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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.

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.

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.

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.

Effects of calmodulin inhibitors in plants. I. Synergism with fusicoccin in the stimulation of growth and H+ secretion and in the hyperpolarization of the transmembrane electric potential

Abstract The effects of some calmodulin-Ca 2+ complex inhibitors (trifluoperazine (TFP), chlorpromazine (CP) and the cis and trans isomers of flupenthixol) on both basal and fusicoccin(FC)-stimulated growth, proton extrusion and transmembrane electrical potential (PD) in different plant materials have been investigated. TFP and CP significantly promoted growth and acid secretion in the materials investigated. The stimulation of acid secretion under basal conditions was small (pea stems and maize coleoptiles) or not significant (maize roots), while it became important when the inhibitors were fed together with FC at suboptimal concentration (namely, concentrations inducing 20–30% of the maximum activation of electrogenic proton extrusion). A similar behaviour was induced by either of the two cis and trans flupenthixol isomers, which resulted equally active. The effect of the calmodulin inhibitors and that of FC at suboptimal concentrations appeared synergistic. The promoting effect of TFP on H + secretion in maize roots was associated with a marked hyperpolarization of the PD. These results provide a first indication that calmodulin might be in some way involved in the regulation of proton secretion and electrogenesis in higher plants.

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+.