Roberta Colombo

Correlation between cell enlargement in pea internode segments and decrease in the pH of the medium of incubation: I. Effects of fusicoccin, natural and synthetic auxins and mannitol

Abstract In pea internode segments the stimulation of cell enlargement by the auxins indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) is accompanied by a significant decrease in the pH of the incubation medium. Fusicoccin (FC) which is more effective than these auxins in stimulating growth, is also more effective in decreasing pH in the medium. No effect on pH is observed in the case of the “anti-auxin” p -chlorophenoxyisobutiric acid (4-CPIB). The decrease of pH which parallels auxin or FC-induced cell enlargement does not depend on changes in the rate of “leakage” from the pea segments of substances buffering in the pH range from 4.5 to 6.5. The “pH decrease” effect of FC and of IAA is not suppressed by the addition to the medium of mannitol at concentrations which inhibit growth without affecting the effect of auxin on cell wall extensibility. These results are interpreted as an indication that the growth-promoting activity of IAA and FC is mediated by the capacity of these compounds to induce a decrease of pH in the cell wall and thus an increase of its plastic extensibility.

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.

Correlation between cell enlargement in pea internode segments and decrease in the pH of the medium of incubation: II. Effects of inhibitors of respiration, oxidative phosphorylation and protein synthesis

Abstract Inhibitors of respiration (CO), oxidative phosphorylation (2,4-dinitrophenol [DNP] and carbonyl cyanide m -chlorophenyl-hydrazone [CCCP]), RNA (actinomycin D [Act D]) and protein synthesis (cycloheximide [CHI] also inhibit, in the excised pea internode test, both auxin- or fusicoccin-stimulated cell enlargement and the progressive decrease of pH in the incubation medium. The inhibition of the “pH effect” is not due to an inhibitor-induced extrusion of buffering substances from the tissues. In the segments prepared from the growing part of the distal internode the markedly greater effect of fusicoccin (FC), as compared to indole-3-acetic acid (IAA), on growth stimulation and on the pH decrease is accompanied by a correspondingly greater increase of respiration. In segments from fully grown internodes, IAA has almost no effect on growth, respiration, and lowering of the pH in the medium, while FC still stimulates respiration and induces the pH decrease effect. These data are interpreted as further support for the hypothesis that the effect of growth-promoting substances on cell enlargement is mediated by their capacity to induce a metabolism-dependent change of H + concentration at the level of the cell wall.

3-O-Methyl glucose uptake stimulation by auxin and by fusicoccin in plant materials and its relationships with proton extrusion.

Auxin and fusicoccin (FC) stimulate the active uptake of 3-O-methyl glucose (3-O-MG) in those materials in which they have been shown to activate an electrogenic proton extrusion (Pisum sativum L. stems, Zea mays L. coleoptiles and roots). In maize roots the curve relating 3-O-MG influx to external concentrations indicated that the values of the apparent Km increase in the 3-O-MG concentration range between 2×10-5 mol l-1 and 2×10-2 mol l-1. FC did not alter the Km values and its stimulating effect was nearly constant at all 3-O-MG concentrations tested. Basal and FC-induced uptake of 3-O-MG appeared associated with a transient proton influx suggesting that also in maize roots a sugar-proton contransport occurs. Diethyl stilbestrol, which inhibits proton extrusion, inhibited also basal and FC-induced 3-O-MG uptake. The data support the view that the stimulation by FC of 3-O-MG uptake is closely related to that of proton extrusion. The stimulation by FC of 3-O-MG uptake cannot be replaced by increasing extracellular proton concentration, nor may be explained only by the FC-induced hyperpolarization of transmembrane potential difference. The hypothesis is proposed that the effect of FC on 3-O-MG uptake depends on an increase of cytoplasmic pH, following the activation of the proton extruding system.

Effect of plasmalemma ATPase inhibitors, diethylstilbestrol and orthovanadate, on fusicoccin-induced H+ extrusion in maize roots

Abstract In order to investigate the possibility that fusicoccin(FC)-induced electrogenic H + extrusion and K + uptake in higher plants depend on the activity of a plasmalemma ATPase, we have studied the effect of two non-mitochondrial membrane-bound ATPase inhibitors, diethylstilbestrol (DES) and vanadate, on the transport of H + and K + in maize root segments. Both DES and vandate strongly inhibited the stimulating effect of FC on H + extrusion and K + uptake. Furthermore, vanadate inhibited the FC-induced H + extrusion in the presence of a lipophilic cation, tributylbenzylammonium (TBBA + ), and in the absence of K + . The parallel determination of ATP and pyruvate levels showed that in our experimental conditions the inhibiting effect of the two substances on transport did not seem to depend on an effect on energy metabolism. The results of this paper seem consistent with the view that FC-stimulated H + extrusion and K + uptake depend on the activation of a plasmalemma ATPase, catalyzing an electrogenic H + uniport, electrically coupled with K + influx.

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+.

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.

Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

SummaryThe patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K2 malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at −100 mV, in 100mm symmetrical KCl was about 160 μS/cm2. The reversal potentials (Erev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K+ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na+ too can flow through the channels. NeitherErev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K+ as well as to Na+. Anions such as Cl− or malate seem to contribute little to the channel current.

Evidence for polyamine channels in protoplasts and vacuoles of Arabidopsisthaliana cells

The presence of ion channels permeable to polyamines in the plasma membrane and tonoplast of Arabidopsis thaliana cultured cells was investigated by means of the patch-clamp technique in the whole-cell configuration. Evidence is shown for channels, activated by depolarizations in protoplasts and by hyperpolarizations in vacuoles, with slow time course of activation, permeable to putrescine, spermidine and spermine.