Fusicoccin Binding Research Articles

The fusicoccin story revisited.

Fusicoccin (FC) is one of the most studied fungal metabolites to date. The finding that the plasma membrane H+-ATPase in combination with 14-3-3 proteins acts as a high-affinity receptor for FC was a breakthrough in the field. Ever since, the binding of FC to the ATPase-14-3-3 receptor complex has taken center stage in explaining all FC-induced physiological effects. However, a more critical review shows that this is not evident for a number of FC-induced effects. This review challenges the notion that all FC-affected processes start with the binding to and activation of the plasma membrane ATPase, and raises the question of whether other proteins with a key role in the respective processes are directly targeted by FC. A second unresolved question is whether FC may be another example of a fungal molecule turning out to be a 'copy' of an as yet unknown plant molecule. In view of the evidence, albeit not conclusive, that plants indeed produce 'FC-like ligands', it is worthwhile making a renewed attempt with modern improved technology to answer this question; the answer might upgrade FC or its structural analogue(s) to the classification of plant hormone.

Stabilization of Protein–Protein Interactions between CaMKK2 and 14–3–3 by Fusicoccins

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates several key physiological and pathophysiological processes, and its dysregulation has been implicated in obesity, diabetes, and cancer. CaMKK2 is inhibited through phosphorylation in a process involving binding to the scaffolding 14-3-3 protein, which maintains CaMKK2 in the phosphorylation-mediated inhibited state. The previously reported structure of the N-terminal CaMKK2 14-3-3-binding motif bound to 14-3-3 suggested that the interaction between 14-3-3 and CaMKK2 could be stabilized by small-molecule compounds. Thus, we investigated the stabilization of interactions between CaMKK2 and 14-3-3γ by Fusicoccin A and other fusicoccanes-diterpene glycosides that bind at the interface between the 14-3-3 ligand binding groove and the 14-3-3 binding motif of the client protein. Our data reveal that two of five tested fusicoccanes considerably increase the binding of phosphopeptide representing the 14-3-3 binding motif of CaMKK2 to 14-3-3γ. Crystal structures of two ternary complexes suggest that the steric contacts between the C-terminal part of the CaMKK2 14-3-3 binding motif and the adjacent fusicoccane molecule are responsible for differences in stabilization potency between the study compounds. Moreover, our data also show that fusicoccanes enhance the binding affinity of phosphorylated full-length CaMKK2 to 14-3-3γ, which in turn slows down CaMKK2 dephosphorylation, thus keeping this protein in its phosphorylation-mediated inhibited state. Therefore, targeting the fusicoccin binding cavity of 14-3-3 by small-molecule compounds may offer an alternative strategy to suppress CaMKK2 activity by stabilizing its phosphorylation-mediated inhibited state.

Effects of fusicoccin on ion fluxes in guard cells

The pharmacology has been further investigated of the two transport systems mediating potassium (rubidium) (K(+)(Rb(+))) release from the guard cell vacuole, responsible, respectively, for the resting efflux and abscisic acid (ABA)-induced transient stimulation of efflux, and for the transient stimulation induced by hypotonic treatment. Here, the effects of fusicoccin and of butyrate-induced cytoplasmic acidification on (86)Rb efflux were measured in isolated guard cells of Commelina communis. Fusicoccin (10 microM) inhibited the resting efflux at the tonoplast and the ABA-induced transient, but had no effect on the hypotonic transient. All three processes were inhibited by cytoplasmic acidification. Fusicoccin did not inhibit efflux at the plasmalemma. As the hypotonic response is inhibited by cytoplasmic acidification but not by fusicoccin, the effect of fusicoccin on the resting efflux and ABA response must be direct, and not the result of fusicoccin-induced cytoplasmic acidification. The collected tonoplast efflux properties resemble those of TPC1 (two-pore channel) rather than TPK1 (two-pore K channel). The flux and TPC1 are both activated by Ca(2+), but inhibited by phenylarsine oxide and by cytoplasmic acidification. The flux is inhibited by fusicoccin. TPC1 is inhibited by 14-3-3 proteins and has the C-terminal sequence STSDT, a type III binding site for 14-3-3 proteins, of the kind involved in fusicoccin binding.

