The Action of Fusicoccin Alone and with Some Plant Growth Substances on Tobacco Tissue Cultures

The interaction of fusicoccin, a terpenoid glucoside produced by Fusicoccum amygdali Del., and some plant growth regulating substances, i.e., indole-3-acetic acid, kinetin, 2,4-dichlorophenoxy-acetic acid and abscisic acid, was investigated on pith and sub-cultured callus cultures of Nicotiana tabacum L. Addition of fusicoccin alone to the basal medium, either in the light or in the dark, caused a fairly good development of tobacco callus. When fusicoccin and kinetin were simultaneously added to the culture medium, the callus growth increased. However, fusicoccin in combination with indole-3-acetic acid caused limited callus development: the tissue appeared brown and reduced in volume. Addition of fusicoccin with 2,4-dichlorophenoxyacetic acid stimulated growth of callus and chlorophyll was formed under light. Finally, abscisic acid did not interfere with the effect of fusicoccin on the callus growth.     

Structural view of a fungal toxin acting on a 14-3-3 regulatory complex

The fungal phytotoxin fusicoccin stabilizes the interaction between the C-terminus of the plant plasma membrane H(+)-ATPase and 14-3-3 proteins, thus leading to permanent activation of the proton pump. This results in an irreversible opening of the stomatal pore, followed by wilting of plants. Here, we report the crystal structure of the ternary complex between a plant 14-3-3 protein, fusicoccin and a phosphopeptide derived from the C-terminus of the H(+)-ATPase. Comparison with the corresponding binary 14-3-3 complexes indicates no major conformational change induced by fusicoccin. The compound rather fills a cavity in the protein-phosphopeptide interaction surface. Isothermal titration calorimetry indicates that the toxin alone binds only weakly to 14-3-3 and that peptide and toxin mutually increase each others' binding affinity approximately 90-fold. These results are important for herbicide development but might have general implications for drug development, since rather than inhibiting protein-protein interactions, which is difficult to accomplish, it might be easier to reverse the strategy and stabilize protein-protein complexes. As the fusicoccin interaction shows, only low-affinity interactions would be required for this strategy.     

Comparison of the responses of corn root tissue to fusicoccin and washing

A comparison has been made of the effects of fusicoccin with those of washing on segments of corn (Zea mays L.) root tissue. Both fusicoccin and washing caused increases in K⁺(⁸⁶Rb) influx, net H⁺ efflux, and electrogenic cell membrane potential, but with no effect on respiration rate. The similarity was most evident with fresh tissue during the initial phases of washing, prior to the developmental changes which augment the anion and general solute transport rates of the tissue. After the development of enhanced transport capacity the proportional response to fusicoccin was much diminished. It is suggested that the fusicoccin-like response to washing may be a manifestation of recovery from injury.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K_d of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K_d 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H⁺-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H⁺-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H⁺-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.     

Modulation of H+-ATPase Activity by Fusicoccin in Plasma Membrane Vesicles from Oat (Avena sativa L.) Roots (A Comparison of Modulation by Fusicoccin,Trypsin, and Lysophosphatidylcholine)

The fungal phytotoxin fusicoccin affects various transport processes in the plasma membrane of plant cells. The plasma membrane (PM) H+-ATPase (EC 3.6.1.35) seems to be the primary target of fusicoccin action. The kinetics of the stimulation of the PM H+-ATPase by fusicoccin was studied in PM vesicles isolated from oat (Avena sativa cv Adamo) roots by aqueous two-phase partitioning. Considerable stimulation of activity was observed only when roots were treated with fusicoccin prior to the PM isolation. Fusicoccin treatment shifted the pH optimum of the ATPase toward more alkaline values and increased Vmax. No effects on Km were observed. Treatment with trypsin resulted in stimulation of ATPase activity in control vesicles but not in the fusicoccin-treated vesicles. The characteristics of stimulation by trypsin in control vesicles were comparable with those of stimulation by fusicoccin. This result and the change of the polypeptide pattern on western blots suggest the involvement of the C-terminal inhibitory domain in the fusicoccin signal transduction chain. On the other hand, stimulation by lyso-PC demonstrated other characteristics than stimulation by fusicoccin. Lyso-PC was able to stimulate ATPase activity at both acidic and alkaline pH values. Kinetic analysis of the pH dependency curves revealed different mechanisms for activation by fusicoccin and by lyso-PC. Whereas fusicoccin shifted the pH dependency of formation of phosphorylated intermediate to more alkaline values, lyso-PC seemed to increase dephosphorylation independently of pH.     

