141 Post-Translational Modification by Transglutaminase of Proteins Involved in Pear Self-Incompatibility R.A. Iorio 1 , D. Serafini-Fracassini 1 , I. Aloisi 1 , S. Del Duca 1 , P. De Franceschi 2 , L. Dondini 2 , S. Sansavini 2 , G. Cai 3 and C. Faleri 3 1 Dipartimento di Biologia, Università di Bologna, Italy 2 Dipartimento di Colture Arboree, Università di Bologna, Italy 3 Dipartimento di Scienze Ambientali, Università di Siena, Italy Keywords: pollen, style, reproduction, Pyrus communis, Rosaceae Abstract Self-incompatibility has been investigated in the cultivar ‘Abbé Fétel’ (Pyrus communis) in order to assign a role to transglutaminase (TGase), an enzyme able to post-translationally forming cross-links among proteins. This enzyme is localised in the pollen cytoplasm where it regulates the polymerisation of cytoskeleton proteins. An extracellular form is also necessary for pollen tube growth. In the self-pollinated style of Abbè Fétel (A x A, incompatible system), the activity of TGase increased when the pollen tube stopped its growth inside the style and the enzyme was immuno-localised around the tube tip. On the contrary in Abbé Fétel styles pollinated with Williams pollen (A x W, compatible system), TGase activity decreased during pollen germination. The TGase gene has been cloned and sequenced from Abbé Fétel pollen and style showing that it shares a high homology with a sequence of apple, whose genome has been recently published, which presents the typical TGase catalytic sequence, and with the TGase of Arabidopsis. This enzyme was expressed during pollen germination inside the style to a similar extent in both systems (A x A and A x W) showing that it is only the activity to have been stimulated by some factor dependent on SI. A molecular approach to clone one of the S-RNase alleles of the cv Abbé Fétel has been performed. This protein, supplied to pollen in the germination medium, was able to inhibit the growth of 50% of the tubes of the homologous pollen. This approach will allow us to verify if an interaction between the two enzymes will occur during SI, as TGase, at least in animal cells, is also able to act as disulphide isomerase modifying the RNase structure. INTRODUCTION Gametophytic self-incompatibility (SI) is one of the mechanisms adopted by plants to prevent self-fertilisation, by blocking the growth of pollen tubes recognised as “self”. Among the known models of interaction between pistil-S and pollen S determinants, the S-RNase-based system has been described in the Rosaceae, Solanaceae and Plantaginaceae. Another model is that of Papaver rhoeas, in which the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca 2+ - dependent signalling pathway which results in cytoskelton modifications and finally leads to programmed cell death (PCD). In fact there is a specific relationship between the polymerization/depolymerization of actin and the onset of PCD of the pollen tube (Thomas et al., 2006; Wheeler et al., 2009). Aggregates of tubulin and punctuate aggregates of actin were also observed in Pyrus communis SI, suggesting a role for the cytoskeleton in Rosaceae SI (Di Sandro et al., 2007), and later this role was verified in vivo and in vitro (Del Duca et al., 2009, 2010). Liu et al. (2007) in Pyrus pyrifolia observed that stylar S-RNases modified the actin cytoskeleton. Recently the role of the cytoskeleton in Pyrinae SI has been reported: the S-RNase acts on depolymerization of actin and formation of high mass aggregates, DNA degradation, collapse of mitochondrial potential, leakage of cyt C, ROS disruption (Wang et al., 2010; Poulter et al., 2011). Thus the Papaver and Pyrinae models share common features of PCD. Previously, in SI Pyrus communis we studied the Ca 2+ -dependent Proc. Intl. Workshop on Floral Biology & S-Incompatibility in Fruit Species Eds.: Sargent et al. Acta Hort. 967, ISHS 2012