Membrane-Bound Receptors for Plant Hormones

There are adequate grounds for believing in the existence of nuclear or cytoplasmic receptors mediating transcriptional or translational effects of plant hormones, and several putative receptor proteins have received limited experimental study [see Venis (1973) for review and also Fox & Erion (1975)l. At the same time many physiological responses to plant hormones, especially to auxins, take place within minutes and would suggest hormonal action at the cell surface, probably the cell membrane. A currently favoured hypothesis of auxin action (Hager et al., 1971) suggests that auxins act as effectors of a membrane-bound adenosine triphosphatase which promotes the transfer of protons outwards across the membrane into the cell wall. There is certainly good experimental evidence with excised tissue sections demonstrating rapid proton extrusion in response to auxin application (Rayle, 1973 ; Cleland, 1973). The studies by Hertel and co-workers (Lembi e t al., 1971 ; Hertel e t al., 1972; Hertel, 1974) demonstrated binding of auxins and an auxin-transport inhibitor to heterogeneous membrane preparations from maize coleoptiles and afforded the first direct evidence for auxin-membrane interaction. The extent of saturable auxin binding relative to nonspecific binding was rather low, even over the wide concentration range studied (>lOOO-fold), permitting only tentative estimates of dissociation constants to be made. With several improvements on the original technique it has proved possible to enhance greatly the amounts of saturable binding and hence obtain more reliable kinetics. We are able to resolve two distinct sets of auxin-binding sites, which differ in their specificities. Homogenates of maize coleoptiles (Zea mays, var. Kelvedon 33) are subjected to differential centrifugation (generally 4000-38000g), yielding a crude membrane preparation. After resuspension in buffer at pH 5.5, the synthetic auxin naphth-I -yl[l-14C]acetic acid is added and the suspension is divided into a series of samples to which unlabelled naphth-l -ylacetic acid is added to the desired final concentration (usual range 0.2-1 PM). Triplicate samples are taken from each sample and membranes are repelleted by high-speed centrifugation. The supernatants are decanted and the drained pellets are rinsed, resuspended in water and counted for radioactivity. Fig. I(a) illustrates the raw data from a typical binding experiment, demonstrating saturation of binding with increasing naphth-1 -ylacetic acid concentration. Scatchard analysis of thesedata (Scatchard, 1949) yields a biphasic plot, indicating the presence of