Physiological and biochemical responses of Quercus pubescens to air warming and drought on acidic and calcareous soils.

The drought- and thermo-tolerant Quercus pubescens, a tree species growing on both acidic and calcareous soils in the sub-Mediterranean region, was exposed to soil drought (-60% to -80% soil water content) and air warming (+1.2 °C daytime temperature), singly and in combination. The experiment was conducted on two natural forest soils with similar texture but different pH (acidic and calcareous soils). The physiological (photosynthesis) and biochemical (antioxidant system) responses of Q. pubescens were investigated. On acidic soil, Q. pubescens had a higher reactive oxygen species (ROS) content than on calcareous soil, confirming that this species is better adapted to the latter soil type. A down-regulation of ascorbate-glutathione cycle enzymes suggests that ROS were used as signalling molecules. Air warming stimulated stomatal opening, while soil drought induced stomatal closure in the late afternoon and reduced Rubisco carboxylation efficiency. Photosynthetic performance in the combined treatment was higher than under single drought stress and similar to control and air warming. Q. pubescens biochemical responses depended on soil pH. On acidic soil, Q. pubescens trees exposed to air warming used ROS as signalling molecules. On calcareous soil, these trees were able to balance both soil drought and air warming stress, avoiding ROS toxic effects by increasing antioxidant enzyme activitiy and maintaining a high enzymatic antioxidant defence. When combined, drought and air warming induced either more severe (higher oxidative pressure and impairment of the light-harvesting complex) or different responses (decline of the thermal energy dissipation capacity) relative to the single stressors. Overall, however, Q. pubescens preserved the functionality of the photosynthetic apparatus and controlled the antioxidant system response, thus confirming its drought and thermo-tolerance and therefore its potential to adapt to the ongoing climate change.

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

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.

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

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.

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.

Mutation in pore domain uncovers cation- and voltage-sensitive recovery from inactivation in KAT1 channel.

Effects of threonine substitution by glutamine at position 256 in the pore of the KAT1 channel have been investigated by voltage-clamp, using heterologous gene expression in Xenopus oocytes. The major discrepancy in T256Q from the wild-type channel (wt) was cation specific. While K(+) currents were reduced in a largely scalar fashion, the NH(4)(+) current exhibited slow, voltage-dependent inhibition during hyperpolarization. The same effects could be induced in wt, or intensified in T256Q, by addition of the impermeant cation methylammonium (MA(+)) to the bath. This stresses that both the mutation and MA(+) affect a mechanism already present in the wt. Assuming that current inhibition could be described as entry of the channel into an inactive state, we modeled in both wt and in T256Q the relaxation kinetics of the clamp currents by a C-O-I gating scheme, where C (closed) and I (inactivated) are nonconductive states, and O is an open state allowing K(+) and NH(4)(+) passage. The key reaction is the transition I-O. This cation-sensitive transition step ensures release of the channel from the inactive state and is approximately 30 times smaller in T256Q compared to wt. It can be inhibited by external MA(+) and is stimulated strongly by K(+) and weakly by NH(4)(+). This sensitivity of gating to external cations may prevent K(+) leakage from cation-starved cells.

A potassium channel protein encoded by chlorella virus PBCV-1.

The large chlorella virus PBCV-1, which contains double-stranded DNA (dsDNA), encodes a 94-codon open reading frame (ORF) that contains a motif resembling the signature sequence of the pore domain of potassium channel proteins. Phylogenetic analyses of the encoded protein, Kcv, indicate a previously unidentified type of potassium channel. The messenger RNA encoded by the ORF leads to functional expression of a potassium-selective conductance in Xenopus laevis oocytes. The channel blockers amantadine and barium, but not cesium, inhibit this conductance, in addition to virus plaque formation. Thus, PBCV-1 encodes the first known viral protein that functions as a potassium-selective channel and is essential in the virus life cycle.

Putrescine Channels in the Plasma Membrane of Arabidopsis thaliana.

The patch-clamp technique was used in the whole-cell configuration to study plasma membrane channels permeable to the diamine putrescine in protoplasts isolated from cultured cells of Arabidopsis thaliana L. Under our experimental conditions, no channels selectively mediating putrescine influx were observed. Inward K+ channels showed a low permeability to putrescine, the permeability ratio of putrescine relative to K+ being around 0.1. Further characterization of the previously identified outward channels mediating putrescine efflux (R. Colombo, R. Cerana, N. Bagni [1992] Biochem Biophys Res Commun 182: 1187-1192) indicated that their activity was regulated by the overall ion concentration of the external medium.

Tonoplast K+ channels in Arabidopsis thaliana are activated by cytoplasmic ATP.

