The RNA dependent DNA methylation pathway is required to restrict SPOROCYTELESS/NOZZLE expression to specify a single female germ cell precursor in Arabidopsis

In higher plants the female germline is formed from the megaspore mother cell (MMC), a single cell in the pre-meiotic ovule. Previously, it was reported that mutants in RNA dependent DNA methylation - RdDM - pathway might be involved in restricting female germline to a single nucellus cell. We show that the DRM methyltransferase double mutant drm1drm2 also presents also presents ectopic enlarged cells, consistent with supernumerary MMC-like cells. In wild-type ovules MMC differentiation required SPOROCYTELESS/NOZZLE (SPL/NZZ), as demonstrated by the spl/nzz mutant failing to develop an MMC. We address the poorly understood upstream regulation of SPL/NZZ in ovules, showing that the RdDM pathway is important to restrict SPL/NZZ expression. In ago9, rdr6 and drm1drm2 mutants, SPL/NZZ is ectopically expressed, suggesting that the mutiple MMC-like cells observed might be due to the ectopic expression of SPL/NZZ. We show that the ovule identity gene SEEDSTICK, directly regulates the AGO9 and RDR6 expression in the ovule and therefore indirectly SPL/NZZ expression. A model is presented describing the network required to restrict SPL/NZZ expression to specify a single MMC.