Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-l-galactose Phosphorylase

The l-galactose (Smirnoff-Wheeler) pathway represents the major route to l-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-l-galactose phosphorylases converting GDP-l-galactose to l-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of l-ascorbate. Here we report that the l-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the l-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-l-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and l-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the l-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells. Background: Ascorbate biosynthesis in plants occurs mainly via the l-galactose pathway. Results:Chlamydomonas reinhardtii VTC2 encodes a GDP-l-galactose phosphorylase whose transcript levels are induced in response to oxidative stress concurrent with increased ascorbate accumulation. Conclusion: Increased oxidative stress in C. reinhardtii results in an enzymatic and non-enzymatic antioxidant response. Significance: First characterization of C. reinhardtii ascorbate biosynthesis and recycling pathways.