Some Properties of a Functional Reconstituted Plasmalemma H-ATPase Activated by Fusicoccin

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H⁺-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V_max of the enzyme. Fusicoccin stimulation of maize H⁺-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).     

Acidification of the Medium Associated with Normal and Fusicoccin-Induced Seed Germination

Fusicoccin, a toxin stimulating cell enlargement and inducing proton extrusion in various plant tissues, has been shown to replace kinetin, gibberellic acid and red light in breaking seed dormancy. It also removes the inhibitory effect of abscisic acid. The present data also show that the stimulating effect of fucicoccin on embryo growth of decoated radish (Raphanus sativus L.) and maize (Zea mays) seeds and on the development of maize embryos is accompanied by an early, significant acidification of the medium. Acidification of the medium is also observed when fusicoccin reverses the abscisic acid-induced inhibition of germination. These results support the hypothesis that the mode of action of fusicoccin in promoting germination involves, as in stimulation of cell enlargement, the activation at the cell membrane level of proton extrusion processes. The physiological significance of fusicoccin-induced release of protons at the onset of germination is discussed in comparison with the results concerning the mechanism of action of fusicoccin on cell enlargement in other plant materials.     

Comparison of the effects of auxin and fusicoccin on phosphate absorption by aged potato tuber discs

Auxin (IAA, 5 × 10⁻⁵ M ) partially prevents the increase in the rate of phosphate uptake during ageing of potato tuber discs ( Solatium tuberosum L. cv. Bintje), whereas fusicoccin (FC, 10⁻⁵ M) stimulates it. After the development of enhanced phosphate transport capacity, the response to fusicoccin is greater than with fresh discs. Complementary experiments on K⁺ (⁸⁶Rb) absorption show that FC also slightly enhances the rate of K⁺ uptake, while IAA has no much effect. It is suggested that IAA acts specifically on the development of a mechanism which occurs during the ageing period, while FC action may be more directly linked to the system of phosphate transport itself.     

Cyclosporin A inhibits programmed cell death and cytochrome c release induced by fusicoccin in sycamore cells

Summary. Programmed cell death plays a vital role in normal plant development, response to environmental stresses, and defense against pathogen attack. Different types of programmed cell death occur in plants and the involvement of mitochondria is still under investigation. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin induces cell death that shows apoptotic features, including chromatin condensation, DNA fragmentation, and release of cytochrome c from mitochondria. In this work, we show that cyclosporin A, an inhibitor of the permeability transition pore of animal mitochondria, inhibits the cell death, DNA fragmentation, and cytochrome c release induced by fusicoccin. In addition, we show that fusicoccin induces a change in the shape of mitochondria which is not prevented by cyclosporin A. These results suggest that the release of cytochrome c induced by fusicoccin occurs through a cyclosporin A-sensitive system that is similar to the permeability transition pore of animal mitochondria and they make it tempting to speculate that this release may be involved in the phytotoxin-induced programmed cell death of sycamore cells. Correspondence and reprints: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.     

Fusicoccin affects cortical microtubule orientation in the isolated epidermis of sunflower hypocotyls

