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Low-dose ionizing radiation generates a hormetic response to modify lipid metabolism in Chlorella sorokiniana – Communications Biology

Chemicals

All chemicals were of ACS purity grade or higher. Chemicals were obtained from Sigma-Aldrich (St. Louis, MI, USA), unless otherwise stated. All experiments were performed using twice-distilled deionized water (18 MΩ) that was obtained by reagent grade water system (Millipore, Billerica, MA, USA).

Strain, growth conditions, and irradiation protocols

Chlorella sorokiniana strain CCAP 211/8 K was obtained from the Culture Collection of Algae and Protozoa (CCAP), Oban, UK. C. sorokiniana was grown in 35 mL aliquots of 3N-BBM + V medium in 100 mL Erlenmeyer flasks that were placed on an orbital shaker (120 rpm) in a growth cabinet with temperature maintained at 22 °C, and a continuous photon flux of 120 μmol/m2/s (Philips MST TL-D Reflex 36W840 1 SLV/25 lamps, Amsterdam, Netherlands). The medium was prepared according to CCAP recipe (https://www.ccap.ac.uk/wp-content/uploads/MR_3N_BBM_V.pdf), with the following composition (final concentration): 8.82 mM NaNO3, 0.17 mM CaCl2, 0.3 mM MgSO4, 0.43 mM K2HPO4, 1.29 mM KH2PO4, 0.43 mM NaCl, trace element solution (containing 12.09 µM Na2EDTA, 2.15 µM FeCl3, 1.24 µM MnCl2, 0.22 µM ZnCl2, 0.05 µM CoCl2, and 0.1 µM Na2MoO4), and 0.12 µg/L vitamin B1 and 0.1 µg/L vitamin B12. The initial pH of the medium was adjusted to pH 7.5. The medium was inoculated with approximately 0.5 × 106 cells/mL. The experiments were conducted in 3 steps to find the optimal time point, doses and dose rates of irradiation. In Step 1, microalgae were irradiated at 3 days after inoculation, in the early exponential phase, and the analyses were performed over 30 days. In Step 2, microalgae were irradiated at 20 days after inoculation, in the early stationary phase, and the analyses were performed at day-10 after irradiation. In Step 3, microalgae were irradiated 20 days after inoculation and detailed analyses were performed after 1 day, with some analyses also performed at day-10. In Step 2 and 3, all flasks were weighed at inoculation and the loss of water by evaporation was corrected with sterile deionized water at day-15. No more than 10% of the total volume was reduced by evaporation. Samples (35 mL) were placed in a glass Petri dish (10 cm diameter) and exposed to a single continuous dose of X-rays using a CellRad system (Faxitron Bioptics LLC, Tucson, AZ, USA), with the following settings: tube power, 750 W; filtration, 1.6 mm Be and 0.5 mm Al; energy, 120 kV. Doses and dose rates were adjusted by changing the current and were measured by a built-in ion chamber dosimeter. Samples were rotated during irradiation. Doses and dose rates of emitted radiation and radiation that was absorbed by the samples (approximately 10% of emitted doses in this setup) are presented in Supplementary Table 1. For simplicity, approximate values of absorbed doses (1, 2, 5, 10, and 20 Gy) and dose rates of irradiation (0.05, 0.25, 0.5 Gy/min) are presented in the text and the figures. Irradiated cultures were placed back into 100 mL Erlenmeyer flasks and grown under the same conditions until further analysis. Treated samples and controls (non-irradiated samples) were always grown in the same batch.

