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 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 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-aminoethyl-ether) {ie166-01},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 Ca2+-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 Ca2+-containing medium and analysed in EGTA, [Ca2+]i was significantly higher (≧ 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.
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Abbreviations
- [Ca2+]i :
-
cytoplasmic calcium ion concentration
- D:
-
darkness
- EGTA:
-
ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid
- λm :
-
transmission center of the interference filter
- R:
-
red light
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Dedicated to Professor H. Mohr on the occasion of his 60th birthday
We gratefully acknowledge the interest shown by Drs. R.M. Brown, Jr. (Dept. Botany, Austin, USA), D.-P. Häder (Botanisches Institut I, Erlangen, FRG) and G. Seitz (Zoologisches Institut II, Erlangen), and their generous provision of laboratory facilities. The authors thank Dr. W. Haupt (Botanisches Institut, Erlangen) for critical reading of the manuscript, Mr. Robert Nagy (Dept. Botany, Austin) and Mr. Egbert Hoiczyk (Botanisches Institut, Erlangen) for valuable technical assistance as well as Mrs. U. Beyerlein (Botanisches Institut, Erlangen) for careful preparation of the drawings. This research was in part supported by the Deutsche Forschungsgemeinschaft (Sche 276/1-1) to R.Sch. and by a NASA grant (NAGW 1519) to S.J.R.
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Scheuerlein, R., Schmidt, K., Poenie, M. et al. Determination of cytoplasmic calcium concentration in Dryopteris spores. Planta 184, 166–174 (1991). https://doi.org/10.1007/BF00197944
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DOI: https://doi.org/10.1007/BF00197944