Graduate Student Whitehead Institute for Biomedical Research Cambridge, Massachusetts
Body of Abstract: The RNA-directed DNA Methylation (RdDM) pathway is the primary mechanism of de novo DNA methylation in plants. Two plant-specific RNA polymerases, RNA Polymerase IV (Pol IV) and RNA Polymerase V (Pol V), are involved in this pathway. RNA Pol IV is required for the production of 24-nt small RNAs (sRNAs) that guide de novo methylation.
Seeds contain a plant embryo that is nourished by the endosperm, a tissue that stores nutrients and also mediates nutrient transfer from maternal tissue during seed development. Mutation of Pol IV results in loss of RdDM and a change in endosperm mRNA and sRNA expression patterns. However, the mechanism by which Pol IV regulates these expression patterns remains unclear. These ongoing projects are intended to elucidate the mechanisms underlying Pol IV action in seeds.
To determine whether Pol IV impacts endosperm mRNA and sRNA expression patterns by acting before or after fertilization, we are identifying the Arabidopsis thaliana seed tissue(s) in which Pol IV activity can complement the endosperm mutant phenotype. We created transgenic lines with NRPD1 expression under the control of promoters specific to the central cell, endosperm, or maternal integuments/seed coat in an nrpd1 mutant background (NPRD1 encodes the largest subunit of Pol IV). Endosperms were dissected from each line and mRNA expression patterns measured by RNA-seq. We aim to identify a tissue in which tissue-specific expression of NRPD1 complements the mutant phenotype, with expression patterns similar to those of the wild-type. If so, this would be an indication of that tissue being the primary location of Pol IV’s action in regulating endosperm expression patterns. This experiment is currently ongoing, and the latest results will be presented.