Basic parameters of microalgal cultures

Cell growth curves were generated according to the changes in optical density that were measured at 750 nm (OD750) with a UV/VIS spectrophotometer (Jenway Genova Plus, Stafordshire, UK), each day for 30 days following the inoculation. OD750 values show significant linear correlation (R2 = 0.975, P < 0.0001) with cell counts per mL (Supplementary Fig. 10). For the measurements, samples were diluted 10× in fresh medium to keep OD750 values below 1. Growth rates in the exponential phase of culture growth were calculated using OD750 values at the start (day 3; X3) and the end (day 20; X20) of the exponential phase, and using the formula (ln(X20) − ln(X3))/17. Cell count was carried out using a Sedgewick-Rafter counting chamber. Microalgal samples (0.5 mL) were treated with Lugol’s iodine solution (10 µL) for 10 min, equally diluted to keep the number of the cells per field in the 10–50 range, and left to settle in the chamber for 5 min. Cells were counted in 10 random fields of view using an optical microscope, and cell density was calculated by multiplying mean cell number per field by dilution factor. For biomass determination, 2 mL aliquots were centrifuged at 5000 g for 5 min, and the supernatant was discarded. Pellets were left to dry at 60 °C for 24 h. The pigments chlorophyll a, chlorophyll b, and carotenoids were extracted according to the following protocol. The pellet of 2 mL sample (collected as for the biomass determination) was resuspended in ice-cold methanol (2 mL), and homogenized in a glass homogenizer on ice for 2 min. The homogenate was supplemented with 8 mL of methanol and left in the dark at 4 °C for 24 h. Absorbance of the solvent extract was measured at 666 nm, 653 nm, and 470 nm. Concentrations of pigments were calculated according to Lichtenthaler and Wellburn55, and normalized to biomass that was determined on the same day. Cell viability was established using the Evans Blue stain, by incubating cells in a 0.05% (w/v) Evans Blue solution for 15 min followed by washing in deionized water56. The proportion of Evans Blue stained cells corresponds to the proportion of non-viable cells. The viability is presented as a percentage of Evans Blue negative cells. At least 100 cells were analyzed per sample.

Fluorescence assay for relative lipid content

Relative lipid content was evaluated using a rapid Nile Red assay to stain lipid droplets57. C. sorokiniana cultures were diluted 20× in 50 mM potassium phosphate buffer (pH = 7.5), so that OD750 does not exceed 0.5. A 0.25% (v/v) bleach solution was added and samples were incubated for 1 min to minimize the interference of chlorophyll58. Samples were washed 2× at 2300 × g for 5 min and resuspended in phosphate buffer with 25% (v/v) DMSO. Nile Red (Acros Organics, Antwerp, Belgium) was added at the final concentration of 50 μg/mL. Samples were incubated in the dark for 10 min. Fluorescence (S1/R1) was measured at 530 nm excitation and 570 nm emission using Fluorolog FL3-221 spectrofluorimeter (Jobin Yvon Horiba, Paris, France), with FluorEssence 3.5 software (Horiba Scientific, Kyoto, Japan). Fluorescence intensity was normalized to mean control fluorescence value on the same experimental day to estimate the relative lipid content in the culture (volumetric lipid content). Additionally, it was normalized to biomass to establish the relative lipid yield in biomass. Values are presented in arbitrary units. Lipid yield values determined by Nile Red fluorescence show a significant positive correlation (R2 = 0.975, P = 0.013) with lipid yield values determined by Soxhlet extraction and gravimetry.

Cell volume

Cell volume was estimated from micrographs made by optical microscopy and TEM. Cross-section areas were used to establish cell radius, and the volume was calculated based on the assumption that C. sorokiniana cells are spherical. Aliquots (10 µL) were smeared on microscopic slides and 5 micrographs (40× magnification) per slide were randomly collected. Micrographs were analyzed in ImageJ image processing software (National Institutes of Health, USA). Cross-section areas were determined using the following ImageJ macro that was optimized by comparison with hand-select analysis: Import image; Image-Type-8-bit; Process-FFT-Bandpass Filter (100 px; 3 px; None; 5%; Autoscale, Saturate); Process-Find Edges; Process-Binary-Make Binary; Process-Binary-Fill Holes; Analyze Particles (200–Infinity; Pixel; Circularity 0.8–1.0). In TEM micrographs (7500× magnification), cross-section areas were established in ImageJ by hand-selected analysis of >25 cells with the nuclear mid-section for each sample.