The V-ATPase from etiolated barley (Hordeum vulgare L.) shoots is activated by blue light and interacts with 14-3-3 proteins

The vacuolar H(+)-ATPase (V-ATPase) is a key enzyme that controls the electrochemical proton potential across endomembranes. Although evidence suggests that V-ATPase is important for photo-morphogenesis, little is known about short-term regulation of V-ATPase upon initiation of the photo-morphogenetic programme by exposure of dark-grown plants to light. In this study, etiolated coleoptiles were given a short blue light treatment and V-ATPase characteristics were determined. The effectiveness of the light treatment was assessed by means of fusicoccin binding to the plasma membrane; this increased 5-fold. The short light treatment also induced a 2-fold to 3-fold increase in the hydrolytic activity of V-ATPase. Members of the 14-3-3 protein family are involved in both blue light perception and the subsequent activation of the P-type ATPase. We provide evidence that 14-3-3 proteins specifically interact with the catalytic A-subunit of the V-ATPase. First, the isolated V1-part of the V-ATPase co-purifies with 14-3-3 on a gel filtration column. Secondly, in an overlay experiment, 14-3-3 interacts with a 68 kDa band that was identified as the V1 A-subunit by mass spectrometry. Thirdly, in 14-3-3 affinity chromatography, both A- and B-subunits of the catalytic moiety of the V-ATPase were identified by matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI TOF/TOF MS) as 14-3-3-interacting proteins. It was shown that the A-subunit can be phosphorylated in vitro by a tonoplast-bound kinase, whose properties are affected by blue light. Taken together, the data show that besides the P- and F-type H(+)-ATPases, the V-type H(+)-ATPase also interacts with 14-3-3 proteins.

Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase.

14-3-3 proteins interact with a novel phosphothreonine motif (Y(946)pTV) at the extreme C-terminal end of the plant plasma membrane H(+)-ATPase molecule. Phosphorylation-independent binding of 14-3-3 protein to the YTV motif can be induced by the fungal phytotoxin fusicoccin. The molecular basis for the phosphorylation-independent interaction between 14-3-3 and H(+)-ATPase in the presence of fusicoccin has been investigated in more detail. Fusicoccin binds to a heteromeric receptor that involves both 14-3-3 protein and H(+)-ATPase. Binding of fusicoccin is dependent upon the YTV motif in the H(+)-ATPase and, in addition, requires residues further upstream of this motif. Apparently, 14-3-3 proteins interact with the unusual epitope in H(+)-ATPase via its conserved amphipathic groove. This implies that very diverse epitopes bind to a common structure in the 14-3-3 protein.

Do 14-3-3 proteins and plasma membrane H+-AtPases interact in the barley epidermis in response to the barley powdery mildew fungus?

14-3-3 proteins form a family of highly conserved proteins with central roles in many eukaryotic signalling networks. In plants, they bind to and activate the plasma membrane H+-ATPase, creating a binding site for the phytotoxin fusicoccin. Barley 14-3-3 transcripts accumulate in the epidermis upon inoculation with the powdery mildew fungus. We have isolated a cDNA encoding a plasma membrane H+-ATPase (HvHAI), which is also induced by powdery mildew attack. The C-terminal domain of this H+-ATPase interacts with 14-3-3 proteins in the yeast two-hybrid system. Inoculation with the powdery mildew fungus, or treatment with fusicoccin, results in an increase in fusicoccin binding ability of barley leaf membranes. Overlay assays show a fungus-induced increase in binding of digoxygenin-labelled 14-3-3 protein to several proteins including a 100 kDa membrane protein, probably the plasma membrane H+-ATPase. These effects are seen specifically in the inoculated epidermis and not in the whole leaf. We propose that 14-3-3 proteins are involved in an epidermis-specific response to the powdery mildew fungus, possibly via an activation of the plasma membrane H+-ATPase.

Regulation of H+Pumping by Plant Plasmalemma under Osmotic Stress: The Role of 14-3-3 Proteins

All higher plants have high-specific sites for binding fusicoccin (FCBS), a metabolite of the fungus Fusicoccum amygdaliDel. These sites are localized on the plasmalemma and produced by the association of the dimers of 14-3-3 proteins with the C-terminal autoinhibitory domain of H+-ATPase. Considering the fusicoccin binding to the plasmalemma as an index characterizing the formation of this complex, we studied the influence of osmotic stress on the interaction between 14-3-3 proteins and H+-ATPase in the suspension-cultured sugar beet cells and protoplasts obtained from them. An increase in the osmolarity of the extracellular medium up to 0.3 Osm was shown to enhance proton efflux from the cells by several times. The number of FCBS in isolated plasma membranes increased in parallel, whereas 14-3-3 proteins accumulated in this membrane to a lesser degree. The amount of H+-ATPase molecules did not change, and the ATP-hydrolase activity changed insignificantly. The data obtained indicate that osmotic stress affects H+-ATPase pumping in the plasmalemma through its influence on the coupling between H+-transport and ATP hydrolysis; 14-3-3 proteins are involved in this coupling. The interaction between the plasmalemma and the cell wall is suggested to be very important in this process.

