Summary of Study ST003035
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001888. The data can be accessed directly via it's Project DOI: 10.21228/M8P728 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003035 |
| Study Title | Central Transcriptional Regulator Controls Growth and Carbon Storage under High Light Stress in Photosynthetic Microalgae Model Strains |
| Study Type | Algae |
| Study Summary | Carbon capture efficiency and biochemical storage are some of the primary drivers of photosynthetic productivity and by extension crop yield. To elucidate the mechanisms governing yield phenotypes and carbon allocation regulatory elements, we selected two microalgae strains as simplified models of photosynthetic crops. The Picochlorum celeri TG2 isolate is one of the fastest growing algae and in this work is juxtaposed to a closely related, slower growing, isolate, TG1, of the same species with less than 2% genomic divergence. Through the application of a comprehensive systems biology light-stress response study, we observed a stark difference in carbon assimilation and storage rates, with the slower growing isolate accumulating almost three times the amount of starch compared to the fast-growing isolate. We characterized the carbon storage rates and allocation dynamics, with metabolic bottlenecks, and transport rates of intermediates underlying the variations in growth and composition in high light using instationary 13C-fluxomics experiments. High light stress analysis of transcriptomic dynamics during acclimation of the strains from low to high light identified a widespread response with up to 73% the annotated gene set significantly differentially expressed after only 1 hour. Broad transcriptional regulatory control was inferred by a rapid depletion of a global diel-responsive transcription factor closely related to a circadian-regulator in plants, as the single most distinct transcription factor. Transferring this factor to the slower variant increased yield, specific growth rate, and carbohydrate accumulation of the selected engineered strain, providing further evidence for a coordinating regulatory mechanism for this complex phenotype. |
| Institute | National Renewable Energy Lab |
| Department | Biosciences |
| Laboratory | Laurens Lab |
| Last Name | Laurens |
| First Name | Lieve |
| Address | 15013 Denver West Paekway, Golden, CO 80401 |
| lieve.laurens@nrel.gov | |
| Phone | +1 720-273-6534 |
| Submit Date | 2023-12-18 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-04-26 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001888 |
| Project DOI: | doi: 10.21228/M8P728 |
| Project Title: | Central Transcriptional Regulator Controls Growth and Carbon Storage under High Light Stress in Photosynthetic Microalgae Model Strains |
| Project Type: | Life Sciences |
| Project Summary: | Carbon capture efficiency and biochemical storage are some of the primary drivers of photosynthetic productivity and by extension crop yield. To elucidate the mechanisms governing yield phenotypes and carbon allocation regulatory elements, we selected two microalgae strains as simplified models of photosynthetic crops. The Picochlorum celeri TG2 isolate is one of the fastest growing algae and in this work is juxtaposed to a closely related, slower growing, isolate, TG1, of the same species with less than 2% genomic divergence. Through the application of a comprehensive systems biology light-stress response study, we observed a stark difference in carbon assimilation and storage rates, with the slower growing isolate accumulating almost three