Christian Eing

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.

Effects of nanosecond pulsed electric field exposure on arabidopsis thaliana

Seven days old seedlings of Arabidopsis thaliana, suspended in a 0.4 S/m buffer solution were exposed to nanosecond pulsed electric fields (nsPEF) with a duration of 10 ns, 25 ns and 100 ns. The electric field was varied from 5 kV/cm up to 50 kV/cm. The specific treatment energy ranged between 100 J/kg and 10 kJ/kg. Due to electroporation of the plasma membrane of the plant cells, the seedlings completely died off, when 100 ns pulses and high electric field pulses were applied. But even at the highest specific treatment energies, 10 ns pulses had no lethal effect on the seedlings. An evaluation of the leaf area 5 and 7 days after pulsed electric field treatment revealed values twice the area of sham treated seedlings up to a specific treatment energy of 4 kJ/kg, when the applied field amplitude was low or the pulse duration 10 ns. A growth stimulating effect after short pulse exposition clearly could be detected. Contrary to the growth inhibiting effect of plasma membrane electroporation on the seedlings, a growth stimulation by nsPEF treatment does not scale with the treatment energy within the applied parameter range.

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.

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.

Pulsed Electric Field Treatment of Microalgae—Benefits for Microalgae Biomass Processing

In this paper, we demonstrate how pulsed electric field (PEF) treatment of microalgae biomass opens promising downstream processing options for an energetic use of algae. The fact that lipid droplets remain intracellular after treatment facilitates selective processing. After separation of the water-soluble cell contents, the algae lipids can be extracted with adequate solvents. The use of the environmental-friendly solvent ethanol results in an extraction yield from wet and dry biomass that, on average, is four times higher compared with untreated samples. Especially, the use of wet biomass opens a promising processing route for an energetic use of microalgae, because the energy consumed conventionally for drying of the biomass is considerably higher than the energy required for PEF treatment, i.e., ~ 1.5 MJ/kg of dry biomass.

Operation of an Electroporation Device for Grape Mash

In the course of the production of red wine, electroporation of mash enables a fast extraction of the red pigments from the skin without remarkable heating of the mash. For white wine, the formation of pores in the cells fosters, among others, the extraction of flavoring substances. During the harvest in 2008, the electroporation device KEA-WEIN has been operated successfully in two wineries. All together, more than 5 m3 of mash has been treated. During this years experiments, some experience in the operation of the electroporation device on-site in a winery has been gained. To facilitate the evaluation of the experimental results, the electroporation device has been equipped with different sensors and measurement systems, e.g., for the power drawn from the grid and the temperature of the mash before and after the electroporation. The data logged during one exemplary run are presented in this paper. Based on the measurements, an estimation about the energy consumption and efficiency of the device has been made. Must and wine made from the grape varieties Pinot Noir and Riesling have been chemically analyzed. Selected data are presented in this paper.

Electric measurement of the electroporation efficiency of mash from wine grapes

The degree of cell opening after electroporation can be derived from an electric impedance measurement in a frequency range between 500 Hz and 10 MHz. For a simple detection in a continuously flowing medium a measurement at a discrete frequency evaluating the phase shift between a measurement voltage across an electrode system and a current flowing through the sample volume has been investigated. The paper describes impedance measurements of the mash of wine grapes. For different grape varieties the frequency dependency of the phase shift has been measured. The data showed that the frequency of maximum phase shift differs with the grape variety. For the mash of wine grapes the measurement results based on the measurement of the complex impedance have been compared to the degree of extraction of color from the peel tissue of red wine grapes as a conventional method to determine the degree of denaturation. The measurements showed a correspondence between the color intensity of the must and the electric measurements.

Electroporation-Assisted Dewatering as an Alternative Method for Drying Plants

During the last few years, electroporation has been introduced to the food-processing industry as an effective method to open cell membranes for the extraction of substances. Currently, it has been investigated whether electroporation is also useful as a part of an energy-efficient drying process for plant material for the production of biofuel. To omit the use of additional water, the material has been placed in contact with the electrodes by means of its own juice after a first pressing step. This paper describes a laboratory-scale parameter study on the electroporation-assisted drying of whole maize plants, grass, and lucerne. The influence of the applied electric-field strength and the number of applied pulses on the drying curves is presented. For the electroporated material, an increased yield of juice during a pressing step after the electroporation and a faster drying have been observed.

Nanosecond Electric Pulses Affect a Plant-Specific Kinesin at the Plasma Membrane

AbstractElectric 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.

Monitoring of Pulsed Electric Field-Induced Abiotic Stress on Microalgae by Chlorophyll Fluorescence Diagnostic

Application of a pulsed electric field (PEF) on biological cells, in general, results in stress reactions of the affected organisms. Depending on pulse parameters, reactions such as growth stimulation or apoptosis can be observed for short pulses on the nanosecond (ns) time scale. Cell inactivation usually occurs at longer pulse duration and appropriate high treatment energy values. In this paper, the impact of short PEFs on chloroplasts of green microalgae Auxenochlorella protothecoides was investigated, with closer inspection of the photosystem II (PS II), located in the thylakoid membrane. For this purpose, a pulse amplitude modulated (PAM) fluorescence diagnostic was employed, which is a common method for monitoring changes in the photosynthesis apparatus. In particular, alterations of the PS II can be identified by fluorescence quenching analysis, sensitively. For PEF treatment of microalgae suspensions, the high-voltage pulse duration was adjusted to 100 and 1000 ns. The treatment energy was varied between 2 and 78 kJ/kg. The electric field amplitude was constant throughout the experiments (ECuv=40 kV/cm). After PEF treatment, the samples were periodically analyzed by chlorophyll fluorescence analysis for 1 h, using the saturation pulse method. For the evaluation of the physiological status of the microalgae, the maximum photochemical quantum yield of PS II, Fv/Fm, was chosen. The obtained results showed that the influence of PEFs on PS II is significant. Contrary to commonly accepted explanations that intracellular organelles are predominantly affected by short ns-pulses, a large influence of PEF exposure on chloroplasts, particularly on PS II, could be identified for longer pulses. In this paper, the diagnostic method, applied pulse protocols, and the results of the PAM fluorescence measurements will be discussed.