Báo cáo lâm nghiệp: "Sexual reproduction in Populus II. Information molecules of the pollen grain"

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Sexual reproduction in Populus II. Information molecules of the pollen grain M. Villar C. Kerhoas 2 M. 1 Gaget 1 C. Dumas l Laboratoire de Reconnaissance Cellulaire et Amélioration des Plantes INRA 23879, Universit! tyon /, 43, bd 11-novembre-19i8, F-69622 Villeurbanne Cedex, and 1NRA, 2 Station dAmelioration des Arbres Forestiers, Ardon, F-45160 Olivet France Introduction Materials and Methods P. nigra, P. deltoides and P. alba branches were Sexual interactions in flowering plants obtained from the INRA Forestry Station in depend upon pollen-pistil biocommunica- Orl6ans (France). Pollen was collected and tions, involving biochemical signals ex- stored in closed vials at -18°C. changed between the mating partners. For cytochemistry, ultrathin pollen sections prepared according to Gaget (1988). They were Such components involved in self-incom- stained with acetate/lead citrate uranyl were patibility are known to be the primary pro- (Reynolds, 1963), and observed at 80 kV with ducts of the S (self-incompatibility) gene. the Hitachi HU 12A microscope at CMEABG, of protein and glycoprotein na- They University of Lyon, France. are (Nasrallah and Nasrallah, 1986; ture Freeze-fracture was performed according to Kerhoas et al. {1987) and replicas were obser- Gaude and Dumas, 1987). On the other ved by transmission electron microscopy hand, no data are available on the pollinic (TEM). signals implicated in interspecific incom- Pollen prints were obtained by placing pollen patibility of self-compatible species. grains onto a formvar-coated grid for 30 min in humid Petri dishes. Grids were then stained Cellular localization and biochemical (uranyl acetate/lead citrate; Reynolds, 1963) identification of pollinic molecules, likely to and observed by TEM. be implicated in interspecific incompatibili- Biochemical analysis: pollen diffusates were ty in Poplars, are presented in this paper. obtained by putting pollen grains in Tris buffer Our model involves 2 species of section (pH 7.5, 25 mM) for 10 min at 4°C. After centri- fugation, the supernatant was filtered and Aigeiros (Populus nigra, P. deltoides) and concentrated by acetone precipitation. The one species of section Leuce (P. alba). resuspended extracts were assayed for total Crosses between species of these 2 bo- proteins, according to Bradford (1976), with tanical sections are strictly incompatible. bovine serum albumin as a standard. Samples
containing 10 0 pg of protein were loaded on an amount of osmiophilic components in the SOS-gel, according to Laemmli’s procedure pollen coat is observed within the arcades (Laemmli, 1970). After running, proteins were of the exine (Fig. 2). These components stained with silver nitrate (Morrissey, 1981). also reactive in the polysaccharide were Proteins were electrophoretically transferred and glycoconjugate detection test (Gaget, onto a nitrocellulose sheet (Towbin et al., 1979) and the glycoproteins revealed by the Con 1988). These components are able to dif- A-peroxidase method (Hawkes, 1982). fuse rapidly from the pollen grain, as shown by pollen prints (not illustrated). Such diffusible materials are of polysac- Results and Discussion charidic nature and contain glycoconju- gates (Gaget, 1988). The freeze-fracture replicas show the presence of microchan- Ultrastructural observations (TEM) show a nels through the fibrillar intine (Fig. 3). bicellular pollen grain containing a vegeta- These structures might be the pathway of tive nucleus, a generative nucleus, the diffusible pollinic components from the cytoplasm of the vegetative cell with vegetative cell to the external surface. numerous amyloplasts and a bilayered Such microchannels have been observed pollen wall with a thin semi-tected exine in the papillar wall of the female partner (Figs. 1 and 2). The presence of a great
and were implicated in the transport of species (present in P. alba). Moreover, recognition proteins to the female surface these bands were also revealed by the Con A-peroxiclase procedure and could (Clarke et al., 1980). be considered as glycoproteins. Globular proteins with molecular mass of 100 000 Da may be able to diffuse across the pollen wall by means of canali- culi whose diameter was estimated to be Conclusion 30 nm. Protein assay showed that, de- pending upon the species used, 5-20% of the pollinic proteins diffuse in 10 min in Our work cleairly demonstrates the exis- Tris buffer, without affecting pollen viability. tence of specific glycoproteins, whose This protein fraction contains numerous presence in pollen is related to sexual incompatibility. Such a study should be protein bands whose molecular masses ranged between 10 000 and 100 000 Da. extended to other species of the 5 sections of the genus Populus. Moreover, extraction Among them, we found some peculiar bands associated with the group of incom- and use of these specific glycoproteins in pollen-pistil recognition could patibility (Fig. 4). Some are specific to implicated Aigeiros species (present in P nigra and attractive tool for overcoming inter- be an P. deltoides). Others are specific to Leuce specific incompatibility in poplars.
Kerhoas C., Gay G. & Dumas C. (1987) A multi- References disciplinary approach to the study of the plasma membrane of Zea mays pollen during controlled 0 dehydration. Planta 171, 1-10 Bradford M.M. (1976) A rapid and sensitive method for the quantitation of microgram quan- Laemmli U.K. (1970) Cleavage of structural pro- tities of protein utilizing the principle of protein teins during the assembly of the head of bacte- dye binding. AnaL Biochem. 72, 249-254 riophage T4. Nature 227, 680-685 Morrissey J.H. (1981 ) Silver stain for proteins in Clarke A.E., Abbot A., Mandel T.E. & Pettitt J.M. polyacrylamide gels: a modified procedure with (1980) Organisation of the wall layers of the enhanced uniform sensitivity. Anal. Biochem. stigmatic papillae of Gladiolus gandavensis: a 0 117, 307-310 freeze fracture study. J. Ultrastruct Res. 73, 269-281 Nasrallah M.E. & Nasrallah J.B. (1986) Molecu- lar Biology of self incompatibility in plants. Gaget M. (1988) Incompatibilit6 intersp6cifique Trends Genet. 2, 239-244 chez Populus: effet Mentor. Ph.D. Thesis, Uni- Reynolds E.S. (1963) The use of lead citrate at versit6 Lyon I, France high pH as an electron opaque stain electron Gaude T & Dumas C. (1987) Molecular and microscopy. J. Cell Biol. 17, 208-2122 cellular events of self-incompatibility. Int Rev. Towbin H., Staehelin T. & Gordon J. (1979) Cytol. 107, 333-366 Electrophoretic transfer of proteins from poly- Hawkes R. (1982) Identification of Con A bind- acrylamide gels to nitrocellulose sheets: proce- ing proteins after SDS gel electrophoresis and dure and some applications. Proc. Natl. Acad. protein blotting. Anal. Biochem. 123, 143-146 Sci. USA 76, 4350-435 4