title: Determination of cytoplasmic calcium concentration in Dryopteris spores : A developmentally non-disruptive technique for loading of the calcium indicator fura-2.
abstract: Germination of Dryopteris spores is mediated by the physiologically active, far-red-absorbing form of phytochrome, Pfr, and external Ca(2+) is necessary for the transduction of the light signal. Because knowledge about the cytoplasmic calcium ion concentration, [Ca(2+)]i, is of great importance for understanding the role of calcium during signal transduction, this value was measured using fura-2 in fern spores undergoing the normal developmental progression into germination. Fura-2 was loaded into the spores by electroporation, which does not disrupt the normal process of germination. The intensity of the fluorescence emission of the loaded fura-2 was analysed by a microspectrophotometric assay of single spores, and successful loading could be obtained by the application of ten electrical pulses (field strength 7.5 kV · cm(-1), half-life (time constant) 230 μs). Fura-2 was alternately excited by light of wavelengths 355 and 385 nm through an inverted fluorescence microscope, and the emitted fura-2 fluorescence was collected by a silicon-intensified video camera. The cytoplasmic calcium ion concentration was calculated from the ratio of the camera output obtained for both wavelengths and displayed by a pseudo-color technique. Spores responded to changes of the extracellular Ca(2+) concentration, and this observation is considered as evidence that fura-2 is loaded into the cytoplasm. The substitution of a low external [Ca(2+)] (1 mM ethyleneglycol-bis(2-aminoethyl-ether) {ie166-01},N'-tetraacetic acid (EGTA)) by 1 mM CaCl2 caused a fast increase of [Ca(2+)]i from approx. 50 nM to above 500 nM. In contrast, the subsequent substitution of CaCl2 by EGTA decreased [Ca(2+)]i again below 100 nM within 0.5 h. Furthermore, the application of ionomycin could initiate a change in [Ca2+]i according to the Ca(2+) gradient established between the extracellular medium and cytoplasm. In spores sown on a Ca(2+)-free medium, [Ca(2+)]i, analysed in a buffer containing EGTA, was found to be around 50 nM during the first days of cultivation, independent of the irradiation protocol. However, if spores were grown in darkness on a Ca(2+)-containing medium and analysed in EGTA, [Ca(2+)]i was significantly higher (≧ 500 nM). In red-light-irradiated spores, [Ca(2+)]i was found to decrease with increasing time after irradiation, and was determined to be less than 100 nM when analysis was done 44 h after germination was initiated by the light treatment.
authors.affiliation: Institut für Botanik und Pharmazeutische Biologie der Universität Erlangen-Nürnberg, Staudtstrasse 5, W-8520, Erlangen, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Scheuerlein
authors.forename: K
authors.initials: K
authors.lastname: Schmidt
authors.forename: M
authors.initials: M
authors.lastname: Poenie
authors.forename: S J
authors.initials: SJ
authors.lastname: Roux