The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.
Inhibition of cdc2 activation by INH/PP2A.