Nanosecond electric pulses affect a plant-specific kinesin at the plasma membrane.

Electric pulses with high field strength and durations in the nanosecond range (nsPEFs) are of considerable interest for biotechnological and medical applications. However, their actual cellular site of action is still under debate--due to their extremely short rise times, nsPEFs are thought to act mainly in the cell interior rather than at the plasma membrane. On the other hand, nsPEFs can induce membrane permeability. We have revisited this issue using plant cells as a model. By mapping the cellular responses to nsPEFs of different field strength and duration in the tobacco BY-2 cell line, we could define a treatment that does not impinge on short-term viability, such that the physiological responses to the treatment can be followed. We observe, for these conditions, a mild disintegration of the cytoskeleton, impaired membrane localization of the PIN1 auxin-efflux transporter and a delayed premitotic nuclear positioning followed by a transient mitotic arrest. To address the target site of nsPEFs, we made use of the plant-specific KCH kinesin, which can assume two different states with different localization (either near the nucleus or at the cell membrane) driving different cellular functions. We show that nsPEFs reduce cell expansion in nontransformed cells but promote expansion in a line overexpressing KCH. Since cell elongation and cell widening are linked to the KCH localized at the cell membrane, the inverted response in the KCH overexpressor provides evidence for a direct action of nsPEFs, also at the cell membrane.

Plant actin controls membrane permeability.

The biological effects of electric pulses with low rise time, high field strength, and durations in the nanosecond range (nsPEFs) have attracted considerable biotechnological and medical interest. However, the cellular mechanisms causing membrane permeabilization by nanosecond pulsed electric fields are still far from being understood. We investigated the role of actin filaments for membrane permeability in plant cells using cell lines where different degrees of actin bundling had been introduced by genetic engineering. We demonstrate that stabilization of actin increases the stability of the plasma membrane against electric permeabilization recorded by penetration of Trypan Blue into the cytoplasm. By use of a cell line expressing the actin bundling WLIM domain under control of an inducible promotor we can activate membrane stabilization by the glucocorticoid analog dexamethasone. By total internal reflection fluorescence microscopy we can visualize a subset of the cytoskeleton that is directly adjacent to the plasma membrane. We conclude that this submembrane cytoskeleton stabilizes the plasma membrane against permeabilization through electric pulses.

A patch clamp study on the electro-permeabilization of higher plant cells: Supra-physiological voltages induce a high-conductance, K+ selective state of the plasma membrane.

Permeabilization of biological membranes by pulsed electric fields ("electroporation") is frequently used as a tool in biotechnology. However, the electrical properties of cellular membranes at supra-physiological voltages are still a topic of intensive research efforts. Here, the patch clamp technique in the whole cell and the outside out configuration was employed to monitor current-voltage relations of protoplasts derived from the tobacco culture cell line "Bright yellow-2". Cells were exposed to a sequence of voltage pulses including supra-physiological voltages. A transition from a low-conductance (~0.1 nS/pF) to a high-conductance state (~5 nS/pF) was observed when the membrane was either hyperpolarized or depolarized beyond threshold values of around -250 to -300 mV and +200 to +250 mV, respectively. Current-voltage curves obtained with ramp protocols revealed that the electro-permeabilized membrane was 5-10 times more permeable to K+ than to gluconate. The K+ channel blocker tetraethylammonium (25 mM) did not affect currents elicited by 10 ms-pulses, suggesting that the electro-permeabilization was not caused by a non-physiological activation of K+ channels. Supra-physiological voltage pulses even reduced "regular" K+ channel activity, probably due to an increase of cytosolic Ca2+ that is known to inhibit outward-rectifying K+ channels in Bright yellow-2 cells. Our data are consistent with a reversible formation of aqueous membrane pores at supra-physiological voltages.

Transmembrane potential measurements on plant cells using the voltage-sensitive dye ANNINE-6.

The charging of the plasma membrane is a necessary condition for the generation of an electric-field-induced permeability increase of the plasmalemma, which is usually explained by the creation and the growth of aqueous pores. For cells suspended in physiological buffers, the time domain of membrane charging is in the submicrosecond range. Systematic measurements using Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) protoplasts stained with the fast voltage-sensitive fluorescence dye ANNINE-6 have been performed using a pulsed laser fluorescence microscopy setup with a time resolution of 5 ns. A clear saturation of the membrane voltage could be measured, caused by a strong membrane permeability increase, commonly explained by enhanced pore formation, which prevents further membrane charging by external electric field exposure. The field strength dependence of the protoplast's transmembrane potential V (M) shows strong asymmetric saturation characteristics due to the high resting potential of the plants plasmalemma. At the pole of the hyperpolarized hemisphere of the cell, saturation starts at an external field strength of 0.3 kV/cm, resulting in a measured transmembrane voltage shift of ∆V(M) = -150 mV, while on the cathodic (depolarized) cell pole, the threshold for enhanced pore formation is reached at a field strength of approximately 1.0 kV/cm and ∆V(M) = 450 mV, respectively. From this asymmetry of the measured maximum membrane voltage shifts, the resting potential of BY-2 protoplasts at the given experimental conditions can be determined to V(R) = -150 mV. Consequently, a strong membrane permeability increase occurs when the membrane voltage diverges |V(M)| = 300 mV from the resting potential of the protoplast. The largest membrane voltage change at a given external electric field occurs at the cell poles. The azimuthal dependence of the transmembrane potential, measured in angular intervals of 10° along the circumference of the cell, shows a flattening and a slight decrease at higher fields at the pole region due to enhanced pore formation. Additionally, at the hyperpolarized cell pole, a polarization reversal could be observed at an external field range around 1.0 kV/cm. This behavior might be attributed to a fast charge transfer through the membrane at the hyperpolarized pole, e.g., by voltage-gated channels.

Xylem ionic relations and salinity tolerance in barley.

Control of ion loading into the xylem has been repeatedly named as a crucial factor determining plant salt tolerance. In this study we further investigate this issue by applying a range of biophysical [the microelectrode ion flux measurement (MIFE) technique for non-invasive ion flux measurements, the patch clamp technique, membrane potential measurements] and physiological (xylem sap and tissue nutrient analysis, photosynthetic characteristics, stomatal conductance) techniques to barley varieties contrasting in their salt tolerance. We report that restricting Na(+) loading into the xylem is not essential for conferring salinity tolerance in barley, with tolerant varieties showing xylem Na(+) concentrations at least as high as those of sensitive ones. At the same time, tolerant genotypes are capable of maintaining higher xylem K(+)/Na(+) ratios and efficiently sequester the accumulated Na(+) in leaves. The former is achieved by more efficient loading of K(+) into the xylem. We argue that the observed increases in xylem K(+) and Na(+) concentrations in tolerant genotypes are required for efficient osmotic adjustment, needed to support leaf expansion growth. We also provide evidence that K(+)-permeable voltage-sensitive channels are involved in xylem loading and operate in a feedback manner to maintain a constant K(+)/Na(+) ratio in the xylem sap.

Nanosecond electric pulses trigger actin responses in plant cells.

We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

TPK1 is a vacuolar ion channel different from the slow-vacuolar cation channel.

TPK1 (formerly KCO1) is the founding member of the family of two-pore domain K(+) channels in Arabidopsis (Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca(2+)- and voltage-dependent outwardly rectifying plasma membrane K(+) channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca(2+) and voltage-dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast (Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100 mM KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca(2+), requiring micromolar concentration for activation. However, in contrast to the SV-type channel, TPK1 exhibits strong selectivity for K(+) over Na(+), and its activity turned out to be independent of the membrane voltage over the range of +/-80 mV. Our data clearly demonstrate that TPK1 is a voltage-independent, Ca(2+)-activated, K(+)-selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.