Elucidating small RNA pathways in Arabidopsis thaliana egg cells

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.