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

Abstract

SummaryAuxin-induced variations of transmembrane potential difference have been shown to be a useful tool for analyzing hormone sensitivity in tobacco protoplasts.Using this technique, we demonstrated that protoplasts derived from wild-type, an auxin-resistant mutant and Agrobacterium-rhizogenes transformed plants differed widely in the sensitivity of their electrical response to naphthalene acetic acid. We have used different antibodies, raised to auxin binding proteins (ABP) from maize coleoptiles, or to the axr' gene product (ABPI), to test whether changes in auxin sensitivity can be correlated with the presence of tobacco proteins immunologically related to this ABP. Titrations indicated that 0.4 nM anti-ABP IgG inhibited 50% of the auxin-specific response of wild-type protoplasts, whereas 0.04 nM or 4 nM anti-ABP IgG were necessary to inhibit the response of mutant and transformed protoplasts, respectively, to the same extent. On wild-type protoplasts, blocking part of the immunoreactive sites with anti-ABP antibodies resulted in a decrease in auxin sensitivity of the electrical response (0.4 nM anti-ABP IgG inducing a 10-fold decrease), whereas addition of maize ABP increased this auxin sensitivity (1 pM ABPl raised the sensitivity more than 1000-fold). The results obtained suggest that the auxin sensitivity detected by our assay system correlates with the amount of tobacco proteins immunologically related to the axr' gene product from maize. A hypothesis accounting for the presence of these proteins at the external surface Of tobacco protoplasts and for the effects of heterologous maize ABP on auxin sensitivity is proposed.

Alterations of Auxin Perception in rolB-Transformed Tobacco Protoplasts (Time Course of rolB mRNA Expression and Increase in Auxin Sensitivity Reveal Multiple Control by Auxin)

“…Protoplasts from tobacco plants transfonned by Agrobacterium rhizogenes T-DNA, especially by the single rolB gene, exhibit an increased sensitivity of their electrical response to auxin (Maurel et al, 1991b). This finding is in agreement with a number of reports in which an increased sensitivity to auxin was described for excised organs, isolated protoplasts, or membrane vesicles from various plant materials containing the whole pRi T-DNA or some of its subfragments (Shen et al, 1988(Shen et al, , 1990; Spano et al, 1988;Barbier-Brygoo et al, 1991;Vansuyt et al, 1992). These findings suggested that morphogenetic effects of pRi T-DNA transformation might involve changes in hormone responsiveness and/or hormone content.…”

“…The E, of protoplasts was measured under the microscope by the microelectrode te$nique as describeli previously (Ephritikhine et al, 1987;Barbier-Brygoo et a]., 1991). For each NAA concentration tested, 15 to 20 individual measurements were performed at room temperature on an aliquot of the protoplast stock solution diluted to 5 X 104 protoplasts/ mL in To medium with the appropriate NAA dose, and a mean E, value was calculated.…”

AUXIN BINDING PROTEIN1: The Outsider

“…In the same year, it was also proven that ABP1 indeed binds auxin (Jones and Venis, 1989), a result that was later corroborated by the crystal structure determination of ABP1 cocrystallized with auxin (Woo et al 2002). ABP1 antibodies and peptides generated in these biochemical studies were increasingly used for physiological studies in heterologous systems, showing auxin induction of cellular processes such as membrane hyperpolarization (Barbier-Brygoo et al, 1991;Venis et al, 1992), K + fluxes (Thiel et al, 1993), or cell expansion, as evidenced by protoplast swelling assays (Steffens et al, 2001). The importance and function of ABP1 for the plant as a whole remained unclear because attempts to modulate ABP1 protein amounts (particularly, its' downregulation) proved difficult in planta.…”

Cellular Responses to Auxin: Division versus Expansion

“…Auxin is one of the major stimuli affecting these mechanisms but it is essential to keep in mind that cell expansion is also under the control of many other stimuli, such as blue light and most of the other phytohormones. Auxin-dependent cell expansion follows a dose-response curve in which high concentrations are inhibitory (Barbier-Brygoo et al 1991;Evans et al 1994). The apparent response-maxima varies according to both the plant and the organ with shoots and roots showing differences in sensitivity of several orders of magnitude.…”