Quantitative Shotgun Proteomics Using a Uniform 15N-Labeled Standard to Monitor Proteome Dynamics in Time Course Experiments Reveals New Insights into the Heat Stress Response of Chlamydomonas reinhardtii

Abstract

Crop-plant-yield safety is jeopardized by temperature stress caused by the global climate change. To take countermeasures by breeding and/or transgenic approaches it is essential to understand the mechanisms underlying plant acclimation to heat stress. To this end proteomics approaches are most promising, as acclimation is largely mediated by proteins. Accordingly, several proteomics studies, mainly based on two-dimensional gel-tandem MS approaches, were conducted in the past. However, results often were inconsistent, presumably attributable to artifacts inherent to the display of complex proteomes via two-dimensional-gels. We describe here a new approach to monitor proteome dynamics in time course experiments. This approach involves full 15N metabolic labeling and mass spectrometry based quantitative shotgun proteomics using a uniform 15N standard over all time points. It comprises a software framework, IOMIQS, that features batch job mediated automated peptide identification by four parallelized search engines, peptide quantification and data assembly for the processing of large numbers of samples. We have applied this approach to monitor proteome dynamics in a heat stress time course using the unicellular green alga Chlamydomonas reinhardtii as model system. We were able to identify 3433 Chlamydomonas proteins, of which 1116 were quantified in at least three of five time points of the time course. Statistical analyses revealed that levels of 38 proteins significantly increased, whereas levels of 206 proteins significantly decreased during heat stress. The increasing proteins comprise 25 (co-)chaperones and 13 proteins involved in chromatin remodeling, signal transduction, apoptosis, photosynthetic light reactions, and yet unknown functions. Proteins decreasing during heat stress were significantly enriched in functional categories that mediate carbon flux from CO2 and external acetate into protein biosynthesis, which also correlated with a rapid, but fully reversible cell cycle arrest after onset of stress. Our approach opens up new perspectives for plant systems biology and provides novel insights into plant stress acclimation.

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

“…A characteristic early response to HS is an immediate arrest of cell division (Figures 2A and 8), which we have observed previously in Chlamydomonas and which is conserved in eukaryotic cells (Richter et al, 2010;Mühlhaus et al, 2011). As the distribution of 1n, 2n, and 4n cells remained almost constant during HS ( Figure 2B), cells apparently were cell cycle arrested in both G1 (unreplicated DNA = 1n) and G2 phases (after DNA replication $ 2n, 4n), perhaps as a consequence of reduced activities of cyclin-dependent kinases (Kühl and Rensing, 2000).…”

“…Chlamydomonas cells were previously shown to fully recover from a 24-h HS treatment at 42°C (Mühlhaus et al, 2011). During this HS and recovery time course, we monitored changes in the proteome, metabolome, and lipidome, as well as cytological and physiological parameters.…”

“…Membrane and soluble proteins were extracted, tryptically digested, and subjected to nano-liquid chromatography-tandem mass spectrometry. Proteins were identified and quantified with the IOMIQS framework (integration of mass spectrometry identification and quantification software) (Mühlhaus et al, 2011). With this approach, a total of 1985 proteins were quantified in samples from at least five out of six time points taken during HS and recovery.…”

“…Recently, we used a shotgun proteomics approach based on 15 N metabolic labeling to monitor changes in abundances of the soluble proteome in heat stressed Chlamydomonas reinhardtii cells during a 3-h time course (Mühlhaus et al, 2011). When comparing results on Chlamydomonas with proteomics studies conducted on various land plants (Skylas et al, 2002;Majoul et al, 2003Majoul et al, , 2004Süle et al, 2004;Ferreira et al, 2006;Lee et al, 2007;Palmblad et al, 2008;Valcu et al, 2008;Xu and Huang, 2008;Scafaro et al, 2010;Xu and Huang, 2010), we concluded that the upregulation of chaperones, FKBP65 orthologs, and thiamine biosynthesis protein and the downregulation of ferredoxin-NADP(H) oxidoreductase, Rubisco, methionine synthase, adenosylhomocysteinase, and S-adenosylmethionine synthetase are conserved in the green lineage.…”

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control

“…Quantitative shotgun proteomics using a uniform 15 N-labeled standard was performed as described by Mühlhaus et al (2011) with some modifications. In brief, one culture of the A31 strain and two cultures of the DCH16 strain were grown for ;10 generations in TAP medium containing 14 N in the case of A31 and 14 N and 15 N-ammonium in the case of DCH16.…”