Single female germline differentiation in Arabidopsis is controlled by RdDM mediated miR160 methylation and ARF10 silencing through ARGONAUTE1

In Arabidopsis, the female germline arises from a pool of seemingly undistinguishable diploid somatic cells in the ovule primordium, one of which becomes the megaspore mother cell (MMC) and acquires the ability to initiate a reproductive developmental program through meiosis. We show herein that precise regulation of AUXIN RESPONSE FACTOR 10 (ARF10) by miR160 - ARGONAUTE1 and the RNA direct DNA methylation (RdDM) system is necessary for proper female germline commitment during the earliest phases of ovule development. A DNA methylome analysis revealed that RdDM mutants exhibited a polymorphic miR160 methylated region, leading to a decrease in pri-pre-miR160 expression levels. Meanwhile, expression analysis using GFP-fusion lines revealed that one of the miR160 targets (ARF10) is active in cells surrounding the differentiated MMC in wild-type contexts, but overexpressed ectopically throughout the whole ovule in miR160-insensitive lines, indicating that miR160 is the controller delimitating ARF10 final expression pattern around the MMC. Moreover, mARF10_GFP lines develop several enlarged nucellar cells with FM identity, incapable of undergoing meiosis but able to develop into supernumerary mature gametophytes with altered polarity and unusual cell identity, a phenotype mimicking aposporous apomixis. Furthermore, mARF10_GFP lines show severe defects in integument development and an early entrance of the MMC into meiosis. Instead, a defective arf10_1 mutant displayed delayed meiosis and 2n/3n gametophytic arrest. We also determined that AGO1 mechanistically mediates the nucellar ARF10 silencing by miR160, since ago1-27 mutant allele induced ectopic nucellar ARF10_GFP expression. Our results indicate in Arabidopsis ARF10 displays a precise expression pattern controlled by the synergic activity of RdDM/miR160/AGO1, which is necessary for proper sexual female identity. The possible involvement of ARF10 in the sexual to agamic transition is discussed here and should be further investigated in apomictic models.