Understanding and engineering cis-regulatory functions in Arabidopsis thaliana

Witham, Samuel (2023) Understanding and engineering cis-regulatory functions in Arabidopsis thaliana. Doctoral thesis, University of East Anglia.

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Nitrate (N) is essential for plant growth and metabolic processes. The use of N-containing fertilisers improved crop yields over several decades but had negative environmental impacts. In response to N-availability, plants alter the expression of thousands of genes, analysis of which enabled the identification of gene regulatory networks (GRNs) containing putative interactions between transcription factors (TFs) and their targets. GRNs contain network motifs that likely affect network dynamics and are important for plant N-responses. Rational engineering of GRNs has the potential to improve plant traits but requires a detailed understanding of network components and their interactions. In this thesis, I characterised interactions within an N-response subnetwork using in vitro protein-DNA relative affinity and in planta gene expression assays (Chapter 3). I investigated the perturbation of this subnetwork using synthetic biology approaches using a CRISPR library to disrupt edges in the promoters/5′ UTRs of four TFs (Chapter 4). Five mutant lines were identified which had altered root growth and gene expression in response to N. A second method of perturbation is the introduction of synthetic genetic feedback controllers built from synthetic elements which are predicted to change network dynamics. To inform the design of synthetic promoters that respond to specific TFs in the N-subnetwork I investigated promoter architectural differences between four groups of genes with different expression patterns (Chapter 5). I successfully built and tested synthetic N-responsive promoters that responded to nitrate and specific TFs in the subnetwork, and synthetic TFs which activated or repressed ARF18 expression. These synthetic elements were co-assembled into synthetic feedback controllers and were transformed into plants (Chapter 6). Together, the work in this thesis demonstrates that the engineering of root growth in response to nitrate can be facilitated by an improved understanding of an N-response subnetwork, providing a path towards the engineering of improved nitrogen-use efficiency.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 06 Dec 2023 09:29
Last Modified: 06 Dec 2023 09:29
URI: https://ueaeprints.uea.ac.uk/id/eprint/93881

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