Effects of nanosecond pulsed electric field exposure on arabidopsis thaliana

ABSTRACT 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 plasmamembrane 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 plasmamembrane electroporation on the seedlings, a growth stimulation by nsPEF treatment does not scale with the treatment energy within the applied parameter range

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

PEF assisted extraction of valuable compounds from microalgae C. vulgaris

In recent years the cultivation and exploitation of Microalgae biomass has stimulated intensive research due both to its high productivity, when compared to agriculturally grown biomass, and the large relative content of more than 50% of valuable cell components, like lipids, proteins, polysaccharides, antioxidants, vitamins and pigments (Goettel et al., 2013; Luengo et al., 2014). However, the extraction of these compounds is limited by the mass transfer of these compounds through cell membranes. In this frame, thanks to the reduction of the resistances to mass transfer due to the induced permeabilization of plant cells, Pulsed Electric Field (PEF) technology can be used as a pre-treatment of concentrated microalgae suspension in order to enhance the extraction yield of valuable compounds (such as antioxidants and colorants) from the inner parts of the cells(Mahnič-Kalamiza et al., 2014). In this work the effect of different PEF treatment intensity on the extraction of valuable compound from microalgae Chlorella Vulgaris was investigated. Culture of C. vulgaris strain inoculated in TAP-medium, were cultivated in batch 26 L photo-bioreactor. Growth conditions were monitored by OD measurements at 750 nm. The algae were harvested after 18-24 days and concentrated up to a final biomass concentration of about 40-50 gdw/kgsus. PEF experiments of different intensities (E=27-35 kV/cm, and WT=50-100- 150 kJ/kg) were carried out in a laboratory scale continuous flow unit. Determinations of time-conductivity profile as well as quantification of dry matter, protein content, total polyphenolics (TP), and antioxidant activity (AA) of the supernatant were performed. Results showed a higher increase of the electrical conductivity of PEF treated suspension, when compared to the untreated sample. Moreover, the PEF treatment increased the dry matter content as well as the amount of proteins released into the supernatant from inside the algae cells. Additionally, increments of total phenolics and antioxidant activity were also detected. The results obtained from this study demonstrate the potential of PEF for improving extraction of compounds of interest from the microalgae C. vulgaris

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

AbstractPermeabilization 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 (~0.1nS/pF) to a high-conductance state (~5nS/pF) was observed when the membrane was either hyperpolarized or depolarized beyond threshold values of around −250 to −300mV and +200 to +250mV, 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 (25mM) did not affect currents elicited by 10ms-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