Abstract
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.
Highlights
Ascorbate biosynthesis in plants occurs mainly via the L-galactose pathway
With the exception of GDP-D-mannose pyrophosphorylase (VTC1), which appears to be missing in Prasinophyceae like Micromonas spp. or Ostreococcus spp., all other enzymes of the L-galactose pathway are conserved in divergent green algae
The absence of VTC1 in Prasinophyceae might be compensated by the operation, in those species, of VTC2 cycles such as those proposed by Laing et al [6] or Wolucka and Van Montagu [47], where L-galactose-1-P would be formed by a GDP-L-galactose transferase activity of VTC2 and GDP-D-mannose formation would be ensured by a hypothetical 2Ј-epimerase from GDP-D-glucose [8]
Summary
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. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are 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. Higher plants facing increased oxidative stress exhibit, in addition to increased VTC2 mRNA and activity levels, elevated transcript abundance for all the enzymes of the vitamin C recycling pathway (ascorbate-glutathione system) in the chloroplast including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GSHR) [2, 17]. This suggests that C. reinhardtii cells respond to oxidative stress by producing more L-ascorbic acid both via de novo synthesis through the L-galactose pathway and via increased recycling
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