ORIGINAL ARTICLE Jan Petra´ sˇek Æ Miroslav Elcˇ kner Æ David A. Morris Eva Zazˇ ı´ malova´ Auxin efflux carrier activity and auxin accumulation regulate cell division and polarity in tobacco cells Received: 19 January 2002 / Accepted: 3 June 2002 / Published online: 14 August 2002 Ó Springer-Verlag 2002 Abstract Division and growth of most types of in vitro- cultured plant cells require an external source of auxin. In such cultures, the ratio of external to internal auxin concentration is crucial for the regulation of the phases of the standard growth cycle. In this report the internal concentration of auxin in suspension-cultured cells of Nicotiana tabacum L., strain VBI-0, was manipulated either (i) by increasing 10-fold the normal concentration of 1-naphthaleneacetic acid (NAA) and 2,4-dichloro- phenoxyacetic acid in the external medium; or (ii) by addition 1-N-naphthylphthalamic acid (NPA; an inhib- itor of auxin efflux and of auxin efflux carrier traffic). Both treatments delayed the onset of cell division for 6–7 days without loss of cell viability. In both cases, cell division activity subsequently resumed coincident with a reduction in the ability of cells to accumulate [ 3 H]NAA from an external medium. Following renewed cell divi- sion, a significant proportion of the NPA-treated cells but not those grown at high auxin concentration, ex- hibited changes in the orientation of new cell divisions and loss of polarity. We conclude that cell division, but not cell elongation, is prevented when the internal auxin concentration rises above a critical threshold value and that the directed traffic of auxin efflux carriers to the plasma membrane may regulate the orientation of cell divisions. Keywords Auxin carrier Æ 1-N-Naphthylphthalamic acid Æ Nicotiana (cell culture) Æ Phytotropin Æ Polar auxin transport Abbreviations 2,4-D: 2,4-dichlorophenoxyacetic acid Æ IAA: indole-3-acetic acid Æ NAA: 1-naphthaleneacetic acid Æ NPA: 1-N-naphthylphthalamic acid Introduction The polar transport of auxins [indole-3-acetic acid (IAA) and related compounds] plays a key role in the regula- tion of auxin-dependent growth and developmental processes. Mediated auxin influx into individual cells is catalysed by specific auxin-anion uptake carriers, whilst efflux is catalysed by a different auxin-anion efflux car- rier system (Rubery and Sheldrake 1974; Raven 1975; reviewed by Goldsmith 1977). Biochemical, physiologi- cal and molecular evidence indicates that the polarity of auxin transport through cells and tissues results from the polarised distribution of auxin efflux carriers in the plasma membrane (reviewed by Bennett et al. 1998; Morris 2000). Available evidence indicates that in con- trast to the auxin uptake carrier, the efflux carrier is a much more complex system consisting of the transport catalyst itself and one or more associated regulatory proteins (Morris et al. 1991; Muday 2000). One of these regulatory proteins is believed to be a specific binding protein for phytotropins [1-N-naphthylphthalamic acid (NPA) and related compounds], which may be associ- ated with the actin cytoskeleton (Muday 2000; Muday and DeLong 2001; Muday and Murphy 2002). Phyto- tropins are potent non-competitive inhibitors of auxin efflux (and, consequently, stimulators of net auxin ac- cumulation) and of polar auxin transport (Rubery 1990). Cell-suspension cultures provide good model systems in which the effects of growth substances on cell division and growth can be studied directly. In such cultures the rate and intensity of cell division are regulated, among other things, by changes in the external and internal concentrations of two essential phytohormones, auxins and cytokinins. Manipulation of the levels of these hormones in tobacco cell cultures (and, consequently, Planta (2002) 216: 302–308 DOI 10.1007/s00425-002-0845-y J. Petra´sˇek Æ M. Elcˇkner Æ D.A. Morris Æ E. Zazˇı´malova´ (&) Institute of Experimental Botany, Rozvojova´ 135, 16502 Prague 6 – Lysolaje, Czech Republic E-mail: eva.zazim@ueb.cas.cz Fax: +420-2-20390474 Present address: D.A. Morris Division of Cell Sciences, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, UK