CHARACTERIZATION OF A CALCIUM-REQUIRING PHASE DURING PHYTOCHROME-MEDIATED FERN-SPORE GERMINATION OF Dryopteris paleacea Sw.*

Abstract— For phytochrome-mediated fern-spore germination in Dryopteris paleacea Sw., initiated by a saturating red light (R) irradiation 20 h after imbibition, an almost absolute requirement for extracellular Ca²+ is found. To investigate the kinetics of this Ca²+ requirement spores were sown on a Ca²+-free medium (Ca²+ < 10^?8M) and Ca²+ was raised to 1 mM at defined periods after R. Alternatively, spores were sown on a Ca²+-containing medium (1 mM) and transferred to a Ca²+-free medium. A clearly defined period for the Ca²+ requirement is found between 30 and 50 h after R, while Ca²+ had to be present in the medium for at least 9 h to obtain half-maximal germination. However, since the kinetics of deprivation and delayed addition of Ca²+ do not provide significantly different results, only a relative short presentation time for extracellular Ca²+ has to be expected at the level of a single cell. Ca²+ sensitivity is determined by the timing of the R irradiation, i.e. the timing of Pfr formation, which has been concluded from the observation that the variation of the imbibition time does not affect the kinetics of Ca²+ requirement and that the temporal application of polyethylene glycol (PEG), which is assumed to interrupt Pfr action, shifts the Ca²+ requirement to delayed intervals. These observations, as well as the fact that the requirement of Ca²+ has been observed for a limited period are interpreted as indirect evidence that Ca²+ action plays the role of a specific link in the phytochrome-mediated signal-transduction chain. The “Ca²+ kinetics” are compared with the kinetics of escape from reversibility by far-red (FR) light and with kinetics of basic cell physiological processes occurring during germination. “Escape kinetics”, indicating “coupling” of Pfr to subsequent dark reactions, are observed at significant earlier intervals, and a gap of about 15 h is found for both the action of Pfr and of Ca²+. Thus, the direct interaction of Pfr with external Ca²+ as a first transduction step can be excluded experimentally. The kinetics of chlorophyll formation are found to be only slightly delayed, whereas the kinetics of mitosis are shifted by approximately 30 h. Almost the same slope is obtained for all kinetics investigated so far and one reaction spans a period of approximately 35 h. Obviously, variability found in the spore population is due to the coupling of Pfr whereas subsequent reactions proceed with almost identical velocity.