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

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