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Title: Structural and functional studies of the plant boron transporter, AtBOR1
Author: Scull, Nicola
ISNI:       0000 0004 7658 9608
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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Boron is an essential nutrient for plant growth however excess levels of boron are toxic. Boron toxicity and deficiency negatively affect many different crop species, highlighting the importance of boron homeostasis in plants. In Arabidopsis thaliana the boron transporter, AtBOR1, is required for normal plant growth at low boron concentrations. AtBOR1 is expressed in the root endodermis and pericycle and actively exports boron from root cells into the xylem. Although a low resolution structure exists for AtBOR1, many questions still remain about the exact substrates of AtBOR1, the location of substrate binding and the mechanism by which boron is transported. A greater knowledge of the structure and function of AtBOR1 could aid the design of genetically engineered crop species resulting in plants with a greater tolerance for extreme boron conditions. The aim of this study was to obtain well diffracting crystals of AtBOR1 as a basis for further structural studies. AtBOR1 was expressed in Saccharomyces cerevisiae and demonstrated boron efflux activity in a yeast BOR1p knockout strain. The protein was successfully purified in mg quantities and shown to be a dimer, the likely physiological state, in detergent based solution. The protein was successfully crystallised and optimisation of the crystallisation conditions produced a crystal that maximally diffracts to 4.2 Å. Based on available structures of AtBOR1 and other related proteins, a range of AtBOR1 mutants were assessed for their affects on stability and function. Additional analysis of two single point mutations that increase plant sensitivity to boron deficiency indicate that these are likely to prevent correct expression and membrane trafficking of AtBOR1. Stability analysis of AtBOR1 in a range of established and novel detergents, identified several detergents that increase AtBOR1 stability compared to DDM.
Supervisor: Byrne, Bernadette Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral