Phaeodactylum tricornutum oxidative stress
A study to investigate redox reactions in Phaeodactylum tricornutum diatom using OxiCAT method.Diatoms are ubiquitous marine photosynthetic eukaryotes that are responsible for about 20% of photosynthesis on Earth and therefore are an important component of the large biogeochemical cycles in the ocean. Very little is known about the molecular mechanisms that mediate cellular stress responses and are therefore responsible for their ecological success. Recent evidences suggest that diatoms may possess a surveillance system based on induction of reactive oxygen species (ROS) which have been implicated in response to various environmental stresses. Here we explored diatom mechanisms of perception of oxidative stress by combining in vivo imaging of organelle-specific redox response with quantification of the whole redox proteome in the model diatom Phaeodactylum tricornutum. Subcellular measurements of the glutathione redox potential using the redox-sensitive GFP (roGFP) sensor revealed distinct compartmentalization in the redox microenvironments. In vivo quantification of the whole thiol-proteome elucidated significant differential oxidation of around 300 redox-sensitive proteins (redoxome) under oxidative stress conditions. Functional assignment of this redoxome revealed involvement of a wide range of cellular functions that include signaling, transcription and translation machinery. We identified a high proportion of redox-sensitive enzymes that regulate key metabolic pathways in diatom biology such as photosynthesis, the urea cycle, photorespiration and lipid biosynthesis. The redoxome analysis revealed enrichment of chloroplast-targeted proteins and their high reactivity under reducing chloroplast microenvironment as determined by the roGFP sensor. Comparative analysis of the diatom redoxome across 48 genomes revealed evolutionary conserved cysteines responsive to oxidative stress across kingdoms. We propose that redox regulation may provide diatoms with important machinery for rapid and reversible responses to multiple environmental cues; therefore this mechanism would be essential for their ecological success in the marine ecosystem. Data Processing, Searching and Analysis: Raw data processing and database searching was performed using Proteinlynx Global Server (IdentityE) version 2.5.2. Database searching was carried out using the Ion Accounting algorithm. Data were searched against a combined target and reversed (decoy) database and the CRAP list of common laboratory contaminants. Trypsin was set as the protease, one missed cleavage was allowed. Each raw data file was searched 4 times with the following modification settings: heavy or light ICAT as fixed modifications; then with heavy or light ICAT as variable modifications. Feature Detection and alignment: Raw data were imported into Rosetta Elucidator System, version 3.3 (Rosetta Biosoftware, Seattle, WA, USA). Elucidator was used for alignment of raw MS1 data in RT and m/z dimensions. Aligned features were extracted and quantitative measurements obtained by integration of three-dimensional volumes (time, m/z, intensity) of each feature as detected in the MS1 scans. Detection of light and heavy pairs: The detection of light and heavy pairs from the MS1 channel in each raw data file was conducted using Rosetta Biosoftware’s Elucidator system, allowing 4 modified cysteines per peptide, with the heavy peak having a mass shift of 9.03 Da. Criteria for the unbiased detection of light and heavy pairs included ±0.2 minutes in the time domain and ±15ppm in the mass domain. Following detection of the pairs, the search results were imported for annotation and the minimum identification score was set to achieve a maximum global false discovery rate of 1%.
Sample Processing Protocol
See details in reference(s) : 24550302
Data Processing Protocol
See details in reference(s) : 24550302
Yishai Levin, Weizmann Institute of Science, Israel
Rosenwasser S, Graff van Creveld S, Schatz D, Malitsky S, Tzfadia O, Aharoni A, Levin Y, Gabashvili A, Feldmesser E, Vardi A. Mapping the diatom redox-sensitive proteome provides insight into response to nitrogen stress in the marine environment. Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2740-5 PubMed: 24550302