Epidermal peels isolated from sunflower hypocotyls provide a convenient model to study the relationship between cortical microtubule orientation and strain rate. Extension of peels can be modulated using chemical treatment and mechanical stress, i.e., by adding a chemical to the incubation medium and applying a load exceeding the yield threshold for irreversible (plastic) strain. In this study, peels were pre-incubated for ca. 12 h (long-term pre-incubation) or for 1 h (short-term pre-incubation). In the long-term pre-incubated peels, fusicoccin applied to the medium neither enhanced the rate of longitudinal plastic strain of loaded peels, nor affected microtubule orientation. However, fusicoccin increased the strain rate of short-term, pre-incubated peels and affected microtubule orientation in both extending (loaded) and non-extending (unloaded) peels. Without fusicoccin, microtubule orientation was generally longitudinal or steep, whereas in fusicoccin-treated unloaded peels it was transverse and oblique microtubules in peel portions corresponding to the apical part of the hypocotyl. Although the frequency of transverse orientation was increased through loading, there was no strong correlation between the rate of fusicoccin-induced strain and microtubule orientation. It is hypothesized that the insensitivity of long-term pre-incubated peels to fusicoccin with respect to strain rate is due to a lack of active plasma membrane H(+) -ATPases. Thus, the sensitivity of short-term, pre-incubated, unloaded (non-extending) peels to fusicoccin, with respect to microtubule orientation, indicates that orientation might be affected by electric currents resulting from fusicoccin stimulation of H(+) -ATPases.     

The phytotoxin fusicoccin promotes platelet aggregation via 14-3-3-glycoprotein Ib-IX-V interaction

The fungal toxin fusicoccin induces plant wilting by affecting ion transport across the plasma membrane of plant cell. The activity of this toxin is so far unknown in humans. In the present study we show that fusicoccin is able to affect the platelet aggregation process. The toxin associates with platelet intracellular binding sites and induces aggregation in platelet-rich plasma in a dose-dependent manner. We identified the adhesion receptor glycoprotein Ib-IX-V as fusicoccin target. The toxin promotes the binding of the regulatory 14-3-3 proteins to glycoprotein Ibα and hampers that to glycoprotein Ibβ subunit. As a result, platelet adhesion to von Willebrand factor is stimulated, leading to platelet spreading and integrin αIIbβ3 activation. We anticipate the present study to be a starting point for future therapeutic use of fusicoccin in genetic bleeding diseases characterized by qualitative or quantitative abnormalities of the platelet membrane-adhesion receptors. Furthermore, the present study also sets the stage for future work to determine the potential pharmacological application of fusicoccin as a drug directed to other 14-3-3-target complexes.     

Studies of the mechanism of action of fusicoccin,the fungal toxin that induces wilting,and its interaction with abscisic acid

Summary Effects of fusicoccin alone and together with abscisic acid were observed on the stomatal complex of Commelina communis. The experimental material consisted of isolated epidermal strips incubated in a medium containing the ions required for stomatal opening. Fusicoccin stimulated opening and this was accompanied by potassium entry into the guard cells, and hydrolysis of the starch in their chloroplasts. Abscisic acid alone inhibited potassium entry and starch hydrolysis, but these effects could be almost entirely overcome by fusicoccin.Attempts were made to measure the solute potential of the guard cells under the various treatments. Abscisic acid clearly increased their solute potential, but no absolute measurements could be made in the presence of fusicoccin owing to a failure of plasmolysis even with mannitol solutions of solute potential as low as —35 bars. Experiments using isotopically labelled mannitol indicated a massive uptake into the epidermis in the presence of fusicoccin.The mechanism of stimulation of stomatal opening by fusicoccin probably depends in part on a stimulation of the normal processes associated with opening in the guard cells, but may also involve release of pressure due to destruction of the surrounding cells. The effectiveness of this toxin under natural conditions may depend on its ability to counteract effects of abscisic acid, the stress hormone that induces stomatal closure.     

Fusicoccin structure-activity relationships: Stimulation of growth by cell enlargement and promotion of seed germination

The activity of a number of fusicoccin derivatives and analogues has been assayed on growth by cell enlargement and on proton extrusion in pea ( Pisum sativum L, cv. Alaska) internode segments, on growth by cell enlargement in isolated squash ( Cucurbita maxima Dechesne in Lam. variety Mantovana) cotyledons, and on germination of light-requiring lettuce ( Lactuca sativa L. cv. Grand Rapids) and of abscisic acid-inhibited radish ( Raphanus sativus L. variety Tondo Rosso Quarantino) seeds. A similar pattern of activity in the different tests was found for most fusicoccin derivatives and analogues. As with fusicoccin, the activity of its derivatives and analogues on growth of pea internodes is paralleled by the activity on the capability of the tissue to acidify the incubation medium.
The obtained data are in full agreement with the view that the activity of fusicoccin on different physiological processes depends on its primary activation of a single system, involved in proton extrusion.     

