The control of ascorbate biosynthesis: the role of light and GDP-L-galactose phosphorylase

Ascorbic acid (vitamin C) is essential for plant growth and is synthesised by plant leaves in large amounts. It is important as an antioxidant (antioxidants are compounds that protect against the damaging effects of reactive oxygen species that are produced from oxygen by all living organisms) and is also needed as an enzyme cofactor for production of collagen (in animals) and hormones (in animals and plants). Understanding the factors that control how much vitamin C accumulates in leaves is important for two reasons. Firstly, it provides vitamin C in the human diet. Secondly, plants with low vitamin C are susceptible to damage caused by various environmental stresses. It has been found that leaf vitamin C content is very closely related to light intensity, most probably because it is needed for protection against potentially damaging reactive oxygen species that are inevitably formed during photosynthesis. This project will determine the biochemical mechanisms that control the concentration of vitamin C in leaves. Part of the research will focus on identifying the contribution of each step in the pathway of vitamin C synthesis to increasing vitamin C in leaves exposed to high light. The other part will be a detailed investigation of two genes (VTC2 and VTC5) which both encode an enzyme involved in vitamin C synthesis (GDP-L-galactose phosphorylase). While the control of the rate of synthesis of products such as vitamin C is generally shared between a number of steps in its synthresis pathway, the evidence strongly implicates VTC2 and perhaps VTC5 as major control points in the pathway. In this project we aim to determine how the activity of VTC2 and VTC5 are controlled in relation to light and photosynthesis and how this contributes, along with other steps in the pathway, to the very precise control of vitamin C concentration in leaves.

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.