Fusicoccin Concentration Research Articles

Effect of K+ and Ca2+ on the indole-3-acetic acid- and fusicoccin-induced growth and membrane potential in maize coleoptile cells.

The role of potassium (K(+)) and calcium (Ca(2+)) in the regulation of plant growth and development is complex and needs a diverse range of physiological studies. Both elements are essential for satisfactory crop production. Here, the effects of K(+) and Ca(2+) ions on endogenous growth and growth in the presence of either indole-3-acetic acid (IAA) or fusicoccin (FC) were studied in maize (Zea mays) coleoptiles. Membrane potentials of coleoptile parenchymal cells, incubated in media containing IAA, FC and different concentrations of K(+) and Ca(2+), were also determined. Growth experiments have shown that in the absence of K(+) in the incubation medium, both endogenous and IAA- or FC-induced growth were significantly inhibited by 0.1 and 1 mM Ca(2+), respectively, while in the presence of 1 mM K(+) they were inhibited only by 1 mM Ca(2+). At 10 mM K(+), endogenous growth and growth induced by either IAA or FC did not depend on Ca(2+) concentration. TEA-Cl, a potassium channel blocker, added 1 h before IAA or FC, caused a reduction of growth by 59 or 45 %, respectively. In contrast to TEA-Cl, verapamil, the Ca(2+) channel blocker, did not affect IAA- and FC-induced growth. It was also found that in parenchymal cells of maize coleoptile segments, membrane potential (Em) was strongly affected by the medium K(+), independently of Ca(2+). However, lack of Ca(2+) in the incubation medium significantly reduced the IAA- and FC-induced membrane potential hyperpolarization. TEA-Cl applied to the control medium in the same way as in growth experiments caused Em hyperpolarization synergistic with hyperpolarization produced by IAA or FC. Verapamil did not change either the Em of parenchymal cells incubated in the control medium or the IAA- and FC-induced membrane hyperpolarization. The data presented here have been discussed considering the role of K(+) uptake channels in regulation of plant cell growth.

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 inhibitor's 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 inhibitor's 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.

The Agrobacterium vitis T‐6b oncoprotein induces auxin‐independent cell expansion in tobacco

Among the Agrobacterium T-DNA genes, rolB, rolC, orf13, orf8, lso, 6b and several other genes encode weakly homologous proteins with remarkable effects on plant growth. The 6b oncogene induces tumors and enations. In order to study its properties we have used transgenic tobacco plants that carry a dexamethasone-inducible 6b gene, dex-T-6b. Upon induction, dex-T-6b plants develop a large array of morphological modifications, some of which involve abnormal cell expansion. In the present investigation, dex-T-6b-induced expansion was studied in intact leaves and an in vitro leaf disc system. Although T-6b and indole-3-acetic acid (IAA) both induced expansion and were non-additive, T-6b expression did not increase IAA levels, nor did it induce an IAA-responsive gene. Fusicoccin (FC) is known to stimulate expansion by increasing cell wall plasticity. T-6b- and FC-induced expansion were additive at saturating FC concentrations, indicating that T-6b does not act by a similar mechanism to FC. T-6b expression led to higher leaf osmolality values, in contrast to FC, suggesting that the T-6b gene induces expansion by increasing osmolyte concentrations. Metabolite profiling showed that glucose and fructose played a major role in this increase. We infer that T-6b disrupts the osmoregulatory controls that govern cell expansion during development and wound healing.

CHANGE IN THE ACTIVITY OF LECTINS IN CELL STRUCTURE OF WINTER WHEAT CROWN UNDER THE ACTION OF LOW-TEMPERATURE HARDENING AND FUSICOCCIN

Haemagglutinating activity was determined in cell walls and total cell organelles of crown cells of Winter wheat (Triticum aestivum L.) plants. The effect of fusicoccin (FC) was investigated using fractions obtained from plants hardened for 7 days at 2°C and from untreated plants. FC concentration (5×10−7m) increased the frost resistance of the plants. The temporal pattern of lectin activity during hardening could be described by a single-peak curve. In the cell wall fraction, the highest activity manifested itself after one-day hardening, and in the fraction of organelles it peaked after five-days hardening. The carbohydrate specificity of lectins also changed during hardening; cell wall lectins completely lost their capacity for interaction with uridine diphosphoglucose, glucose 6-phosphate, D-galactosamine, and N-acetylglucosamine and the lectins of organelles retained some affinity only for amino sugars. After hardening the test plants, the activity of the lectins increased substantially in the cell walls and plastids, decreased in the nuclei, and was practically flat in mitochondria and microsomes. Consequently, low temperature and FC with their antistress effect improved frost resistance and stimulated the activity of the lectins of some cell structures of the tillering node of winter wheat. A similar action of low temperature and FC in increasing the activity of lectins of plastids was found. Further information was obtained on the subcellular localization of lectins providing additional information on their possible participation in the development of frost resistance of winter wheat.

