A comparison has been made of the developmental gradients along a mung bean (Vigna radiata L.) hypocotyl of the growth rate, plasma membrane ATPase, and fusicoccin-binding protein (FCBP) activity to determine whether they are interrelated. The hook and four sequential 7.5 millimeter segments of the hypocotyl below the hook were cut. A plasma membrane-enriched fraction was isolated from each section by aqueous two-phase partitioning and assayed for vanadate-sensitive ATPase and FCBP activity. Each gradient had a distinctive and different pattem. Endogenous growth rate was maximal in the second section and much lower in the others. Vanadate-sensitive ATPase activity was maximal in the third section, but remained high in the older sections. Amounts of ATPase protein, shown by specific antibody binding, did not correlate with the amount of vanadate-sensitive ATPase activity in the three youngest sections. FCBP activity was almost absent in the first section, then increased to a maximum in the oldest sections. These data show that the growth rate is not determined by the ATPase activity, and that there are no fixed ratios between the ATPase and FCBP.As cells develop, they undergo changes in physiological potential and in their spectrum of proteins. The challenge is to relate changes in proteins to changes in physiological potential. Cells in a hypocotyl go through a period of elongation prior to maturation (7). This elongation is due, at least in part, to auxin-induced wall acidification, mediated by the PM2 ATPase (1 1). The first question addressed here is whether the peak in elongation in mung bean hypocotyls is paralleled by a peak of PM ATPase activity. Because hypocotyl development progresses from apex to base, with the youngest, meristematic cells contained in the hook, the growth rate and PM ATPase activity were compared in successive sections along the hypocotyl, starting with the hook region.Proton excretion via the PM ATPase is greatly enhanced in most higher plant cells by the fungal toxin FC (19,24 does not bind to the catalytic peptide of the ATPase, but to a separate FCBP (1,5,6,20). It has been suggested that the FCBP is a constitutive regulatory subunit of the ATPase (5, 10); if so, one would expect a constant ratio between ATPase and FCBP activities. The second question addressed here is the quantitative relationship between these two activities along the mung bean hypocotyl. Mung beans ( Vigna radiata L.) were selected because of the large body of information about other developmental gradients along its hypocotyl (7), and the relative ease of obtaining PM using aqueous twophase partitioning (21, 31). MATERIALS AND METHODS Plant MaterialMung bean (Vigna radiata L. cv Berken) seeds were obtained from Burpee Seed Co. Seeds were soaked with aeration overnight, then dark-grown at 25°C on soaked and drained coarse vermiculite. Three hundred milliliters ofa 1 mm CaSO4 solution were added to the vermiculite to prevent dampingoff symptoms. Hypocotyls were harvested after 5 d, when their lengths wer...

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...