The unicellular green alga C. reinhardtii strain CC-125 cells (1.5 × 10⁶) were cultured in 50 mL liquid tris-acetate-phosphate (TAP) media in 250 mL flasks (Harris, 1989) by shaking at 25°C and 140 rpm under constant white light of 90–100 μmol photons m⁻² s⁻¹. In addition to the wild type (WT), two transgenic C. reinhardtii lines (AtTHI-OE1 and AtTHI-OE2) harboring the Arabidopsis thaliana Thionin 2.1 gene, which encodes antibacterial peptides, thionins, were used to investigate genotype-dependent radiation-sensitivity. Cells were harvested at 800 × g, dispensed, and subjected to X- or γ-irradiation as described below. The subsequent post-irradiation cultivations of the cells were performed in 10 mL fresh TAP media in 50 mL conical tubes, with an initial optical density of 0.05 at 750 nm for mock (or control) under the same culture conditions. The basal TAP medium was supplemented with 5 or 10 mM NaHCO₃ to evaluate the synergistic effect of X-rays and sodium bicarbonate on cell growth.

Mid-exponential phase C. reinhardtii cells with an approximate optical density of 0.6 OD₇₅₀ were harvested from 10 mL cultures in 50 mL conical tubes, resuspended in 1.8 mL of TAP medium, and dispensed into three 1.5 mL microcentrifuge tubes. Samples were subjected to X-irradiation of 3, 6, 12, or 18 Gy for 11.1, 22.2, 44.4, or 66.6 min at 160 kV and 1 mA, or 25, 50, or 100 Gy for 4.05, 8.1, or 16.2 min at 160 kV and 10 mA using a cabinet type X-ray machine (CP-160, Faxitron X-ray LLC, Lincolnshire, IL, USA). In addition, X-irradiation was repeated two or three times at 2-day intervals. For γ-irradiation, the mid-exponential phase C. reinhardtii cells from 50 mL cultures in 250 mL flasks were equally dispensed into two 50 mL conical tubes without concentrating and exposed to γ-rays of 3, 6, 12, 25, 50, or 100 Gy for 1 h, which were generated from a 3 kCi ⁶⁰Co source at the Advanced Radiation Technology Institute (Jeollabuk-do, Korea). The absorbed radiation dose for each sample was determined using a 5 mm-diameter alanine dosimeter (Bruker Instruments, Rheinstetten, Germany), as described previously (Song et al., 2016).

Cell density was used to represent the relative growth of the mock (or control) and irradiated cells. The cell densities of C. reinhardtii cultures in liquid TAP media were obtained by measuring the OD₇₅₀ at 2 days or consecutively for 5 days after each X- or γ-irradiation. For the survival rate, the mock and X-irradiated cells (1 × 10³–1 × 10⁴ cells) were spread on solid TAP medium plates and incubated at 25°C for 7 days under constant white light of approximately 90–100 μmol photons m⁻² s⁻¹. After incubation, visible colonies were counted and used to calculate the relative survival rates of mock and irradiated cells.

For RNA isolation, the harvested and frozen cells (1 × 10⁸) were resuspended in 1 mL of TRIzol reagent (Invitrogen, CA, United States), vortexed for 10 min, incubated for 5 min at 25°C, and centrifuged for 10 min at 4°C and 12,000 × g. The supernatant was mixed with 250 µL of chloroform (Sigma-Aldrich, MO, United States) by vortexing for 2 min, combined with an equal volume of phenol-chloroform (1:1 v/v; Sigma-Aldrich, MO, United States) after centrifugation, mixed by vortexing for 2 min, and centrifuged again. It was finally mixed with an equal volume of isopropanol, incubated for 45 min at 4°C, and centrifuged for 20 min at 4°C and 12,000 × g. The resultant RNA pellet was washed twice with 800 µL 75%(v/v) ethanol, dissolved in DEPC-treated water, and used for RNA-Seq and quantitative RT-PCR analyses.