times the amount of starch compared to the fast-growing isolate. We characterized the carbon storage rates and allocation dynamics, with metabolic bottlenecks, and transport rates of intermediates underlying the variations in growth and composition in high light using instationary 13C-fluxomics experiments. High light stress analysis of transcriptomic dynamics during acclimation of the strains from low to high light identified a widespread response with up to 73% the annotated gene set significantly differentially expressed after only 1 hour. Broad transcriptional regulatory control was inferred by a rapid depletion of a global diel-responsive transcription factor closely related to a circadian-regulator in plants, as the single most distinct transcription factor. Transferring this factor to the slower variant increased yield, specific growth rate, and carbohydrate accumulation of the selected engineered strain, providing further evidence for a coordinating regulatory mechanism for this complex phenotype. |
| Institute: | National Renewable Energy Lab |
| Department: | Biosciences |
| Laboratory: | Laurens Lab |
| Last Name: | Laurens |
| First Name: | Lieve |
| Address: | 15013 Denver West Parkway, Golden, Colorado, 80401, USA |
| Email: | lieve.laurens@nrel.gov |
| Phone: | +1 720-273-6534 |
| Funding Source: | This work was financially supported by a collaborative research and development agreement with ExxonMobil Technology and Engineering Co. (EMTEC) |
| Contributors: | Steichen, S, Deshpande, A., Mosey, M., Loob, J., Douchi, D., Knoshaug, E.P., Brown, S, Nielsen, R., Weissman, J., Carrillo, L.R., Laurens, L.M.L. |
Subject:
| Subject ID: | SU003149 |
| Subject Type: | Plant |
| Subject Species: | Picochlorum celeri |
Factors:
Subject type: Plant; Subject species: Picochlorum celeri (Factor headings shown in green)
| mb_sample_id | local_sample_id | Strain | Time (s) |
|---|---|---|---|
| SA328731 | TG1-MYB99_R3_0 | TG1-MYB99 | 0 (no label) |
| SA328732 | TG1-MYB99_R2_0 | TG1-MYB99 | 0 (no label) |
| SA328733 | TG1-MYB99_R1_0 | TG1-MYB99 | 0 (no label) |
| SA328734 | TG1-MYB99_R1_180 | TG1-MYB99 | 180 |
| SA328735 | TG1-MYB99_R3_180 | TG1-MYB99 | 180 |
| SA328736 | TG1-MYB99_R2_180 | TG1-MYB99 | 180 |
| SA328737 | TG1-MYB99_R2_30 | TG1-MYB99 | 30 |
| SA328738 | TG1-MYB99_R3_30 | TG1-MYB99 | 30 |
| SA328739 | TG1-MYB99_R1_30 | TG1-MYB99 | 30 |
| SA328740 | TG1-MYB99_R3_300 | TG1-MYB99 | 300 |
| SA328741 | TG1-MYB99_R1_300 | TG1-MYB99 | 300 |
| SA328742 | TG1-MYB99_R2_300 | TG1-MYB99 | 300 |
| SA328743 | TG1-MYB99_R1_60 | TG1-MYB99 | 60 |
| SA328744 | TG1-MYB99_R2_60 | TG1-MYB99 | 60 |
| SA328745 | TG1-MYB99_R3_60 | TG1-MYB99 | 60 |
| SA328746 | TG1-MYB99_R1_600 | TG1-MYB99 | 600 |
| SA328747 | TG1-MYB99_R2_600 | TG1-MYB99 | 600 |
| SA328748 | TG1-MYB99_R3_600 | TG1-MYB99 | 600 |
| SA328749 | TG1_R3_0 | TG1 | 0 (no label) |
| SA328750 | TG1_R1_0 | TG1 | 0 (no label) |
| SA328751 | TG1_R2_0 | TG1 | 0 (no label) |
| SA328752 | TG1_R3_180 | TG1 | 180 |
| SA328753 | TG1_R2_180 | TG1 | 180 |
| SA328754 | TG1_R1_180 | TG1 | 180 |
| SA328755 | TG1_R3_30 | TG1 | 30 |
| SA328756 | TG1_R1_30 | TG1 | 30 |
| SA328757 | TG1_R2_30 | TG1 | 30 |
| SA328758 | TG1_R3_300 | TG1 | 300 |
| SA328759 | TG1_R1_300 | TG1 | 300 |
| SA328760 | TG1_R2_300 | TG1 | 300 |
| SA328761 | TG1_R1_60 | TG1 | 60 |
| SA328762 | TG1_R3_60 | TG1 | 60 |
| SA328763 | TG1_R2_60 | TG1 | 60 |
| SA328764 | TG1_R1_600 | TG1 | 600 |
| SA328765 | TG1_R3_600 | TG1 | 600 |
| SA328766 | TG1_R2_600 | TG1 | 600 |
| SA328767 | TG2_R1_0 | TG2 | 0 (no label) |
| SA328768 | TG2_R3_0 | TG2 | 0 (no label) |
