Systems Biology Project

Title The control of ascorbate biosynthesis: the role of light & GDP-L-Galactose Phosphorylase

Principal Investigator Professor Nick Smirnoff

Summary The ascorbate concentration in Arabidopsis leaves is very finely controlled in relation to light and photosynthetic activity. The following approaches will be used to determine how ascorbate biosynthesis and turnover are controlled in relation to light. The research programme falls into two parts. The first is a detailed analysis of ascorbate synthesis via the GDP-mannose pathway to determine which steps are affected by light. Secondly, because strong a priori evidence implicates GDP-L-galactose phosphorylase (encoded by VTC2 and VTC5), as an important control point, the control of the activity and expression in relation to light will be investigated. 1. The response of the ascorbate biosynthesis pathway to light will be measured by assay of intermediates, enzyme activity/kinetics properties and gene expression. LC-ESI-MS/MS will be used for new sensitive metabolite and enzyme assays. Steady state fluxes measured by 14C-labelling. 2. The properties of VTC2 and VTC5 in relation to control of ascorbate synthesis will be investigated by the following approaches: Dynamics and location of expression with promoter::luc constructs (including truncated promoters); identification of interacting partners by yeast 2 hybrid and TAP-tagging; subcellular localisation by immunolocalisation of myc-tagged protein and phenotypic characterisation of T-DNA/RNAi mutants in genes of interacting proteins.

Funding body BBSRC

Timescale 2009 to 2012