|M.Sc Student||Kristina Vragovic|
|Subject||Precise Mapping of Growth Hormone Effect by Cell-Specific|
Gene Activation Response
|Department||Department of Biology||Supervisor||Professor Savaldi-Goldste Sigal|
|Full Thesis text|
The activity of plant hormones in specific cells drives growth of entire organs via a yet unresolved mechanism. It was shown that the BR receptor, BRI1, active specifically in the epidermis of Arabidopsis primary root is sufficient to control root meristem size and this by providing a non-autonomous signal to the inner root tissues. However, the nature of the signal remains unknown. One approach towards dissecting this tissue-to-tissue communication is to delineate the direct (epidermal) and systemically regulated (inner cells) target genes in response to epidermal BRI1 activity. Hence, we have established a reproducible system for analysis of cell-type specific gene expression based on immunopurification of polysome-associated mRNA from specific root cells followed by RNA-seq.
In order to isolate polysomal mRNA populations from specific root cells we produced a set of transgenic lines expressing the FLAG-epitope tagged ribosomal protein L18 (HF-RPL18) under control of different cell-type specific promoters. We immunopurified the epitope-tagged polysomes from cells with anti-FLAG M2 affinity gel and isolated the associated mRNA with conventional methods using organic solvents. Enrichment of tissue-specific gene markers in polysomal mRNA pools isolated from cells expressing the HF-RPL18 confirmed its cell-type specific expression in transgenic lines and therefore allowed us to analyze translating mRNA populations at cellular resolution. We further showed that immunopurification of polysomal mRNA enables detection of fast BR-responsive genes and provided evidence for utilisation of HF-RPL18 transgenic lines for high resolution profiling of early cellular responses to BRs. Finally, by generating combinatorial genetic crosses with targeted expression of HF-RPL18 and BRI1 in different cell types we identified both non-autonomous and autonomous cell-specific responses to epidermal BRI1 activity. Our data thus demonstrate how cell-specific responses to BRs, otherwise masked at whole-organ level, can be uncovered with use of the established system. Importantly, this research reinforces the system’s potential in elucidating the spatiotemporal mechanisms of BR-mediated control of root growth in combination with high-throughput translatome profiling technology.