Phytotropins such as 1-N-naphthylphthalamic acid (NPA) strongly inhibit auxin efflux, but the mechanism of this inhibition remains unknown. Auxin efflux is also strongly decreased by the vesicle trafficking inhibitor brefeldin A (BFA). Using suspension-cultured interphase cells of the BY-2 tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line, we compared the effects of NPA and BFA on auxin accumulation and on the arrangement of the cytoskeleton and endoplasmic reticulum (ER). The inhibition of auxin efflux (stimulation of net accumulation) by both NPA and BFA occurred rapidly with no measurable lag. NPA had no observable effect on the arrangement of microtubules, actin filaments, or ER. Thus, its inhibitory effect on auxin efflux was not mediated by perturbation of the cytoskeletal system and ER. BFA, however, caused substantial alterations to the arrangement of actin filaments and ER, including a characteristic accumulation of actin in the perinuclear cytoplasm. Even at saturating concentrations, NPA inhibited net auxin efflux far more effectively than did BFA. Therefore, a proportion of the NPA-sensitive auxin efflux carriers may be protected from the action of BFA. Maximum inhibition of auxin efflux occurred at concentrations of NPA substantially below those previously reported to be necessary to perturb vesicle trafficking. We found no evidence to support recent suggestions that the action of auxin transport inhibitors is mediated by a general inhibition of vesicle-mediated protein traffic to the plasma membrane.

Institute of Experimental Botany The Academy of Sciences of the Czech Republic Rozvojová 135 CZ 16502 Prague 6 Czech Republic

Zobrazit více v PubMed

An G. High efficiency transformation of cultured tobacco cells. Plant Physiol. 1985;79:568–570. PubMed PMC

Batoko H, Zheng HQ, Hawes C, Moore I. A Rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants. Plant Cell. 2000;12:2201–2217. PubMed PMC

Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA, Walker AR, Schulz B, Feldmann KA. Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science. 1996;273:948–950. PubMed

Bernasconi P, Patel BC, Reagan JD, Subramanian MV. The N-1-naphthylphthalamic acid-binding protein is an integral membrane protein. Plant Physiol. 1996;111:427–432. PubMed PMC

Boevink P, Martin B, Oparka K, Santa Cruz S, Hawes C. Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A. Planta. 1999;208:392–400.

Boevink P, Oparka K, Santa Cruz S, Martin B, Betteridge A, Hawes C. Stacks on tracks: The plant Golgi apparatus traffics on an actin/ER network. Plant J. 1998;15:441–447. PubMed

Boevink P, Santa Cruz S, Hawes C, Harris N, Oparka KJ. Virus-mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plant cells. Plant J. 1996;10:935–941.

Butler JH, Hu SQ, Brady SR, Dixon MW, Muday GK. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls. Plant J. 1998;13:291–301. PubMed

Cande WZ, Goldsmith MHM, Ray PM. Polar auxin transport and auxin-induced elongation in the absence of cytoplasmic streaming. Planta. 1973;111:279–296. PubMed

Cox DN, Muday GK. NPA binding activity is peripheral to the plasma membrane and is associated with the cytoskeleton. Plant Cell. 1994;6:1941–1953. PubMed PMC

Davies PJ. The plant hormone concept: concentration, sensitivity and transport. In: Davies PJ, editor. Plant Hormones: Physiology, Biochemistry and Molecular Biology. Ed 2. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1995. pp. 13–38.

Delbarre A, Muller P, Guern J. Short-lived and phosphorylated proteins contribute to carrier-mediated efflux, but not to influx, of auxin in suspension-cultured tobacco cells. Plant Physiol. 1998;116:833–844. PubMed PMC

Delbarre A, Muller P, Imhoff V, Guern J. 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. Planta. 1996;198:532–541. PubMed

Depta H, Eisele K-H, Hertel R. Specific inhibitors of auxin transport: action on tissue segments and in vitro binding to membranes from maize coleoptiles. Plant Sci Lett. 1983;31:181–192.

