A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

Target of Rapamycin Inhibition in Chlamydomonas reinhardtii Triggers de Novo Amino Acid Synthesis by Enhancing Nitrogen Assimilation.

The Target of Rapamycin (TOR) kinase is a central regulator of growth and metabolism in all eukaryotic organisms, including animals, fungi, and plants. Even though the inputs and outputs of TOR signaling are well characterized for animals and fungi, our understanding of the upstream regulators of TOR and its downstream targets is still fragmentary in photosynthetic organisms. In this study, we employed the rapamycin-sensitive green alga Chlamydomonas reinhardtii to elucidate the molecular cause of the amino acid accumulation that occurs after rapamycin-induced inhibition of TOR. Using different growth conditions and stable 13C- and 15N-isotope labeling, we show that this phenotype is accompanied by increased nitrogen (N) uptake, which is induced within minutes of TOR inhibition. Interestingly, this increased N influx is accompanied by increased activities of glutamine synthetase and glutamine oxoglutarate aminotransferase, the main N-assimilating enzymes, which are responsible for the rise in levels of several amino acids, which occurs within a few minutes. Accordingly, we conclude that even though translation initiation and autophagy have been reported to be the main downstream targets of TOR, the upregulation of de novo amino acid synthesis seems to be one of the earliest responses induced after the inhibition of TOR in Chlamydomonas.

The target of rapamycin kinase affects biomass accumulation and cell cycle progression by altering carbon/nitrogen balance in synchronized Chlamydomonas reinhardtii cells.

Several metabolic processes tightly regulate growth and biomass accumulation. A highly conserved protein complex containing the target of rapamycin (TOR) kinase is known to integrate intra- and extracellular stimuli controlling nutrient allocation and hence cellular growth. Although several functions of TOR have been described in various heterotrophic eukaryotes, our understanding lags far behind in photosynthetic organisms. In the present investigation, we used the model alga Chlamydomonas reinhardtii to conduct a time-resolved analysis of molecular and physiological features throughout the diurnal cycle after TOR inhibition. Detailed examination of the cell cycle phases revealed that growth is not only repressed by 50%, but also that significant, non-linear delays in the progression can be observed. By using metabolomics analysis, we elucidated that the growth repression was mainly driven by differential carbon partitioning between anabolic and catabolic processes. Accordingly, the time-resolved analysis illustrated that metabolic processes including amino acid-, starch- and triacylglycerol synthesis, as well RNA degradation, were redirected within minutes of TOR inhibition. Here especially the high accumulation of nitrogen-containing compounds indicated that an active TOR kinase controls the carbon to nitrogen balance of the cell, which is responsible for biomass accumulation, growth and cell cycle progression.

Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii.

Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15 million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems.

Protocol: a fast, comprehensive and reproducible one-step extraction method for the rapid preparation of polar and semi-polar metabolites, lipids, proteins, starch and cell wall polymers from a single sample.

BACKGROUND: The elucidation of complex biological systems requires integration of multiple molecular parameters. Accordingly, high throughput methods like transcriptomics, proteomics, metabolomics and lipidomics have emerged to provide the tools for successful system-wide investigations. Unfortunately, optimized analysis of different compounds requires specific extraction procedures in combination with specific analytical instrumentation. However, the most efficient extraction protocols often only cover a restricted number of compounds due to the different physico-chemical properties of these biological compounds. Consequently, comprehensive analysis of several molecular components like polar primary metabolites next to lipids or proteins require multiple aliquots to enable the specific extraction procedures required to cover these diverse compound classes. This multi-parallel sample handling of different sample aliquots is therefore not only more sample intensive, it also requires more time and effort to obtain the required extracts. RESULTS: To circumvent large sample amounts, distributed into several aliquots for the comprehensive extraction of most relevant biological compounds, we developed a simple, robust and reproducible two-phase liquid-liquid extraction protocol. This one-step extraction protocol allows for the analysis of polar-, semi-polar and hydrophobic metabolites, next to insoluble or precipitated compounds, including proteins, starch and plant cell wall components, from a single sample. The method is scalable regarding the used sample amounts but also the employed volumes and can be performed in microcentrifuge tubes, enabling high throughput analysis. The obtained fractions are fully compatible with common analytical methods, including spectroscopic, chromatographic and mass spectrometry-based techniques. To document the utility of the described protocol, we used 25 mg of Arabidopsis thaliana rosette leaves for the generation of multi-omics data sets, covering lipidomics, metabolomics and proteomics. The obtained data allowed us to measure and annotate more than 200 lipid compounds, 100 primary metabolites, 50 secondary metabolites and 2000 proteins. CONCLUSIONS: The described extraction protocol provides a simple and straightforward method for the efficient extraction of lipids, metabolites and proteins from minute amounts of a single sample, enabling the targeted but also untargeted high-throughput analyses of diverse biological tissues and samples.

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.