Auxin Effect on the Transmembrane Potential Difference of Wild-Type and Mutant Tobacco Protoplasts Exhibiting a Differential Sensitiity to Auxin

Abstract

The effects of 1-naphthaleneacetic acid (NAA) and other auxin analogs on the transmembrane potential difference (Em) were compared on tobacco protoplasts isolated from two genotypes differing in their sensitivity to auxins. For both types, NAA modifies Em by inducing at low doses a hyperpolarization, the amplitude of which increased with auxin concentration. Above an optimal concentration this hyperpolarization was reduced and even nullified. However, for the mutant type, this electrical response was shifted toward higher NAA concentrations, as its growth response. In the presence of structural analogs of auxin which have been showed to modify the dose-response curve for growth, the Em was altered: the growth-stimulatory molecule (picloram) initiated hyperpolarization, whereas the growth-inhibitory substance (4-bromophenylacetic acid) caused depolarization. These results provide evidence for a specific action of auxin at the membrane level related to its biological activity.There is considerable evidence for interactions between auxin and membranes. Ultrastructural alterations were induced by auxin in isolated and in situ soybean plasma membranes (22). Modifications of the physical properties of membranes after auxin treatment have been reported. These include an increase in the microviscosity of lipid bilayers (17) or plasma membrane vesicles (13) and changes in electrostatic surface properties of protoplasts (29). Finally, a specific binding of auxin to membranes isolated from maize coleoptiles (9) and tobacco cell suspensions (30) tiles (24).Causal links between these effects of auxin on membrane properties and the biological activity of auxin are difficult to establish. Very recently, binding of auxin at the plasmalemma of epidermal cells has been reported to be related to the elongation and the curling of corn coleoptiles (18). In other studies, the auxin-induced proton extrusion has been correlated to the elongation rate of shoots (12, 14, 25) and the control of root growth (10, 21) on the basis of similarities between the timing of the responses, the dependency upon auxin concentration, and the specificity of the response to auxin analogs. Such results provide evidence for the existence of a relationship between the action of auxin on membranes and the overall activity of this hormone on cell enlargement. Up to now, the involvement of hormoneinduced modifications of membranes into the control of cell division and differentiation has not been directly demonstrated.A novel opportunity to study the basis ofthe biological activity ofauxins at the cellular level was recently offered by the selection of a NAA-tolerant mutant from tobacco mesophyll protoplasts (5, 23). The wild-type (clone D8) and the mutant (clone 36) differ in their sensitivity to auxins as measured by the ability of the cells derived from protoplasts to proliferate in the presence of NAA. In this paper we test the hypothesis that the wild-type and mutant protoplasts differ in their membrane properties. A comparative study of the effe...

Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts

“…Such a depolarized state of protoplasts was recently shown to result from an increased anion efflux through the plasma membrane, fed by the leakage of chloride from the electrode tip (Shen et a/., 1990). Despitc this drawback, microelectrodes filled with 1 M KCI were used in this study, as they provided reliable monitoring of the auxin-induced variations of the Em of isolated protoplasts (Ephritikhine et a/., 1987;Shen eta/., 1988Shen eta/., , 1990). …”

“…Tobacco protoplasts isolated from mesophyll cells have been shown to respond to several active auxin analogs by changing their transmembrane Em (Ephritikhine et a/., 1987). To address the relevance of these measurements, we have investigated the specificity of both inactive and active auxins.…”

“…The auxin-induced electrical response displays a bell-shaped dose-response curve (Ephritikhine eta/., 1987;Shen eta/., 1988), from which the sensitivity of protoplasts to auxins can be deduced and expressed as the hormone concentration inducing the maximal membrane hyperpolarization. Using this membrane response, tobacco leaf protoplasts isolated from an auxinresistant mutant were shown to be 10-times less sensitive to auxin than wild-type protoplasts (Ephritikhine et a/., 1987); in contrast, protoplasts isolated from roots of different species transformed by Agrobacterium rhizogenes were 100-1 000-times more sensitive to the hormone than protoplasts isolated from normal roots (Shen eta/., 1988(Shen eta/., , 1990. In this report, we demonstrate that antibodies directed against the ABP from maize or the maize axr' gene product (ABPI ; Hesse eta/., 1989) recognize an immunologically related protein on tobacco protoplasts.…”

“…In the search for a cellular assay for auxin activity, we demonstrated that auxin induces variations of the log [effector] ( M ) transmembrane Em (electrical potential difference) of isolated protoplasts (Ephritikhine et a/., 1987). This membrane response of protoplasts to auxin, which is specifically inhibited by antibodies directed against auxin binding proteins (ABP) from maize coleoptiles (BarbierBrygoo et a/., 1989), and can be fully induced by nonpermeating auxin analogs (Venis et a/., 1990), probably involves the occupancy of an auxin-responsive protein at the cell surface.…”

“…However, the mechanisms of auxin action remain to be determined. Experimental and theoretical approaches to understanding auxin action have caused a debate concerning the relative importance of variations in auxin concentration versus differential sensitivity of different plant cells to this phytohormone (Cleland, 1983;Firn, 1986;Guern, 1987;Trewavas, 1982Trewavas, , 1983. Experimental evidence with different plant systems failed to demonstrate clear correlations between phytohormone concentrations and changes in development, suggesting that phytohormone concentration was not rate limiting.…”