Forward Genetic Studies Towards the Understanding of the Molecular Mechanisms Underlying Floral Meristem Determinacy and Small RNA Function in Arabidopsis

Forward genetics is a powerful tool to identify genes involved in particular biological processes. In my thesis work, I participated in forward genetic screens to identify genes involved in two biological processes in plants, stem cell maintenance in the floral meristem and small RNA biogenesis/function. First, I characterized a Polycomb (PcG) gene, CURLY LEAF (CLF) as a factor required for floral meristem termination. A mutation in CLF enhances the floral determinacy defects of ag-10, a weak allele in AGAMOUS (AG), a gene essential for floral stem cell termination. CLF acts in the AG pathway to repress the stem cell identity gene, WUSCHEL (WUS) to result in floral stem cell termination. In addition, I show that this role of CLF reflects the role of the PcG complex in the control of floral meristem determinacy. Taken together, I provide a link between epigenetic regulation and stem cell maintenance in the floral meristem. Second, I identified two genes, MED20a and STERILE APETALA (SAP) that promote small RNA biogenesis and modulate small RNA activities, respectively. MED20a is a subunit of the Mediator complex, which is thought to bridge transcription factors and the RNA Polymerase II (Pol II) transcriptional machinery. In med20a and mutants in two other Mediator subunits, med17 and med18, miRNA accumulation is generally reduced. In the med20a mutant, the levels of pri-miRNA are also lower and the promoter activity of MIR167a is reduced, suggesting that the Mediator promotes MIR gene expression at the transcriptional level. I show that SAP is a negative regulator of the activities of a subset of miRNAs. In sap mutants, the mRNA and protein levels of several miRNA target genes are reduced despite no changes in miRNA accumulation. In addition, some miRNAs are more active in sap mutants, resulting in more 3' cleavage products from target mRNAs. This indicates that SAP negatively regulates the activities of a subset of miRNAs at the posttranscriptional level. These studies extend our understanding of the dynamic control of small RNA biogenesis and function