Starch content

Relative starch content in microalgal cultures was estimated using a rapid assay based on Lugol’s staining of starch59. Microalgal culture samples (200 μL) were placed into 96-well microplates. Lugol’s solution (5 μL) was added and the suspensions were mixed. Each stained sample had an unstained pair. Optical density at 660 nm (OD660) was measured using a microplate reader. Relative starch content was calculated as follows: ODLugol stained sample – ODunstained sample – ODLugol – ODwater. The obtained values were further normalized to the mean control value, and to biomass to measure relative starch yield. The results were presented in arbitrary units. Next, to determine absolute starch yield, microalgal culture samples (4 mL) were centrifuged at 5000 g for 5 min, pellets were dried at 60 °C for 24 h, and resuspended in 80% (v/v) ethanol (500 µL). Biomass was homogenized by 4 × 15 s mixing at 30 Hz with 5 mm stainless steel beads in mixer mill (MM400, Retsch, Haan, Germany). The samples were then incubated in 80% (v/v) ethanol at 85 °C for 5 min to remove the pigments (the procedure was repeated until the pellet became colorless). Starch yield in biomass was determined with a Total Starch Assay Kit (Megazyme International Ireland Ltd., Wicklow, Ireland), according to the modified AOAC 996.11 method60. Pellets were resuspended in a mixture of 80% (v/v) ethanol (200 µL) and DMSO (500 µL), and incubated at 90 °C for 1 h. Thermostable α-amylase was diluted 30-fold in 50 mM MOPS buffer (pH = 7) with 5 mM CaCl2 and added to samples (30 units). The samples were incubated at 90 °C for 15 min. Finally, 500 µL of 50 mM sodium acetate buffer (pH = 4.5) with 5 mM CaCl2 and 10 µL of amyloglucosidase (33 units) was added and the samples were incubated at 50 °C for 1 h. To measure released glucose, supernatant was separated from the pellet by centrifugation 13000 × g for 10 min, mixed with GOPOD reagent from the kit in 1:5 ratio, and incubated at 50 °C for 20 min. Samples were cooled to room temperature and absorbance was measured at 508 nm. Supernatant was diluted prior to mixing with GOPOD reagent to fit the standard curve that was made using a serial dilution of glucose solution from the kit. Concentrations were calculated from the standard curve.

Lipid yield and fatty acids profile analysis

Samples were centrifuged at 5000 × g for 5 min, and the pellets were left to dry at 50 °C for 24 h. Biomass was homogenized with mortar and pestle, and samples were merged to obtain at least 170 mg of biomass for each extraction. Lipids were extracted using Soxhlet (SOX406 Semi-Automatic Soxhlet Fat Analyzer, Hanon, Beijing, China), with the following settings: solvent mixture, chloroform:methanol 2:1 (v/v); temperature, 80 °C; time, 4 h. Before and after the extraction, samples were dried at 50 °C for 12 h and cooled in desiccator with silica gel. The analysis of fatty acid methyl ester (FAME) profiles involved transesterification through acidic methanolysis and gas chromatography-mass spectrometry (GC-MS). Extracted lipids (30 mg) were dissolved in 6 mL of methanol with 2-3 drops of concentrated sulfuric acid. The mixture was refluxed at 80 °C for 2 h and then pH was adjusted to 7 using NaHCO3 solution (0.1 g/mL of water). FAMEs were collected using hexane (4 × 6 mL). The hexane layer was collected with a Pasteur pipette, and dried with 15 g of anhydrous Na2SO4 for 15 min. The solution was filtered to remove the drying agent, and the solvent is removed in a rotary film evaporator working at 40 °C under reduced pressure. The FAME extract was dissolved in dichloromethane (5 mg/mL) and purified by vigorous mixing with activated charcoal (20 mg/mL) and Sephadex A25 (6 mg/mL). The analysis was performed using a GC-MS QP2010 plus, equipped with an AOC 5000 injector (Shimadzu, Kyoto, Japan), and FAME column (Phenomenex, L = 30 m, ID = 0.25 mm, df = 0.50 µm), and using GCMSsolution Ver. 2 software (Shimadzu). Samples (1 µL) were injected in the split mode (1:30), with the injector temperature set to 250 °C. Mass spectra were acquired in EI mode ( ± 70 eV) in the m/z range 50–500 amu (SCAN) mode. Helium (99.999%) was used as a carrier gas with a flow rate of 1.34 mL/min. The column was heated linearly from 100 °C (hold 2 min) to 240 °C with a gradient of 3 °C/min and hold at 240 °C for 5 min. Ion source temperature was set to 240 °C; interface temperature to 260 °C. Identification of constituents was performed by comparing their mass spectra to those from NIST05, Wiley8 and FFNSC3 libraries, using different search engines, and a set of FAME standards in Supelco® 37 Component FAME Mix that was dissolved in hexane (1 mg/mL). Quantitative data were obtained from GC peak area by the method of area normalization then the results were expressed relative to cell biomass. Cetane numbers were calculated using the formula and reference numbers presented by Knothe61. Cetane numbers for C15:1, C17:0, and C16:3 are not available and were taken to be the same as for C16:1, C16:0, and C18:3, respectively.

