After fertilization, the basic body plans of both plants and animals are established during early embryo development. However, despite the fundamental importance of this formative phase of the plant’s life to society's understanding of developmental biology and agricultural practices, the molecular mechanisms that generate the most basic cell-types in plants remain largely uncharacterized. Small RNAs are short non-coding RNAs that regulate gene expression in plants and animals. Although small RNAs are essential for proper gene regulation and cellular differentiation, little is known regarding their embryonic functions, especially in plants. Arabidopsis thaliana (Arabidopsis) is a highly suitable model system to study the regulatory roles of small RNAs because of the abundance of genetic resources and available (genome-wide) data. Moreover, Arabidopsis embryos undergo invariant division patterns and individual cells rapidly differentiate from each other to generate the most basic plant cell-types arranged in correct positions. Early Arabidopsis embryos are therefore morphologically simple structures composed of diverse cell types making them ideal for determining the influence of small RNAs on fundamental cellular differentiation and reprogramming events.
The objectives of the proposed research were designed to assess the regulatory roles of small RNAs in establishing the basic body plan in plant embryos. By utilizing a combination of conventional and novel next-generation sequencing technologies and genetic approaches, we have identified and characterized the small RNAs present in developing embryos including microRNAs (miRNAs) and small interfering RNAs (siRNAs). We found that multiple miRNAs mediate the cleavage and repression of at least 59 transcripts including 30 encoding transcription factors. Moreover, several of these miRNA-mediated repression of transcription factors are dynamic across cell types and individually required for the proper morphogenesis of several embryonic cell lineages. This indicates that in addition to transcriptional regulation, post-transcriptional regulation of transcription factors is critical for embryonic pattern formation in plants. Additionally, compared to other tissues we found that siRNAs are highly enriched in embryos where they direct the (re-)methylation of transposable elements in the new generation. siRNA-directed methylation of transposable elements helps ensure that they remain immobilized and thus preserves genomic integrity. Interestingly, we found that chromatin and sRNAs form a feedback loop to provide a balance between cellular growth and genome defense. In addition to generating biological insights, we have developed various genome-wide technologies suitable for a broad range of eukaryotic species including low-input profiling of small RNA populations and the 5’ ends of RNAs, synthetic RNAs for normalization of genome-wide small RNA sequencing data, and software to inspect the purity of RNA sequencing data, as well as transcriptome assembly. Altogether, the experiments funded by this action allowed the development of tools which in turn yielded insights into how small RNAs help establish the basic body plan and nascent epigenome of early embryos. Our work has helped clarify important questions in small RNA and reproductive biology, and will serve as a foundation for future research.