The pro Gene Causes an Enhanced Cell Expansion Response to Fusicoccin in Tomato

The response to fusicoccin (FC) of the elongated tomato mutant procera was tested. Hypocotyl sections from etiolated procera seedlings responded to a range of FC concentrations similar to that of normal sections, but at a given FC concentration the mutant sections exhibited about 40% greater elongation than normal. The initial rates of elongation in FC were similar for normal and procera sections, but elongation continued at a high rate for a longer period in the mutant. Measurements of outer cortical cells demonstrated that the enhanced elongation of the procera hypocotyl sections was due to enhanced cell expansion.

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.

Regulation of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase by the Plasmalemma Proton Pump in Acer pseudoplatanus L. Cultured Cells

The involvement of the plasmalemma proton pump in the fusicoccin-dependent activation of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase) and of ethylene production in cultured Acer cells has been investigated. A good correspondence between the degree of acidification of the medium and the increase of ethylene production was observed for a wide range of stimulating fusicoccin concentrations. Vanadate and Erythrosin B, known as inhibitors of the plasmalemma proton pump, inhibited both the acidification of the medium and the stimulation of ethylene production; 0.6 M mannitol did not inhibit the «basal» production of ethylene either in hormone depleted cells or in cells re-supplied with growthpromoting 2,4-dichlorophenoxyacetic acid (2,4-D). On the contrary, mannitol practically suppressed the stimulation of ethylene production by fusicoccin but the acidification of the medium was only slightly reduced. Vanadate did not influence the cellular level of ACC. Mannitol almost doubled the level of ACC in the fusicoccin treated cells. The data are discussed in terms of regulation by the plasmalemma proton pump of an ACC oxidase presumably associated to the plasma membrane.

Isolation and biochemical characterization of fusicoccin-insensitive variant lines of Arabidopsis thaliana (L.) Heynh

Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H+-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a 3H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.

Phytochrome‐mediated closure of Albizzia lophantha leaflets as affected by sorbitol, mannitol and fusicoccin

The effect of sorbitol and mannitol and their interaction with the effect of fusiccocin has been investigated in phytochrome‐mediated nyctinastic closure of Albizzia lophantha leaflets. Treatments were commenced 2 h into the photoperiod. Pairs of leaflets were excised, floated on test solutions, and transferred to darkness immediately after red or far red irradiation. Leaflet angles were measured in darkness for 3 h. Fusicoccin (1 to 100 μM) inhibited nyctinastic closure and enhanced reopening in the presence of Pft (far‐red‐absorbing form of phytochrome). Fusicoccin effects on far red treated leaflets were not constant and whichever fusicoccin concentration was applied, the effects were small. These results confirm the role of the plasma membrane proton pump in extensor cells during opening. Sorbitol and mannitol (25–100 mM) increased nyctinastic closure for about 2 h and then enhanced reopening in the presence of Pfr. When sugar alcohols were applied together with fusicoccin, sorbitol and mannitol enhanced the fusicoccin inhibition of closure and a marked synergistic effect was observed. These results indicate that both fusiccocin and the reduction of external osmotic potential caused by sorbitol and mannitol supply may stimulate the activity of the extensor H+ pump promoting the reopening.

Characteristics and implications of prolonged fusicoccin‐induced growth of Avena coleoptile sections

A study has been made of the prolonged growth of Avena coleoptile sections in response to fusicoccin (FC), a phytotoxin that promotes apoplastic acidification. The final amount of FC-induced growth is a function of the FC concentration. Removal of the epidermis speeds up the initial rate of elongation and shortens the duration of the response, without affecting the total amount of extension. A suboptimal FC concentration (7 x 10(-8) M) which induces the same rate of proton excretion as does optimal indoleacetic acid (IAA) (1 x 10(-5) M), causes elongation which is 60-75% of that induced by IAA in 4 h or 50-65% in 7 h. This suggests that acid-induced extension could make a major contribution to auxin-induced growth for at least 7 h.

Synergistic Effect of Light and Fusicoccin on Stomatal Opening

Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K(+) channel blocker, tetraethyl-ammonium(+) (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (-60 millivolts) at which K(+) channels are inactivated. These results are indicative of FC activation of a H(+) pump. FC effects on the magnitude of inward and outward K(+) currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H(+) ATPase of guard cells is a primary locus for the FC effect on stomatal apertures.

