A MICROSCOPY-PAM Chlorophyll Fluorometer (Heinz Walz GmbH, Effeltrich, Germany) was combined with a patch–clamp set-up (Hamill et al., 1981) in order to carry out chlorophyll fluorescence measurements and ion channel recordings on single V. faba guard cell protoplasts. After breaking the membrane under the patch-pipette and establishment of the whole-cell configuration (t = 0.5 min), dark–light induction curves were measured with repetitive application of saturation pulses to estimate the effective quantum yield of energy conversion at photosystem II reaction centres and the relative electron transport rate (Genty et al., 1989; Schreiber et al., 1994). K+-channel activities in the plasma membrane of the same protoplast were determined right after recordings of the photosynthetic electron transport. Following the electrophysiological recordings, the measurements on the electron transport of the chloroplasts was repeated.
Chlorophyll fluorescence was measured as previously described (Goh et al., 1999; Schreiber, 1998). This system is based on an epifluorescence microscope (Type Axiovert, Carl Zeiss GmbH, Oberkochen, Germany), with a single blue light (470 nm) emitting diode (LED) acting as source of modulated measuring light and a photomultiplier serving as fluorescence detector. The MICROSCOPY-PAM was operated in conjunction with a Pentium II PC and the WinControl-software (Walz). While actinic light intensity was 67 µmol m−2 sec−1 PAR, measuring light intensity was 0.8 µmol m−2 sec−1 and saturation pulse intensity amounted to 4280 µmol m−2 sec−1, as measured with a special pin-hole microquantum-sensor (Walz).
Ion currents were recorded using an EPC-7 patch–clamp amplifier (HEKA, Lambrecht, Germany). Whole-cell data were low-pass filtered with an eight-pole Bessel filter at a cut-off frequency of 2 kHz. Data were sampled at 2.5 fold filter frequency, digitised (ITC-16, Instrutech Corp., Elmont, NY, USA), stored on hard disk and analysed with software of Instrutech Corp. on a Power-MacIntosh (Gravis TT200, Gravis GmbH, Berlin). Patch-pipettes were prepared from Kimax-51 glass (Kimble products, Vineland, NY, USA) and coated with silicone (Sylgard 184 silicone elastomer kit, Dow Corning, USA). In order to determine membrane potentials the command voltages were corrected off-line for liquid junction potentials according to Neher (Neher, 1992). The standard pipette solution (cytoplasm) contained 150 mm K-gluconate, 2 mm MgCl2, 3 mm CaCl2, 5 mm EGTA, ±2 mm MgATP, 10 mm Hepes–Tris pH 7.4. The bathing medium contained 30 mm K-gluconate, 1 mm CaCl2, 10 mm Mes–Tris pH 5.6. For measurements in the presence of ADP- and Pi-containing solutions, 2 mm MgATP were replaced by 2 mm K2-ADP in addition to K2HPO4, and 2 mm MgCl2 were added to give a final concentration of 4 mm Mg2+. All solutions were adjusted to 400 mos mol kg−1 using d-sorbitol. Chemicals were obtained from Sigma (Sigma Chemie, Deisenhofen, Germany).