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item Vaughn, Kevin

Submitted to: Protoplasma
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/3/2005
Publication Date: 5/30/2006
Citation: Vaughn, K.C., Renzaglia, K.S. 2006. Structural and immunocytochemical characterization of the gingko biloba l. sperm motility apparatus. Protoplasma 227:165-173.

Interpretive Summary: Almost nothing is known as to how the plant cells organize their microtubules, which are the target sites for over one quarter of the herbicides. To investigate this question, we used microscopic techniques to identify proteins in the sperm cells of ginkgo to identify the proteins present in a given structure. Gingko sperm was chosen because it is the most primitive seed plant still alive. The data from this study indicate that the calcium binding protein centrin is a critical component and that a modification to tubulin protein is critical in forming stable arrays. These latter are resistant to microtubule disrupter herbicides.

Technical Abstract: Ginkgo and the cycads are the extant only seed plants with motile sperm cells. However, there has been no immunocytochemical characterization of these gametes to determine if they share characteristics with the flagellated sperm found in bryophytes and pteridophytes or might give clues as to the relationships to nonflagellated sperm in all other seed plants. To determine characteristics of proteins associated with the motility apparatus in these motile sperm, we probed thin sections of developing spermatogenous cells of Ginkgo with antibodies to acetylated and tyrosinated tubulin, monoclonal antibodies that recognize mammalian centrosomes, and centrin. The blepharoplast that occurs as a precursor to the motility apparatus consists of an amorphous core, pitted with cavities containing microtubules and a surface studded with probasal bodies. The probasal bodies and microtubules within the blepharoplast cavities are labeled with antibodies specific to acetylated tubulin. Positive but weak reactions of the blepharoplast core occur with the centrosome-reactive antibodies MPM-2 and C-9. Reactions to centrin antibodies are negative at this developmental stage. From this pre-motile apparatus structure, an assemblage of ~1000 flagella and associated structures arise as the precursor to the motility apparatus for the sperm. The flagellar apparatus consists of a three-layered multilayered structure (MLS) that subtends a layer of spline microtubules, a zone of amorphous material similar to that in the blepharoplast, and the flagellar band. Centrin antibodies reacts strongly with the MLS, the transition zone of the flagella and with fibrillar material near the flagellar base at the surface of the amorphous material. Both the spline microtubules and all of the tubules in the flagella react strongly with the antibodies to acetylated tubulin. These localizations are consistent with the localizations of these components in pteridophyte and bryophyte spermatogenous cells, although the blepharoplast material surrounding and connecting flagellar bases does not occur in the seedless (non-seed) land plants. These data indicate that despite the large size of Ginkgo gametes and the taxonomic separation between pteridophytes and Ginkgo, similar proteins in gametes of both groups perform similar functions and are therefore homologous among these plants. Moreover, the presence of acetylated tubulin in bands of microtubules may be a shared character with more derived nonflagellated sperm of other conifers and angiosperms.