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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - sRNA-EMB (Small RNA regulation of the body plan and epigenome in Arabidopsis embryos)

Teaser

Summary

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 plantâ€™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 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 are designed to assess the regulatory roles of small RNAs in establishing the basic body plan in plant embryos. We are utilizing a combination of modified next-generation sequencing technologies and genetic approaches to identify and characterize the small RNAs present in developing embryos. Altogether, the experiments being funded by this action are expected to yield novel insights into how small RNAs help establish the basic body plan of early embryos, and thus clarify some of the most outstanding questions in small RNA and reproductive biology fields. Moreover, our research should open up new avenues for exploring the regulatory roles of these fascinating molecules during the earliest stages of life.

Work performed

Overall, our ERC-funded project is well on-track to accomplish the originally proposed objectives. We currently have several manuscripts describing our progress during the first reporting period that have been published or are being prepared for submission. Because our research results are unpublished, only a very general overview is provided below to protect the confidential nature of our work. During the first 30 months of the project, we have developed a few biotechnology applications/tools that increase our ability to study genome-wide molecular biology phenomena in a quantitative manner from extremely low amounts of material. For instance, we developed exogenous small RNA spike-in oligonucleotides that allow for absolute normalization of small RNA sequencing (sRNA-Seq) data (Lutzmayer et al. [2017] Scientific Reports). These sRNA spike-ins facilitate comparisons of small RNA levels across different tissue types and genotypes, as well as genome-wide estimations of sRNA:mRNA stoichiometries. We also produced a statistical tool that revealed the presence of substantial RNA contamination from maternal tissues in nearly all published Arabidopsis endosperm and early embryo transcriptomes (Schon and Nodine [2017] The Plant Cell). Not only is this a useful tool for the community to ensure the generation of accurate datasets, but we also found that maternal RNA contamination in previously published datasets had been repeatedly misinterpreted as epigenetic phenomena including the magnitude and maternal bias of imprinted genes in seeds.

Final results

Molecular biology approaches are increasingly being applied to specific cell-types and even individual cells to more precisely understand complex gene regulatory mechanisms at the cellular level. The applications and tools that we are developing are therefore not only instrumental to our ability to accomplish our project\'s stated aims, but are expected to be generally helpful to research groups studying cell-type specific processes. For example, the sRNA spike-in method we developed can be used to improve the interpretation of small RNA-related data that are routinely generated and used in both basic and applied research. Therefore, this work may also have a broad impact extending from fundamental molecular biology research to clinical assessment of molecular markers related to disease-afflicted cell-types. Moreover, our statistical tool to detect contamination in seed transcriptome datasets has large implications in the field of epigenetic imprinting. Because methods that we are developing are broadly applicable, we expect them to have an impact beyond the results originally expected from the aims stated in the original proposal.