Although the calcium requirement of phytochrome-mediated fern spore germination and early rhizoid growth is well established, the calcium-binding proteins that serve as transducers for these responses are not known. Here we report the presence of annexin-like proteins in germinating spores of Dryopteris filix-mas (L.) Schott and Anemia phyllitidis (L.) Sw. and evidence that they may be important participants in early photomorphogenic changes in gametophytes. Immunolocalization and immunoblot assays of these proteins were carried out using polyclonal antibodies raised either against a 35-kDa annexin-like protein from pea or against anchorin CII from chicken. Western-blot analysis showed that crude protein extracts obtained from both species after red-light treatment contained two cross-reactive protein bands with molecular weights around 70 kDa. These proteins were annexin-like in that they bound to a phosphatidylserine affinity column in a calcium-dependent fashion. Using this column, two protein bands around 70 kDa, i.e. 67 and 73 kDa, were partially purified together with proteins at 36 kDa and a doublet at 54 kDa. Proteins of these latter molecular weights are suggested to be members of the annexin family, but no cross-reactivity could be found between these and the two antibodies used in our investigations. Immunodetectable levels of these proteins were observed only after light-mediated induction of spore germination. Imaging of the immuno-localization patterns observed with both antibodies showed that the annexin-like proteins are concentrated at the extreme tips of the rhizoids in D. filix-mas and A. phyllitidis during rhizoid initiation and all stages of elongation. We suggest that these proteins may play a major role in the tip-oriented exocytosis events that are critical for the initiation and growth of fern rhizoids.
Spore germination in Dryopteris filix‐mas occurs via a cascade of cellular responses, and chlorophyll formation, mitosis or rhizoid elongation are commonly used as parameters to determine spore germination. Detailed investigations of these parameters led to the hypothesis that they are regulated by different, independent phytochrome‐mediated responses. This concept could be confirmed, as is described in this paper which demonstrates that perception of light via phytochrome occurs within two different phases separated in time. Presence of the far‐red absorbing phytochrome form, Pfr, for 36 h, induces chlorophyll formation and the first unequal cell division, by which a rhizoid initial and a protonemal initial are formed (first phytochrome‐mediated response). However, rhizoid elongation requires a second period of Pfr, presence (second phytochrome‐mediated response). There is a clear temporal distinction between the first and the second phytochrome‐mediated response with respect to the coupling of Pfr to the transduction chain; Pfr is unable to induce rhizoid growth until 60 h after the start of the first red irradiation. The effectivity of Pfr for inducing the second response shows an optimum at ca 96 h after the beginning of the presence of Pfr; thereafter, it declines slowly. The fluence‐response relationship and the presence of red/far‐red reversibility demonstrate that rhizoid elongation is a low‐fluence response mediated by phytochrome and is independent of the first phytochrome response.
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