Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq analysis of the transcriptome from Sulfur Deprivation Chlamydomonas cells

ABSTRACT: The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells; the mutant is null for the regulatory serine-threonine kinase SNRK2.1, which is required for acclimation to sulfur deprivation. The transcriptome analyses involved microarray hybridization and RNA-seq technology; RT-qPCR evaluation of the data obtained by these techniques showed that RNA-seq is significantly more quantitative than microarray hybridizations. Sulfur-deprivation-responsive transcripts included those encoding proteins involved in sulfur acquisition and assimilation, recycling of sulfur-containing amino acids, synthesis of reduced sulfur metabolites and cofactors, and modification of cellular structures such as the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that cells deprived of sulfur favors accumulation of proteins with fewer sulfur-containing amino acids. Most of these sulfur-deprivation responses are controlled by the SNRK2.1 kinase. Furthermore, the snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfur-depleted conditions that are not observed in wild-type cells. Many of these responses are likely to be elicited by singlet O2 accumulation in the mutant cells. The transcriptome results for the wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as the cells become depleted for sulfur, and reveal aspects of acclimation that are likely critical for cell survival. The three supplementary files GSE17970_supplemental_table_*.xls below include results of the differential expression analysis (expression estimates, fold changes and p-values), and different clusters of functionally related genes. Chlamydomonas strains used for this study were D66 (wt strain; nit2 cw15 mt+), ars11 (snrk2.1cw15mt+), 21gr (wt strain; nit5 mt-) and sac1 (sac1mt+). The ars11 strain was designated as the snrk2.1 mutant throughout since the lesion is in the SNRK2.1 gene. Cells were cultured under continuous light of ~60 M-NM-

ORGANISM(S): Chlamydomonas reinhardtii

SUBMITTER: Arthur Grossman

PROVIDER: E-GEOD-17970 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

RNA-seq analysis of sulfur-deprived Chlamydomonas cells reveals aspects of acclimation critical for cell survival.

González-Ballester David D Casero David D Cokus Shawn S Pellegrini Matteo M Merchant Sabeeha S SS Grossman Arthur R AR

The Plant cell 20100629 6

The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells. This mutant is null for the regulatory Ser-Thr kinase SNRK2.1, which is required for acclimation of the alga to sulfur deprivation. The transcriptome analyses used microarray hybridization and RNA-seq technology. Quantitative RT-PCR evaluation of the results obtained by these techniques showed that RNA-seq reports a larger dynamic range of expression levels ...[more]

PMID: 20587772

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Project description:Interest in exploiting algae as a biofuel source and the role of nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can be easily manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements on the ionomes of cells would result in optimized cell growth. We characterized the ionomes of multiple wild-type Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel our measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between laboratories using the same supplement. Visual observation did not reveal defects in cell motility or mating efficiency. Ni2+-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased amount of Ni2+ supplementation due to the introduction of an EDTA buffer system in the revised medium. Equilibrium modeling of the revised supplement predicts less metal precipitation in the revised medium. Other advantages include more facile preparation of trace element stock solutions that can readily be adapted for deficiency studies, a reduction in total chemical use, a more consistent batch-to-batch formulation, and long-term stability (up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient-deficiencies. In N and S deficiency, cells accumulate TAG as well in the new medium as previously demonstrated. Fe and Zn deficiency also induced TAG accumulation as suggested by Nile Red and Bodipy staining. This ionomic approach can be used to efficiently optimize culturing conditions for other algal species to improve growth and assay cell physiology. Sampling of Chlamydomonas CC-1021 (2137) cultivated in TAP medium supplemented with a revised trace element recipe based on ionomic data.

Project description:Zinc is an essential nutrient because of its role in catalysis and in stabilizing protein structure, but excess zinc can also be deleterious. Four nutritional zinc states have been identified in the alga Chlamydomonas reinhardtii: zinc toxic, zinc replete, zinc deficient and zinc limited. Growth is inhibited in zinc-limited and zinc toxic cells relative to zinc-replete cells, while zinc-deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition responsive changes in gene expression. We identified a subset of genes encoding zinc-handling components, including ZIP family transporters and candidate zinc chaperones. In addition, we noted an impact on two other regulatory pathways, the carbon concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc-deficiency, as a likely consequence of reduced carbonic anhydrase activity, which is validated by mass spectrometry and immunoblot analysis of Cah1, Cah3 and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in air in zinc limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. Surprisingly, we noted also that the Crr1 regulon, which responds to Cu limitation, is also turned on in zinc deficiency, and in fact, Crr1 is required for growth in zinc-limiting conditions. Zinc deficient cells are functionally copper deficient, as evidenced by reduced plastocyanin abundance, even though they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester Cu in a bio-unavailable form, perhaps to prevent mis-metallation of critical zinc sites. Zn-limited wild-type cells were generated by transfer of cells from the first round of growth in medium with no supplemental Zn into TAP medium supplemented or not with 2.5 ÂµM Zn-EDTA The control samples for this study are represented in GSE25622

Dataset Information

RNA-seq analysis of the transcriptome from Sulfur Deprivation Chlamydomonas cells

Publications

RNA-seq analysis of sulfur-deprived Chlamydomonas cells reveals aspects of acclimation critical for cell survival.

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