Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components.     

The Arabidopsis concentration-dependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis

Arabidopsis ATP-binding cassette B4 (ABCB4) is a root-localised auxin efflux transporter with reported auxin uptake activity in low auxin concentrations. Results reported here demonstrate that ABCB4 is a substrate-activated regulator of cellular auxin levels. The contribution of ABCB4 to shootward auxin movement at the root apex increases with auxin concentration, but in root hair elongation assays ABCB4-mediated uptake is evident at low concentrations as well. Uptake kinetics of ABCB4 heterologously expressed in Schizosaccharomyces pombe differed from the saturation kinetics of AUX1 as uptake converted to efflux at threshold indole-3-acetic acid (IAA) concentrations. The concentration dependence of ABCB4 appears to be a direct effect on transporter activity, as ABCB4 expression and ABCB4 plasma membrane (PM) localisation at the root apex are relatively insensitive to changes in auxin concentration. However, PM localization of ABCB4 decreases with 1-naphthylphthalamic acid (NPA) treatment. Unlike other plant ABCBs studied to date, and consistent with decreased detergent solubility, ABCB4(pro) :ABCB4-GFP is partially internalised in all cell types by 0.05% DMSO, but not 0.1% ethanol. In trichoblasts, ABCB4(pro) :ABCB4-GFP PM signals are reduced by >200 nm IAA and 2,4-dichlorophenoxyacetic acid (2,4-D). In heterologous systems and in planta, ABCB4 transports benzoic acid with weak affinity, but not the oxidative catabolism products 2-oxindole-3-acetic-acid and 2-oxindole-3-acetyl-β-D-glucose. ABCB4 mediates uptake, but not efflux, of the synthetic auxin 2,4-D in cells lacking AUX1 activity. Results presented here suggest that 2,4-D is a non-competitive inhibitor of IAA transport by ABCB4 and indicate that ABCB4 is a target of 2,4-D herbicidal activity.     

Properties of auxin binding sites in different subcellular fractions from maize coleoptiles

In-vitro binding of labeled auxins to sedimentable particles was tested in subcellular fractions from homogenates of maize (Zea mays L.) coleoptiles. The material was fractionated by differential centrifugation or on sucrose density gradients. It was confirmed that the major saturable binding activity (site I) for 1-naphthyl[1-¹⁴C]acetic acid is associated with vesicles derived from the endoplasmatic reticulum. A second type of specific auxin binding (site II) could be distinguished by several criteria, e.g. by the low affinity towards phenylacetic acid. The particles carrying site II could be clearly separated from markers of the endoplasmatic reticulum, the plasmalemma, the mitochondria and the nuclei, while their density as well as sedimentation velocity correlated with particle-bound acid phosphatase, indicating a localization at the tonoplast. In contrast to site I, binding at site II was hardly affected by a supernatant factor and by sulfhydryl groups. However, the specificity pattern of site II towards auxins and auxin analogs was very similar to that of site I tested in the presence of supernatant factor. The existence of a third auxin receptor localized in plasma membrane-rich gradient fractions was indicated by a preferential in-vitro binding of 2,4-dichlorophenoxyacetic acid.Abbreviations 1-NAA 1-naphthyl acetic acid - 2-NAA 2-naphthyl acetic acid - IAA 3-indolyl acetic acid - PAA phenyl acetic acid - 2,4-D 2,4-D-dichlorophenoxy acetic acid - D-2,4-DP dichlorophenoxy isopropionic acid - NPA 1-N-naphthyl phthalamic acid - ER endoplasmatic reticulum - SF supernatant factor     

Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology

Summary Mutant lines of Arabidopsis thaliana resistant to the artificial auxin 2,4-dichloro phenoxyacetic acid (2,4-D) were isolated by screening for growth of seedlings in the presence of toxic levels of 2,4-D. Genetic analysis of these resistant lines indicated that 2,4-D resistance is due to a recessive mutation at a locus we have designated Axr-1. Mutant seedlings were resistant to approximately 50-fold higher concentrations of 2,4-D than wild-type and were also resistant to 8-fold higher concentrations of indole-3-acetic acid (IAA) than wild-type. Labelling studies with (¹⁴C)2,4-D suggest that resistance was not due to changes in uptake or metabolism of 2,4-D. In addition to auxin resistance the mutants have a distinct morphological phenotype including alterations of the roots, leaves, and flowers. Genetic evidence indicates that both auxin resistance and the morphological changes are due to the same mutation. Because of the pleiotropic morphological effects of these mutations the Axr-1 gene may code for a function involved in auxin action in all tissues of the plant.     

Incubation of corn coleoptiles with auxin enhances in-vitro fusicoccin binding

Binding of fusicoccin (FC) to microsomal preparations of corn (Zea mays L.) coleoptiles is enhanced after incubation of the tissue with indole-3-acetic acid (IAA). Treatment of the kinetic data according to Scatchard shows that the enhancement is a consequence of an increase in the number of high-affinity FC-binding sites without changes of their K_D. The minimal effective concentration of IAA is 10^-7 M; above 10^-5 M the effect declines. The stimulation is insensitive to protein-synthesis inhibitors (cycloheximide and puromycin). The same effect is observed with the synthetic auxins 2,4-dichlorophenoxyacetic acid and naphtalene-1-acetic acid while it is abolished by the auxin antagonists naphtalene-2-acetic acid and p-chlorophenoxyisobutyric acid. Since the above effect is only observed with intact tissue and not after incubation of IAA with microsomal preparations, a direct interaction of IAA with the FC-binding sites is ruled out and an alternative mechanism must be sought.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FC fusicoccin - [³H]FC ³H-labeled dihydrofusicoccin - IAA indole-3-acetic acid - 1-NAA naphtalene-1-acetic acid - 2-NAA naphtalene-2-acetic acid - PCIB p-chlorophenoxyisobutyric acid     

