Nicoletta Beffagna

Stimulating effect of fusicoccin on K-activated ATPase in plasmalemma preparations from higher plant tissues

Abstract Fusicoccin (FC), a diterpene glucoside very active in stimulating proton/K + exchange in higher plant tissues, is shown to increase in vitro the K + Mg 2+ -stimulated ATPase activity of plasmalemma-enriched membrane preparations from maize coleoptiles and from spinach leaves. The concentration/activity curve of the effect of FC on ATPase closely parallels that of the in vivo effect on K + uptake. DAK, a derivative of FC inactive in vivo on K + uptake and associated responses, is also inactive on the ATPase activity in vitro. These results are consistent with the hypothesis that the stimulating effects of FC on proton extrusion and cation uptake are due to its capacity to activate at cell membrane level a H + /K + exchange system depending on ATP as an energy source.

Inhibiting effect of fusicoccin on abscisic acid-induced proline accumulation in barley leaves

Abstract Abscisic acid (ABA) treatments induce in barley leaf fragments an increase in proline with a maximum at 10 −4 M ABA. This effect is suppressed by the presence of fusicoccin (FC). FC induces an acidification on the incubation medium paralleled by an increase in cell sap pH; both of these effects of FC are significantly antagonized by the simultaneous addition of ABA. The antagonism between FC and ABA on the proline accumulation process and on the external pH changes suggests that both of these substances can influence H + secretion, presumably by acting on the proton pump, and that the consequent changes in intracellular pH might affect some metabolic process and thus influence proline metabolism.

Solubilisation and partial purification of a fusicoccin-receptor complex from maize microsomes

Abstract Fusicoccin (FC) specifically bound to microsomal preparations from maize coleoptiles can be partially solubilised by treatments with Triton X-100, deoxycholate (DOC), SDS, urea, sodium perchlorate and trypsin. In the cases of the treatments with Triton X-100, deoxycholate, percholate or trypsin a consistent fraction of the solubilised FC appears still bound to a macromolecular component. On Sephadex G-200 filtration the perchlorate-solubilised FC-macromolecule complex shows a well-defined peak, corresponding to a molecular weight of approx. 80 000. Acrylamide disc gel electrophoresis of the bound FC-containing fractions from Sephadex chromatography seperates bound FC in a single, narrow peak corresponding to a minor protein peak. These data are interpreted as suggesting that the high-affinity, low-reversibility FC-macromolecule complex is due to the interaction between FC and a single receptor protein, localised at the plasmamembrane, and possibly involved in the physiological activity of the toxin.

Cation-activated ATPase activity of plasmalemma-enriched membrane preparations from maize coleoptiles.

The ATPase activity present in plasmalemma-enriched preparations from maize coleoptiles shows an optimum at pH 6, a strong dependence on Mg2+, and is stimulated by K+ and other monovalent cations, both organic and inorganic. The activation of ATPase by K+ obeys Michaelis Menten kinetics, saturation being reached at 50 mM K+ concentration. K+, Mg2+-stimulated ATPase activity is strongly inhibited by N,N′-dicyclohexylcarbodiimide and by diethylstilbestrol and, to a lesser extent, by octylguanidine.

Distribution of fusicoccin bound in vivo among subcellular fractions from maize coleoptiles

Abstract Maize coleoptile segments were treated in vivo with 10 −6 M radioactive fusicoccin ([ 3 H]FC), and its distribution in various subcellular fractions was investigated. Recovery of bound [ 3 H]FC was maximal in the microsomal fraction, lower in the mitochondrial, and almost negligible in the ribosomes and soluble proteins fractions. Binding was readily labile at 60°C, but not at 0–4°C, and apparently non-covalent. In sucrose density gradients of the microsomal preparations the profile of bound FC coincided with that of DES-sensitive, K + -dependent (plasmalemma) ATPase; in those of the mitochondrial fraction bound FC peaked in a region different from that of cytochrome oxidase, and presumably corresponding to plasmalemma contamination. The subcellular distribution and the general characteristic of the in vivo binding of FC are similar to those of the high-affinity, low-reversibility binding previously investigated in vitro, thus suggesting that the same receptor is involved in the two cases.

On the relationship between ATPase and fusicoccin binding capacity of crude and partially-purified microsomal preparations from maize coleoptiles

Abstract The behaviour of ATPase activity and of in vitro binding of fusicoccin (FC) in particulate and partially solubilised microsomal preparations from maize coleoptiles was investigated. In the particulate system diethylstilbestrol (DES) and Cercospora beticola toxin (CBT) parallely inhibited FC binding and dicyclohexy-carbodiimide (DCCD)-sensitive ATPase activity. Among the solubilising treatments sodium deoxycholate (DOC) and trypsin solubilised equal fractions of bound FC and of ATPase activity, while sodium perchlorate, incubation at pH 8.8 and EDTA in the absence of Mg 2+ were much more active in solubilising the ATPase than the bound FC. Disc gel electrophoresis of the microsomal perchlorate extract led to the separation of the FC-receptor complex from the ATPase activity. The sensitivity of ATPase to DCCD and to DES was not influenced by solubilisation with DOC, but was almost completely suppressed by that with perchlorate, while solubilisation by incubation at pH 8.8 suppressed the sensitivity to DCCD but not that to DES. These data suggest the presence in the plasma membrane of a multi-unit ATPase system coordinating distinct components, some of which would be able to interact with FC and with the inhibitors in such a way as to influence the activity of the catalytic sub-unit.

The chl1 Arabidopsis mutant impaired in the nitrate-inducible NO3− transporter has an acidic intracellular pH in the absence of nitrate

Summary Seedlings of wt and chl1 , an Arabidopsis thaliana mutant impaired in nitrate transport, were grown in a nitrate-free medium, and a comparison was made between the values of cell sap pH, cytoplasmic pH and malate content detected in the two strains. In this condition, in which the presence of the mutation should not have any influence, cytoplasmic and cell sap pH of wt were higher than those of chl1 either when measured at the end of the growth period or after treatments in different nitrate-free media. The low intracellular pH detected in chl1 was not apparently due to a reduced activity of the plasmalemma H + -ATPase, since the cytoplasmic alkalinization following the activation of the H + pump by K + was larger in chl1 than in wt , the amplitude of the resulting pH change substantially depending on the actual value of cytosolic pH at the beginning of the treatment with K + for both strains. The intracellular pH differences detected between wt and chl1 show that this mutant, defined as impaired in the nitrate-inducible NO 3 − transporter, exhibits phenotypic expression in the absence of nitrate too, thus suggesting that some other system, possibly involved in intracellular pH regulation, might be indirectly influenced by the mutation.

Changes of Vacuolar and Cytoplasmic pH Associated with the Activation of the H+ Pump in Elodea Leaves

The widespread presence of an ATP-driven H+ pump operating at the plasmalemma of higher plant cells is well established by in vivo and by in vitro work (Marre and Ballarin-Denti, 1985). In recent investigations attention has been centered on the relationships between the activity at this pump, the activity of the biochemical PEP-malate pH-stat and the regulation of intracellular pH (Marre et al., 1986 b).

Fusicoccin-induced catalase inhibitor is produced independently of H+-ATPase activation and behaves as an organic acid.

The phytotoxin fusicoccin (FC) was found to induce an increase in apoplastic H₂O₂ content in Arabidopsis thaliana cells, apparently linked to the presence of an as yet unidentified catalase inhibitor detectable even in the external medium of FC-treated cells. This study, aimed to further characterize the inhibitors features, shows that (1) FC-induced H₂O₂ accumulation increases as a function of FC concentration and correlates to the amount of inhibitor released at apoplastic level. The pattern of H+ efflux, conversely, does not fit with that of these two parameters, suggesting that neither the production nor the release of the catalase inhibitor is linked to the main role of FC in activating the plasma membrane (PM) H+-ATPase; (2) treatment with 10 µM erythrosine B (EB) early and totally inhibits net H+ and K+ fluxes across the PM, indicative of the H+ pump activity; nevertheless, also in these conditions a huge FC-induced H₂O₂ accumulation occurs, confirming that this effect is not related to the FC-induced PM H+-ATPase activation; (3) the inhibitors release increases with time in all conditions tested and is markedly affected by extracellular pH (a higher pH value being associated to a larger efflux), in agreement with a weak acid release; and (4) the inhibitor can be almost completely recovered in a CH₂C₂-soluble fraction extracted from the incubation medium by sequential acid-base partitioning which contains nearly all of the organic acids released. These final results strongly suggest that the metabolite responsible for the FC-induced catalase inhibition belongs to the organic acid class.