Characterization of cyclin-dependent kinases and their expression in developing maize endosperm

by Dante, Ricardo Augusto.

During maize endosperm development, cells switch from mitotic into endoreduplication cell cycles, concomitant with the differentiation of starchy endosperm cells. The identity and role of cyclins and cyclin-dependent kinases (CDKs) that control these cell cycles are poorly understood. I identified and characterized maize cyclins of the D- (CYCD2;1 and CYCD5;1) and A1-types (CYCA1;3) that are expressed in endosperm. CYCA1;3 RNA decreased sharply as the endosperm transitioned from the mitotic to the endoreduplication stage, whereas CYCD2;1 and CYCD5;1 transcripts declined gradually. Polyclonal antibodies against CYCA1;3, CYCD5;1, and CYCD2;1 and a B-type homologue, CYCB1;3, were used to characterize cyclin protein accumulation, the associated CDK activity, and spatial localization of cyclins in endosperm. Except for CYCA1;3, cyclin protein levels were nearly constant or decreased slightly throughout development. CDK activity associated with CYCA1;3 was most abundant 7 days after pollination (DAP), while that associated with CYCB1;3, CYCD2;1 and CYCD5;1 peaked at 11 DAP. CYCA1;3 appeared to be most abundant in the cytoplasm of non-endoreduplicating cells. CYCB1;3 and CYCD2;1 were found throughout the endosperm, while CYCD5;1 was mostly 12 localized in aleurone and subaleurone cells. Stability assays using in vitrosynthesized cyclins showed that CYCA1;3, CYCB1;3, CYCD2;1 and CYCD5;1 were degraded to a large extent via the proteasome in mitotic but not in endoreduplicating endosperm extracts. The results suggest that cyclin proteolysis may not play a major role in controlling endoreduplication cell cycles in maize endosperm. I identified and characterized maize CDKs of the A- (CDKA;3) and B- (CDKB1;1 and CDKB2;1) types that are expressed in developing endosperm. Their transcripts were more abundant during the mitotic than the endoreduplication stage of development. Polyclonal antibodies that specifically recognized CDKA;3 and CDKA;1, and CDKB1;1, were obtained. A-type CDK protein levels were nearly constant, while those of CDKB1;1 decreased abruptly during endosperm development. CDKB1;1 extracted from 9- DAP endosperm bound p13suc1 in vitro, suggesting that CDKB1;1 contributes to mitotic CDK activity at early stages of endosperm development. In coexpression experiments in Drosophila S2 cells, CDKA;1 and CDKA;1 formed catalytically active complexes with CYCD2;1 and inactive complexes with CYCB1;3 and CYCD5;1. 13