Mutations in components of small RNA (sRNA) silencing pathways also influence gametophyte initiation. Ovules lacking function of ARGONAUTE9 (AGO9), RNA-DEPENDENT RNA POLYMERASE6 (RDR6) or SUPPRESSOR OF GENE SILENCING3 produce multiple functional megaspore-like cells in pre-meiotic ovules

INTRODUCTION Unlike animals, in which male and female gametes are derived from germline stem cells, plants produce their gametes from somatic cells that are directed onto specialised pathways based on their position within the reproductive organ, rather than their lineage (Huala and Sussex, 1993; Nonomura et al., 2007). Formation of the female gametophyte (FG) in Arabidopsis ovules begins with megasporogenesis when a single sub-epidermal somatic cell differentiates from nucellar tissue located at the distal tip of the immature ovule, and is specified as a megaspore mother cell (MMC; Fig. 1A,B). The diploid MMC subsequently initiates meiosis to produce four haploid megaspores (Fig. 1C). The most proximal of these, termed the functional megaspore, initiates megagametogenesis and undergoes three rounds of syncytial mitosis, eventually giving rise to the mature female gametophyte (Fig. 1A). Transcriptome profiling in Arabidopsis has identified pathways acting in the megaspore mother cell (Schmidt et al., 2011) and in dividing or mature female gametophytes (Yu et al., 2005; Johnston et al., 2007; Jones-Rhoades et al., 2007). Little is known, however, about pathways involved in the transition from megasporogenesis to megagametogenesis. Mutations in the INNER NO OUTER and WUSCHEL (WUS) transcription factors induce somatic defects in ovule tissues that compromise early stages of gametophyte development, suggesting that inter-regional signalling is important (Elliott et al., 1996; Lieber et al., 2011). Mutations in components of small RNA (sRNA) silencing pathways also influence gametophyte initiation. Ovules lacking function of ARGONAUTE9 (AGO9), RNA-DEPENDENT RNA POLYMERASE6 (RDR6) or SUPPRESSOR OF GENE SILENCING3 produce multiple functional megaspore-like cells in pre-meiotic ovules that express the pFM1:GUS marker (Olmedo-Monfil et al., 2010). AGO9 protein binds 24 nt small interfering RNAs (siRNAs) and accumulates in nucellar epidermal cells, suggesting that it promotes a non-cell-autonomous signal that restricts gametophyte identity in sub-epidermal cells (Olmedo-Monfil et al., 2010). In this study, Arabidopsis ovules were dissected by laser capture microdissection (LCM) and profiled to identify pathways involved in the transition from megasporogenesis to megagametogenesis. Analyses revealed genes upregulated in distal parts of the ovule, including the nucellus and megaspores, relative to other ovule tissues. Characterisation of a unique insertion allele for one of the genes, ARGONAUTE5 (AGO5), suggested that sRNA pathways acting in somatic nucellar cells promote the initiation of megagametogenesis in the functional megaspore. This was supported by tissue-specific expression of viral RNAi suppressor proteins, and was shown to be independent of AGO9. These results indicate that at least two somatic sRNA pathways contribute to gametophyte development in Arabidopsis. One pathway restricts reproductive potential to the functional megaspore and another promotes the initiation of megagametogenesis in this cell. 1CSIRO Plant Industry, Waite Campus, Hartley Grove, Urrbrae SA 5064, Australia. 2College of Life Science, Capital Normal University, Xi San Huan Bei Lu 105, Beijing, 100037, China. 3CSIRO Plant Industry, Black Mountain Laboratories, Clunies Ross Street, Black Mountain ACT 2601, Australia.