Dataset Information

Identification of Reference and Biomarker Proteins in Chlamydomonas reinhardtii Cultured under Different Stress Conditions.

ABSTRACT: Reference proteins and biomarkers are important for the quantitative evaluation of protein abundance. Chlamydomonasreinhardtii was grown under five stress conditions (dark, cold, heat, salt, and glucose supplementation), and the OD750 and total protein contents were evaluated on days 0, 1, 2, 4, and 6 of culture. Antibodies for 20 candidate proteins were generated, and the protein expression patterns were examined by western blotting. Reference protein(s) for each treatment were identified by calculating the Pearson's correlation coefficient (PCC) between target protein abundance and total protein content. Histone H3, beta tubulin 1 (TUB-1), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL), and mitochondrial F1F0 ATP synthase subunit 6 (ATPs-6) were the top reference proteins, because they were expressed stably under multiple stress conditions. The average relative-fold change (ARF) value of each protein was calculated to identify biomarkers. Heat shock protein 90B (HSP90B), flagellar associated protein (FAP127) and ATP synthase CF0 A subunit (ATPs-A) were suitable biomarkers for multiple treatments, while receptor of activated protein kinase C1 (RCK1), biotin carboxylase (BCR1), mitochondrial phosphate carrier protein (MPC1), and rubisco large subunit N-methyltransferase (RMT1) were suitable biomarkers for the dark, cold, heat, and glucose treatments, respectively.

SUBMITTER: Shi J

PROVIDER: S-EPMC5578208 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Identification of Reference and Biomarker Proteins in Chlamydomonas reinhardtii Cultured under Different Stress Conditions.

Shi Jianan J Huang Teng T Chai Shuaijie S Guo Yalu Y Wei Jian J Dou Shijuan S Li Liyun L Liu Guozhen G

International journal of molecular sciences 20170822 8

Reference proteins and biomarkers are important for the quantitative evaluation of protein abundance. Chlamydomonasreinhardtii was grown under five stress conditions (dark, cold, heat, salt, and glucose supplementation), and the OD750 and total protein contents were evaluated on days 0, 1, 2, 4, and 6 of culture. Antibodies for 20 candidate proteins were generated, and the protein expression patterns were examined by western blotting. Reference protein(s) for each treatm ...[more]

PMID: 28829403

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Project description:Acclimation to singlet oxygen was shown to induce various oxidative stress response genes of which some were also strongly overexpressed in the singlet oxygen resistant mutant sor1. Because sor1 was also more tolerant to other oxidative and electrophilic stress conditions, and because many of the sor1 overexpressed genes are known to be involved in the detoxification of reactive electrophile species, the response of the C. reinhardtii wild-type strain to various oxidative and electrophilic stress conditions was determined. Therefore, cultures were exposed to the reactive oxygen species-producing photosensitizer neutral red, the organic hydroperoxide tert-butylhydroperoxide, the photosynthetic herbicide 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the lipophilic electrophile 2(E)-Hexenal for two hours and the global genetic response was analyzed. Cluster analysis revealed the most similar expression pattern between DBMIB and 2(E)-Hexenal and to a lower degree between NR and tBOOH. Still, there were many common induced genes including several of the oxidative stress response and detoxification genes overexpressed in the sor1 mutant. The 4A+ wild-type strain was grown mixotrophically in a Tris-acetate phosphate to a density of 2x10^6 cells/ml. Then cultures were split into 20 ml subcultures, and exposed to either of the four chemicals DBMIB (2 µM), 2(E)-Hexenal (0.3 mM), neutral red (NR, 3 µM), tert-butylhydroperoxide (tBOOH, 100 µM) or no chemical (control) for 2 hours, in three independent biological replicates. The cells of each replicate were harvested by centrifugation and total RNA was isolated using the RNeasy Mini Kit (Qiagen). DNA microarrays were performed using the ‘One-Color Microarray-Based Gene Expression Analysis’ system and a custom made 4 × 44 K ‘Chlamydomonas Whole Genome DNA Microarrays’ (Agilent Technologies) containing 15143 specific probes designed based on the Chlamydomonas version 4.0 transcript models provided by the DOE Joint Genome Institute (JGI), with an average of three replicates for each probe

Project description:Chlamydomonas reinhardtii is one of the most important model organisms nowadays phylogenetically situated between higher plants and animals (Merchant et al. 2007). Stress adaptation of this unicellular model algae is in the focus because of its relevance to biomass and biofuel production. Here, we have studied cold stress adaptation of C. reinhardtii hitherto not described for this algae whereas intensively studied in higher plants. Toward this goal, high throughput mass spectrometry was employed to integrate proteome, metabolome, physiological and cell-morphological changes during a time-course from 0 to 120 h. These data were complemented with RT-qPCR for target genes involved in central metabolism, signaling, and lipid biosynthesis. Using this approach dynamics in central metabolism were linked to cold-stress dependent sugar and autophagy pathways as well as novel genes in C. reinhardtii such as CKIN1, CKIN2 and a hitherto functionally not annotated protein named CKIN3. Cold stress affected extensively the physiology and the organization of the cell. Gluconeogenesis and starch biosynthesis pathways are activated leading to a pronounced starch and sugar accumulation. Quantitative lipid profiles indicate a sharp decrease in the lipophilic fraction and an increase in polyunsaturated fatty acids suggesting this as a mechanism of maintaining membrane fluidity. The proteome is completely remodeled during cold stress: specific candidates of the ribosome and the spliceosome indicate altered biosynthesis and degradation of proteins important for adaptation to low temperatures. Specific proteasome degradation may be mediated by the observed cold-specific changes in the ubiquitinylation system. Sparse partial least squares regression analysis was applied for protein correlation network analysis using proteins as predictors and Fv/Fm, FW, total lipids, and starch as responses. We applied also Granger causality analysis and revealed correlations between proteins and metabolites otherwise not detectable. Twenty percent of the proteins responsive to cold are uncharacterized proteins. This presents a considerable resource for new discoveries in cold stress biology in alga and plants.

Dataset Information

Identification of Reference and Biomarker Proteins in Chlamydomonas reinhardtii Cultured under Different Stress Conditions.

Publications

Identification of Reference and Biomarker Proteins in Chlamydomonas reinhardtii Cultured under Different Stress Conditions.

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