The effect of ATP on the slow vacuolar cation channels in the tonoplast of suspension-cultured cells of Arabidopsis thaliana L. was examined using the patch clamp technique in the whole-vacuole configuration. The channels were specifically activated by cytoplasmic ATP, other nucleotides (ADP, GTP and nonhydrolizable ATP analogues) being ineffective.

Evidence for polyamine channels in protoplasts and vacuoles of Arabidopsis thaliana 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.

Inward Rectifying K Channels in the Plasma Membrane of Arabidopsis thaliana.

Ion channels in the plasma membrane of protoplasts isolated from cultured cells of Arabidopsis thaliana were studied by means of the patch-clamp technique applied in the whole-cell configuration. In some protoplasts, depolarizing pulses and, in other protoplasts, hyperpolarizing pulses elicited time-dependent currents; both kinds of current were only rarely observed in the same protoplast. The hyperpolarization-activated inward rectifying currents, the focus of this paper, appeared to be due to the relatively slow opening of channels (activation time constant = 150 to 300 milliseconds), which closed at positive potentials. The reversal potential of this current, measured in the presence of different ion concentrations (symmetrical or asymmetrical K(+) and Cl(-) or gluconate), was always close to the electrochemical equilibrium potential of K(+). The currents were inhibited by 10 millimolar tetraethylammonium, a K(+) channel blocker. These data show that the hyperpolarization-activated currents flow through K(+) channels, which can provide a pathway for the passive diffusion of K(+) down its electrochemical gradient.

Regulation of tonoplast k channels by voltage in the range of physiological electric potentials.

The presence of tonoplast ion channels regulated by voltage in the physiological range of transtonoplast electric potential was studied in isolated vacuoles from Acer pseudoplatanus cultured cells. In symmetrical KCl or K-gluconate depolarizing pulses induced instantaneously developing, decaying outward currents, while in symmetrical tetramethylammonium chloride these currents were absent. The outward currents were reduced if the depolarizations were applied from a holding potential of +30 millivolts and increased upon depolarizations from a holding potential of -30 millivolts and even more from a holding potential of -50 millivolts. These results indicate that the outward currents are due to K(+) movement through channels which are open around 0 millivolt and close at positive potentials. These K(+) channels, regulated in the range of the physiological electric potentials reported for the vacuoles in situ, are likely the same K(+) channels activated by hyperpolarizations which we have previously described (R Colombo, R Cerana, P Lado, A Peres [1988] J Membr Biol 103: 227-236).

Tributylbenzylammonium (TBBA(+))-dependent fusicoccin (FC)-induced H(+) extrusion in maize roots: relationship between the stimulating effects of TBBA(+) on H (+) extrusion and on Cl (-) efflux.

The mechanism of the stimulating effect of lipophilic cations on H(+) extrusion in maize root segments (Zea mays L.) has been investigated. The measurement of the uptake of [(3)H]tributylbenzylammonium ([(3)H]TBBA(+)), the most active lipophilic cation on H(+) extrusion, indicated that although a relevant fraction of TBBA(+) taken up by the tissue is adsorbed to cell surfaces, a fraction of the cation enters the cells. However no correlation was observed between the rate of TBBA(+) uptake and that of H(+) extrusion. On the other hand, the lipophilic cations active on H(+) extrusion (TBBA(+) and dibenzyldimethylammonium (DDA(+))), in the presence of fusicoccin (FC), induced under the same conditions an efflux of Cl(-), while tetramethylammonium (TMA(+)), inactive on H(+) extrusion, did not. The stimulation of Cl(-) efflux by TBBA(+) was independent of the anion present in the medium and was inhibited by Na-orthovanadate, an inhibitor of plasma membrane ATPase and of TBBA(+)-induced H(+) extrusion. These results suggest that the stimulation of H(+) extrusion by TBBA(+) depends on its effect on Cl(-) efflux rather than on its penetration into the cells.

Relationship between ATP Level and Activity of Fusicoccin-stimulated H/K-Exchange System in Plant Tissues.

The treatment with fusicoccin causes a slight but significant decrease (about 15%) in the ATP level in pea-internode and maize-coleoptile segments. This decrease is detectable within 15 minutes and is accompanied by a parallel increase in O(2) uptake. Sodium azide inhibits O(2) uptake and completely blocks the stimulation of O(2) uptake by fusicoccin in both pea and coleoptile segments. Benzohydroxamic acid does not affect either basal or fusicoccin-induced O(2) uptake in maize-coleoptile sections. The drop of ATP level induced by various treatments (sodium arsenate, 2-deoxyglucose, limiting O(2), and anaerobiosis) is accompanied by a parallel inhibition of K(+) uptake in maize coleoptiles treated with or without fusicoccin. These results are consistent with the hypothesis that ATP is the energy source for the fusicoccin-activated H(+)/K(+)-exchange system.