Gametogenesis-related small RNAs and Argonaute Proteins in Arabidopsis thaliana

The life cycle of flowering plants alternates between a diploid multicellular sporophyte generation and a highly reduced haploid gametophyte generation located within the reproductive tissues of the sporophyte. Emerging evidence indicates that the transition from sporophyte to a gametophytic life phase and the acquisition of reproductive fate is marked by extensive epigenetic reprogramming employing distinct members of the ARGONAUTE (AGO) family of proteins with roles in RNA-directed DNA methylation (RdDM) and heterochromatin formation. Nevertheless, almost nothing is known about RNA silencing machinery components and small noncoding RNAs associated with epigenetic reprogramming and the acquisition of gametic cell fate. The aim of this work was the detailed investigation of two RdDM-associated Argonaute genes (AGO8 and AGO9) regarding their expression pattern and function in the female reproductive lineage of Arabidopsis thaliana. Furthermore, a specialized Arabidopsis cell line with an egg cell-like transcriptome was generated to enable the identification of small RNAs and RNA silencing machinery components potentially associated with female reproductive fate. RT-PCR based expression studies were performed for all ten Arabidopsis AGO genes, confirming that AGO5, AGO8 and AGO9 are predominantly expressed in floral tissues containing reproductive lineages, while other AGO genes are more or less ubiquitously expressed. Protein abundance was investigated by Western Blots using peptide antibodies available for six AGOs andp09 revealed that AGO5 and AGO9 are strongly represented in reproductive organs and in the egg cell-like callus. To investigate AGO8 and AGO9 promoter activities and the presence of AGO8 and AGO9 proteins in more detail during female gametophyte development, transgenic Arabidopsis reporterlines were generated and whole-mount immunolocalization experiments were performed. These studies showed that the AGO8 promoter is only active in the egg cell. Nevertheless, AGO8 expression only yielded aberrant spliced mRNA and a GFP-AGO8 fusion protein was not detectable in egg cells expressing AGO8p:GFP-AGO8, suggesting that AGO8 represents a pseudogene. AGO9 promoter-reporter lines showed activity in the nucellus cells, the megaspore mother cell (MMC) and the functional megaspore (FM) of young ovules. In the mature female gametophyte, AGO9 promoter activity was detectable in the egg cell, the central cell and the chalazal region of the ovule. This is in remarkable contrast to results obtained by AGO9 whole-mount immunolocalization and expression of the GFP-AGO9 fusion protein: AGO9 was neither detectable in the MMC and FM nor in the egg cell nor central cell. The occurrence of aberrant splicing by intron retention in AGO9 mRNA, emerging during ovules maturation, and the results obtained from ectopic expression of correctly spliced AGO9 cDNA in the egg cell suggests that both, developmentally regulated differential splicing and decreased AGO9 protein stability is responsible for the lack of AGO9 protein in the egg cell. A few plants were obtained expressing GFP-AGO9 in the egg cell, which did, however, not correlate with developmental defects in the female gametophyte or in developing seeds. The impact of ectopic GFP-AGO9 on DNA methylation in the egg cell remains to be investigated. To identify protein coding genes and small noncoding RNAs possibly involved in the acquisition of female reproductive fate or in egg cell specification a specialized transgenic Arabidopsis cell line with egg cell-like expression profile was established by ectopically expressing the transcription factor RKD2. Compared to the control cell line 5,511 genes were classified as differentially expressed by RNA-seq. Quantitative real-time PCR with eighteen selected genes confirmed mRNA-Seq expression data. A global look at genes involved in small RNA pathways revealed several differentially expressed genes in RKD2-induced cell line, including upregulated genes encoding the DNA methyltransferase DOMAINS REARRANGED METHLYTRANSFERASE 1 (DRM1), the DOUBLE-STRANDED RNA BINDING PROTEIN 3 (DRB3), the HISTONE DEACETLYLASE 18 (HDA18), the putative chromatin remodeling protein CHROMATIN REMODELING 34 (CHR34) and a so far undescribed DNA-directed RNA polymerase V subunit 5A-like gene. Small RNA profiling in the RKD2-induced cell line revealed noticeable differences compared to the control callus. The most abundant group of small RNAs in the control callus are miRNAs, whereas the biogenesis of siRNAs from transposable element (TE) transcripts is dominant in the egg cell-like callus. TE-derived siRNAs of the Ty3/Gypsy superfamily of long terminal repeat retrotransposons, containing members of the ATHILA, ATLANTYS, and ATGP family, form the largest fraction of siRNAs, supporting the idea that the plant reproductive lineage is protected by siRNAs against the activity of TEs. This is in line with the observation that aligned reads to respective TE transcripts are not present in the mRNA-Seq data of the egg cell-like cell line. Differential expression analysis of miRNAs revealed 96 miRNAs being at least up- or down regulated with a log2 fold change of 2, including only 9 miRNAs upregulated in the RKD2-induced callus. Furthermore, 32 novel miRNAs were discovered, with 20 miRNAs being derived from known miRNA precursors while 12 miRNAs are completely novel. Target gene predictions of differential expressed miRNAs revealed several inverse correlations between miRNA and mRNA target expression levels in both the egg cell-like callus and the control callus. The discovery of so far undescribed transcripts in the egg cell-like cell line encoding small RNA pathway components involved in chromatin modification, together with the identification of differentially expressed known and novel small noncoding RNAs open up interesting possibilities for future investigations regarding their contribution to the acquisition of reproductive fate and egg cell specification.