Patch‐clamp analysis establishes a role for an auxin binding protein in the auxin stimulation of plasma membrane current in Zea mays protoplasts

Abstract

The electrical response of Zea mays protoplasts to different auxins and to antibodies raised against an ER‐located auxin binding protein from maize (Zm‐ERabp1), was investigated using the patch‐clamp technique (whole‐cell configuration). Following a lag‐phase of 30–40 seconds, indole‐3‐acetic acid and 1‐naphthylacetic acid induced an outwardly directed current of positive charge in a concentration‐dependent manner. This current was further increased by the fungal toxin fusicoccin (FC). The current was observed only in the presence of Mg2+‐ATP in the patch‐pipette and was abolished after addition of erythrosin B, an inhibitor of H+‐ATPase, to the protoplasts indicating that the plasma membrane H+‐ATPase is activated by auxins and fusicoccin. Addition of antibodies directed against Zm‐ERabp1 abolished the current induced by auxins, without affecting the response of protoplasts to fusicoccin. Antibodies directed against a peptide representing part of the putative auxin binding domain of Zm‐ERabp1 showed auxin agonist activity, stimulating an outwardly directed membrane current in the absence of auxin. These results suggest that (i) Zm‐ERabp1 or antigenically related proteins represent a site for auxin perception through which the plasma membrane H+‐ATPase is activated, and (ii) that the activation of the H+‐ATPase by such proteins is initiated from outside the plasma membrane.

Cellular Responses to Auxin: Division versus Expansion

“…Various lines of evidence, mainly relying on electrophysiological experiments and use of a whole series of ABP1 molecular tools (specific monoclonal and polyclonal antibodies, carboxy-terminal synthetic peptides, recombinant wild-type and mutated proteins) support the hypothesis that ABP1 is involved in the initial perception of auxin at the outer face of the plasma membrane and induces the activation of H þ ATPase and K þ inward channels (Rück et al 1993;Thiel et al 1993;Barbier-Brygoo et al 1996;Fellner et al 1996;Leblanc et al 1999a;Leblanc et al 1999b;David et al 2001) (Fig. 3).…”

“…This rapid effect is believed to result from the activation of a proton pump ATPase at the plasma membrane, inducing extrusion of H þ , extracellular acidification, activation of expansins, and subsequent wall loosening. Activation of the plasma membrane H þ ATPase causes hyperpolarization of the membrane potential and activation of voltage-dependent K þ inward channels (Rück et al 1993;Philippar et al 1999;Thiel and Weise 1999;Philippar et al 2004). Uptake of K þ is likely to contribute to the water uptake necessary to sustain expansion.…”

ABP1 is required for organized cell elongation and division in Arabidopsis embryogenesis

“…An informative array of compounds was used to propose a model of the auxin-binding site indicating the structural constraints expected for a receptor (Edgerton et al 1994). Antibodies directed against ABP1 as well as ABP1 peptide mimetics modulate auxin-regulated plasma membrane hyperpolarization, the earliest event in auxin-induced cell wall loosening (Barbier-Brygoo et al 1989Ruck et al 1993;Thiel et al 1993;LeBlanc et al 1999). Furthermore, ectopic and inducible expression of ABP1 confers auxin-dependent cell expansion in cells normally lacking auxin responsiveness (Jones et al 1998).…”

A new tool for plant cell biology: in vivo antibody uptake in plant protoplasts

“…In plants, the penetration of large molecules into cells is strongly limited by the presence of the cell wall, whose porosity limit for globular proteins has been estimated at about 120 kDa (Baron-Epel et al 1988). To date, in vivo experiments with antibodies used as blocking agents were limited to studies on transmembrane receptors in symbiosomes (Moreau et al 2002) or in protoplasts (Barbier-Brygoo et al 1989;Barbier-Brygoo et al 1991;Rück et al 1993;Leonhardt et al 2001), or to the use of Fab fragments of lower molecular weight (Gehring et al 1998). In this article, we report on the penetration of antibodies into living protoplasts and on their use as blocking agents to study the function of intracellular target proteins.…”