Project 1 - The roles of multiple isoforms of pyrophosphate:fructose 6-phosphate phosphotransferase in Arabidopsis
The aim of this work is to examine the roles of multiple isoforms of pyrophosphate:fructose 6-phosphate phosphotransferase (PFP) in carbohydrate metabolism in arabidopsis plants. PFP catalyses the reversible interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate using PPi as the phosphoryl donor. The enzyme is ubiquitous in higher plants, where it is confined to the cytosol, and is extremely sensitive to activation by the signal metabolite fructose 2,6-bisphosphate. It is a heterotetramer composed of equal quantities of two similar polypeptides, PFPa and PFPb. The arabidopsis genome contains four closely related genes thought to encode two isoforms of each subunit. The role of the different subunits in catalysis and activation remains speculative, and the significance of multiple forms of each is unknown.
This project will test the proposal that different isoforms of PFP have unique metabolic roles and will establish the physiological function of the exchange between hexoseP and trioseP pools catalysed by PFP. The location of the isoforms of the two polypeptides in different organs and cell types of the plant will be studied by RNA blots and by expression of GUS-promoter fusions in transgenic plants. Particular emphasis will be placed on establishing whether expression of both PFPa and PFPb is required to obtain activity, and determining the extent to which specific combinations of PFPa and PFPb isoforms are co-expressed in different tissues. We will characterise the metabolic phenotypes of arabidopsis knockout mutants for each of the four PFP isoforms. Analysis of the GARLIC database suggests that appropriate knockout lines exist for each of the four genes in the Syngenta T-DNA collection, although if necessary other tagged collections are available for screening. In addition to monitoring for gross morphological and developmental effects, each mutant will be assessed for changes in primary carbohydrate metabolism in tissues in which expression of PFP isoforms has been affected. Standard metabolic characterisation will be based on analysis of the metabolism of radiolabelled substrates and enzymic/HPLC measurement of metabolic intermediates. The extent of hexoseP/trioseP exchange, known to be catalysed by PFP, will be determined by NMR analysis following metabolism of [1-13C]glucose. This will establish the functions of PFP and indicate whether each isoform has a distinct role in carbohydrate metabolism. Subsequently, if appropriate, knockout mutants will be crossed to generate lines expressing only defined PFPa/PFPb isozyme pairs. PFP will be purified from each of the four possible genotypes and used to study the influence of isozyme composition on the kinetic properties of the enzyme. These analyses will indicate whether distinct kinetic properties are likely to be important in determining the roles of specific isoforms. Additionally, the consequences of changes in PFP composition on cellular gene expression will be assessed by DNA microarray analysis using cDNA synthesised from each of the mutant lines. By identifying the range of genes with altered expression in these lines, this analysis will define the functional consequences of perturbing hexoseP/trioseP exchange catalysed by PFP and the broader effects of changes in PFP isoform composition.
Recent relevant publications
Fernie, A.R., Roscher, A., Ratcliffe, R.G. and Kruger, N.J. (2001) Fructose 2,6-bisphosphate activates pyrophosphate:fructose 6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells. Planta 212, 250-263.
Theodorou, M.E. and Kruger, N.J. (2001) Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate: fructose 6-phosphate 1-phosphotransferase. Planta 213, 147-157. Sweetlove, L.J., Kruger, N.J. and Hill, S.A. (2001) Starch synthesis in transgenic potato tubers with increased 3-phosphoglyceric acid content as a consequence of increased 6-phosphofructokinase activity. Planta 213, 478-482.
Barratt, D.H.P., Barber, L., Kruger, N.J., Smith, A.M., Wang, T.L. and Martin, C. (2001) Multiple, distinct isoforms of sucrose synthase in pea. Plant Physiology 127, 655-664.
Fernie, A.R., Roscher, A., Ratcliffe, R.G. and Kruger N.J. (2002) Activation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase by increased fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells. Physiologia Plantarum (in press).
Funding status: Applied for BBSRC Special studentship. Eligible for BBSRC/NERC Quota studentship
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