TRANSGLUTAMINASE-CATALYZED MODIFICATION OF CYTOSKELETAL PROTEINS BY POLYAMINES DURING THE GERMINATION OF MALUS-DOMESTICA POLLEN

We investigated polyamine linkage to differ ent structural proteins in pollen of Malus domestica Borkh. cv Red Chief at different phases of germination. This linkage has the characteristics of covalent linkages , indeed, it could be catalyzed by transglutaminase (TGase; EC 2.3.2.1 3). This assumption is supported by: (1) formation of a labelled TCA p ellet and selective labelling of endogenous proteins by covalent bindi ng of radioactive polyamines and (2) cross-reactivity of two different polyclonal antibodies against mammalian TGases; western blot analysis allowed us to detect a protein of about 80 kDa in both rehydrated ung erminated and germinated pollen. TGase activity was high at 90 min aft er germination and was influenced by Ca2+ supply only in the rehydrate d ungerminated pollen. Extraction by Triton X-100 suggests that pollen TGase was at least partially membrane-bound. The enzyme catalyzed the incorporation of polyamines mainly into proteins having a molecular m ass of 43 kDa and 52-58 kDa in both ungerminated and germinated pollen . These bands matched immunolabelled spots identified by mouse monoclo nal anti-actin and anti-alpha-tubulin antibodies. Supplying exogenous actin and tubulin in a cell-free extract of rehydrated ungerminated an d germinated pollen enhanced the activity. Autoradiography of the SDS- PAGE of these samples clearly showed that both actin and tubulin were substrates of TGase. Thus, the pollen TGase may be involved in the rap id cytoskeletal rearrangement which takes place during rehydration of ungerminated pollen and organization and growth of pollen tubes.