Effect of fusicoccin and gibberellic acid on primary dormant Avena fatua caryopses

Fusicoccin induced germination in dormant and partially afterripened dormant caryopses of Avena fatua L. The rate of caryopsis germination was slower and final percentage germination lower in the highly dormant inbred line M73 at a given concentration of fusicoccin than in the dormant caryopses of line AN265. Gibberellic acid was more effective than fusicoccin in breaking dormancy in both lines. Promotion of germination of dormant caryopses by fusicoccin was inhibited by a 6-day pretreatment with (2-chloroethyl)trimethylammonium chloride.
The basal rate of proton efflux from embryos isolated from dormant and fully afterripened line AN265 caryopses was similar. Addition of fusicoccin increased the rate of proton efflux from the isolated embryos of dormant and afterripened caryopses by nearly 400%. Gibberellic acid had no effect on the rate of proton extrusion. The uptake of ⁸⁶Rb⁺ in dormant and afterripened A. fatua embryos was similar after a 2 h uptake period. The addition of fusicoccin to the medium doubled the uptake of ⁸⁶Rb⁴ by dormant and afterripened embryos. Gibberelleic acid had no effect on the uptake of ⁸⁶Rb⁺ by isolated embryos from either dormant or afterripened caryopses. The experimental results indicate that gibberellic acid is more versatile in its action than fusicoccin, and gibberellic acid may facilitate dormant A. fatua caryopsis germination by stimulating mechanisms other than the direct H⁺ efflux and K⁺ uptake at the membrane level.     

Fusicoccin and Air Pollutant Injury to Plants : Evidence for Enhancement of SO(2) but Not O(3) Injury

Garden peas (Pisum sativum L. cv Alsweet) and a tomato mutant (Lycopersicon esculentum Mill. var flacca) were sprayed with fusicoccin, a fungal toxin affecting membrane transport properties, before exposure to SO₂ or O₃. Tomatoes treated with 10 micromolar fusicoccin and exposed to SO₂ (0.6 microliter per liter for 2 hours) exhibited twice as much foliar necrosis as untreated plants exposed to SO₂. Peas treated with fusicoccin and exposed to SO₂ (0.7 to 1.0 microliter per liter for 2 hours) exhibited 2 to 6 times more injury than untreated plants exposed to SO₂. Peas treated with fusicoccin and exposed to O₃ had less injury than untreated plants exposed to O₃ (0.1 to 0.3 microliter per liter for 2 hours). Several lines of evidence suggested that the fusicoccin enhancement of SO₂ injury is not the result of increased gas exchange, i.e. the tomato mutant has permanently open stomata under all conditions, and in peas fusicoccin had no effect on SO₂ or H₂O flux in plants exposed to 0.12 microliter per liter SO₂. However, a 21% greater leaf conductance in fusicoccin treated versus untreated plants indicated the possibility of some differences in gas exchange for peas exposed to 1.0 microliter per liter SO₂.     

ACTION OF FUSICOCCIN ON THE POTASSIUM BALANCE OF GUARD CELLS OF PHASEOLUS VULGARIS

Fusicoccin induces stomatal opening in both the light and dark. The stomatal aperture and K content of guard cells was measured to determine whether the action of fusicoccin in inducing stomatal opening is directly related to the uptake of K by the guard cells. Both detached and attached epidermis was treated with fusicoccin and the K content was determined by staining with cobalt sodium nitrite or by electron probe microanalysis. The K content of guard cells in detached epidermal strips floated on 10 μm fusicoccin in 10 mm KCl and aqueous CH₃OH (0.02%, v/v) increased in the light and dark as the stomata opened. After exposure to fusicoccin for 6 hr in the light, however, the stomata were closed and no K could be detected in the guard cells. The K content of guard cells of attached epidermis painted with fusicoccin also increased as the stomata opened, but the concentration of K in the subsidiary cells was not significantly altered by fusicoccin-stimulated opening. Moreover, painting with fusicoccin did not significantly change the Ca and P content of the guard or subsidiary cells. Stomata of epidermal strips, opened to their maximum width by fusicoccin, showed only a small and temporary closure when transferred to a solution of 10 μm abscisic acid. The use of metabolic inhibitors suggested that energy for the uptake of the K may be provided by both photophosphorylation and oxidative phosphorylation.     