Some Properties of a Functional Reconstituted Plasmalemma H+-ATPase Activated by Fusicoccin

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H(+)-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V(max) of the enzyme. Fusicoccin stimulation of maize H(+)-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).

Fusicoccin action in cell-suspension cultures of Corydalis sempervirens Pers.

Mid-log-phase cell suspensions of Corydalis sempervirens Pers., when incubated in micromolar or submicromolar concentrations of fusicoccin, strongly acidified the culture medium. High-affinity fusicoccin-binding sites were found in microsomes prepared from these cells using the radioligand [(3)H]-9'-norfusicoccin-8'-alcohol. Binding was saturable with an apparent dissociation constant (K d) of 2.8 nM, a pH optimum of 6.0, a temperature optimum of 35° C and was rapid (t1/2 = 8 min). The site abundance was 0.76±0.17 pmol · (mg of protein)(-1). In the same membrane preparations, the K(+), Mg(2+)-ATPase (EC 3.6.1.3) was characterized. The enzyme was highly vanadate-sensitive (IC50=6.5 μM) and nucleotide-specific (ATP≫NTP), had a pH optimum of 6.2, an apparent K m for ATP of 0.23±0.12 mM, and V max of 10.6±1.8 nkat (mg of protein)(-1). Fusicoccin doubled V max and lowered, by a factor of 2, the apparent K m for ATP of the enzyme when the cells were incubated with the toxin for 30 min prior to homogenization of the cells. The stimulation of the enzyme was also pronounced when fusicoccin was added to the homogenization medium just prior to homogenization of the cells, but was slight to zero when the toxin was added at the microsomal stage. The pronounced stimulatory effect of fusicoccin on the ATPase was seen at pH 7.1, i.e. at a pH typical for the cytoplasmic compartment, but was not detectable at pH 6.2, the pH optimum of the enzyme. The implications of these findings for an understanding of fusicoccin action are discussed.

The role of pH and ion transport in oligosaccharide‐induced proteinase inhibitor accumulation in tomato plants

Abstract. The induction of proteinase inhibitor (PrI) activity in young tomato plants by wounding and oligosaccharides has been shown to be prevented by pretreatment of the plants with phenolic acids such as aspirin. Aspirin applied over a wide range of concentrations is able to inhibit PrI induction by pectic fragments. The possibility that other weak acids may also inhibit PrI induction was investigated. Isobutyric acid and trimethylacetic acid were shown to be less effective as inhibitors than aspirin, and weak bases were without effect. However, it was demonstrated that various agents known to influence ATPase activity and intracellular pH were able to inhibit PrI induction, and in particular the striking effect of low concentrations of fusicoccin on PrI induction was noted.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K(d) of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K(d) 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H(+)-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H(+)-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H(+)-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.

Evidence that H + extrusion in Elodea densa leaves is mediated by an ATP-driven H + pump

Data are presented on the ATP-driven proton extrusion mechanism in Elodea densa leaves, in the absebce of photosynthetic activity. In this material, in the dark, or in the light with photosynthesis inhibited by DCMU, H + extrusion at pH 6 was very low in the absence of permeating cations and became significant (approx. 1.8 μmol/g fresh wt.) in the presence of K +. Rb + and, much less so, Na +, could partially substitute for K + while Li +, Cs +, Ca 2+, Co 2+, Ni 2+ were ineffective. Fusicoccin (FC), a toxin which stimulates the plasmalemma H + ATPase activity in several plant tissues, strongly increased H + extrusion only in the presence of K +, Rb +, and, to a much lesser extent, Na +. The capacity of cations to stimulate FC-induced H + extrusion decreased in the same order as their E m -depolarizing activity (K + > Rb + ⪢ Na +). Increasing concentrations of FC, between 10 −7 and 5 × 10 +4 M, showed a saturating effect for H + extrusion at 10 −4 M FC. The stimulating effect of FC on H + extrusion was accompanied by a hyperpolarization of the transmembrane electrical potential (by approx. 30 mV), larger in the absence than in the presence of K +, and by a marked increase of K + uptake. FC-induced H + extrusion was strongly inhibited by orthovanadate and erythrosin B, two inhibitors of the plasmalemma H + transporting ATPase. The activity of an ATPase and an ATP-dependent H + transport, both sensitive to vanadate and erythrosin B, were also demonstrated in membrane vesicle preparations. These results are interpreted as indicating the presence in E. densa leaves of an electrogenic H + extrusion mechanism which appears identical to the K +-dependent, FC-sensitive, ATP-driven proton pump described in several terrestrial plant cells.

