Rationales and Approaches for Studying Metabolism in Eukaryotic Microalgae

The generation of efficient production strains is essential for the use of eukaryotic microalgae for biofuel production. Systems biology approaches including metabolite profiling on promising microalgal strains, will provide a better understanding of their metabolic networks, which is crucial for metabolic engineering efforts. Chlamydomonas reinhardtii represents a suited model system for this purpose. We give an overview to genetically amenable microalgal strains with the potential for biofuel production and provide a critical review of currently used protocols for metabolite profiling on Chlamydomonas. We provide our own experimental data to underpin the validity of the conclusions drawn

Methods for analysis of protein glutathionylation and their application to photosynthetic organisms

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed

System-wide detection of protein-small molecule complexes suggests extensive metabolite regulation in plants

Protein small molecule interactions are at the core of cell regulation controlling metabolism and development. We reasoned that due to the lack of system wide approaches only a minority of those regulatory molecules are known. In order to see whether or not this assumption is true we developed an effective approach for the identification of small molecules having potential regulatory role that obviates the need of protein or small molecule baits. At the core of this approach is a simple biochemical co-fractionation taking advantage of size differences between proteins and small molecules. Metabolomics based analysis of small molecules co-fractionating with proteins identified a multitude of small molecules in Arabidopsis suggesting the existence of numerous, small molecules/metabolites bound to proteins representing potential regulatory molecules. The approach presented here uses Arabidopsis cell cultures, but is generic and hence applicable to all biological systems

Chloroplast DnaJ-like proteins 3 and 4 (CDJ3/4) from Chlamydomonas reinhardtii contain redox-active Fe-S clusters and interact with stromal HSP70B

International audienceIn this study we report on the identification and characterization of three novel chloroplast-targeted J-domain proteins, CDJ3-5, which in addition to their J-domains contain bacterial-type ferredoxin domains. In the databases we could identify homologs of CDJ3-5 in green algae, moss and higher plants, but not in cyanobacteria. Phylogenetic analyses allowed distinguishing two clades containing CDJ3/4 and CDJ5 that must have diverged early in an ancestor of the green lineage and have further diversified later on. Molecular and biochemical analysis of CDJ3 and CDJ4 from Chlamydomonas reinhardtii revealed that both are weakly expressed proteins that appear to be localized to the stroma and to thylakoid membranes, respectively. The low transcript levels of the CDJ3 and CDJ4 genes declined even further in the initial phase of heat shock, but CDJ3 transcript levels strongly increased after dark-to-light shift. Accordingly, the Arabidopsis ortholog of CDJ5 was also found to be light inducible and to be under strong circadian control. CDJ3 and CDJ4 proteins could both be expressed in Escherichia coli with redox-active Fe-S clusters. In vitro crosslinking studies demonstrated that CDJ3 and CDJ4 interact with chloroplast HSP70B in the ATP state, presumably as dimers, and immunoprecipitation studies showed that CDJ3/4 were in common complexes with HSP70B also in Chlamydomonas cell extracts. Finally, CDJ3 was found in complexes with apparent molecular masses of ~550 to 2800 kDa that appeared to contain RNA. We speculate that CDJ3-5 might represent redox switches that act by recruiting HSP70B for the reorganization of regulatory protein complexes

Systems-Wide Analysis of Acclimation Responses to Long-Term Heat Stress and Recovery in the Photosynthetic Model Organism Chlamydomonas reinhardtii

We applied a top-down systems biology approach to understand how Chlamydomonas reinhardtii acclimates to long-term heat stress (HS) and recovers from it. For this, we shifted cells from 25 to 42°}C for 24 h and back to 25{°C for >=8 h and monitored abundances of 1856 proteins/protein groups, 99 polar and 185 lipophilic metabolites, and cytological and photosynthesis parameters. Our data indicate that acclimation of Chlamydomonas to long-term HS consists of a temporally ordered, orchestrated implementation of response elements at various system levels. These comprise (1) cell cycle arrest; (2) catabolism of larger molecules to generate compounds with roles in stress protection; (3) accumulation of molecular chaperones to restore protein homeostasis together with compatible solutes; (4) redirection of photosynthetic energy and reducing power from the Calvin cycle to the de novo synthesis of saturated fatty acids to replace polyunsaturated ones in membrane lipids, which are deposited in lipid bodies; and (5) when sinks for photosynthetic energy and reducing power are depleted, resumption of Calvin cycle activity associated with increased photorespiration, accumulation of reactive oxygen species scavengers, and throttling of linear electron flow by antenna uncoupling. During recovery from HS, cells appear to focus on processes allowing rapid resumption of growth rather than restoring pre-HS conditions.GlossaryHSheat stressERendoplasmic reticulumPSIIphotosystem IITAGtriacylglycerolPLBprolamellar bodyLC-MSliquid chromatography-mass spectrometryLHClight-harvesting complexPSIphotosystem IGC-MSgas chromatography-mass spectrometryGPGglycerophosphoglycerolDGTSdiacylglycerol trimethyl homoserinePEphosphatidyl ethanolamineMGDGmonogalactosyl diacylglycerolDGDGdigalactosyl diacylglycerolSQDGsulfoquinovosyl diacylglycerolPGphosphatidyl glycerolDAGdiacylglycerolFAfatty acidTCAtricarboxylic acidROSreactive oxygen speciesBCAAbranched-chain amino acidLEFlinear electron flowTAPTris-acetate-phosphatePIpropidium diiodideIOMIQSintegration of mass spectrometry identification and quantification softwar