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

Recently, effects of pulsed electric fields on plant cells have become an interesting topic of research in plant electrophysiology. In analogy to results obtained with mammalian cells, systematic measurements using tobacco protoplasts in suspension have been performed using a Pulsed Laser Fluorescence Microscopy (PLFM) setup. Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) has been chosen due to its well-known properties as a model in plant physiology. Furthermore, it exhibits several advantages such as a short cell cycle and the absence of potentially disturbing chlorophyllic fluorescence. In contrast to mammalian cells, the field-strength-dependence of the protoplast´s transmembrane potential shows strong asymmetric saturation characteristics due to the influence of the higher resting potential. At the hyperpolarized hemisphere of the cell, saturation initiates at field strengths in the order of 0.6...0.7 kV/cm, while on the cathodic (depolarized) side the threshold for pore formation is reached at lower fields due to the superposition of the resting potential. Pulse-train experiments reveal that a subsequent re-charging of cell membranes to the same value is possible within a few seconds. This indicates a fast full recovery process of the cell membrane. Obviously, irreversible pores from preceding pulses do not influence a subsequent charging process. To determine the azimuthal dependence of the transmembrane potential, the relative fluorescence change has been measured in angular intervals of 10deg along the circumference of the cell. Field-strength-dependencies for several angular bins will be presented. In addition, high- resolution time-courses of the transmembrane potential following an external field pulse have been measured.