Rationales and Approaches for Studying Metabolism in Eukaryotic Microalgae

Veyel, Daniel; Erban, Alexander; Fehrle, Ines; Kopka, Joachim; Schroda, Michael

doi:10.3390/metabo4020184

Cited by 18 publications

(16 citation statements)

References 166 publications

(285 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas hereafter) has long served as a model organism for studies of photosynthesis (Moroney et al ., ), cellular motility (Pedersen et al ., ), and, more recently, potential biofuel synthesis (Radakovits et al ., ; Veyel et al ., ). Among its useful features are its publicly available nuclear genome sequence and the ability to transform all three of its genomes (nuclear, mitochondrial and chloroplast) with foreign DNA (Boynton et al ., ; Grossman, ; Merchant et al ., ).…”

Section: Introductionmentioning

confidence: 97%

Improved and versatile viral 2A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii

et al. 2015

View full text Add to dashboard Cite

SUMMARYA significantly improved viral 2A peptide system for dependable high-level expression of dicistronic genes in Chlamydomonas reinhardtii has been developed. Data are presented demonstrating that use of an especially proficient 'extended FMDV 2A' coding region allows production of two independent protein products from a dicistronic gene with almost complete efficiency. Importantly, results are also presented that demonstrate the utility of this 2A system for efficient high-level expression of foreign genes in C. reinhardtii, which has not previously been reliably achievable in this algal model system. To expand the versatility of the 2A expression system, a number of commonly used selectable marker proteins were assessed for their compatibility with the extended FMDV 2A peptide. Additional experiments demonstrate the feasibility and utility of 2A-containing dicistronic systems that rely on a strong conditional promoter for transcriptional control and a low-expression marker gene for selection. This strategy allows easy and efficient delivery of genes of interest whose expression levels require regulation either to mitigate potential toxicity or allow differential expression under controlled experimental conditions. Finally, as an additional practical demonstration of the utility of the extended FMDV 2A system, confocal fluorescence microscopy is used to demonstrate that native and foreign proteins of interest bearing post-translational remnants of the extended FMDV 2A peptide localize correctly to various cellular compartments, including a striking demonstration of the almost exclusive localization of the Rubisco small subunit protein to the pyrenoid of the C. reinhardtii chloroplast in cells maintained under ambient CO 2 concentrations.

show abstract

Section: Introductionmentioning

confidence: 97%

Improved and versatile viral 2A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii

et al. 2015

View full text Add to dashboard Cite

show abstract

“…For the profiling of polar metabolites, cells were harvested by fast filtration and polar metabolites were extracted with cold methanol/chloroform, derivatized by methoxamination and silylation, and analyzed by gas chromatography-mass spectrometry (GC-MS) (Lisec et al, 2006;Veyel et al, 2014a). Excluding standards, 106 polar analytes were identified by matching reconstructed spectra against the Golm Metabolome Database Schauer et al, 2005;Hummel et al, 2010) using the TagFinder software (Luedemann et al, 2008).…”

Section: Analysis Of the Dynamics Of Polar Metabolitesmentioning

confidence: 99%

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

et al. 2014

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Though we used centrifugation to harvest the cells, alternate harvesting strategies can be used for harvesting of the samples. However, it is important to note that different harvesting strategies are known to influence the metabolic state of the cells 37 hence, consistent harvesting approach must be used for all experimental samples. Secondly, it is important to avoid drying of the upper non-polar phase containing chlorophyll, since this can influence the levels of dissolved chlorophyll in the solvent affecting the normalization factor for the samples.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, elucidating molecular changes of specific metabolic pathways or cellular processes. Similarly, these high-throughput data can allow identification of targets for the metabolic engineering and refine or test predictions from genome-scale metabolic models 37,39 .…”

Section: Discussionmentioning

confidence: 99%

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga <em>Chlamydomonas reinhardtii</em>

Mubeen

Giraldi

Jüppner

et al. 2019

JoVE

View full text Add to dashboard Cite

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 indepth 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. Video Link The video component of this article can be found at https://www.jove.com/video/59547/ 10 , here, the quality of the proteomic samples obtained from the same pellet is also demonstrated.

show abstract

Rationales and Approaches for Studying Metabolism in Eukaryotic Microalgae

Cited by 18 publications

References 166 publications

Improved and versatile viral 2A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii

Improved and versatile viral 2A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii

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

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga <em>Chlamydomonas reinhardtii</em>

Contact Info

Product

Resources

About