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

Abstract

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