Targeting of auxin carriers to the plasma membrane: differential effects of brefeldin A on the traffic of auxin uptake and efflux carriers

Monensin and brefeldin A (BFA), inhibitors of Golgi-mediated protein secretion, rapidly perturb the transport catalytic activity of specific plasma membrane-associated efflux carriers for indole-3-acetic acid (IAA) and inhibit polar transport of IAA. To determine if these responses result solely from perturbation of the efflux carrier or whether specific auxin uptake carrier function is also affected, the influence of BFA on the cellular transport of a range of auxins with contrasting affinities for specific auxin uptake and efflux carriers was investigated in zucchini (Cucurbita pepo L.) hypocotyl tissue. In-flight addition of BFA (3 · 10⁻⁵ mol · dm⁻³) caused a rapid (lag < 10 min) and substantial (fourfold) increase in the rate of [1-¹⁴C]IAA net uptake by zucchini hypocotyl tissue. In the presence of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA; 3 · 10⁻⁶ mol · dm⁻³), BFA slightly reduced the rate of [1-¹⁴C]IAA net uptake. Stimulation of [1-¹⁴C]IAA net uptake by BFA was concentration-dependent. In the absence of BFA, net uptake of [1-¹⁴C]IAA exhibited the characteristic biphasic response to increasing concentrations of competing cold IAA but in the presence of BFA, [1-¹⁴C]IAA uptake decreased smoothly with increase in concentration of competing unlabelled IAA, indicating a loss of auxin efflux carrier activity but retention of functional uptake carriers. The half-time for mediated efflux of [1-¹⁴C]IAA from preloaded zucchini tissue was substantially increased by BFA (t_1/2 = 51 min, controls; 107 min, BFA-treated). Treatment with BFA and/or NPA did not significantly affect the net uptake by, or efflux from, zucchini tissue of [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([1-¹⁴C]2,4-D), a substrate for the auxin uptake carrier but not the auxin efflux carrier. Uptake of [1-¹⁴C]2,4-D declined smoothly with increasing concentrations of competing unlabelled IAA whether or not BFA was included in the uptake medium, confirming the failure of BFA to perturb auxin uptake carrier function. Transport of 1-[4-³H]naphthaleneacetic acid (1-NAA) exhibited little response to BFA or NPA, confirming that it is only a weakly transported substrate for the efflux carrier in zucchini cells. Received: 12 November 1997 / Accepted: 27 January 1998     

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.     

Flavonoids and flavonoid sulphates as probes of auxin-transport regulation in Cucurbita pepo hypocotyl segments and vesicles

The accumulation of IAA by sealed microsomal vesicles prepared from hypocotyls of dark-grown Cucurbita pepo L. (zucchini) seedlings was stimulated by N-1-naphthylphthalamic acid (NPA: an inhibitor of carrier-mediated auxin efflux and hence of polar auxin transport) as well as by quercetin and certain other flavonoids with a specificity pattern similar to that previously shown for their NPA-like effects on auxin transport and inhibition of NPA binding to saturable sites. In contrast, putatively nonpenetrant negatively charged quercetinsulphate esters did not stimulate such auxin accumulation although they were able to oppose stimulation by NPA or quercetin itself. However, the binding of NPA to hypocotyl microsomes was 30- to 80-fold more strongly inhibited by the quercetin sulphates than by unsubstituted quercetin. As with vesicles, net IAA uptake by hypocotyl segments (2 mm) from dark-grown zucchini was stimulated less effectively by quercetin-sulphate esters than by quercetin itself. We discuss the implications of these observations for the accessibility of the NPA receptor from cell wall or cytoplasm and for the coupling of its occupancy to inhibition of the auxin efflux carrier.Abbreviations ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - NPA N-1-naphthylphthalamic acid - PMSF phenylmethylsulphonyl fluoride This work was supported by a Studentship (I.J.F.) from the Science and Engineering Research Council and by the Gatsby Charitable Foundation. We are particularly grateful to Dr. W. Michalke for a preprint and permission to use his method of microsome preparation in advance of publication.     

Physiological evidence that the primary site of auxin action in maize coleoptiles is an intracellular site

To locate functionally the primary site of auxin action in growing cells, the pool of auxin relevant to induction of growth in maize (Zea mays L.) coleoptile sections was determined. A positive correlation was consistently noted between growth and intracellular levels of indole-3-acetic acid (IAA), i.e. growth appears to be relatively independent of the external level of IAA. N-1-Naphthylphthalamic acid (NPA), a potent inhibitor of auxin transport, was used to enhance accumulation of IAA in coleoptile cells. From the use of NPA, it is shown that: 1) increasing the accumulation of IAA in cells, while the external concentration is held constant, resulted in a concomitant increase in growth, and 2) blocking the exit of IAA from cells with NPA sustained an IAA-induced growth response in the absence of externally applied IAA. Furthermore, the absence of any alterations in auxin binding to microsomal fractions by NPA indicates that the action of NPA in causing enhancement of auxin-induced growth is based upon its inhibition of efflux of IAA from the cells. This research was supported by National Science Foundation grant No. DMB 8515925. The careful assistance of Laurie Brulport is gratefully acknowledged.     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

A microtechnique for the analysis of free and conjugated indole-3-acetic acid in milligram amounts of plant tissue using a benchtop gas chromatograph-mass spectrometer

