Localization of membrane-associated calcium during development of fucoid algae using chlorotetracycline

During the first day of development, fertilized eggs of fucoid algae generate an embryonic axis and commence rhizoid growth at one pole. Using Fucus distichus (L.) Powell, F. vesiculosus L. and Pelvetia fastigiata (J.Ag.) DeTony we have investigated the role of calcium in axis formation and fixation as well as in tip growth. The intracellular distribution of membrane-associated calcium was visualized with the fluorescent calcium probe chlorotetracycline (CTC). Punctate fluorescence associated with organelle-like structures was found in conjunction with diffuse staining at all developmental stages. This membrane-associated calcium remained uniformly distributed throughout the cortical cytoplasm while the axis was established, but increased in the rhizoid protuberance at germination. In subsequent development, fluorescence was restricted to the cortical cytoplasm at the elongating tip and at sites of crosswall biosynthesis.The requirement for Ca(2+) uptake during development was investigated through inhibition studies; influx was impaired with transport antagonists or by removal of extracellular calcium. Both treatments curtailed germination and tip elongation but had little effect on axis polarization. Reductions in external calcium that interfered with elongation also markedly reduced the apical CTC fluorescencence, indicating that calcium uptake and localization are prerequisites for tip growth. This apical Ca(2+) is probably involved in the secretory process that sustains tip elongation. By contrast, calcium was not implicated in the generation of an embryonic axis.