Involvement of 14-3-3 proteins in the osmotic regulation of H+-ATPase in plant plasma membranes.

Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H+-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H+ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes: the 14-3-3 content in the membranes increased 2- to 3-fold, while the H+-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H+-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H+ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins.

Phenylarsine oxide inhibits the fusicoccin-induced activation of plasma membrane H(+)-ATPase.

To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H(+)-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H(+)-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H(+)-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H(+)-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H(+)-ATPase.

A Phosphopeptide Corresponding to the Cytosolic Stretch Connecting Transmembrane Segments 8 and 9 of the Plasma Membrane H+-ATPase Binds 14-3-3 Proteins and Inhibits Fusicoccin-Induced Activation of the H+-ATPase

: A putative consensus domain for binding of 14-3-3 proteins to the plasma membrane (PM) H+-ATPase was identified in the highly-conserved sequence RSR(p)SWSF [where (p)S is Ser776 of the maize isoform MHA2], localized in the cytosolic stretch connecting transmembrane segments 8 and 9. A 15 amino acid biotinylated phosphopeptide comprising this motif: i) bound a recombinant 14-3-3 protein, ii) inhibited fusicoccin-induced stimulation of the PM H+-ATPase activity both in PM isolated from germinating radish (Raphanus sativus L.) seedlings and in ER isolated from Saccharomyces cerevisiae expressing AHA1 (an isoform of Arabidopsis thaliana PM H+-ATPase), and iii) inhibited fusicoccin binding to PM isolated from germinating radish seedlings. The corresponding non-phosphorylated peptide was inactive in all the performed assays. Together, these results suggest that the cytosolic strand connecting transmembrane segments 8 and 9 of the PM H+-ATPase is a 14-3-3 binding site which might cooperate with the C-terminal domain of the'enzyme in generating a stable association between the H+-ATPase and 14-3-3 protein.

Role of the 14-3-3 proteins in the regulation of H +-ATPase activity in the plasma membrane of suspension-cultured sugar beet cells under cold stress

All higher plants possess highly specific binding sites for fusicoccin, a metabolite of the fungus Fusicoccum amygdali Del. These sites are harboured in the plasma membranes and formed by a 14-3-3 protein dimer associated with the C-terminal autoinhibitory domain of H +-ATPase. We considered the fusicoccin binding to plasma membranes to be an indicator of complexation between the 14-3-3 dimer and H +-ATPase, we assessed the effect of cold stress on the interaction of these proteins in suspension-cultured sugar beet cells and protoplasts derived from these cells. In both objects, upon lowering the temperature to 0–4°C, a portion of the cytoplasmic 14-3-3 proteins became associated with the plasma membrane, which showed an increasing amount of ATPase/14-3-3 complexes and enhanced ATPase activity. Association between ATPase and 14-3-3 resulted in a several-fold rise in the H + efflux from protoplasts and intact cells. We suppose that regulation of the H + pumping under changing external conditions may be based on the interaction between H +-ATPase and the 14-3-3 proteins.

Complementation of the Saccharomyces cerevisiaePlasma Membrane H+-ATPase by a Plant H+-ATPase Generates a Highly Abundant Fusicoccin Binding Site

Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme. The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo. In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity fusicoccin binding site that is exceptionally abundant. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs. The existence of a (plant) PMA2.(yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase.14-3-3 complex formation nor FC binding activity could be observed. Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs. Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding.

Fusicoccin Effect on the in Vitro Interaction between Plant 14-3-3 Proteins and Plasma Membrane H+-ATPase

A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H+-ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H+-ATPase. The binding was dependent on Mg2+ and was strongly increased by fusicoccin. Controlled trypsin digestion of H+-ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the C-terminal domain of H+-ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.

The 14-3-3 proteins associate with the plant plasma membrane H(+)-ATPase to generate a fusicoccin binding complex and a fusicoccin responsive system.

The plasma membrane H(+)-ATPase in higher plants has been implicated in nutrient uptake, phloem loading, elongation growth and establishment of turgor. Although a C-terminal regulatory domain has been identified, little is known about the physiological factors involved in controlling the activity of the enzyme. To identify components which play a role in the regulation of the plant H(+)-ATPase, a fusicoccin responsive yeast expressing Arabidopsis plasma membrane H(+)-ATPase AHA2 was employed. By testing the fusicoccin binding activity of yeast membranes, the C-terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin receptor, a component of which was the 14-3-3 protein. ATP hydrolytic activity of AHA2 expressed in yeast internal membranes was activated by all tested isoforms of the 14-3-3 protein of yeast and Arabidopsis, but only in the presence of fusicoccin, and activation was prevented by a phosphoserine peptide representing a known 14-3-3 protein binding motif in Raf-1. The results demonstrate that the 14-3-3 protein is an activator molecule of the H(+)-ATPase and provides the first evidence of a protein involved in activation of plant plasma membrane H(+)-ATPase.

