MIR822 modulates monosporic female gametogenesis through an ARGONAUTE9-dependent pathway in Arabidopsis thaliana

In the ovule of flowering plants, the establishment of the haploid generation occurs when a somatic subepidermal cell specified as the gametophytic precursor differentiates into a Megaspore Mother Cell (MMC) and initiates meiosis. As most flowering plants, Arabidopsis thaliana (Arabidopsis) undergoes a monosporic type of gametogenesis as three meiotically derived cells degenerate without further division, and a single one – the functional megaspore (FM) - divides mitotically to form the female gametophyte. The genetic basis and molecular mechanisms that control monosporic gametogenesis remain largely unknown. In Arabidopsis, ARGONAUTE proteins are involved the control of megasporogenesis. In particular, mutations in ARGONAUTE9 (AGO9) lead to the ectopic differentiation of gametic precursors that can give rise to apomeiotically derived female gametophytes. Here, we show that Arabidopsis plants carrying loss-of-function mutations in the AGO9-interacting microRNA miR822a give rise to extranumerary surviving megaspores that acquire a FM identity and divide without giving rise to differentiated female gametophytes. The overexpression of three miR822a target genes encoding Cysteine/Histidine-Rich C1 domain proteins (At5g02350, At5g02330 and At2g13900) results in defects equivalent to those found in mir822 plants. All three miR822a targets are overexpressed in ago9 mutant ovules, confirming that miR822a acts through an AGO9-dependent pathway to negatively regulate Cysteine/Histidine-Rich C1 domain proteins and restricts the survival of meiotically derived cells to a single megaspore. Our results identify a microRNA-dependent mechanism that is involved in the control of megaspore degeneration and the most prevalent form of female gametogenesis in flowering plants. ### Competing Interest Statement The authors have declared no competing interest.