The effects of podolactone-type inhibitors on fusicoccin-induced growth and proton efflux

Podolactones A and E and lycoritidinol inhibit growth induced by fusicoccin in dwarf pea ( Pisum sativum L.) hooks and tips; however the inhibition is reduced at the highest fusicoccin concentrations. In short term experiments (3 h) on pea stem tissues, growth and proton efflux induced by fusicoccin are only partially inhibited by podolactone A and lycoricidinol. Auxin-induced growth and proton efflux are completely suppressed by 10 μM lycoricidinol. The inhibitors do not affect ATP levels.     

The role of darkness, GA and fusicoccin (FC) in breaking photodormancy in Phacelia tanacetifolia seeds

The germination of the negatively photoblastic seeds of Phacelia tanacetifolia Benth. (cv. Bleu Clair) is promoted by gibberellic acid and fusicoccin. In the dark, or in the light in the presence of fusicoccin, seed germination is accompanied by an increase of gibberellic acid-like substances. In these conditions, the inhibition of the synthesis of gibberellic acid-like substances does not prevent seed germination, but it affects the growth and the survival of the seedlings. Seed germination, growth, and survival of seedlings are discussed in relation to phytochrome, fusicoccin, and gibberellic acid-iite substances.     

Fusicoccin receptors: perception and transduction of the fusicoccin signal

Fusicoccin receptors are proteins that are widespread in plasmamembrane of higher plants. They act as a perception system forthe fungal metabolite fusicoccin, a toxin affecting plasma membranetransport by the stimulation of proton ATPase. FC representsa unique tool to elucidate the regulatory mechanisms underlyingtransport processes at the plasma membrane for its ability toelicit several hormone-like responses rapidly. This review willsummarize the studies so far reported on localization, biochemicalproperties and purification of fusicoccin receptors and willalso deal with more recent data on the role played by 14-3-3-likeproteins in fusicoccin signalling. Key words: Fusicoccin, receptors, H⁺–ATPase, 14-3-3 proteins     

Fusicoccin A,a Phytotoxic Carbotricyclic Diterpene Glucoside of Fungal Origin,Reduces Proliferation and Invasion of Glioblastoma Cells by Targeting Multiple Tyrosine Kinases

Glioblastoma multiforme (GBM) is a deadly cancer that possesses an intrinsic resistance to pro-apoptotic insults, such as conventional chemotherapy and radiotherapy, and diffusely invades the brain parenchyma, which renders it elusive to total surgical resection. We found that fusicoccin A, a fungal metabolite from Fusicoccum amygdali, decreased the proliferation and migration of human GBM cell lines in vitro, including several cell lines that exhibit varying degrees of resistance to pro-apoptotic stimuli. The data demonstrate that fusicoccin A inhibits GBM cell proliferation by decreasing growth rates and increasing the duration of cell division and also decreases two-dimensional (measured by quantitative video microscopy) and three-dimensional (measured by Boyden chamber assays) migration. These effects of fusicoccin A treatment translated into structural changes in actin cytoskeletal organization and a loss of GBM cell adhesion. Therefore, fusicoccin A exerts cytostatic effects but low cytotoxic effects (as demonstrated by flow cytometry). These cytostatic effects can partly be explained by the fact that fusicoccin inhibits the activities of a dozen kinases, including focal adhesion kinase (FAK), that have been implicated in cell proliferation and migration. Overexpression of FAK, a nonreceptor protein tyrosine kinase, directly correlates with the invasive phenotype of aggressive human gliomas because FAK promotes cell proliferation and migration. Fusicoccin A led to the down-regulation of FAK tyrosine phosphorylation, which occurred in both normoxic and hypoxic GBM cell culture conditions. In conclusion, the current study identifies a novel compound that could be used as a chemical template for generating cytostatic compounds designed to combat GBM.