Pyruvate, orthophosphate dikinase and phospho<i>enol</i>pyruvate carboxykinase both catalyse the synthesis of phospho<i>enol</i>pyruvate. Single and double <i>Arabidopsis PPDK </i>and <i>PCK1 </i>T-DNA insertional knock-out mutants were used to investigate whole plant enzyme pools. Vein specific <i>Arabidopsis</i> knock-down lines were created by using a combined approach of RNAi and enhancer trapping to reduce activity of the two enzymes in a cell-specific manner. Developmental analyses of the two mutant classes indicated an important role for both enzymes during mature aerial tissue development and early seedling growth. Metabolic profiling of mature leaves of vein specific knock-down lines suggested that veinal pools of the two enzymes may be involved in amino acid interconversions in these tissues. Microarray analysis of etiolated knock-out seedlings implied that PPDK may act to supplement the gluconeogenic role of PEPCK during seedling establishment under limiting conditions. PPDK is regulated by a bifunctional regulatory protein which catalyses inactivation of PPDK via phosphorylation, and its activation via dephosyphorylation. Both activities proceed via atypical mechanisms, but despite these unusual properties, little is known about the structure of RP. The location of the unidentified kinase and dephosphorylase domains of <i>Arabidopsis </i>RP, and the mechanism of interaction with PPDK, were investigated using mutagenised AtRP1 and chimaeric AtRP1-AtRP2 variants. Results did not support the proposal that separate sites are involved in RP kinase and dephosphorylase activities, or the current view that a putative P-loop is likely to be only critical for kinase activity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:596198 |
| Date | January 2010 |
| Creators | Astley, H. M. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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