Tracing the turnover of vitamin C in harvested salad tissues

BACKGROUND Vitamin C (= L-ascorbate + dehydro-L-ascorbate) is widely recognised as a major benefit of eating salads and fruits. Ascorbate is relatively unstable in the plant and may therefore be lost post-harvest. Its breakdown products include oxalic and tartaric acids: astringent and potentially harmful components of some edible plants e.g. rhubarb and spinach. Increasing the vitamin C content of edible plants such as salads could in principle involve promoting ascorbate biosynthesis or inhibiting its degradation. Much is now known about the biosynthesis, but the degradative pathway(s) remain unclear. Elucidation of the breakdown pathways could promote an understanding not only of how to maintain the vitamin C content of salads post-harvest, but also of the roles of ascorbate in plants, e.g. to explain the intriguing positive correlation between growth rate and ascorbate oxidase activity. Some of the metabolic steps between ascorbate and oxalate were recently elucidated [Green & Fry (2005) Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate. Nature 433:83]. We showed that this pathway operates in the apoplast, proceeds via at least 4 novel intermediates and involves at least 3 new enzyme activities. Two by-products ('C' and 'E') remain unidentified. More recently we have shown that plant cells respond to oxidative stresses by specifically exporting ascorbate into the cell wall, thus potentially initiating a cascade of downstream metabolic steps [Parsons & Fry (2010) ROS-induced release of intracellular ascorbate in plant cell-suspension cultures and evidence for pulsing of net release rate. New Phytol 187:332]. In the plant, ascorbate has wide-ranging roles: protecting against ozone and UV damage; in cell signalling; generating H2O2; controlling cell growth; controlling lignification; and as a source of new metabolites which may themselves have botanical and nutritional significance. We are now in an excellent position to extend our studies by identifying some of the remaining unknown metabolites, characterising their biological role, and charting alternative routes of ascorbate catabolism. STUDENT'S WORK PROGRAMME * Characterising the unidentified ascorbate metabolite E and its lactone C. * Detecting and characterising ascorbate metabolites in living salad plants, especially oxalyl-esters and diesters. * Exploring possible trans-oxalylation reactions in the leaf cell wall, which may generate polysaccharide-oxalate bonds. * Determining the breakdown products formed when ascorbate protects leaves against ozone and UV damage. * Investigating the further metabolic and cellular fates of threonate and oxalate in salad plants. * Determining the factors that control the ratio of oxidative : non-oxidative degradation of dehydroascorbate. METHODS Salad plants will be grown and packed under commercially realistic conditions at Vitacress. Extracts will be analyses by HPLC (Rezex column) and high-voltage electrophoresis at Edinburgh. Pathways will be traced by in-vivo radiolabelling. [14C]Oxalate and [14C]ascorbate are available commercially. [14C]Dehydroascorbate will be prepared from [14C]ascorbate by the student. L-[2,3-3H]Threonate, L-[2,3-3H]tartaric acid and L-[6-3H]galactonolactone have been synthesised by Steve Fry in preliminary work leading to this proposal. FUTURE DIRECTION OF THE WORK The project is designed as ground-breaking work on the discovery and fate of vitamin C metabolites in salads - a surprisingly little-explored field. When the metabolites have been characterised and placed in the correct metabolic sequence, we would be in an excellent position to begin a new line of enquiry: characterising new enzymes involved (oxalylesterases, dehydroascorbate oxidase and oxalyltransferases) and their genes.