Polyamines Conjugated to Pollen Proteins by a Novel Extracellular Transglutaminse Activity Are Involved In Plant Reproduction

There is currently an intense interest in understanding how fertilisation in flowering plants is controlled because of the crucial importance of sexual reproduction in plant lifecycles. The regulation of fertilization in higher plants has proved to be remarkably complex, involving controls of various and direct interactions between male and female gametophytes, which still remain an enigma. Reproduction in higher plants requires at the base of the style the directed growth of pollen tubes through a Ca2+-dependent reorganisation of the cytoskeleton (tubulin is modified via polyamines by transglutaminase1) and an intense movement of pollen organelles along microfilaments. The novelty of the research consists in the detection of the first extracellular plant TGase, and demonstration that its extracellular protein crosslinking activity is essential for pollen germination. Transglutaminases (TGase) are a family of enzymes widespread throughout a range of different organisms. TGase are able to post-translationally modify proteins by forming bridges between protein-bound glutaminyl residues and primary amines such as -lysine residues or polyamines. The first intracellular plant TGase has been recently sequenced2. Inhibition of transamidating activity either by a monoclonal anti-TGase antibody (ID10 Mab) or site directed inhibitors (R283 and R281) blocked pollen tube growth (see Fig. 1), while TGase mediated incorporation of the X6-press GFP substrate into the pollen tube cell wall enhanced tube growth. The extracellular TGase enzyme, immunolocalised by laser scanning confocal microscopy on the surface of the pollen tube, was also capable of crosslinking labelled polyamines into extracellular proteins in immediate contact with the pollen tube. Contribution of TGase-mediated crosslinking in cell wall formation was confirmed by the immunodetection of endogenous (-glutamyl)lysine crosslinks. Immunoblotting with the anti-TGase antibody revealed the presence of two 81 and 75 kDa immunoreactive proteins in the low-speed supernatant of germinated pollen and in the germination medium. Taken together, these findings suggest that the enzyme function is important both in the stabilisation of the pollen tube cell wall and in the attachment of the pollen tube to the female style. Given the ability of the extracellular TGase to incorporate labelled substrates into extracellular proteins distinct from those of the pollen tube and the fact that the extracellular matrix is a key regulator of the male and female gametophyte communication, we propose that TGase may also be involved in the structural adhesion occurring between the pollen cell wall and the stylar ECM