TEM analysis

C. sorokiniana cells were collected by centrifugation at 5000 × g for 5 min and fixed overnight in 0.1 M phosphate buffer (pH = 7.2) containing 3% (v/v) glutaraldehyde (Serva, Heidelberg, Germany) and 1% (v/v) paraformaldehyde (pH = 6.9) at 4 °C. Post-fixation was performed with 1% (w/v) osmium tetroxide (Serva) in 0.1 M phosphate buffer (pH = 7.2) at room temperature for 2 h. Samples were dehydrated in a graded acetone series and embedded in resin for soft blocks (AGR1031, Agar Scientific, Stansted, UK). Thin sections (70 nm), obtained with a Leica UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany), were stained with uranyl acetate and lead acetate and observed at 60 kV using a JEOL JEM-1010 TEM (JEOL, Tokyo, Japan) with an XR16 CCD camera and AMT Image Capture Engine (Advanced Microscopy Techniques, Woburn, MA, USA). The analysis of micrographs that included cell cross-section area, number of lipid droplets and starch granules per cell cross-section, and the total areas of all lipid droplets and starch granules in the cross-sections, was performed using ImageJ. At least 25 randomly selected cells with the nuclear mid-section in 3 independent replicates were analyzed for each treatment.

EPR spectroscopy

C. sorokiniana cells were collected by centrifugation at 5000 × g for 5 min and washed 3× with water to remove extracellular EPR-active metals. Each cell pellet (100 mg) was mixed with 100 µL of water, placed into quartz cuvettes (Wilmad-Lab Glass, Vineland, NJ, USA), and quickly frozen in cold isopentane. EPR spectra were recorded at 19 K on a Bruker Elexsys II E540 spectrometer with XEPR software, operating at X-band (9.4 GHz), with an Oxford Instruments ESR900 helium cryostat, at the EPR Laboratory, Faculty of Physical Chemistry, University of Belgrade. The experimental parameters were: microwave power, 3.17 mW; scan time, 2 min; modulation amplitude, 0.5 mT; modulation frequency, 100 kHz; number of accumulations, 4. Signal amplitude vs. power plot was built to establish power range that avoids saturation. All spectra were baseline corrected.

