Project number: 2021/43/D/NZ1/00154
Financed by: National Science Center in Poland
Project leader: PhD Anna M. Wójcik
Social Media: Find us on Twitter: #FANSinSE
11.2022-11.2025
The project aims at the identification of the new, cell-specific genetic elements engaged in the formation of pluripotent stem cells from somatic cells of Arabidopsis thaliana. The totipotency of the plant somatic cells is reflected during the induction of somatic embryogenesis (SE) when somatic cells change their developmental pathway and became fully functional embryos (somatic embryos). SE provides a useful system in studies on molecular factors determining the pluripotency of somatic cells. To achieve the set in the project goal, we are going use, already adopted by the PI, a new procedure enabling identification, isolation, and transcriptomic analysis of the homogenous population of specific SE-responsive cells. The cell-specific RNA will be used for complex transcriptomic analysis in order to identify the elements of a genetic network that control the embryogenic transition during SE induction. The functional analyses of candidate genes and sRNA molecules from transcriptomic analyses will be carried out during the project. Moreover, pioneer transcriptomic data obtained from somatic embryos at different developmental stages will be compared with the existing data from Arabidopsis zygotic embryos.
A fundamental limitation in experimental approaches to identify molecular determinants of SE is the difficulty to separate and analyse exclusively responsive and reprogramming cells at an early state after induction. To date, the analyses rely on RNA isolation techniques from cultures where embryogenic cells are only a small percentage. To answer the questions about transcriptomic changes during the formation of pluripotent stem cells during SE induction and somatic embryo development, we are going to perform complex transcriptomic analysis in which the fluorescence activated nuclear sorting (FANS) method will be used. FANS allows efficient purification of very few fluorescence-tagged nuclei from a large background of non-labeled tissue (Gutzat and Mittelsten Scheid, 2020). The embryogenic direct SE culture, in which somatic embryos are produced rapidly and efficiently will be used (Gaj, 2001). The analysis of several time points during SE culture with the FANS method will give us a unique opportunity to get insights into dynamic transcriptomic changes associated with embryogenic induction. To analyze the transcriptomes of the FANS-isolated nuclei of embryogenic cells, we are going to apply protocols for a low-input RNA and small RNA sequencing (RNA-seq; sRNA-seq). Such protocols have been successfully developed for the analysis of zygotic embryogenesis in Arabidopsis (Plotnikova et al. 2018, Hoffman et al. 2019, Kao et al. 2021).
In order to verify the role of candidate genes and sRNAs chosen by transcriptomic analyses and selected by their known role in the maintenance of meristematic stem cells different transgenic lines with modulated activity of relevant genes will be used. Moreover, the obtained data from cell-specific transcriptomes of somatic embryos during their development are planned to be compared with the existing transcriptomic data from zygotic embryo development (Hofmann et al. 2019, Kao et al. 2021). The results of the project will extend knowledge of the mechanism controlling somatic cell pluripotency contributing to the progress in many areas of biotechnology and medicine. The implementation of the FANS for studies on SE allows for pioneer analyses on specific cell fractions undergoing SE, which will significantly broaden the knowledge of the molecular basis of plant cell totipotency, including economically important species. Additionally, the planned in project comparison may be a key element to the ongoing discussion on the convergence of genetic mechanisms involved in zygotic and somatic embryogenesis in plants.