Raffaella Cerana

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.

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.

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.

Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells

Programmed cell death (PCD) plays a vital role in plant development and is involved in defence mechanisms against biotic and abiotic stresses. Different forms of PCD have been described in plants on the basis of the cell organelle first involved. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin (FC) induces cell death. However, only a fraction of the dead cells shows the typical hallmarks of animal apoptosis, including cell shrinkage, chromatin condensation, DNA fragmentation and release of cytochrome c from the mitochondrion. In this work, we show that the scavenging of nitric oxide (NO), produced in the presence of FC, by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and rutin inhibits cell death without affecting DNA fragmentation and cytochrome c release. In addition, we show that FC induces a massive depolymerization of actin filaments that is prevented by the NO scavengers. Finally, the addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of cPTIO on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that besides an apoptotic-like form of PCD involving the release of cytochrome c, FC induces at least another form of cell death, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD.

Comparison between the effects of fusicoccin, Tunicamycin, and Brefeldin A on programmed cell death of cultured sycamore (Acer pseudoplatanus L.) cells

Summary.Programmed cell death occurs in plants during several developmental processes and during the expression of resistance to pathogen attack (i.e., the hypersensitive response). An unsolved question of plant programmed cell death is whether a unique signaling pathway or different, possibly convergent pathways exist. This problem was addressed in cultured sycamore (Acer pseudoplatanus L.) cells by comparing the effects of fusicoccin, Tunicamycin and Brefeldin A, inducers of programmed cell death with well-defined molecular and cellular targets, on some of the parameters involved in the regulation of this process. In addition to cell death, the inducers are able to stimulate the production of H2O2, the leakage of cytochrome c from mitochondria, the accumulation of cytosolic 14-3-3 proteins, and changes at the endoplasmic reticulum level, such as accumulation of the molecular chaperone binding protein and modifications in the organelle architecture. Interestingly, no additive effect on any of these parameters is observed when fusicoccin is administered in combination with Tunicamycin or Brefeldin A. Thus, these inducers seem to utilize the same or largely coincident pathways to induce programmed cell death and involvement of the endoplasmic reticulum, in addition to that of mitochondria, appears to be a common step.

Fusicoccin induces in plant cells a programmed cell death showing apoptotic features

Summary. Programmed cell death plays a pivotal role in several developmental processes of plants and it is involved in the response to environmental stresses and in the defense mechanisms against pathogen attack. It has not yet been defined which part of the death signalling mechanism and which molecules involved in programmed cell death are common to animals and plants. In this paper we show that fusicoccin, a well-known phytotoxin, induces a strong acceleration in the appearance of Evans Blue-stainable (dead) cells in sycamore (Acer pseudoplatanus L.) cultures. This fusicoccin-induced cell death shows aspects common to the form of animal programmed cell death termed apoptosis: i.e., cell shrinkage, changes in nucleus morphology, increase in DNA fragmentation detectable by a specific immunological reaction, and presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Since fusicoccin has a well-identified molecular target, the plasma membrane H+-ATPase, and thoroughly investigated physiological effects, this toxin appears to be a useful tool to study the transduction of death signals leading to programmed cell death in plants.

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.

Cyclosporin A inhibits programmed cell death and cytochrome c release induced by fusicoccin in sycamore cells

Summary.Programmed cell death plays a vital role in normal plant development, response to environmental stresses, and defense against pathogen attack. Different types of programmed cell death occur in plants and the involvement of mitochondria is still under investigation. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin induces cell death that shows apoptotic features, including chromatin condensation, DNA fragmentation, and release of cytochrome c from mitochondria. In this work, we show that cyclosporin A, an inhibitor of the permeability transition pore of animal mitochondria, inhibits the cell death, DNA fragmentation, and cytochrome c release induced by fusicoccin. In addition, we show that fusicoccin induces a change in the shape of mitochondria which is not prevented by cyclosporin A. These results suggest that the release of cytochrome c induced by fusicoccin occurs through a cyclosporin A-sensitive system that is similar to the permeability transition pore of animal mitochondria and they make it tempting to speculate that this release may be involved in the phytotoxin-induced programmed cell death of sycamore cells.

Effects of Cercospora beticola toxin on ATP level of maize roots and on the phosphorylating activity of isolated pea mitochondria

Abstract The Cercospora beticola toxin (CBT), an inhibitor of membrane transport phenomena, did not significantly affect ATP and pyruvate levels of maize roots at concentrations up to 40 μg/ml. In contrast, in isolated mitochondrial preparations from pea stem, CBT severely inhibited oxidative phosphorylation at concentrations higher than 10 μg/ml. The oligomycin-sensitive ATPase activity of these mitochondrial preparations was completely inhibited by 10 μg/ml CRT. The results suggest that CBT fed to root segments at concentrations from 10 to 40 μg/ml does not influence the energy supply linked to oxidative phosphorylation, but it markedly inhibits mitochondrial functions in cell free systems and, in particular, mitochondrial ATPase and thus phosphorylation. The lack of effect of CBT on ATP and pyruvate levels of root segments is interpreted as being due to the lack of penetration of the toxin in the intact cells. These data are thus consistent with the view that the inhibiting action of CBT on cellular ion transport depends on its direct action on some systems located on the plasmalemma.