| SA328769 | TG2_R2_0 | TG2 | 0 (no label) |
| SA328770 | TG2_R2_180 | TG2 | 180 |
| SA328771 | TG2_R1_180 | TG2 | 180 |
| SA328772 | TG2_R3_180 | TG2 | 180 |
| SA328773 | TG2_R3_30 | TG2 | 30 |
| SA328774 | TG2_R2_30 | TG2 | 30 |
| SA328775 | TG2_R1_30 | TG2 | 30 |
| SA328776 | TG2_R1_300 | TG2 | 300 |
| SA328777 | TG2_R3_300 | TG2 | 300 |
| SA328778 | TG2_R2_300 | TG2 | 300 |
| SA328779 | TG2_R2_60 | TG2 | 60 |
| SA328780 | TG2_R1_60 | TG2 | 60 |
| SA328781 | TG2_R3_60 | TG2 | 60 |
| SA328782 | TG2_R3_600 | TG2 | 600 |
| SA328783 | TG2_R2_600 | TG2 | 600 |
| SA328784 | TG2_R1_600 | TG2 | 600 |
| Showing results 1 to 54 of 54 |
Collection:
| Collection ID: | CO003142 |
| Collection Summary: | P. celeri strains TG1, TG2, and TG1-MYB99 were adapted to HL intensity (1000 μmol m−2 s−1 PAR) by ensuring chlorophyll concentration was maintained below 0.75 µg/mL for several days prior to any transient labeling experiment. A 2 L volume was inoculated on the day prior to the transient labeling experiment in the SAGE photobioreactor and total organic carbon (TOC) measurements were taken 2 hours before and at the start of the experiment to determine the specific growth rate. OD750 of the cultures were always maintained <0.3 for the transient labeling experiment. The culture was then split equally into 6 photobioreactor positions, each of which served as a time point for the transient labeling experiment. Transient labeling was achieved by turning off the pH control (CO2 supply) and by the addition of NaH13CO3 (Cambridge Isotope Laboratories, Andover, MA) up to a final concentration of 11.76 mM simultaneously with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Sigma Aldrich, St. Louis) up to a final concentration of 5 mM. NaH13CO3 was preferred to 13CO2 to avoid gas-liquid mass transfer limitations and achieve rapid equilibrium. Preliminary experiments were performed to determine the concentration of NaH13CO3 and HEPES buffer that need to be added simultaneously to achieve a step change from 12C to 13C, maintain pH <7.5, as well as be able to minimize contribution of unlabeled carbon in the labeling dynamics. Once the 13C pulse was introduced, each identical culture was harvested at different time points, namely 0 (no label pulse), 30, 60, 180, 300, and 600s by rapidly filtering on a 9 cm Fisherbrand glass microfiber filters followed by washing with 15 mL of 0.2 M ammonium bicarbonate in 5% methanol kept in an ice bath and quenching in liquid nitrogen. |
| Collection Protocol Filename: | Sample_Collection_AD.pdf |
| Sample Type: | Algae |
Treatment:
| Treatment ID: | TR003158 |
| Treatment Summary: | Once the 13C pulse was introduced, each identical culture was harvested at different time points, namely 0 (no label pulse), 30, 60, 180, 300, and 600s by rapidly filtering on a 9 cm Fisherbrand glass microfiber filters followed by washing with 15 mL of 0.2 M ammonium bicarbonate in 5% methanol kept in an ice bath and quenching in liquid nitrogen. |
Sample Preparation:
| Sampleprep ID: | SP003155 |
| Sampleprep Summary: | Filters with biomass were kept on dry ice and 26 µL of 100 µM 1-13C Leucine was added as the internal standard. The label 1-13C Leucine was chosen as the internal standard since contribution of 1-13C Leucine from the transient labeling experiment was minimal in this time scale of the experiment. The filters were then folded and stored in 15 mL centrifuge tubes on dry ice. Extraction was performed by first adding 2 mL of precooled methanol followed by maceration till the filter was a pulp. This was followed by addition of 2 mL pre-cooled chloroform and further maceration. Once the extraction was complete with the methanol:chloroform mixture, 5 mL chloroform was added followed by 1 mL of 0.05% ammonium hydroxide (pH ~ 10.4) for phase separation. The extracts were then gently vortexed for a minimum of 15s followed by centrifugation at 4o C at 2500 g. The clear phase on the top was then removed and filtered using a syringe filter, diluted in acetonitrile (ACN) (3 ACN: 1 Extract) and transferred to LC-MS vials prior to injection. |