Transcriptomic analysis

C. sorokiniana cells were collected by centrifugation at 5000 × g for 5 min, washed 3× with ice-cold deionised water, snap-frozen in liquid N2, and stored at −80 °C until further analysis. RNA extraction was performed by the addition of 1 mL TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA USA), which was added to each sample and left to incubate at room temperature for 5 min. Chloroform (200 µL) was added to each sample for further extraction. Samples were briefly vortexed and left to stand for additional 5 min. To isolate RNA from DNA, proteins and other cell debris, the samples were centrifuged for 15 min at 12000 g and 4 °C. The upper layer was then aliquoted, taking care not to disturb the lower layers. To precipitate the RNA, 500 µL of isopropanol was added to each sample and left for 15 min at room temperature. To collect the precipitated RNA, samples were centrifuged for 10 min at 12000 g and 4 °C and the supernatant was removed. The RNA pellet was washed two times with 1 mL of 75% ethanol (v/v) and centrifugation at 12,000 × g for 5 min at 4 °C. Samples were left on ice for 30 min to allow any remaining ethanol to evaporate. The RNA pellet was then resuspended in 50 µL sterile deionized H2O. The total RNA samples were submitted to the Faculty of Biology, Medicine and Health Genomic Technologies Core Facility, University of Manchester, for cDNA library preparation and sequencing. Quality and integrity of the RNA samples were assessed using a 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA) and then libraries were generated using the TruSeq® Stranded mRNA assay (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s protocol. RNA samples with a RIN value > 7 were used for library generation. RIN values were: Control-1, 8.7; Control-2, 9.4; Control-3, 9.1; Irradiated-1, 8.2; Irradiated-2, 7.1, Irradiated-3, 7.8. Briefly, total RNA (0.1–4 µg) was used as input material from which polyadenylated mRNA was purified using poly-T, oligo-attached, magnetic beads. The mRNA was then fragmented using divalent cations under elevated temperature and then reverse transcribed into first strand cDNA using random primers. Second strand cDNA was then synthesized using DNA Polymerase I and RNase H. Following a single ‘A’ base addition, adapters were ligated to the cDNA fragments, and the products were then purified and enriched by PCR to create the final cDNA library. Adapter indices were used to multiplex libraries, which were pooled prior to cluster generation using a cBot instrument. The loaded flow-cell was then paired-end sequenced (76 + 76 cycles, plus indices) on an Illumina HiSeq4000 instrument. Finally, the output data was demultiplexed (allowing one mismatch) and BCL-to-Fastq conversion performed using Illumina’s bcl2fastq software, version 2.20.0.422. All sequence reads were pre-processed using the Trimmomatic filter software62 to remove adapters and contaminants from the data. After the data was cleaned, reads were mapped and counted to a reference genome assembly (version 2) of C. sorokiniana UTEX 1602 (https://www.ncbi.nlm.nih.gov/assembly/GCA_002245835.2) generated by Arriola et al. 23. Mapping was performed using STAR mapping software63 whilst the read counting was performed using the htseq-count script tool in HTSeq software64. Finally, normalization and differential expression calculations were performed using DESeq2 software65. Transcript abundance was presented as normalized counts derived from the DEseq2. Heatmaps were generated using Morpheus software (software.broadinstitute.org/Morpheus) and clustered using k-means analysis, which allowed demonstration of strong clustering between independent replicate samples, showing that the transcriptional changes and the metabolic response to low-dose irradiation was reproducible (Supplementary Fig. 11). The C. sorokiniana gene transcript annotation data was obtained from JGI PhyoCosm (https://phycocosm.jgi.doe.gov/Chloso1602_1/Chloso1602_1.home.html). For all transcripts that showed significant differential expression between the irradiated versus control treatments (false discovery rate (FDR) < 0.05; with a FDR adjusted p value generated as described66), transcript annotation was further manually validated, including by use of BLASTx comparisons with annotated sequences from the Chlamydomonas reinhardtii CC-4532 v6.1 genome annotation (https://phytozome-next.jgi.doe.gov/info/CreinhardtiiCC_4532_v6_1), using the BLAST tools on the JGI Phytozome genomics portal. KEGG annotation (https://www.genome.jp/kegg/annotation/) was used to further determine functional classes (Supplementary Data 1), while KEGG Mapper (https://www.genome.jp/kegg/mapper/)67 was used to map transcripts to lipid metabolism pathways (Supplementary Fig. 7 and Supplementary Data 1). Multiple sequence alignments to determine distinct gene isoforms were performed using translated amino acid sequences and Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) using default settings. Conserved amino acids of CrPSR1 and the C. sorokiniana PSR1 orthologue (C2E21_4446) were visualized using Easy Sequencing in PostScript (ESPript; https://espript.ibcp.fr/ESPript/ESPript/)68 with standard default parameters.

Quantitative reverse transcription PCR (qPCR)

RNA was extracted from C. sorokiniana cells as described above. RNA was treated with RQ1 DNase (Promega, Madison, WI, USA) and cDNA synthesis was performed using a Superscript III reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA USA) and an oligo(dT) primer (Promega, Madison, WI, USA). The qPCR was prepared using 100 ng of cDNA in triplicate (technical replicates) of three biological replicates of the control and irradiated samples in a 20 µL sample containing 10 µL of SensiFAST SYBR Hi ROX kit (Meridian Bioscience, Cincinnati, OH, USA) and 1 mM of each oligonucleotide primer (Eurofins Genomics, Ebersberg, Germany). Primer sequences are shown in Supplementary Table 2. The reaction was performed using a StepOnePlus™ Real-Time PCR machine with StepOne™ software v2.3. The C. sorokiniana 18 S rRNA gene (GenBank accession number KR904895) was used as a normalization control gene. Relative gene expression was determined using the 2−∆∆CT method69.

Statistics and reproducibility

All experiments were performed in at least biological triplicates. The exact numbers of biological replicates in different experiments are described in each figure and are available alongside the raw source data shown in the Source Data file (Supplementary Data 2). Values are presented as means ± standard error. Differences between treated samples and controls were tested using a non-parametric two-tailed or one-tailed Mann–Whitney U test, as appropriate. Results were considered to be statistically significant if p < 0.05. All individual p values are listed in Supplementary Data 2. Statistical analysis was performed in STATISTICA 8.0 (StatSoft Inc., Tulsa, OK, USA). OD750 data were fitted using sigmoidal fit. The goodness of fits was evaluated by R2 (the adjusted R-square value), which was > 0.95 for all sets of analyzed data.

Ethics and inclusion statement

The author list includes contributors from the locations where the research was conducted, who participated in study conception, study design, data collection, analysis, and interpretation of the findings.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.