Abundant expression of maternal siRNAs is a conserved feature of seed development

Abstract

Small RNAs are abundant in plant reproductive tissues, especially 24-nt short interfering (si)RNAs. Most 24-nt siRNAs are dependent on RNA Pol IV and RDR2, and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.

Embryo CHH hypermethylation is mediated by RdDM and is autonomously directed in Brassica rapa

“…Communication between maternal and filial tissues, as well as between the embryo and endosperm, is essential to coordinate the development of a seed [37,38]. Small RNAs have been proposed to move between seed tissues and to establish robust methylation of transposons at this transition between generations [22,[28][29][30][31].…”

“…To further probe the possibility that maternal sporophytically-derived siRNAs might trigger hypermethylation of the embryo, we assessed DNA methylation levels at previously-defined "siren" loci [31]. These loci produced over 90% of the 24-nt siRNAs in maternal integuments and are also the most highly-expressed siRNA loci in endosperm.…”

“…We calculated enrichment of genomic features like small RNA loci, TEs, and genes within the CHH hyper-DMWs using BEDTools intersect [52,53]. Transposable elements were annotated as in [31]. We considered features to be overlapping if there was at least 1 nucleotide shared.…”

“…Methylation over pre-defined siren loci [31] was determined with BEDTools intersect [52] and a custom Python script [(bedgraph_methylation_by_bed.py) developed previously [31] in the lab.…”

“…In Arabidopsis, the developing endosperm is demethylated at sequences that show hypermethylation in the embryo, leading to the hypothesis that siRNAs produced in the endosperm might move to the embryo to direct methylation [22,[28][29][30]. Movement of siRNAs between the maternal integuments and the filial tissues has also been proposed [31]. However, embryos produced through somatic embryogenesis also display hypermethylation, despite lack association with either endosperm or maternal integuments [27].…”