Fusicoccin binding to its plasma membrane receptor and the activation of the plasma membrane H(+)-ATPase. IV. Fusicoccin induces the association between the plasma membrane H(+)-ATPase and the fusicoccin receptor.

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H(+)-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H(+)-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H(+)-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H(+)-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H(+)-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H(+)-ATPase antiserum and with an anti-14-3-3 antiserum indicated an FC-induced association of FCBP with the PM H(+)-ATPase. Analysis of the activation state of PM H(+)-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.

Topology and target interaction of the fusicoccin‐binding 14‐3‐3 homologs of Commelina communis

Upon binding to a high‐affinity plasma membrane (PM) protein (a member of the 14‐3‐3 family of regulatory proteins), the fungal phytotoxin fusicoccin (FC) activates the H+‐ ATPase by hindering the inhibitory interaction of the enzyme’s C‐terminus with its catalytic site. Protease protection experiments carried out with sealed PM vesicles of different orientation proved that the FC‐binding site faces the cytoplasmic surface of the membrane. The in vivo induced activation of the H+‐ATPase by FC was retained during solubilization of PM proteins. Two‐dimensional gel systems combining a native separation of membrane protein complexes with a denaturing dimension as well as high‐performance anion‐exchange chromatography proved the existence of a labile ATPase:14‐3‐3 complex in plasma membranes. Stabilization of this complex could be achieved by FC treatment in vivo or in vitro. Mild proteolytic removal of the C‐terminal auto‐inhibitory domain of the H+ATPase liberated apparent hydrophobic 14‐3‐3 isoforms from the membrane in soluble form. During size exclusion chromatography of the proteolytically released proteins, co‐elution of 14‐3‐3 dimers, protein‐bound FC and the C‐terminus of the H+ATPase was observed. Moreover, the data suggest that 14‐3‐3 dimers themselves are not able to bind FC. Based on these results, it is proposed that the ‘FC receptor’ is represented by a labile complex between a 14‐3‐3 dimer and the H+‐ATPase whose formation is part of a mechanism regulating ATPase‐activity under physiological conditions. In our working model, binding of FC stabilizes this labile complex, thus leading to a strong and persistent activation of the H+‐ATPase in vivo. The possibility that the C‐terminus of the enzyme represents the binding domain for 14‐3‐3 homologs is discussed.

Plant cell pH-static circuit mediated by fusicoccin-binding proteins

On sugar beet protoplasts that carry two types of fusicoccin-binding sites, a pH downshift in a physiological range (7.0–6.6) markedly enhanced the efficiency of fusicoccin (FC) binding, mainly owing to increased avidity of low-affinity FC-binding sites. This may allow the FC-binding proteins to act as pH-sensitive modulators of cell activity, for instance, via plasma membrane H+-ATPase or potassium channels. © 1997 Federation of European Biochemical Societies

Does plasma membrane H+‐ATPase activation by fusicoccin involve protein kinase?

Sugar beet (Beta vulgaris L.) root suspension‐cultured cells were converted to protoplasts which responded to fusicoccin (FC) by a rise in cytoplasmic pH (pHcyt) averaging 0.25 units in the fluorimetric assay. This effect was blocked by erythrosin B, a specific inhibitor of the plasma membrane H+‐ATPase. A protein kinase inhibitor, staurosporine also caused cytosolic alkalinization that was sensitive to H+‐ATPase inhibitors. Most strikingly, the effect of staurosporine was suppressed by fusicoccin and vice versa. Addition of okadaic acid, entailing overall protein phosphorylation, also led to H+‐ATPase activation, whereupon fusicoccin lost its effect on proton transport. In parallel, kinetic and inhibitor analyses demonstrated that FC binding to the protoplast plasma membrane involved two sites with dissociation constants of 1 nM and 0.2 μM and was indifferent to phosphorylation and dephosphorylation inhibitors. Thus, it could be concluded that (1) the effect of FC on cytoplasmic pH probably depends on the phosphorylation state of plasma membrane proteins and may have either sign; (2) the activation of H+‐ATPase by FC most likely proceeds directly through conformational receptor‐enzyme interaction.

A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?).

A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase.