During Higher Plant reproduction, pollen is transported to the style of flower in order to allow the gamia between the female gamete located inside the ovule and the male gamete located in a long pollen tube, which develops inside the stylar tissues. Development of the pollen tube is allowed when compatible varieties are crossed, whereas in incompatible or auto-incompatible crosses the tube cannot develop or is blocked during growth. The self-incompatibility (SI) response is a specie-specific and genetically-controlled mechanism. Gametophytic SI is dependent from mechanisms blocking pollen growth at the level of the upper third of the style. In Rosaceae, the stylar S locus encodes for glycoproteins showing ribonuclease (S-RNases) activity, even if also other genes, still to be identified, are involved in the regulation of this molecular mechanism. The involvement of putative pollen determinants in incompatibility has been investigated. During pollen growth, the cytoskeleton undergoes a dynamic reorganization. Microfilaments and motor proteins are responsible for the apical growth and transport of vesicles and organelles. Microtubules, co-localized with microfilaments, are involved, with motor proteins (i.e., dynein, kinesin, myosin) in the migration of male gamete and in cytoskeleton organization. The cytoskeleton role in SI phenomena is scarcely known. Solely in incompatible papaver tubes, F-actin foci are formed due to a not characterized crosslinking mechanism, able to block tube elongation and causing tube PCD. The pear self-incompatibility in two varieties of Pyrus communis (Abbé Fétel and William) has been investigated in order to identify the S-alleles and pollen putative determinant. Transglutaminase (TGase) is recognised as a modulator of in vitro pollen growth as its inhibition blocks the tube apical growth. Two main hypotheses on the action mechanism of TGase in pollen growth have been explored. The first is related to a possible TGase action as PDI, in controlling the interaction among S-RNases and F-boxes products that results in a protein complex development during SI process. The second hypothesis is related to its regulation of cytoskeleton structural organisation and motility by acting as a cross-linking agent. In the self-pollinated style (incompatible system), the activity of TGase is stimulated in comparison to style-pollinated with compatible pollen (compatible system), and high molecular mass cross-linked products were formed; high mass aggregates of tubulin, and punctuate aggregates of actin were therein observed by confocal laser microscopy. Also polyamine distribution and their cross-linking appear to be different in the two systems. To clarify the involvement of this enzymatic activity in pollen incompatibility, this enzyme has been purified from pollen either of Malus and Pyrus showing a great similarity for several parameters and sequence. In vitro experiments have been performed by incubating purified pollen TGase with tubulin and actin in the presence or absence of the polyamine putrescine. TGase was able to induce the formation of high mass aggregates of tubulin, potentially indicating a likely involvement of TGase in the cytoskeletal protein organisation. Moreover, additional studies performed to evaluate the microtubule motility in vitro in the presence of kinesin demonstrated that both the binding affinity and the velocity of microtubules on kinesin is strongly reduced. Studies performed by incubating actin with myosin showed that the binding affinity and the ATPase activity of myosin are reduced when actin is incubated with TGase. These data confirm that pollen TGase modifies the dynamic of the cytoskeleton, as already proposed for mammal cell types (Eligula et al., 1998). In vivo studies showed that the TGase presence increased during the growth in incompatible pollen tube. Its localization looks to be cytosolic at 24 h of germination, whereas accumulated prevalently into the c...

Transglutaminases and reproduction: possibile roles of pollen transglutaminases in incompatibility phenomena.

SERAFINI FRACASSINI, DONATELLA;DI SANDRO, ALESSIA;DEL DUCA, STEFANO;DONDINI, LUCA;DELLA MEA, MASSIMILIANO;DE FRANCESCHI, PAOLO;SANSAVINI, SILVIERO
2007

Abstract

During Higher Plant reproduction, pollen is transported to the style of flower in order to allow the gamia between the female gamete located inside the ovule and the male gamete located in a long pollen tube, which develops inside the stylar tissues. Development of the pollen tube is allowed when compatible varieties are crossed, whereas in incompatible or auto-incompatible crosses the tube cannot develop or is blocked during growth. The self-incompatibility (SI) response is a specie-specific and genetically-controlled mechanism. Gametophytic SI is dependent from mechanisms blocking pollen growth at the level of the upper third of the style. In Rosaceae, the stylar S locus encodes for glycoproteins showing ribonuclease (S-RNases) activity, even if also other genes, still to be identified, are involved in the regulation of this molecular mechanism. The involvement of putative pollen determinants in incompatibility has been investigated. During pollen growth, the cytoskeleton undergoes a dynamic reorganization. Microfilaments and motor proteins are responsible for the apical growth and transport of vesicles and organelles. Microtubules, co-localized with microfilaments, are involved, with motor proteins (i.e., dynein, kinesin, myosin) in the migration of male gamete and in cytoskeleton organization. The cytoskeleton role in SI phenomena is scarcely known. Solely in incompatible papaver tubes, F-actin foci are formed due to a not characterized crosslinking mechanism, able to block tube elongation and causing tube PCD. The pear self-incompatibility in two varieties of Pyrus communis (Abbé Fétel and William) has been investigated in order to identify the S-alleles and pollen putative determinant. Transglutaminase (TGase) is recognised as a modulator of in vitro pollen growth as its inhibition blocks the tube apical growth. Two main hypotheses on the action mechanism of TGase in pollen growth have been explored. The first is related to a possible TGase action as PDI, in controlling the interaction among S-RNases and F-boxes products that results in a protein complex development during SI process. The second hypothesis is related to its regulation of cytoskeleton structural organisation and motility by acting as a cross-linking agent. In the self-pollinated style (incompatible system), the activity of TGase is stimulated in comparison to style-pollinated with compatible pollen (compatible system), and high molecular mass cross-linked products were formed; high mass aggregates of tubulin, and punctuate aggregates of actin were therein observed by confocal laser microscopy. Also polyamine distribution and their cross-linking appear to be different in the two systems. To clarify the involvement of this enzymatic activity in pollen incompatibility, this enzyme has been purified from pollen either of Malus and Pyrus showing a great similarity for several parameters and sequence. In vitro experiments have been performed by incubating purified pollen TGase with tubulin and actin in the presence or absence of the polyamine putrescine. TGase was able to induce the formation of high mass aggregates of tubulin, potentially indicating a likely involvement of TGase in the cytoskeletal protein organisation. Moreover, additional studies performed to evaluate the microtubule motility in vitro in the presence of kinesin demonstrated that both the binding affinity and the velocity of microtubules on kinesin is strongly reduced. Studies performed by incubating actin with myosin showed that the binding affinity and the ATPase activity of myosin are reduced when actin is incubated with TGase. These data confirm that pollen TGase modifies the dynamic of the cytoskeleton, as already proposed for mammal cell types (Eligula et al., 1998). In vivo studies showed that the TGase presence increased during the growth in incompatible pollen tube. Its localization looks to be cytosolic at 24 h of germination, whereas accumulated prevalently into the c...
International Congress on Biogenic Amines: Biological and Clinical Perspectives
95
97
D. Serafini-Fracassini; A. Di Sandro; S. Del Duca; L.Dondini; M. Della Mea; P. De Franceschi; C. Faleri; G. Cai; S.Sansavini
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/56818
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