Partial Characterization of Fusicoccin Binding to Receptor Sites on Oat Root Membranes

Abstract

The possibility that fusicoccin (FC) binds to plasma membrane-associated ATPases of oat (cv. Victory) roots has been examined. Specific FC-binding in viro is localized primarily on plasma membrane-enriched fractions. This FC-binding is greatly reduced by pretreatment of the membrane vesicles at temperatures above 45 C or with trypsin, and the same treatments cause the release of already bound FC. These results support the idea that the FC receptor is a protein located on the plasma membrane.Both active ATPases and FC-bndlng proteins were solubilized using 1% Triton X-100. When this material was fractionated using gel chromatography, the ATPase activity could be separated from the FC-bnding proteins. The identity of the FC-binding proteins is discussed with regard to the extensive evidence which supports the involvement of plasma membrane-ATPase H+/K+ pumps in FC-stimulated aciification and K+ uptake.FC3, a phytotoxin isolated from the fungus Fusicoccum amygdali (2), rapidly stimulates net H+ efflux and K+ uptake in practically all higher plant tissues so far examined (17). FC has also been reported to stimulate plant cell enlargement (4,19,24), to cause rapid stomatal opening (25), and to promote seed germination (13). It has been suggested that the effects ofFC on these important physiological processes may be a consequence of an FC-activated increase in H /K+ exchange across the plasma membrane (16). Inasmuch as, in some instances, FC mimics the action of natural plant hormones (17), an understanding ofthe mechanism ofaction of FC may aid not only in the study of membrane transport in plants but also in studies on the mechanism of action of plant hormones.The rapidity of the effects of FC on H+ and K+ transport (5, 21) and on hyperpolarization of the membrane potential (7, Seeds ofA vena sativa L cv. Victory were germinated and grown hydroponically using the culture techniques of Hodges and Leonard (9). The oat seeds were grown in the dark for 4 to 5 days at about 25 C over a vigorously aerated solution of I mm CaSO4. Whole roots (5-10 cm long) were excised and washed two to three times with cold distilled H20 prior to homogenization.Alternatively, seeds were sown on moist Vermiculite and grown in the dark at 28 C for 4 to 5 days. Intact seedlings were gently removed, and the roots were washed in distilled H20 to remove the Vermiculite. The excised roots were washed three times with cold distilled H20 before homogenization. Bacterial contamination of the oat roots grown in this manner was determined, and it was found that, after three washes in distilled H20, bacterial levels were reduced to about 104 bacteria/g fresh weight of tissue (Table I). No detectable differences in amount or activity of ATPases of the PM-enriched fractions were found between roots grown hydroponically and in Vermiculite.Membrane Fraction Preparation. The procedures of Hodges and Leonard (9) were used to isolate membrane fractions, and all operations were carried out at 0 to 5 C. The roots were first chopped up with a razor ...

Evidence for a Cl−-Stimulated MgATPase Proton Pump in Oat Root Membranes

“…Seeds of Avena sativa L. cv. Victory were germinated and grown on moist vermiculite as previously described (27). Intact 5-d-old seedlings were gently removed, and the roots were washed in distilled H20 to remove the vermiculite.…”

“…ATPase activity was assayed at 25°C as previously described (27 (24). ["C]Imidazole has previously been used to monitor ATP-generated pH gradients in membrane vesicles from mucosa (23) and Neurospora plasma membranes (25).…”

Fusicoccin Activity and Binding in Arabidopsis thaliana

“…As shown in Figure 2, the specific binding of (2), corn coleoptiles (4), Viciafaba leaf tissue (6), and oat roots (13).…”

“…Evidence from both in vivo and in vitro experiments supports the hypothesis that FC2 primarily acts by stimulating the activity of ATPase proton pumps in the plasma membrane of plant cells (7,9,10). The putative receptor site for FC is not, however, the ATPase, but another protein located in the plant plasma membrane (13,14). Recent evidence suggests that FC may also cause a blockage of a K+ efflux channel in the plasma membrane in addition to stimulating the proton pump (3).…”

Purification and photoaffinity labeling of fusicoccin receptors from maize

“…Attempts to solubilise and purify FC receptors have been reported since 1979, using different procedures on various plant tissues (Pesci et al, 1979b;Stout and Cleland 1980;Aducci et al, 1982). From these studies the estimated molecular mass of the receptors was 60-100 kDa.…”