Mendes, M.A.Petrella, R.Cucinotta, M.Vignati, E.Gatti, S.Pinto, S.C.Bird, D.C.Gregis, V.Dickinson, H.Tucker, M.R.Colombo, L.2021-09-072021-09-072020Development, 2020; 147(23):dev194274.-1-dev194274.-110950-19911477-9129http://hdl.handle.net/2440/131873In 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 drm1drm2 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 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 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, SEEDSTICK, directly regulates AGO9 and RDR6 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.en© 2020. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.Female germline; MADS-box transcription factors; MMC; RdDM; SPOROCYTELESS; SEEDSTICKThe RNA-dependent DNA methylation pathway is required to restrict SPOROCYTELESS/NOZZLE expression to specify a single female germ cell precursor in ArabidopsisJournal article100003047810.1242/dev.1942740005994607000112-s2.0-85098472069557101Bird, D.C. [0000-0001-7071-7937]Tucker, M.R. [0000-0003-4661-6700]