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, the products of this activity may provide strength to the pollen tube migrating through the style.

Key words:

• Transglutaminase
• pollen
• fluorescein-cadaverine

Acknowledgements

We are grateful to Professor Martin Griffin (School of Life and Health Sciences, Aston University, Birmingham) for providing the TG inhibitor 281. This work was supported by a grant from the “British Council/Ministero dell'Istruzione, dell'Universita’ e della Ricerca” to EAMV and ADS and by the PRIN project 2005–2007 to Professor D. Serafini Fracassini.