The TREX Complex Regulates Megaspore Mother Cell Specification Through The TAS3-ARF3 Module

In most sexually reproducing plants,a single somatic,sub-epidermal cell in an ovule is selected to differentiate into a megaspore mother cell(MMC),which is committed to giving rise to the female germline.However,it remains unclear how intercellular signaling among somatic cells results in only one cell in the sub-epidermal layer to differentiate into the MMC.The previous studies indicates that RDR6 plays important role in MMC formation.The mutation in RDR6 results in multiple MMCs in ovule premordia.To identify additional genes functioning in MMC differentiation,we conducted an ethylmethanesulfonate mutagenesis screen for enhancers and suppressors of the rdr6-11 mutant.In this study,we uncovered a critical role of the Arabidopsis THO complex in preventing ectopic MMC formation.We showed that the THO complex and RDR6 control MMC differentiation through the TAS3-derived ta-siRNA,tasiR-ARF,which spatially restricts ARF3 expression in ovule primordia.The main results are bellows:Mutations in TEX1,a component of the THO/TREX complex,led to the formation of multiple MMCs,which were able to initiate gametogenesis.We demonstrated that only one MMC has the ability to undergo meiosis among the multiple MMC.We studied the localization of the TEX1 protein using a TEX1-green florescence protein(GFP)fusion driven by the TEX1 promoter.At the megasporogenesis stage,TEX1 accumulated in the nuclei of the epidermal cell layer of ovules.At the megagametogenesis stage,the TEX1-GFP fusion protein was located in the nuclei of female gametophyte.After cellularization of the embryo sac,the expression of TEX1 ceased in a step-wise manner along the micropylar-chalazal axis.Besides,the mutations in another two components of THO/TREX complex HPR1 and TH06 also resulted multiple MMC in ovule primodia,which suggested that TEX1 exerted its function in MMC specification through the THO/TREX complex.We demonstrated that TEX1 controls MMC differentiation by promoting the biogenesis of TAS3 derived ta-siRNAs,which repress ARF3 expression.The TEX1 protein was present in epidermal cells but not in the germline,and through TAS3-derived ta-siRNAs,restricted ARF3 expression to the medio domain of ovule primordia.Supernumerary MMC formation was induced by ectopic expression of ARF3 in lateral epidermal cells in a TAS3 ta-siRNA-insensitive mutant,suggesting that TEX1 and TAS3-mediated restriction of ARF3 expression limits excessive MMC formation non-cell autonomously.Our findings reveal the role of a small RNA pathway regulating MMC specification in Arabidopsis.