Manipulating the subcellular localisation of cell cycle regulators to break cell size dependency in the Arabidopsis Root

Williams, Emma 2023. Manipulating the subcellular localisation of cell cycle regulators to break cell size dependency in the Arabidopsis Root. MPhil Thesis, Cardiff University. Item availability restricted.

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Abstract

Cell growth and cell division are fundamental processes that govern the size and specialised functions of individual cells and multi-cellular organisms. Regulation occurs at two transitions in the plant cell cycle: G1/S and G2/M. These checkpoints are controlled by proteins which coordinate a sequence of phosphorylation events, eliciting transcriptional changes required for DNA synthesis and mitosis. Cell size ontrol in Arabidopsis thaliana has been demonstrated at G1/S, where a molecular mechanism involving the dilution of a cell cycle inhibitor’s concentration facilities cell cycle progression and corrects asymmetric cell size divisions. This is termed “sizer” cell size control. However, the existence of a similar mechanism at G2/M is in plants is unproven. This study presents a strategy to experimentally test the relationship between protein concentration and cell cycle progression, with an aim to identify potential G2/M sizer proteins. To test the effect of changing the concentration of potential sizer proteins on cell size, an inducible dexamethasone (DEX) system was implemented. GLUCOCORTICOID RECEPTOR (GR) reporter proteins, fused to GFP in both the N and C terminal position were constructed determine the optimal expression cassette design. Six Genes of Interest (GOIs) with potential sizer roles at G2/M were successfully cloned into the system, one of which, (MYB3R3) was analysed in detail. Five homozygous inducible MYB3R3 lines were optimised for induction and examined for changes in root length and cortex cell size. Results revealed that p35s::MYB3R3-GFP-GR roots induced at 100 M DEX for 4 days had significantly larger cell sizes compared to their non-induced counterparts, as well as wildtype and reporter control plants. Further, after 15 days, reductions were seen in the biomass of roots and leaves. These results imply a role in cell size control for MYB3R3 where its concentration must dilute below a threshold for division. Overall, this approach provides a system for identifying GOIs with sizer functions, with future applications in elucidating effects on cell cycle progression and gene expression correlated to cell size.

Item Type:	Thesis (MPhil)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	31 May 2024
Last Modified:	31 May 2024 10:31
URI:	https://orca.cardiff.ac.uk/id/eprint/169349

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