Depta H, Rubery PH. A comparative study of carrier participation in the transport of 2,3,5-triiodobenzoic acid, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid by Cucurbita pepo L. hypocotyl segments. J Plant Physiol. 1984;115:371–387. PubMed

Dixon MW, Jacobson JA, Cady CT, Muday GK. Cytoplasmic orientation of the naphthylphthalamic acid-binding protein in zucchini plasma membrane vesicles. Plant Physiol. 1996;112:421–432. PubMed PMC

Friml J, Palme K. Polar auxin transport—old questions and new concepts? Plant Mol Biol. 2002;49:273–284. PubMed

Gälweiler L, Guan C, Müller A, Wisman E, Mendgen K, Yephremov A, Palme K. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science. 1998;282:2226–2230. PubMed

Geldner N, Friml J, Stierhof YD, Jürgens G, Palme K. Auxin transport inhibitors block PIN 1 cycling and vesicle trafficking. Nature. 2001;413:425–428. PubMed

Gil P, Dewey E, Friml J, Zhao Y, Snowden KC, Putterill J, Palme K, Estelle M, Chory J. BIG: a calossin-like protein required for polar auxin transport in Arabidopsis. Genes Dev. 2001;15:1985–1997. PubMed PMC

Goldsmith MHM. The polar transport of auxin. Annu Rev Plant Physiol. 1977;28:439–478.

Haseloff J, Siemering KR, Prasher DC, Hodge S. Removal of a cryptic intron and subcellular localisation of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA. 1997;94:2122–2127. PubMed PMC

Hasenstein KH, Blancaflor EB, Lee JS. The microtubule cytoskeleton does not integrate auxin transport and gravitropism in maize roots. Physiol Plant. 1999;105:729–738. PubMed

Hawes C, Saint-Jore C, Martin B, Zheng HQ. ER confirmed as the location of mystery organelles in Arabidopsis plants expressing GFP! Trends Plant Sci. 2001;6:245–246. PubMed

Henderson J, Satiat-Jeunemaitre B, Napier R, Hawes C. Brefeldin A-induced disassembly of the Golgi apparatus is followed by disruption of the endoplasmic reticulum in plant cells. J Exp Bot. 1994;45:1347–1351.

Hu SQ, Brady SR, Kovar DR, Staiger CJ, Clark GB, Roux SJ, Muday GK. Identification of plant actin binding proteins by F-actin affinity chromatography. Plant J. 2000;24:127–137. PubMed

Kakimoto T, Shibaoka H. Actin filaments and microtubules in the preprophase band and phragmoplast of tobacco cells. Protoplasma. 1987;140:151–156.

Katekar GF, Geissler AE. Auxin transport inhibitors: IV. Evidence of a common mode of action for a proposed class of auxin transport inhibitors: the phytotropins. Plant Physiol. 1980;66:1190–1195. PubMed PMC

Li HM, Altschmied L, Chory J. Arabidopsis mutants define downstream branches in the phototransduction pathway. Genes Dev. 1994;8:339–349. PubMed

Lomax TL, Muday GK, Rubery PH. Auxin transport. In: Davies PJ, editor. Plant Hormones: Physiology, Biochemistry and Molecular Biology. Ed 2. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1995. pp. 509–530.

Luschnig C. Auxin transport: why plants like to think BIG. Curr Biol. 2001;11:831–833. PubMed

Mizuno K. Induction of cold stability of microtubules in cultured tobacco cells. Plant Physiol. 1992;100:740–748. PubMed PMC

Morris DA. Transmembrane auxin carrier systems—dynamic regulators of polar auxin transport. Plant Growth Regul. 2000;32:161–172. PubMed

Morris DA, Robinson JS. Targeting of auxin carriers to the plasma membrane: differential effects of brefeldin A on the traffic of auxin uptake and efflux carriers. Planta. 1998;205:606–612.

Morris DA, Rubery PH, Jarman J, Sabater M. Effects of inhibitors of protein synthesis on transmembrane auxin transport in Cucurbita pepo L. hypocotyl segments. J Exp Bot. 1991;42:773–783.

Müller A, Guan CH, Gälweiler L, Tänzler P, Huijser P, Marchant A, Parry G, Bennett M, Wisman E, Palme K. AtPIN2 defines a locus of Arabidopsis for root gravitropism control. EMBO J. 1998;17:6903–6911. PubMed PMC

Muday GK. Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton. J Plant Growth Regul. 2000;19:385–396. PubMed

Muday GK, DeLong A. Polar auxin transport: controlling where and how much. Trends Plant Sci. 2001;6:535–542. PubMed

Muday GK, Murphy AS. An emerging model of auxin transport regulation. Plant Cell. 2002;14:293–299. PubMed PMC

Nagata T, Nemoto Y, Hasezava S. Tobacco BY-2 cell line as the “Hela” cell in the cell biology of higher plants. Int Rev Cytol. 1992;132:1–30.

