Daniel Veyel

Systems Analysis of the Response of Photosynthesis, Metabolism, and Growth to an Increase in Irradiance in the Photosynthetic Model Organism Chlamydomonas reinhardtii

Systems analysis reveals that Chlamydomonas reinhardtii responds flexibly to an increase in light intensity. Rising metabolite levels and posttranslation regulation facilitate a rapid increase in the rate of carbon fixation and a slightly delayed increase in the rate of growth, while slower changes in protein abundance adjust allocation and relieve potential bottlenecks under the new conditions. We investigated the systems response of metabolism and growth after an increase in irradiance in the nonsaturating range in the algal model Chlamydomonas reinhardtii. In a three-step process, photosynthesis and the levels of metabolites increased immediately, growth increased after 10 to 15 min, and transcript and protein abundance responded by 40 and 120 to 240 min, respectively. In the first phase, starch and metabolites provided a transient buffer for carbon until growth increased. This uncouples photosynthesis from growth in a fluctuating light environment. In the first and second phases, rising metabolite levels and increased polysome loading drove an increase in fluxes. Most Calvin-Benson cycle (CBC) enzymes were substrate-limited in vivo, and strikingly, many were present at higher concentrations than their substrates, explaining how rising metabolite levels stimulate CBC flux. Rubisco, fructose-1,6-biosphosphatase, and seduheptulose-1,7-bisphosphatase were close to substrate saturation in vivo, and flux was increased by posttranslational activation. In the third phase, changes in abundance of particular proteins, including increases in plastidial ATP synthase and some CBC enzymes, relieved potential bottlenecks and readjusted protein allocation between different processes. Despite reasonable overall agreement between changes in transcript and protein abundance (R2 = 0.24), many proteins, including those in photosynthesis, changed independently of transcript abundance.

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

Early acclimation responses of Chlamydomonas to long-term heat stress are directed toward restoration of protein homeostasis and membrane fluidity involving the redirection of photosynthetic electron flow from carbon fixation to saturated fatty acid synthesis, while late responses deal with the depletion of electron sinks. During recovery, cells aim at a rapid resumption of cell division/growth. 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.

Synthesis and Use of Stable-Isotope-Labeled Internal Standards for Quantification of Phosphorylated Metabolites by LC-MS/MS

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique for measuring metabolites. However, coeluting components in tissue extracts can interfere with ionization at the interface of the LC and MS/MS phases, causing under- or overestimation of metabolite concentrations. Spiking of samples with known amounts of stable-isotope-labeled internal standards (SIL-IS) allows measurements of the corresponding metabolites to be corrected for such matrix effects. We describe criteria for selection of suitable SIL-IS and report the enzymatic synthesis and purification of nine SIL-IS for hexose-, pentose-, and triose-phosphates, UDP-glucose, and adenosine monophosphate (AMP). Along with commercially available SIL-IS for seven other metabolites, these were validated by LC-MS/MS analyses of extracts from leaves, nonphotosynthetic plant tissues, mouse liver, and cells of Chlamydomonas reinhardtii, Escherichia coli and bakers yeast (Saccharomyces cerevisiae). With only a few exceptions, spiking with SIL-IS significantly improved the reproducibility of LC-MS/MS-based metabolite measurements across a wide range of extract dilutions, indicating effective correction for matrix effects by this approach. With use of SIL-IS to correct for matrix effects, LC-MS/MS offers unprecedented scope for reliable determination of photosynthetic and respiratory intermediates in a diverse range of organisms.

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response (HSR) are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotolerance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenuation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cells entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP70B-antisense strains.

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.

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.

Affinity purification with metabolomic and proteomic analysis unravels diverse roles of nucleoside diphosphate kinases

We demonstrate that affinity purification is suited for analysing protein–small molecule interactions. Analysis of protein–protein–small molecule complexes of Arabidopsis nucleoside diphosphate kinases gave new insight into their function and regulation.

Identification and Validation of Protein-Protein Interactions by Combining Co-immunoprecipitation, Antigen Competition, and Stable Isotope Labeling

Co-immunoprecipitation (coIP) in combination with mass spectrometry (MS) is a powerful tool to identify potential protein-protein interactions. However, unspecifically precipitated proteins usually result in large numbers of false-positive identifications. Here we describe a detailed protocol particularly useful in plant sciences that is based on (15)N stable isotope labeling of cells, (14)N antigen titration, and coIP/MS to distinguish true from false protein-protein interactions.

Interaction of 2',3'-cAMP with Rbp47b plays a role in stress granule formation

2′,3′-cAMP associates with Arabidopsis Rbp47b and plays a role in stress granule formation. 2′,3′-cAMP is an intriguing small molecule that is conserved among different kingdoms. 2′,3′-cAMP is presumably produced during RNA degradation, with increased cellular levels observed especially under stress conditions. Previously, we observed the presence of 2′,3′-cAMP in Arabidopsis (Arabidopsis thaliana) protein complexes isolated from native lysate, suggesting that 2′,3′-cAMP has potential protein partners in plants. Here, affinity purification experiments revealed that 2′,3′-cAMP associates with the stress granule (SG) proteome. SGs are aggregates composed of protein and mRNA, which enable cells to selectively store mRNA for use in response to stress such as heat whereby translation initiation is impaired. Using size-exclusion chromatography and affinity purification analyses, we identified Rbp47b, the key component of SGs, as a potential interacting partner of 2′,3′-cAMP. Furthermore, SG formation was promoted in 2′,3′-cAMP-treated Arabidopsis seedlings, and interactions between 2′,3′-cAMP and RNA-binding domains of Rbp47b, RRM2 and RRM3, were confirmed in vitro using microscale thermophoresis. Taken together, these results (1) describe novel small-molecule regulation of SG formation, (2) provide evidence for the biological role of 2′,3′-cAMP, and (3) demonstrate an original biochemical pipeline for the identification of protein-metabolite interactors.

PROMIS, global analysis of PROtein–metabolite interactions using size separation in Arabidopsis thaliana

Small molecules not only represent cellular building blocks and metabolic intermediates, but also regulatory ligands and signaling molecules that interact with proteins. Although these interactions affect cellular metabolism, growth, and development, they have been largely understudied. Herein, we describe a method, which we named PROtein–Metabolite Interactions using Size separation (PROMIS), that allows simultaneous, global analysis of endogenous protein–small molecule and of protein–protein complexes. To this end, a cell-free native lysate from Arabidopsis thaliana cell cultures was fractionated by size-exclusion chromatography, followed by quantitative metabolomic and proteomic analyses. Proteins and small molecules showing similar elution behavior, across protein-containing fractions, constituted putative interactors. Applying PROMIS to an A. thaliana extract, we ascertained known protein–protein (PPIs) and protein–metabolite (PMIs) interactions and reproduced binding between small-molecule protease inhibitors and their respective proteases. More importantly, we present examples of two experimental strategies that exploit the PROMIS dataset to identify novel PMIs. By looking for similar elution behavior of metabolites and enzymes belonging to the same biochemical pathways, we identified putative feedback and feed-forward regulations in pantothenate biosynthesis and the methionine salvage cycle, respectively. By combining PROMIS with an orthogonal affinity purification approach, we identified an interaction between the dipeptide Tyr–Asp and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. In summary, we present proof of concept for a powerful experimental tool that enables system-wide analysis of PMIs and PPIs across all biological systems. The dataset obtained here comprises nearly 140 metabolites and 5000 proteins, which can be mined for putative interactors.