| Sampleprep Protocol Filename: | Sample_Extraction_AD.pdf |
| Processing Storage Conditions: | Described in summary |
| Extraction Method: | Methanol-Chloroform-Water |
| Extract Storage: | -80℃ |
| Sample Spiking: | 1-13C-Leucine |
Combined analysis:
| Analysis ID | AN004976 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | Waters ACQUITY Premier BEH Amide (150 x 2.1mm, 1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE |
| Units | Raw Area Count |
Chromatography:
| Chromatography ID: | CH003757 |
| Chromatography Summary: | The metabolites were separated using hydrophilic interaction chromatography (HILIC) using a BEH Amide column (1.7 µm, 2.1 mm X 150 mm, ACQUITY Premier BEH Amide, Waters Corporation) via a gradient method. Solvent A comprised of 20 mM ammonium acetate and 15 mM ammonium hydroxide in 97% 18.2 mΩ-cm water and 3% acetonitrile whereas solvent B comprised of 20 mM ammonium acetate and 15 mM ammonium hydroxide in 95% acetonitrile and 5% 18.2 mΩ-cm water. All the chemicals used were LC-MS grade. The chromatography method used a constant flow rate of 0.2 mL/min and a linear gradient to enable separation of a wide range of metabolites followed by a column regeneration step as follows: 90% solvent B for 1 min followed by a linear gradient down to 75% solvent B for 23 min, a linear gradient down to 45% solvent B in 2 min, a linear gradient down to 25% B in 4 min, followed by a step to the starting composition of 90% B and hold for 6 min for column regeneration for a total run time of 36 min. The column temperature was maintained at 25°C and injection volume was set to 20 µL. Metabolite extracts were diluted such that the final injection solvent was 75% acetonitrile. Data was collected using a Thermo Scientific Q-Exactive mass spectrometer in negative ion mode. The scan ranged from 75 to 1000 m/z with a resolution of 140,000. AGC target was set to 3e6 while maximum IT was 200 ms. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY Premier BEH Amide (150 x 2.1mm, 1.7um) |
| Column Temperature: | 25 |
| Flow Gradient: | 90% solvent B for 1 min followed by a linear gradient down to 75% solvent B for 23 min, a linear gradient down to 45% solvent B in 2 min, a linear gradient down to 25% B in 4 min, followed by a step to the starting composition of 90% B and hold for 6 min for column regeneration for a total run time of 36 min |
| Flow Rate: | 0.2 mL/min |
| Internal Standard: | 1-13C Leucine |
| Solvent A: | 20 mM ammonium acetate and 15 mM ammonium hydroxide in 97% 18.2 mΩ-cm water and 3% acetonitrile |
| Solvent B: | 20 mM ammonium acetate and 15 mM ammonium hydroxide in 95% acetonitrile and 5% 18.2 mΩ-cm water |
| Randomization Order: | Samples were randomized |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004716 |
| Analysis ID: | AN004976 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Data was collected using a Thermo Scientific Q-Exactive mass spectrometer in negative ion mode. The scan ranged from 75 to 1000 m/z with a resolution of 140,000. AGC target was set to 3e6 while maximum IT was 200 ms. |
| Ion Mode: | NEGATIVE |
