Small RNA pathway components and small RNA profiles of flowering plant egg cells are largely unexplored, mainly because they are not easily accessible but deeply buried inside the ovary. We describe here the utilization of proliferating callus tissue that adopted transcriptome features of Arabidopsis egg cell as a tool to explore small RNA pathway components and small RNA profiles in egg cells. We furthermore complement our studies with mRNA-Seq data from isolated Arabidopsis egg cells and provide data validation by promoter-reporter studies and whole mount in situ hybridization. Sequencing of small RNA libraries demonstrate the predominance of TE-derived siRNAs in the egg cell-related callus. TE-features and expression profiles suggest post-transcriptional silencing of activated Gypsy-like LTR retrotransposons, whereas the majority of class II DNA transposons belonging to Copia, CACTA, hAT-like and Mutator superfamilies are subjected to transcriptional silencing. Small RNA-seq furthermore led to the identification of differentially expressed known and novel miRNAs whose expression in the egg cell was verified by small RNA whole mount in situ hybridization. Both the strong expression of miRNAs in the egg-cell-adjoining synergids and the secretion of miRNAs into the micropyle suggest hitherto undescribed roles for these accessory cells in intercellular communication with the egg cell and the arriving pollen tube. In conclusion, our datasets provide valuable and comprehensive resources to study small RNA pathways and small-RNA-mediated epigenetic reprogramming during egg cell differentiation and the onset of plant embryogenesis.

During double fertilization in Arabidopsis thaliana, the egg cell secretes small cysteine-rich EC1 (egg cell 1) proteins, which enable the arriving sperm pair to rapidly interact with the two female gametes. EC1 proteins are members of the large and unexplored group of ECA1 (early culture abundant 1) gametogenesis-related family proteins, characterized by a prolamin-like domain with six conserved cysteine residues that may form three pairs of disulfide bonds. The distinguishing marks of egg-cell-expressed EC1 proteins are, however, two short amino acid sequence motifs present in all EC1-like proteins. EC1 genes appear to encode the major CRPs (cysteine-rich proteins) expressed by the plant egg cell, and they are restricted to flowering plants, including the most basal extant flowering plant Amborella trichopoda. Many other ECA1 gametogenesis-related family genes are preferentially expressed in the synergid cell. Functional diversification among the ECA1 gametogenesis-related family is suggested by the different patterns of expression in the female garnetophyte and the low primary sequence conservation.