Early quantitative method for measuring germination in non‐green spores of Dryopteris paleacea using an epifluorescence‐microscope technique

Abstract

A method is described to determine germination by blue-light excited red fluorescence in the positively photoblastic spores of Dryopteris paleacea Sw. This fluorescence is due to chlorophyll as evidenced from 1) a fluorescence-emission spectrum in vivo, where a bright fluorescence around 675 nm is obtained only in red light (R)-irradiated spores and 2) in vitro measurements with acetone extracts prepared from homogenized spores. Significant amounts of chlorophyll can be found only in R-treated spores; this chlorophyll exhibits an emission band around 668 nm, when irradiated with 430 nm light at 21 degrees C. Compared to other criteria for germination, such as swelling of the cell, coat splitting, greening, and rhizoid formation, which require longer periods after induction for their expression, chlorophyll fluorescence can be used to quantify germination after two days. This result is confirmed by fluence-response curves for R-induced spore germination; the same relationship between applied R and germination is obtained by the evaluation with the epifluorescence method 2 days after the light treatment as compared with the evaluation with bright-field microscopy 5 days after the inducing R. Using this technique we show for the first time that Ca2+ contributes to the signal-transduction chain in phytochrome-mediated chlorophyll synthesis in spores of Dryopteris paleacea.

The Role(s) of Calcium Ions in Phytochrome Action

“…Using an epifluorescence microscope or a spectroscopic determination of extracted chlorophyll it is possible to determine the first step of spore germination 2 days after light treatment (Scheuerlein et al, 1988). Both in vivo and in vitro determined chlorophyll synthesis occurs only if Ca2+ is present in the surrounding medium (Scheuerlein et al, 1988). The half-maximal response was observed in a medium containing 3 p M free Ca2+ and the optimum was reached around 100 p M .…”

“…The effect of phytochrome and Ca2+ on germination of Dryopteris paleacea spores has been extensively studied by Scheuerlein and colleagues (Durr and Scheuerlein, 1990;Scheuerlein et al, 1988Scheuerlein et al, , 1989. In non-green Dryopteris spores the first visible evidence of germination is the appearance of chlorophyll synthesis.…”

“…In non-green Dryopteris spores the first visible evidence of germination is the appearance of chlorophyll synthesis. Using an epifluorescence microscope or a spectroscopic determination of extracted chlorophyll it is possible to determine the first step of spore germination 2 days after light treatment (Scheuerlein et al, 1988). Both in vivo and in vitro determined chlorophyll synthesis occurs only if Ca2+ is present in the surrounding medium (Scheuerlein et al, 1988).…”

Polar distribution of annexin-like proteins during phytochrome-mediated initiation and growth of rhizoids in the ferns Dryopteris and Anemia

“…Germination of fern spores results in rhizoid initiation in both Dryopteris and Anemia, and it requires activation of the phytochrome system and a source of external calcium (Scheuerlein et al 1988; Haas et al 1992). The rhizoid initial is a cell which elongates by apical extension like pollen tubes, root hairs, and fungal hyphae.…”

Determination of cytoplasmic calcium concentration in Dryopteris spores

“…Unloaded spores showed a weak autofluorescence due to the spore coat (see Scheuerlein et al 1988). The fluorescence-excitation spectrum between 300 and 400 nm, obtained for single-cell measurements, had a shallow maximum around 370 nm (Fig.…”