E. Marrè

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.

The proton pumps of the plasmalemma and the tonoplast of higher plants

Studies on intact cells, membrane vesicles, and reconstituted proteoliposomes have demonstrated in higher plants the existence of an ATP-driven electrogenic proton pump operating at the plasmalemma. There is also evidence of a second ATP-driven H+ pump localized at the tonoplast. The characteristics of both these ATP-driven pumps closely correspond to those of the plasmalemma and tonoplast proton pumps ofNeurospora and yeasts.

Transmembrane potential increase induced by auxin, benzyladenine and fusicoccin. Correlation with proton extrusion and cell enlargement

Abstract Previous work showed that the stimulation of cell enlargement by indole-3-acetic acid (IAA) and fusicoccin (FC), in internode segments, and by cytokinins and FC, in isolated cytyledons, is accompanied by an apparent extrusion of protons. The results reported herein show that the proton extrusion effect of these growth-promoting agents, in pea internode segments as well as in squash cotyledons, is accompanied by a significant increase of the negative transmembrane potential. Both effects are already clearly recognizable a few minutes after the beginning of the treatment and are significantly reduced by the phosphorylation uncoupler carbonyl cyanide m- chlorophylhydrazone (CCCP) and by the protein synthesis inhibitors cycloheximide (CH) and puromycin. These data are in agreement with the hypothesis that stimulation of cell enlargement is mediated by the activation of a metabolism-dependent proton extrusion mechanism. The increase of negative intracellular potential is interpreted as a consequence of proton translocation. The effect of protein synthesis inhibitors is interpreted as evidence that a rapidly turning over protein is involved in the process of ion transport at the cell membrane level.

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.

Characteristics of fusicoccin-induced changes of transmembrane potential and ion uptake in maize root segments

Abstract The effects of fusicoccin (FC) on the transmembrane electric potential difference (PD) of cortical cells of isolated apical root segments was investigated under various conditions of osmotic pressure, temperature, ionic composition and pH of the incubation medium, and compared with the effects of this substance on the uptake of K + , Rb + , Na + and Cs + ions. FC induced a rapid hyper-polarization of the negative PD, ranging from 20 to 50 mV depending on the ionic composition of the medium. With 5·10 −5 M FC maximal PD value was reached in ca. 6 min. Changing the osmotic pressure of the medium from 0.01 to 0.25 osmolar (mannitol) progressively inhibited elongation without affecting the PD change thus showing that the effect of FC does not depend on the increase of cell enlargement. Low temperature (6°C) and the phosphorylation uncoupler ( p -trifluoromethoxy)-carbonyl cyanide-phenylhydrazone (FCCP) rapidly and severely decreased both normal and FC-induced PD. FC stimulated the rate of monovalent cation uptake. In the 1–10 mM concentration range this effect was much larger for K + and Rb + than for Na + and Cs + . K + and (to a lesser extent) Rb + at concentrations higher than 5·10 −4 M induced a rapid depolarization, larger in the presence than in the absence of FC, while Na + , Cs + and Li + at concentrations up to 10 −2 M did not significantly influence PD. No significant PD changes were observed when the anion Cl − was substituted with SO 4 2− or NO 3 2− . Increasing proton concentration in the medium from pH 6 to pH 4 induced a significant hyperpolarization, probably due to the inhibition of K + uptake by low pH. These data are compared with other results showing the remarkable activity of FC in stimulating proton secretion in root segments, and they are interpreted as indicating that FC activates in roots, as in a wide variety of other plant organs, an electrogenic mechanism of proton extrusion deeply involved in cation transport. The hypothesis is proposed that the differential activity of the monovalent cations in inducing depolarization of PD might depend on their different affinity for the proton/cation electrogenic exchange mechanism.

Fusicoccin-induced, K+-stimulated proton secretion and acid-induced growth of apical root segments

Abstract 1. (1) Fusicoccin (FC) markedly stimulates proton secretion in isolated apical segments from pea, bean and maize roots. This effect of FC is already detectable after 1 min, and reaches a maximal rate approx. 10 min after the addition of the glucoside. 2. (2) FC-induced proton secretion is strongly stimulated by the presence in the medium of K+ (and not by that of Na+ or Li+) at concentrations higher than 2·10−4 M, while it is not significantly influenced by substitution of Cl− with the slowly penetrating SO4= or benzenesulfonate anions. Net K+ uptake is significantly stimulated by FC. 3. (3) Experiments of titration at constant pH in the range between pH 5.9 and 4 show that the rate of proton secretion in the absence of FC strongly depends on external pH. In contrast, FC-induced increase of the rate of proton extrusion appears very little affected by external pH in the same pH range. 4. (4) FC markedly stimulates elongation of the root segments. This effect is enhanced by the presence of K+ (but not of Na+ or Li+) in the medium. In the absence of FC low pH values of the medium strongly promote irreversible elongation of apical segments of maize roots. Maximal elongation rate is observed at pH 3.7. The effect of acid on elongation is consistently lesser than that of FC, and is not additive to it. The presence of K+ (and not of Na+) in the medium strongly increases FC-induced elongation at pH 6, while it has little effect at pH 3.7. These results are interpreted as indicating that FC stimulates in roots the same energy-dependent proton secretion mechanism previously shown to be activated by FC in stem, coleoptiles, leaves, cotyledons and embryos from dormant seeds. In roots, as in stem and coleoptiles, FC-induced stimulation of cell enlargement seems largely, but not completely explained by its capacity to decrease the pH in the cell wall space.

Localization of “in vitro” binding of the fungal toxin fusicoccin to plasma-membrane-rich fractions from corn coleoptiles

Abstract Radioactive fusicoccin ([3H]FC) associates in vitro with subcellular, post-mitochondrial particles from corn coleoptiles, as shown by the appearance of radioactivity in high speed pellets. This binding is specific, not readily chaseable, and saturable by 10−4M unlabelled FC. Auxins do not cross-compete for these sites. The binding is heat-labile at 60°C and has a pH optimum around 6. Differential centrifugation and isopycnic sucrose density gradients with parallel determination of markers indicate the localization of the FC binding sites at the plasmalemma. This is in agreement with the known in vivo effects of this fungal toxin.

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.