Nebenführ A, Gallagher LA, Dunahay TG, Frohlick JA, Mazurkiewicz AM, Meehl JB, Staehelin LA. Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system. Plant Physiol. 1999;121:1127–1141. PubMed PMC

Olyslaegers G, Verbelen JP. Improved staining of F-actin and co-localization of mitochondria in plant cells. J Microsc Oxford. 1998;192:73–77.

Petrášek J, Elčkner M, Morris DA, Zažímalová E. Auxin efflux carrier activity and auxin accumulation regulate cell division and polarity in tobacco cells. Planta. 2002;216:302–308. PubMed

Raven JA. Transport of indoleacetic acid in plant cells in relation to pH and electrical potential gradients, and its significance for polar IAA transport. New Phytol. 1975;74:163–174.

Richards S, Hillman T, Stern M. Mutations in the Drosophila pushover gene confer increased neuronal excitability and spontaneous synaptic vesicle fusion. Genetics. 1996;142:1215–1223. PubMed PMC

Ritzenthaler C, Nebenführ A, Movafeghi A, Stussi-Garaud C, Behnia L, Pimpl P, Staehelin LA, Robinson DG. Reevaluation of the effects of brefeldin A on plant cells using tobacco bright yellow 2 cells expressing Golgi-targeted green fluorescent protein and COPI antisera. Plant Cell. 2002;14:237–261. PubMed PMC

Robinson JS, Albert AC, Morris DA. Differential effects of brefeldin A and cycloheximide on the activity of auxin efflux carriers in Cucurbita pepo L. J Plant Physiol. 1999;155:678–684.

Rubery PH. Phytotropins: receptors and endogenous ligands. Symp Soc Exp Biol. 1990;44:119–146. PubMed

Rubery PH, Sheldrake AR. Carrier-mediated auxin transport. Planta. 1974;188:101–121. PubMed

Ruegger M, Dewey E, Hobbie L, Brown D, Bernasconi P, Turner J, Muday G, Estelle M. Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects. Plant Cell. 1997;9:745–757. PubMed PMC

Satiat-Jeunemaitre B, Steele C, Hawes C. Golgi-membrane dynamics are cytoskeleton dependent: a study on Golgi stack movement induced by brefeldin A. Protoplasma. 1996;191:21–33.

Saint-Jore CM, Evins J, Batoko H, Brandizzi F, Moore I, Hawes C. Redistribution of membrane proteins between the Golgi apparatus and endoplasmatic reticulum in plants is reversible and not dependent on cytoskeletal networks. Plant J. 2002;29:661–678. PubMed

Sussman MR, Gardner G. Solubilization of the receptor for N-1-naphthylphthalamic acid. Plant Physiol. 1980;66:1074–1078. PubMed PMC

Swarup R, Friml J, Marchant A, Ljung K, Sandberg G, Palme K, Bennett M. Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Genes Dev. 2001;15:2648–2653. PubMed PMC

Waller F, Riemann M, Nick P. A role of actin-driven secretion in auxin-induced growth. Protoplasma. 2002;219:72–81. PubMed

Wick SM, Seagull RW, Osborn M, Weber K, Gunning BES. Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells. J Cell Biol. 1981;89:685–690. PubMed PMC

Wilkinson S, Morris DA. Targeting of auxin carriers to the plasma membrane: effects of monensin on transmembrane auxin transport in Cucurbita pepo L. tissue. Planta. 1994;193:194–202.

Tubulin is actively exported from the nucleus through the Exportin1/CRM1 pathway

ACCERBATIN, a small molecule at the intersection of auxin and reactive oxygen species homeostasis with herbicidal properties

cis-Cinnamic Acid Is a Novel, Natural Auxin Efflux Inhibitor That Promotes Lateral Root Formation

Auxin flow-mediated competition between axillary buds to restore apical dominance

Inhibitors of plant hormone transport

The Allelochemical MDCA Inhibits Lignification and Affects Auxin Homeostasis

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics

NtGNL1a ARF-GEF acts in endocytosis in tobacco cells

Overexpression of the auxin binding protein1 modulates PIN-dependent auxin transport in tobacco cells

Formins: emerging players in the dynamic plant cell cortex

Auxin transport at cellular level: new insights supported by mathematical modelling

Auxin influx inhibitors 1-NOA, 2-NOA, and CHPAA interfere with membrane dynamics in tobacco cells

Cytokinins modulate auxin-induced organogenesis in plants via regulation of the auxin efflux

Intranuclear accumulation of plant tubulin in response to low temperature