Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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 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 Smirnoff-Wheeler 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. We have also shown that enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, Mn superoxide dismutase, dehydroascorbate reductase) are up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a key enzyme in ascorbate biosynthesis in green algae and together with components of the ascorbate recycling system represents the major route in providing protective levels of ascorbate in oxidatively stressed algal cells. Our results suggest that C. reinhardtii cells exposed to oxidative stress conditions produce more ascorbate both by de novo synthesis (Smirnoff-Wheeler pathway) and by recycling via the ascorbate-glutathione cycle. Sampling of Chlamydomonas 2137 exposed to hydrogen peroxide

ORGANISM(S): Chlamydomonas reinhardtii

SUBMITTER: Sabeeha Merchant

PROVIDER: E-GEOD-34826 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

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:The absence of oxygen (O2) is a stress condition for aerobic organisms and requires extensive acclimation responses. Previously, Chlamydomonas reinhardtii has been used as a reference organism for understanding these acclimation responses. In this work, we use RNA-Seq for a whole genome view of the acclimation of the organism to dark-anoxic conditions. To distinguish the responses dependent on the COPPER RESPONSE REGULATOR 1 (CRR1), which is also involved in hypoxic gene regulation, we compared the transcriptome of crr1 mutants to that of complemented strains. Nearly 10% of the genome (~ 1,400 genes) are affected by hypoxia based on pairwise comparisons of all strains and two time-points. Comparing transcript profiles from early (hypoxic) with those from late (anoxic) time-points indicated that the cells activated oxidative energy generation pathways before employing fermentative enzymes. Probable substrates included not only carbohydrates but also amino acids and fatty acids (FAs). Lipid profiling of the C. reinhardtii cells revealed that they degraded FAs but also accumulated triacylglycerols (TAGs). In contrast to N-deprived cells, the TAGs accumulating in hypoxic cells are enriched in desaturated FAs, which distinguishes the contribution of individual pathways for Chlamydomonas TAG accumulation. In crr1 mutants, about 140 genes were aberrantly regulated , re-affirming the importance of CRR1 for the hypoxic response, but indicating also the contribution of additional O2-sensors and signaling strategies to account for the remaining differentially regulated transcripts. We conclude that nitric oxide (NO) dependent signaling cascades, employing both known and novel components, are operative in C. reinhardtii. The transcriptome of four different Chlamydomonas strains (wild type CC-124, crr1 mutant, crr1:CRR1 rescued strain and crr1dCys rescued strain) are profiled by RNA-Seq in the dark at different times after the transition from light-oxic to dark-anoxic conditions

Project description:When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen-starved in acetate and then "boosted" after two days with additional acetate, the cells become "obese" after eight days, with triacylglyceride-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, sta6 and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the two days prior and two days post boost, and the data were compared with published reports using other strains and growth conditions. During the two hours post boost, ~425 genes are up-regulated M-bM-^IM-%2-fold and ~875 genes are down-regulated M-bM-^IM-%2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes in the glyoxylate and Calvin Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boost. Four genes--encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)--are selectively up-regulated in sta6. Although the bulk rate of acetate depletion from the medium is not boost-enhanced, three candidate acetate permease-encoding genes in the GPR1_FUN34_YaaH superfamily are boost-up-regulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is down-regulated by boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in sta6. Here we report studies on gene-expression patterns during the path to obesity. The Merchant/Pellegrini and Los Alamos laboratories recently generated and analyzed RNA-Seq transcriptomes of cw15, sta6, and several complemented sta6 strains during two days of N-starvation (0M-bM-^FM-^R48 h). In collaboration with these groups, the Goodenough lab generated a second pair of transcriptomes using cw15 and sta6, tracing 0M-bM-^FM-^R48 h gene expression patterns under a different set of culture conditions and taking the time course out to 96 h, with an intervening acetate boost. Analysis of these data was deeply informed by cross-comparisons with the Blaby et al. data. Three additional RNA-Seq studies of wild-type strains were also considered.

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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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