Molecular and phenotypic characterisation of plant cation exchangers

  • James Connorton

    Student thesis: Phd


    The cation exchanger (CAX) proteins are an important six-membered family of Arabidopsis antiporters thought to provide metal tolerance and to be key components in resetting cytosolic Ca2+ levels following a signalling event. CAX1 and CAX3 in particular have been implicated in this role and are thought to functionally associate although the expression profiles of the two genes are distinct to shoot and root tissue respectively under most circumstances. Here the CAX1-CAX3 interaction is confirmed through in vivo bimolecular fluorescence complementation and shown to localise to the tonoplast. Furthermore the site of the interaction is partially mapped to an N-terminal region distinct from the previously-identified N-terminal regulatory region through studies of the molecular behaviour of split-protein constructs. The co-localisation of CAX1 and CAX3 is confirmed through fluorescent-protein tagged recombinant protein analysis and preliminary data suggesting an additional, nuclear location for CAX1 is presented. An 11-transmembrane domain model is given for the membrane topology of CAX1 based on combination of experimental analysis through partial proteolysis of tagged recombinant proteins and computer-based analysis. In addition the physiological significance of CAX1, CAX2 and CAX3 is examined through the analysis of single and double knockout mutant plants and a CAX1-overexpressor plant in response to metal stress. Previously unknown phenotypes with regard to metal sensitivities, germination profiles and stress-induced membrane permeability characteristics are identified and discussed. These studies are interpreted with a view to further establishing the relationship between the structure and regulation of the CAX proteins and their function.
    Date of Award1 Aug 2011
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorClifford Bray (Supervisor) & Jon Pittman (Supervisor)

    Cite this