Dissecting the mechanisms governing meiotic progression

Meiosis is an essential prerequisite of sexual reproduction as it leads to the formation of haploid gametes from diploid precursor cells. The sequential chromosome segregation that occurs during meiosis requires extensive remodeling of cell cycle machinery. Regulatory mechanisms that define and control meiotic divisions are still rather poorly understood, particularly in multicellular organisms in which meiosis is confined to a few highly specialized cells. Here we propose to examine mechanisms that regulate meiotic progression in the model plant Arabidopsis thaliana. The entry points for this project are two proteins that play an important role in meiotic progression. TDM1 is a plant-specific protein deficiency that leads to an attempted division of unreplicated haploid nuclei (third meiotic division). The second protein is the conserved nonsense-mediated mRNA decay factor SMG7. SMG7 appears to act in the same pathway as TDM1 with its mutation causing unique meiotic arrest in anaphase II. We predict that these proteins are essential for the transition from meiosis to subsequent haploid mitoses by facilitating downregulation of meiotic CDK activity. We propose to delineate the regulatory network that involves TDM1 and SMG7 proteins and to define the core components of the cell cycle machinery that drive meiotic progression. First, we want to identify and functionally characterize cyclins and CDKs that are active during meiosis. To this end, we will use protein immunolocalization to survey expression of all Arabidopsis A- and B- type cyclins and analyze mutants in the promising candidate genes. Second, we want to decipher the molecular mechanism of TDM1 action. Specifically, we will conduct genetic and biochemical experiments to test whether TDM1 acts as an inhibitor of meiotic cyclin-CDK complexes. Finally, we want to determine the role of SMG7 in meiosis by performing a forward genetic screen to identify putative targets of SMG7 regulation. Elucidating pathways that regulate meiotic divisions is essential for full understanding of plant sexual reproduction, which is important for the development of new breeding strategies.

Meiosis is specialized cell division required for formation of haploid gametes from diploid precursor cells. Mechanisms that define and control meiotic division are still not well understood, particularly in multicellular organisms in which meiosis is confined to a few highly specialized cells. In this project we elucidated regulation of meiosis in the model plant Arabidopsis thaliana. We examined regulatory circuit consisting of the nonsense mediated RNA decay (NMD) factor SMG7, plant specific protein TDM1 and cyclin TAM, which are important for proper execution of meiotic divisions. We predicted that this circuit directly affects activity of meiotic cyclin-dependent kinases. To define the core meiotic CDK oscillator, we identified Arabidopsis cyclins expressed in meiosis. Curiously, none of the cyclin was essential for meiotic progression arguing that plant meiosis is driven by unorthodox cell cycle regulators. To discover novel genes involved in meiosis, we performed a suppressor screen for mutations alleviating reduced fertility of smg7 mutants. We recovered a large number of mutant lines with restored fertility and our mapping efforts yielded several promising candidate genes which will be analyzed in our future work. In our attempt to decipher the role of SMG7, we made an unexpected discovery that nonsense mediated RNA decay contributes to plant pathogen defense by regulating levels of immune receptors in response to infection. This is one of the first examples demonstrating functional significance of NMD-mediated regulation of physiological transcripts.