A microtechnique was developed for the quantification of indole-3-acetic acid (IAA) in plant samples of one milligram fresh weight or less. The method permitted quantification of both free and conjugated IAA using a benchtop gas chromatograph-mass spectrometer. New methods for sample purification with high recovery at microscale levels, together with simple changes that result in enhanced sensitivity of the instrumentation, allowed for a significant reduction in the amount of plant material required for analysis. Single oat (Avena sativa L.) coleoptile tips could be studied with this method and were found to contain free and total IAA levels of 137 and 399 pg · mg⁻¹ fresh weight, respectively. A single 5-d-old Arabidopsis thaliana (L.) Heynh. seedling was shown to contain 61 pg · mg⁻¹ fresh weight free IAA and 7850 pg · mg⁻¹ fresh weight of total IAA following basic hydrolysis. This microtechnique provides a way to accurately measure IAA levels in very small structures and individual seedlings, thus making it a valuable research tool for elucidating the role and distribution of auxin in relation to growth and development. Received: 1 May 1994 / Accepted: 25 June 1997     

Characterization of the growth and auxin physiology of roots of the tomato mutant,diageotropica

Roots of the tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([³H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [³H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.Abbreviations BCA bicinchoninic acid - IAA indole 3-acetic acid - dgt diageotropica - IC₅₀ concentration for 50% inhibition of growth - NPA N-1-naphthylphthalamic acid - SCB-1 semicarbazone 1 This research was supported by grants from Sandoz Agro, Inc. (G.K.M), the National Aeronautics and Space Administration (NASA) and the National Science Foundation (T.L.L), and NASA (D.L.R.).     

Arabidopsis AtGSTF2 is regulated by ethylene and auxin, and encodes a glutathione S-transferase that interacts with flavonoids

Expression of the Arabidopsis glutathione S-transferase (GST) gene AtGSTF2 is induced by several stimuli, but the function of this GST remains unknown. We demonstrate that AtGSTF2 expression is also induced by glutathione, paraquat, copper, and naphthalene acetic acid (NAA) via a mechanism independent of ethylene perception, as determined by analysis of the ethylene-insensitive etr1 mutant. Deletion analyses identified two promoter regions important for regulation of AtGSTF2 expression in response to several of these inducers. Previous studies have suggested that AtGSTF2 interacts with indole-3-acetic acid (IAA) and the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA). We show that recombinant AtGSTF2 directly binds IAA, NPA, and the artificial auxin NAA. As NPA may act as an endogenous flavonoid regulator of auxin transport, competition between NPA and flavonoids for binding to AtGSTF2 was examined. Both quercetin and kaempferol competed with NPA for AtGSTF2 binding, indicating that all three compounds bind AtGSTF2 at the same site. In transgenic Arabidopsis seedlings, AtGSTF2::GUS expression occurred at the root-shoot transition zone and was induced in this region, as well as at the root distal elongation zone, after treatment with IAA. In wild-type seedlings, AtGSTF2 is localized near the plasma membrane of cells in the root-shoot transition zone. However, both AtGSTF2::GUS expression and localization of AtGSTF2 protein were disrupted in flavonoid-deficient tt4 seedlings. Our results indicate that AtGSTF2 is involved not only in stress responses but also in development under normal growth conditions.     

Gravity-induced modification of auxin transport and distribution for peg formation in cucumber seedlings: possible roles for CS-AUX1 and CS-PIN1

Cucurbit seedlings potentially develop a peg on each side of the transition zone between the hypocotyl and root. Seedlings grown in a horizontal position suppress the development of the peg on the upper side of the transition zone in response to gravity. It is suggested that this suppression occurs due to a reduction in auxin levels to below the threshold value. We show in this study that the free indole-3-acetic acid (IAA) content is low, while IAA conjugates are significantly more abundant in the upper side of the transition zone of gravistimulated seedlings, compared to the lower side. A transient increase in mRNA of the auxin-inducible gene, CS-IAA1, was observed in the excised transition zone. The result suggests that the transition zone is a source of auxin. Cucumber seedlings treated with auxin-transport inhibitors exhibited agravitropic growth and developed a peg on each side of the transition zone. Auxin-transport inhibitors additionally caused an increase in CS-IAA1 mRNA accumulation at the transition zone, indicating a rise in intracellular auxin concentrations due to a block of auxin efflux. To study the involvement of the auxin transport system in peg formation, we isolated the cDNAs of a putative auxin influx carrier, CS-AUX1, and putative efflux carrier, CS-PIN1, from cucumber (Cucumis sativus L.) plants. Both genes (CS-AUX1 in particular) were auxin-inducible. Accumulation of CS-AUX1 and CS-PIN1 mRNAs was observed in vascular tissue, cortex and epidermis of the transition zone. A reduced level of CS-AUX1 mRNA was observed in the upper side of the gravistimulated transition zone, compared with the lower side. It is therefore possible that a balance in the activities of auxin influx and efflux carriers controls intracellular auxin concentration at the transition zone, which results in lateral placement of a peg in cucumber seedlings.Abbreviations HFCA 9-hydroxyfluorene-9-carboxylic acid - IAA indole-3-acetic acid - NPA 1-N-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid