Franca Rasi-Caldogno

Evidence for the coupling of proton extrusion to K+ uptake in pea internode segments treated in fusicoccin or auxin

Abstract When pea internode segments are incubated in a K + -deficient medium, a rapid increase of K + in the medium is observed in the first 3 h, followed by a progressive decrease in the following period. Fusicoccin (FC) and, to a much lesser extent, indole-3-acetic acid (IAA), increase the initial K + outburst, while in the second phase they accelerate the reabsorption of K + . Since the initial K + outburst takes place also if the segments are incubated in buffered solutions at progressively decreasing pH, its enhancement by FC is interpreted as a consequence of the acidification of the free space. Such an acidification makes available, for both uptake into the cells and diffusion into the medium, a consistent amount of the cations retained by non-exchangeable anions in the Donnan free space (DFS). The proton extrusion promoted by FC or IAA is differentially affected by the presence of monovalent cations in the medium. K + and to a lesser extent Na + stimulate it while Cs + and Li + have little activity or inhibit the reaction. The order of activity of these ions roughly corresponds to their capacity to activate a K + -activated ATPase present in “plasmalemma” preparations from pea internode segments. Dicyclohexycarbodiimide (DCCD), an inhibitor of K + -ATPases, also inhibits growth and proton extrusion. If segments were partially depleted of the K + in the DFS by preincubation with either growth-promoting substances or in acidic buffers, and then transferred into fresh medium, the FC- and IAA-promoted proton extrusion was strongly stimulated by the presence in the fresh medium of K + , while Na + appeared much less active. A satisfactory correlation between the stimulatory effect on proton extrusion and that on K + uptake appears to exist even in the very first phase of treatment with FC, provided the segments are preincubated in buffers at pH 5. This treatment eliminates the initial K + outburst that otherwise would mask the stimulation of K + uptake. Under such conditions a proton extrusion/K + uptake ratio close to unity was found.

Correlation between proton extrusion and stimulation of cell enlargement effects of fusicoccin and of cytokinins on leaf fragments and isolated cotyledons

Abstract Fusicoccin (FC), a diterpene glucoside which stimulates growth of pea internode segments and of leaf fragments, is shown to induce in isolated cotyledons from germinating squash and radish seeds, an effect on cell enlargement markedly greater than that of benzyladenine (BA) and kinetin. The stimulation of cell enlargement induced in leaf fragments by FC, and in cotyledons by FC or by the cytokinins, is accompanied by a proportional rapid decrease in the pH of the medium, similar to the one previously observed when the pea internode segments are treated with auxin or with FC. This apparent proton extrusion accompanying growth stimulation by auxins, cytokinins and FC, is markedly reduced by the phosphorylation uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP) and by the protein synthesis inhibitors cycloheximide (CH) and puromycin (PUR). A comparison of these results with those of a parallel investigation shows that the effects of growth stimulators and of the inhibitors on cell enlargement and on proton extrusion are accompanied by corresponding changes in the negative transmembrane electric potential. A model is proposed for the interpretation of the interrelationship between these effects.

Fusicoccin stimulates the H+-ATPase of plasmalemma in isolated membrane vesicles from radish.

The effect of fusicoccin on the plasmalemma H+-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H+-transport is mediated only by the plasmalemma H+-ATPase. Fusicoccin stimulated the plasmalemma H+-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of delta psi and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H+-ATPase was evident at pH 6.6.

Two distinct proton translocating ATPases are present in membrane vesicles from radish seedlings

Two distinct systems of Mg‐ATP‐dependent transport of protons have been put in evidence in microsomal vesicles from radish seedlings by investigating the effects of vanadate and of NO− 3 on the initial rate of acridine orange accumulation. One system is vanadate‐resistant and is inhibited by NO− 3 (I 50 1.3 mM), the other is inhibited by vanadate (I 50 43 μM) and unaffected by NO− 3. The two systems differently distribute along a continuous sucrose density gradient: the NO− 3‐sensitive system of Mg‐ATP‐dependent transport of protons peaks at d = 1.13–1.14, while the vanadate‐sensitive one peaks at d = 1.16–1.17.

Identification of the plasma membrane Ca2+-ATPase and of its autoinhibitory domain

The effect of controlled proteolysis on the plasma membrane (PM)Ca2+-ATPase was studied at the molecular level in PM purified from radish (Raphanus sativus L.) seedlings. Two new methods for labeling the PM Ca2+-ATPase are described. The PM Ca2+-ATPase can be selectively labeled by treatment with micromolar fluorescein isothiocyanate (FITC), a strong inhibitor of enzyme activity. Both inhibition of activity and FITC binding to the PM Ca2+-ATPase are suppressed by millimolar MgITP. The PM Ca2+-ATPase maintains the capability to bind calmodulin also after sodium dodecyl sulfate gel electrophoresis and blotting; therefore, it can be conveniently identified by 125l-calmodulin overlay in the presence of calcium. With both methods a molecular mass of 133 kD can be calculated for the PM Ca2+-ATPase. FITC-labeled PM Ca2+-ATPase co-migrates with the phosphorylated intermediate of the enzyme[mdash]labeled by incubation with [[gamma]-32P]GTP in the presence of calcium[mdash]on acidic sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Controlled trypsin treatment of purified PM determines a reduction of the molecular mass of the PM Ca2+-ATPase from 133 to 118 kD parallel to the increase of enzyme activity. Only the 133-kD but not the 118-kD PM Ca2+-ATPase binds calmodulin. These results indicate that trypsin removes from the PM Ca2+-ATPase an autoinhibitory domain that contains the calmodulin-binding domain of the enzyme.

Effects of monovalent cations on IAA- and FC-stimulated proton-cation exchange in pea stem segments

Abstract 1. (1) At relatively low (1–10 mM) salt concentration in the medium, proton extrusion in normal, auxin (IAA)- and fusicoccin (FC)-treated pea internode segments is markedly stimulated by K+ and, at a lesser extent, by Rb+, while Na+ and other monovalent cations show little or no effect. At high (100 mM) concentration also Na+ stimulates H+ extrusion, and the effects of K+, Rb+ and Na+ become quantitatively similar. The stimulation of H+ extrusion by monovalent cations is clearly synergistic with the one induced by either IAA or FC. 2. (2) Both IAA and (much more) FC markedly enhance the rates of K+ and of Na+ uptake in pea internode segments. Both growth promoters strongly increase the selectivity of the uptake system for K+, as compared with Na+, at low but not at high salt concentrations. Under all conditions tested a qualitative correlation exists between K+ and Na+ uptake rate and the stimulating effect of these cations on proton extrusion. The cation uptake/titratable H+ extrusion ratio is decreased by either IAA or FC. The value of this ratio is always larger than 1, and increases with the increase of salt concentration in the medium. This may suggest that re-absorption and/or reassociation of an aliquot of extruded protons with extruded −OH or weak base mask a consistent fraction of the proton efflux. These results are consistent with the hypothesis that IAA and FC, although acting on different primary receptors, activate the same proton/monovalent cation antiport mechanism endowed with a high affinity for K+.

Active transport of Ca2+ in membrane vesicles from pea: Evidence for a H+/Ca2+ antiport

Two non mitochondrial systems involved in ATP-dependent Ca2+ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca2+ accumulation system, which shows the characteristics of an ATP-dependent H+/Ca2+ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO4-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg2+ or Mn2+. A second system of ATP-dependent Ca2+ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO4-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg2+ or Mn2+. This system is interpreted as corresponding to the (Ca2+ + Mg2+)-ATPase described by Dieter, P. and Marme, D. ((1980) Planta 150, 1–8).

Effect of cycloheximide on IAA- or FC-induced cell enlargement in pea internode segments

Abstract In pea internode segments cycloheximide (CH) at concentration above 50 μg/ml almost completely inhibits protein sysnthesis without inducing relevant effects on both respiration and phosphorylation. Treatment with CH completely suppresses the effects of indole-acetic acid (IAA) on cell enlargement, QO2, H+ extrusion and transmembrane electric potential (PD), while it only partially inhibits the effects of fusicoccin (FC) on the same parameters. The rate of CH-resistant FC-induced growth (ca. 30% of the value in the absence of CH) remains constant for at least 5 h. The data indicate that some short-lived protein(s) is limiting the full activity of the mechanism mediating the action of IAA and FC on cell membrane activity and cell enlargement. The finding that a consistent part of FC-induced growth is still observed under conditions of blocked protein synthesis suggests that FC effects do not directly depend on de novo protein synthesis. The much more stringent requirement for protein synthesis in the case of IAA-induced effects on H+ extrusion, PD, QO2 and growth suggests that some short-lived protein(s) may represent an obbligatory step in the mechanism of action of the natural hormone.

Controlled Proteolysis Activates the Plasma Membrane Ca2+ Pump of Higher Plants (A Comparison with the Effect of Calmodulin in Plasma Membrane from Radish Seedlings).

The effects of calmodulin and of controlled trypsin treatments on the activity of the Ca2+ pump were investigated in plasma membrane purified from radish (Raphanus sativus L.) seedlings. Treatment of the plasma membrane with ethylenediaminetetra-acetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Ca2+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Ca2+ pump activity was maximal at low free Ca2+ (2-5 [mu]M) and decreased with the increase of free Ca2+ concentration. The Ca2+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the Ca2+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free Ca2+ concentration in the assay medium. Moreover, the Ca2+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin.

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.