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 Ca2+ is necessary for the transduction of the light signal.  Because knowledge about the cytoplasmic calcium ion concentration, [Ca2+]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 microseconds).  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 Ca2+ concentration, and this observation is considered as evidence that fura-2 is loaded into the cytoplasm.  The substitution of a low external [Ca2+] (1 mM ethyleneglycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA)) by 1 mM CaCl2 caused a fast increase of [Ca2+]i from approx. 50 nM to above 500 nM.  In contrast, the subsequent substitution of CaCl2 by EGTA decreased [Ca2+]i again below 100 nM within 0.5 h.  Furthermore, the application of ionomycin could initiate a change in [Ca2+]i according to the Ca2+ gradient established between the extracellular medium and cytoplasm.  In spores sown on a Ca(2+) -free medium, [Ca2+]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, [Ca2+]i was significantly higher (> or = 500 nM).  In red-light-irradiated spores, [Ca2+]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, Federal Republic of 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
mesh_terms: D002118:Calcium / Q000032:analysis* / Q000502:physiology; D002614:Chelating Agents; D003593:Cytoplasm / Q000737:chemistry* / Q000502:physiology; D018274:Electroporation / Q000379:methods*; D005456:Fluorescent Dyes; D016257:Fura-2; D018525:Germination / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D015759:Ionomycin; D008027:Light; D010834:Phytochrome / Q000528:radiation effects; D010944:Plants / Q000737:chemistry / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013170:Spores / Q000737:chemistry / Q000187:drug effects / Q000502:physiology
chemical_list: D002614:Chelating Agents; D005456:Fluorescent Dyes; D010834:Phytochrome; D015759:Ionomycin; D002118:Calcium; D016257:Fura-2