No abstract

l h e phytotoxin fusicoccin (FC), after binding to a plasma membrane-localized receptor, causes higher plant cells to excrete protons. Ligand-binding analysis has been used to show that the plasma membrane of mung bean (Vigna radiata 1.) hypocotyls contains both high-affinity (HA) and low-affinity (LA) binding sites for FC. The effect of tissue maturation on these sites was determined on isolated membrane vesicles from the meristematic region (hook) and the elongation zone and from mature hypocotyl tissues. In the meristematic region the HALA ratio was 1:20. As hypocotyl tissues matured, the site density of HA increased and there was no change in LA density, so that the HALA ratio increased to 1:2 in mature tissues. FC-induced proton excretion correlates with the HA density, not the LA density. When sedions isolated from each region were incubated with FC prior to isolation of membranes, there was an apparent conversion of LA to HA sites during the first 90 min in all regions. During the next 1 to 3 h there was a further 2.5-to 3-fold increase in binding sites in all regions, accompanied by a slight decline in dissociation constant. l h e increase in binding sites, but not the apparent conversion of LA to HA, was partly blocked by cycloheximide. lhese data suggest that FC alters FC-binding protein activity in two ways: first, by causing an increase in affinity for FC of preexisting LA receptors, and second by inducing the synthesis of additional FC receptors. This apparent up-regulation of a phytotoxin receptor by its ligand in plants has not previously been reported.The phytotoxin FC, produced by the fungus Fusicoccum amygdali Del., has the ability to cause proton excretion from a11 higher plants so far tested (Marré, 1979). After binding to a 30.5-to 32.5-kD receptor (FCBP) (de Boer et al., 1989;Feyerabend and Weiler, 1989) on the PM, it activates the PM H+-ATPase, apparently by relieving the inhibition caused by the autoinhibitory C-terminal domain of the ATPase (Pugliarello et al., 1992;Johansson et al., 1993).Our previous study (Basel and Cleland, 1992) , 1982;Meyer et al., 1989;Schulz et al., 1990; Abramycheva et al., 1993). Because our previous FCBP assay would have measured only HA sites, this study was undertaken to determine whether mung bean hypocotyls possess both HA and LA FC-binding sites and, if so, whether the distributions of HA and LA sites were the same. Preliminary experiments indicated that, when hook sections from mung bean hypocotyls were treated with FC, there was no detectable inaease in proton excretion during the 1st h, as expected from the apparent lack of FCBP in this region. But after the 1st h, FC-enhanced H+ excretion commenced, suggesting that FC might have up-regulated the ability of these cells to bind FC. To test these possibilities, ligandbinding analysis (Scatchard, 1949) has been used with microsoma1 and PM vesicles prepared from freshly cut sections of the mung bean hypocotyl or prepared after preincubation of sections for up to 6 h with or without FC. The Scatchard ana...