Protoplasma

, Volume 219, Issue 1, pp 99-105

First online:

Observation of polarity induction by cytochemical localization of phenylalkylamine-binding sites in regenerating protoplasts of the moss Physcomitrella patens

  • S. C. BhatlaAffiliated withPlant Biotechnology, Institute of Biology II, University of Freiburg, Freiburg
  • , J. KiesslingAffiliated withPlant Biotechnology, Institute of Biology II, University of Freiburg, Freiburg
  • , R. ReskiAffiliated withPlant Biotechnology, Institute of Biology II, University of Freiburg, Freiburg

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Summary.

Different external (e.g., light) and internal (e.g., auxin and calcium gradients) factors control differentiation of the moss protonema. The present investigations demonstrate that exogenously applied auxin, the pharmacological blockade of auxin efflux by naphthylphthalamic acid, and treatment with (−)bepridil, a calcium channel antagonist, inhibit protoplast division without affecting protoplast viability in the moss Physcomitrella patens. A fluorescently labelled phenylalkylamine (DM-Bodipy PAA), another calcium channel antagonist, was used as a probe for in vivo labelling of phenylalkylamine(PAA)-binding sites. The specificity of this binding was demonstrated by competition with (−)bepridil. Confocal laser scanning microscopy visualized PAA-binding sites on the plasma membrane and along the nuclear membrane as uniformly distributed clusters. During asymmetric division of P. patens protoplasts, however, fluorescence labelling particularly increases at the membrane invagination and later along the plate separating the new cells. Intracellular localization of PAA-binding sites, probably at the membranes of vesicles and vacuoles, significantly increases in the smaller daughter cell, destined to later form a polar outgrowth, the first chloronema cell. Thus, a system was established to visualize early events in P. patens protoplast polarization at the subcellular level.

Keywords: Auxin; Calcium channel; Cell polarity; Phenylalkylamine binding site; Physcomitrella patens; Polarity induction.