Polyamines, their analogs, and transglutaminase effects on apical growth of the pollen tube

Polyamines, their analogs, and transglutaminase effects on apical growth of the pollen tube Stefano Del Duca1, Rosa Anna Iorio1, Iris Aloisi1 , Donatella Serafini-Fracassini1, Claudia Faleri 2, Vincenzo Tumiatti3, Anna Minarini3 and Giampiero Cai2 1 Dipartimento di Biologia, Università di Bologna, Via Irnerio 42, Bologna (Italy) 2 Dipartimento Scienze Ambientali, Università di Siena, via Mattioli 4, Siena (Italy) 3 Dipartimento di Scienze Farmaceutiche, Università di Bologna, via Belmeloro 6, Bologna (Italy). e-mail: stefano.delduca@unibo.it Sexual reproduction of flowering plants depends on delivery of the male gamete to the egg cell, which occurs through a polarized growth, located exclusively at the tip of the pollen tube, growing from the pollen grain, distinguished by very fast rates and extended lengths. Pollen germination is characterised by the continuous rebuilding of the cell wall and apical migration of the cytoplasm sustained by cytoskeleton re-organisation. Polyamines (PAs) are essential for cell growth in animals and plants. Early evidence showed that PAs are metabolised and essential factors during pollen tube emergence (Bagni et al. 1981). We investigated the effect of natural and synthetic PAs to check the effect on the apical growth of the pollen tube. We observed that among the natural PAs, spermine inhibited the growth from 10 M onwards. Among the synthetic ones, BD 23, an aromatic derivative of Spm also showed similar effects. The morphology of the tubes showed loss of polarity and enlarged tip. The effects of PAs are related, at least in part to the inhibition of the ROS synthesis, that, at physiological concentration, support the apical growth. As TGase mediates some of the effects of PAs by covalently binding them to proteins, the activity of this enzyme was examined and confirmed by detection of Ca2+-activated TGase activity (Del Duca et al. 1997) and by identification of actin and tubulin as substrates of purified pollen TGase (Del Duca et al. 2009). The enzyme was found in two distinct location: as a soluble and a cell wall associated form (Di Sandro et al. 2010). Fluorescently-labeled of TGase products were essentially present at the apex of pollen tubes, in a region close to the pollen grain and in the pollen grain (Iorio et al. 2008). While the presence and activity of cytoplasmic TGase is likely to be related to the regulation of processes possibly associated with the cytoskeleton activity, the extracellular TGase might provide strength to the pollen tube cell wall during tube migration through the style (Di Sandro et al. 2010). Here, we investigate in detail the localization and distribution of TGase in growing pollen tubes of pear (Pyrus communis). We used specific antibodies that cross-react with TGase of pollen tubes to localize the enzyme in different membrane compartments and in the cell wall of pollen tubes. Use of specific inhibitors indicated that the delivery of extracellular TGase is dependent on both actin filaments and membrane dynamics. Analysis by bidimensional electrophoresis showed that distinct TGase isoforms are associated with different cell compartments. The membrane TGase is likely to be associated with both Golgi-derived structures and the plasma membrane. The distribution of cytoplasmic TGase is dependent on the cytoskeleton while a different TGase form is actively transported via a membrane/cytoskeleton-based transport system and is secreted in the cell wall of pollen tubes, suggesting a Golgi-based exocytosis. Acknowledgements. This research has been supported by the RFO 2010. 1 Bagni, N., Adamo, P. and Serafini-Fracassini, D. (1981) RNA, proteins and polyamines during tube growth in germinating apple pollen. Plant Physiol. 68: 727–730. 2 Folk, J.E., Park, M.H., Chung, S.I., Schrode, J., Lester, E.P. and Cooper, H. (1980) Polyamines as physiological substrates for transglutaminases. J. Biol. Chem.: 255, 3695-3700. 3 Del Duc..