Expression of Factors of DNA Methylation in A. thaliana reproductive structures

RNA-directed DNA methylation (RdDM) is a small RNA-mediated epigenetic pathway involved in transcriptional repression of both transposons and genes. The expression of genes involved in this pathway is required for proper differentiation of gametophyte precursors in A. thaliana ovules. Members of the gene family Factors of DNA Methylation (FDM), together with their closely related IDN2, are dsRNA-binding proteins that regulate RdDM at chromatin targets. Recently we found that several FDM-like transcripts are differentially expressed in ovules collected from aposporous apomictic H. perforatum accessions. To gain further insight into the activity of FDM-like genes in gamete formation, we studied the expression of IDN2 and five FDM genes in A. thaliana reproductive structures. Microarray data indicate that expression of FDM genes is higher in flowers at stage 9 and decreases throughout flower development. Among the different flower parts, their expression is enriched in carpels at stage 12 and 15 and, within the pistil, it appears to be higher in the ovary. A protocol allowing reverse-transcription quantitative PCR (RT-qPCR) directly from micro-dissected ovules, without RNA extraction, was developed to monitor the expression levels of FDM genes in ovules at different developmental time points. In doing so, we could confirm the expression of IDN2 together with FDM1, FDM2 and FDM5 in ovules at different time points corresponding to sporogenesis and gametogenesis. Knockout and reporter lines were investigated to address the role of FDM genes in gametes formation and seed production. In doing so, we found that the A. thaliana IDN2 knockout line displays a significant reduction in the number of vital seeds. Similar phenotypes were observed when other effectors of the RdDM pathway were knocked out. Results on FDM expression contribute in paving the way toward a better comprehension of genetic and molecular basis of gametes formation and seed production.