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Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-L-galactose phosphorylase.

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.

SUBMITTER: Urzica EI

PROVIDER: S-EPMC3340187 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-L-galactose phosphorylase.

Urzica Eugen I EI Adler Lital N LN Page M Dudley MD Linster Carole L CL Arbing Mark A MA Casero David D Pellegrini Matteo M Merchant Sabeeha S SS Clarke Steven G SG

The Journal of biological chemistry 20120305 17

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 reinhard ...[more]

PMID: 22393048

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Project description:Ascorbic acid (AsA) is a widespread antioxidant in living organisms, and plays essential roles in the growth and development of animals and plants as well as in the response to abiotic stress tolerance. The GDP-L-galactose phosphorylase (GGP) is a key regulatory gene in plant AsA biosynthesis that can regulate the concentration of AsA at the transcriptional and translational levels. The function and regulation mechanisms of GGP have been well understood; however, the molecular evolutionary patterns of the gene remain unclear. In this study, a total of 149 homologous sequences of GGP were sampled from 71 plant species covering the major groups of Viridiplantae, and the phylogenetic relationships, gene duplication and molecular evolution analyses of the genes were systematically investigated. Results showed that GGP genes are present throughout the plant kingdom and five shared whole-genome duplications and several lineage-specific whole-genome duplications were found, which led to the rapid expansion of GGPs in seed plants, especially in angiosperms. The structure of GGP genes was more conserved in land plants, but varied greatly in green algae, indicating that GGP may have undergone great differentiation in the early stages of plant evolution. Most GGP proteins had a conserved motif arrangement and composition, suggesting that plant GGPs have similar catalytic functions. Molecular evolutionary analyses showed that GGP genes were predominated by purifying selection, indicating that the gene is functionally conserved due to its vital importance in AsA biosynthesis. Most of the branches under positive selection identified by the branch-site model were mainly in the chlorophytes lineage, indicating episodic diversifying selection may contribute to the evolution of GGPs, especially in the chlorophyte lineage. The conserved function of GGP and its rapid expansion in angiosperms maybe one of the reasons for the increase of AsA content in angiosperms, enabling angiosperms to adapt to changing environments.

Project description:BACKGROUND:Ascorbate is a powerful antioxidant in plants and an essential micronutrient for humans. The GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting enzyme of the L-galactose pathway-the dominant ascorbate biosynthetic pathway in plants-and is a promising gene candidate for increasing ascorbate in crops. In addition to transcriptional regulation, GGP production is regulated at the translational level through an upstream open reading frame (uORF) in the long 5'-untranslated region (5'UTR). The GGP genes have yet to be identified in bread wheat (Triticum aestivum L.), one of the most important food grain sources for humans. RESULTS:Bread wheat chromosomal groups 4 and 5 were found to each contain three homoeologous TaGGP genes on the A, B, and D subgenomes (TaGGP2-A/B/D and TaGGP1-A/B/D, respectively) and a highly conserved uORF was present in the long 5'UTR of all six genes. Phylogenetic analyses demonstrated that the TaGGP genes separate into two distinct groups and identified a duplication event of the GGP gene in the ancestor of the Brachypodium/Triticeae lineage. A microsynteny analysis revealed that the TaGGP1 and TaGGP2 subchromosomal regions have no shared synteny suggesting that TaGGP2 may have been duplicated via a transposable element. The two groups of TaGGP genes have distinct expression patterns with the TaGGP1 homoeologs broadly expressed across different tissues and developmental stages and the TaGGP2 homoeologs highly expressed in anthers. Transient transformation of the TaGGP coding sequences in Nicotiana benthamiana leaf tissue increased ascorbate concentrations more than five-fold, confirming their functional role in ascorbate biosynthesis in planta. CONCLUSIONS:We have identified six TaGGP genes in the bread wheat genome, each with a highly conserved uORF. Phylogenetic and microsynteny analyses highlight that a transposable element may have been responsible for the duplication and specialized expression of GGP2 in anthers in the Brachypodium/Triticeae lineage. Transient transformation of the TaGGP coding sequences in N. benthamiana demonstrated their activity in planta. The six TaGGP genes and uORFs identified in this study provide a valuable genetic resource for increasing ascorbate concentrations in bread wheat.

Dataset Information

Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-L-galactose phosphorylase.

Publications

Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-L-galactose phosphorylase.

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