The effects of podolactone‐type inhibitors on fusicoccin‐induced growth and proton efflux

Podolactones A and E and lycoritidinol inhibit growth induced by fusicoccin in dwarf pea (Pisum sativum L.) hooks and tips; however the inhibition is reduced at the highest fusicoccin concentrations. In short term experiments (3 h) on pea stem tissues, growth and proton efflux induced by fusicoccin are only partially inhibited by podolactone A and lycoricidinol. Auxin‐induced growth and proton efflux are completely suppressed by 10 μM lycoricidinol. The inhibitors do not affect ATP levels.

Ca2+-Counteracted Changes of Membrane Functions Induced by Calmodulin Antagonists in Red Beet Storage Tissue, in Pea and Maize Tissues Treated with Optimal Fusicoccin Concentrations

Previous work shows that the two calmodulin antagonists trifluoperazine (TFP) and chlorpromazine (CP), applied in vivo to isolated segments from Pea stems maize coleoptiles and roots, enhance acid secretion and hyperpolarize the transmembrane electric potential difference (PD) (maize roots) in the absence and, more markedly, in the presence of a fusicoccin (FC) concentration inducing half maximal stimulation of electrogenic H+ extrusion. The present data show that, when the two drugs are supplied in the presence of optimal FC concentrations, their stimulating effect on acid secretion disappears and in the roots becomes inhibitory and is associated with a depolarization of the PD. A similar inhibition of acid secretion and depolarization of PD is induced by TFP and CP in red beet root disks. TFP and CP induce an increase of the leakage of ninhydrin-reacting substances in Pea stem and maize root segments and of betacyanine in red beet root disks both in the absence and in the presence of FC. These responses to the calmodulin antagonists are partially counteracted by 0.5 mM CaSO4. No correlation is observed between the changes in the leakage of amino acids induced by the drugs and the simultaneous effects of these drugs on acid secretion under the different conditions. These results suggest that the two calmodulin antagonists, when applied with PC at optimal concentration, inhibit electrogenic H+ extrusion. The question remains open, however, whether this effect depends on the inactivation of the Ca2+-calmodulin complex or on consequent changes of free Ca2+ concentration or on non-specific modifications of the membrane structure.

Effect of Fusicoccin on Dark 14CO2 Fixation by Vicia faba Guard Cell Protoplasts

When Vicia faba guard cell protoplasts were treated with fusicoccin, dark (14)CO(2) fixation rates increased by as much as 8-fold. Rate increase was saturated with less than 1 micromolar fusicoccin. Even after 6 minutes of dark (14)CO(2) fixation, more than 95% of the incorporated radioactivity was in stable products derived from carboxylation of phosphoenolpyruvate (about 50% and 30% in malate and aspartate, respectively). The relative distribution of (14)C among products and in the C-4 position of malate (initially more than 90% of [(14)C]malate) was independent of fusicoccin concentration. After incubation in the dark, malate content was higher in protoplasts treated with fusicoccin. A positive correlation was observed between the amounts of (14)CO(2) fixed and malate content.It was concluded that (a) fusicoccin causes an increase in the rate of dark (14)CO(2) fixation without alteration of the relative fluxes through pathways by which it is metabolized, (b) fusicoccin causes an increase in malate synthesis, and (c) dark (14)CO(2) fixation and malate synthesis are mediated by phosphoenolpyruvate carboxylase.

Evaluation of H+ Secretion Relative to Zeatin-Induced Growth of Detached Cucumber Cotyledons

Cytokinins promote expansion of cotyledons detached from seedlings of more than a dozen species. The zeatin-enhanced expansion of cucumber (Cucumis sativus L. cv Marketer) cotyledons was investigated. In addition, whether acid secretion is involved in wall loosening accompanying such accelerated growth was evaluated. For cotyledons abraded with carborundum or cut into either eight or 18 pieces, we detected no zeatin-enhanced acidification of the growth medium during growth periods of 3 days. Measurements of pH values on each surface of zeatin-treated, abraded cotyledons after 3 days of growth also showed no detectable acidification caused by the hormone. Furthermore, with several buffers at pH values ranging from 5 to 8, growth of nonabraded, abraded, or cut cotyledons with or without zeatin was independent of external pH. However, experiments restricted to about 12 hours indicated that certain acidic buffers enhanced growth of cotyledons cut into 18 pieces. Lastly, concentrations of fusicoccin that caused growth promotion equal to that of zeatin initiated substantial acidification of the medium. Collectively, these data suggest that zeatin-induced expansion of detached cucumber cotyledons is independent of H(+) secretion.