Poster: Cell Cycle & Cytokinesis
Abs #
1097: Expression of a dominant negative mutant of cyclin-dependent kinase A (ZmCDKA) reduces DNA endoreduplication during maize endosperm development
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Presenter: |
Leiva-Neto, Joao T, joao@email.arizona.edu |
Authors | Leiva-Neto, Joao T (A) Grafi, Gideon (B) Sabelli, Paolo A (A) Dante, Ricardo A (A) Coelho, Cintia M (A) Maddock, Sheila (C) Gordon-Kamm, William J (C) Larkins, Brian A (A) | | Affiliations: |
(A): Department of Plant Sciences - University of Arizona,
(B): Department of Plant Sciences - The Weizmann Institute of Science
(C): Pioneer Hi-Bred International Inc.
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| Web Site: | http://www.ag.arizona.edu/research/larkinslab/ | |
The study of maize endosperm is appealing because of its importance in agriculture, and the insight it provides regarding important biological processes. During endosperm development, cells transition from a mitotic to an endoreduplication cell cycle, and this coincides with an increase in cell size and accumulation of storage proteins and starch. In this study, we generated transgenic maize plants over-expressing HA epitope-tagged maize cyclin-dependent kinase A (HA-ZmCDKA) or a dominant negative form (HA-ZmCDKA D146N) of this enzyme. Both genes were ectopically expressed using a highly active endosperm-specific promoter (27-kD gamma-zein). Anti-HA and anti-CDKA immunoblots showed high levels of transgenic protein accumulation beginning around 12 days after pollination (DAP). Histone H1 kinase assays on HA immunoprecipitates revealed the point mutation (HA-ZmCDKA D146N) completely abolished kinase activity. Additionally, a considerable reduction of p13suc1 adsorbed kinase activity was observed in HA-ZmCDKA D146N endosperms. Flow-cytometric analysis performed on developing HA-ZmCDKA D146N endosperms showed a significant reduction in DNA endoreduplication, when compared to control endosperms segregating on the same ear. By 18 DAP, the mean ploidy of HA-ZmCDKA D146N endosperms (5.9 C) was 50% of the control endosperms (11.9 C), and the maximal ploidies observed were 24C and 96C, respectively. This dramatic reduction in endopolyploidy was reflected in nuclear size, as seen in DAPI-stained endosperm sections analyzed by fluorescence microscopy. A detailed characterization of the anatomical and physiological effects created by the reduced level of endoreduplication in HA-ZmCDKA D146N endosperms will be reported.