Inhibition of the indole-3-acetic acid-induced epinastic curvature in tobacco leaf strips by 2,4-dichlorophenoxyacetic acid

It has been reported that auxin induces an epinastic growth response in plant leaf tissues. Leaf strips of tobacco (Nicotiana tabacum L. 'Bright Yellow 2') were used to study the effects of indole-3-acetic acid (IAA), the principal form of auxin in higher plants, and a synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), on epinastic leaf curvature. Incubation of leaf strips with 10 micro M IAA resulted in a marked epinastic curvature response. Unexpectedly, 2,4-D showed only a weak IAA-like activity in inducing epinasty. Interestingly, the presence of 2,4-D resulted in inhibition of the IAA-dependent epinastic curvature. In vivo Lineweaver-Burk kinetic analysis clearly indicated that the interaction between IAA and 2,4-D reported here is not a result of competitive inhibition. Using kinetic analysis, it was not possible to determine whether the mode of interaction between IAA and 2,4-D was non-competitive or uncompetitive. 2,4-D inhibits the IAA-dependent epinasty via complex and as yet unidentified mechanisms.     

The electrical response of maize to auxins

The electrical response of Zea mays coleoptiles and suspension cultured cells to several growth-promoting auxins (IAA, IBA, 2,4-D, 2,4,5-T, 1-NAA) and some of their structural analogues (2,3-D, 2-NAA) has been tested. In coleoptile two typical electrical responses to IAA are observed: an immediated rapid depolarization, and a hyperpolarization following 7-10 minutes after the first external addition of IAA. Of the other tested compounds only 1-NAA significantly depolarized the cells, whereas all auxins as well as the analogues evoked delayed hyperpolarizations. In contrast, the suspension cells were not hyperpolarized by any of the tested compounds, but were strongly depolarized by IAA, 1-NAA, and to a lesser extent by 2-NAA. In these cells IAA and 1-NAA induced inwardly directed currents of positive charge which both saturated around 12 mA/m2. The strong pH-dependence together with the half-maximal currents 0.49 microM IAA and 0.76 microM 1-NAA point to a symport of the anions with at least 2H+. The delayed plasma membrane hyperpolarization is a different response, and seems to be initiated by the protonated auxin species. In accordance with the current literature, it is interpreted as consequence of a stimulated proton extrusion. The finding that all tested compounds evoked a hyperpolarization, makes this response unspecific. It is concluded that a stimulation of proton extrusion is a necessary, but not sufficient step to induce elongation growth.     

A soluble auxin-binding protein from mung bean hypocotyls has indole-3-acetaldehyde reductase activity

To clarify the roles of auxin-binding proteins (ABPs) in the action of auxin, soluble auxin-binding proteins were isolated from an extract of etiolated mung bean hypocotyls by affinity chromatography on 2,4-dichlorophenoxyacetic acid (2,4-D)-linked Sepharose 4B. A 39-kDa polypeptide was retained on the affinity column and eluted with a solution containing IAA or 2,4-D, but not with a solution containing benzoic acid. The protein was then purified by several column-chromatographic steps. The apparent molecular mass of the protein was estimated to be 77 kDa by gel filtration and 39 kDa by SDS-PAGE. We designated this protein ABP39. The partial amino acid sequences of ABP39, obtained after chemical cleavage by CNBr, revealed high homology with alcohol dehydrogenase (ADH; EC 1.2.1.1). While the ABP39 was not capable of oxidizing ethanol, it did catalyze the reduction of indole-3-acetaldehyde (IAAld) to indole-3-ethanol (IEt) with an apparent K_m of 22 μ M. The IAAld reductase (EC 1.2.3.1) is specific for NADPH as a cofactor. The ABP39 also catalyzed the reduction of other aldehydes, such as acetaldehyde, benzaldehyde, phenylacetaldehyde and propionealdehyde. Indole-3-aldehyde was a poor substrate. The enzyme activity was inhibited by both indole-3-acetic acid and 2,4-D in a competitive manner. Therefore, the enzyme is considered to be retained on the affinity column by recognition of auxin structure.     

Increased auxin efflux in the IAA-overproducing sur1 mutant of Arabidopsis thaliana: A mechanism of reducing auxin levels?

With the aim of investigating the mechanisms that maintain auxin homeostasis in plants, we have monitored the net uptake and metabolism of exogenously supplied indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA) in seedlings of wild type and the IAA-overproducing mutant sur1 of Arabidopsis thaliana . Tritiated IAA and NAA entered the seedling tissues within minutes and were mostly accumulated as metabolites, probably amino acid and sugar conjugates. The mutant seedlings were marked by a strong increase of [³H]IAA metabolism and a reduction of the accumulation levels of both free [³H]IAA and [³H]NAA. The same characteristics were observed in wild-type seedlings grown on 5 μ M picloram. We measured [³H]NAA uptake in the presence of high concentrations of unlabeled NAA or the auxin efflux carrier inhibitor naphthylphthalamic acid (NPA). This abolished the difference in free [³H]NAA accumulation between the mutant or picloram-treated seedlings and wild-type seedlings. These data indicated that active auxin efflux carriers were present in Arabidopsis seedling tissues. Picloram-treated seedlings and seedlings of the IAA-overproducing mutant sur1 displayed increased auxin efflux carrier activity as well as elevated conjugation of IAA. There is previous evidence to suggest that conjugation is a means to remove excess IAA in plant cells. Here, we discuss the possibility of efflux constituting an additional mechanism for regulating free IAA levels in the face of an excess auxin supply.