Visualisation of transglutaminase-mediated cross-linking activity in germinating pollen by laser confocal microscopy

Transglutaminases (TG) are a multigenic family of calcium-dependent protein cross-linking enzymes, which are present in mammalian and plant cells. We have previously reported the presence of TG in the cytosol and, more recently, in the cell wall of Malus domestica pollen, which may be involved in pollen germination and pollen-stylar interactions. In this report we describe a simple method for the visualisation of extracellular pollen TG activity in situ, during pollen germination. The method is based on the incorporation of a fluorescently labelled exogenous amine substrate of TG (fluorescein-cadaverine) into pollen endogenous substrates by pollen TG. Following the in situ TG activity reaction, the presence of crosslinked pollen proteins was visualised in the fixed specimens of germinated pollen by laser confocal microscopy. Our data indicate the presence of TG cross-linking activity at the apical part of the pollen tube, in the region of tube proximal to the grain and in the pollen grain. 2