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Female germline cells in flowering plants differentiate from somatic cells to produce specialized reproductive organs, called ovules, embedded deep inside the flowers. We investigated the molecular basis of this distinctive developmental program by performing single-cell RNA sequencing (scRNA-seq) of 16,872 single cells of Arabidopsis thaliana ovule primordia at three developmental time points during female germline differentiation. This allowed us to identify the characteristic expression patterns of the main cell types, including the female germline and its surrounding nucellus. We then reconstructed the continuous trajectory of female germline differentiation and observed dynamic waves of gene expression along the developmental trajectory. A focused analysis revealed transcriptional cascades and identified key transcriptional factors that showed distinct expression patterns along the germline differentiation trajectory. Our study provides a valuable reference dataset of the transcriptional process during female germline differentiation at single-cell resolution, shedding light on the mechanisms underlying germline cell fate determination. Zhimin Hou, Yanhui Liu et al. used single cell RNA-seq to analyze the model organism, Arabidopsis thaliana, at three stages during female germline differentiation. They reconstructed the continuous trajectory of female germline differentiation, providing a valuable reference for future investigation of germline cell fate determination in A. thaliana. [ABSTRACT FROM AUTHOR]

Summary In most sexually reproducing plants, a single somatic, sub-epidermal cell in an ovule is selected to differentiate into a megaspore mother cell, 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 differentiating into the megaspore mother cell. Here we uncovered a role of the THO complex in restricting the megaspore mother cell fate to a single cell. Mutations in TEX1 , HPR1 , and THO6 , components of the THO/TREX complex, led to the formation of multiple megaspore mother cells, which were able to initiate gametogenesis. We demonstrated that TEX1 repressed the megaspore mother cell fate by promoting the biogenesis of TAS3 -derived trans -acting small interfering RNA (ta-siRNA), which represses ARF3 expression. The TEX1 protein was present in epidermal cells, but not in the germline, and, through TAS3 -derived ta-siRNA, restricted ARF3 expression to the medio domain of ovule primordia. Expansion of ARF3 expression into lateral epidermal cells in a TAS3 ta-siRNA-insensitive mutant led to the formation of supernumerary megaspore mother cells, suggesting that TEX1- and TAS3- mediated restriction of ARF3 expression limits excessive megaspore mother cell formation non-cell-autonomously. Our findings reveal the role of a small-RNA pathway in the regulation of female germline specification in Arabidopsis . [ABSTRACT FROM AUTHOR]