For RNA-Seq, three biological replicates from different batches were prepared for transcriptome analysis. Total RNA was quantified using the Invitrogen Quant-IT™ RiboGreen™ RNA Assay Kit, and its integrity was assessed on the Agilent TapeStation system (Agilent Technologies, Santa Clara, CA, USA) using the RNA ScreenTape. RNA-Seq paired-end libraries were prepared using the Illumina TruSeq Stranded Total RNA Library Prep Plant Kit (Illumina, San Diego, CA, USA). The libraries were quantified using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) and qualified using the Agilent TapeStation system with the D1000 ScreenTape. Sequencing of paired-end libraries was performed using the Illumina NovaSeq 6000 System (Illumina, Inc., San Diego, CA, USA).

The quality of the raw reads (FASTQ) was evaluated using the FastQC v0.11.5, and ‘dirty’ reads were removed using the Trimmomatic v0.36 to decrease data noise before downstream analysis. Briefly, the reads were subjected to the standard quality control (QC) criteria for trimming and cleaning as follows: (1) reads that aligned to primers and/or adaptors, (2) reads with over 50% low-quality bases (quality value ≤ 5) in one read, and (3) reads with over 10% unknown bases (N bases). After filtering, the remaining ‘clean’ reads were stored in FASTQ format. All high-quality clean reads were then mapped to the NCBI Chlamydomonas reinhardtii v5.5 or the Phytozome Chlamydomonas reinhardtii v6.1 reference genome using HISAT2 v2.2.1 (Kim et al., 2015a) to generate a BAM file. The BAM files were sorted and indexed with Samtools, and the number of reads matching each gene in the Chlamydomonas reinhardtii genome was counted using HTSeq-count v0.11.3.

Differentially expressed genes (DEGs) were identified using the read count data. Briefly, the read count data were filtered [read count < 1 (3 of 3)], and the count per million (CPM) was normalized using EdgeR software. Finally, the read count of each gene in every sample was normalized with that of the mock sample to calculate the fold change in gene expression between mock and test samples. Log₂-transformed fold-change values > 1 were considered differentially expressed between the mock and test samples. Functional enrichment analysis of DEGs for Gene Ontology (GO) and KEGG pathway were conducted using the Gene Ontology Database and DAVID, respectively (Huang et al., 2009). The DEGs for this analysis were obtained using the Phytozome Chlamydomonas reinhardtii v6.1 reference genome.

For quantitative RT-PCR, RNA isolation was performed as described in RNA-Seq analysis. cDNA was synthesized from 1 μg of each RNA sample using oligo(dT) primers and the LaboPass™ cDNA Synthesis Kit (Cosmo Genetech, Seoul, Korea). Subsequent quantitative PCR (qPCR) amplification cycle conditions were 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, 57°C for 10 s, and 72°C for 1 min using the CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with the iTaq Universal SYBR^® Green Supermix (Bio-Rad Laboratories) and gene-specific primers ( Supplementary Table 1 ). The relative expression level of each gene was calculated between mock and X- or γ-irradiated samples using the comparative C_T method (Liu and Saint, 2002). The expression data of the three biological replicates were normalized to that of the endogenous reference gene CrTUBA1.

The quantitative analysis of apoptotic and necrotic dead cells was conducted using the flow cytometer Muse ™ Cell Analyzer (Merck Millipore, Billerica, MA, USA) and the Muse™ Annexin V and Dead Cell Assay Kit (MCH100105; Merck Millipore) according to the manufacturer’s instructions. The mock and irradiated C. reinhardtii cells were harvested at 6, 24, and 48 h after X-irradiation, washed twice with Dulbecco’s PBS, stained with Muse™ Annexin V and Dead Cell Reagent at a final concentration of 5 × 10⁶ cells mL ⁻¹, and finally subjected to the apoptotic cell death assay using 5,000 cells for each sample. Apoptotic cell death was expressed as the proportion of living, early/late apoptotic, and dead cells, which were determined using Muse™ Cell Analyzer software (Muse 1.1.2; Merck Millipore).

Chlorophyll fluorescence in C. reinhardtii cells was measured and analyzed in a 96-well plate using the IMAGING-PAM chlorophyll fluorometer (WALZ, Effeltrich, Germany) as described previously (Kim et al., 2009) to evaluate photosynthetic activity. The cells (1 × 10⁸) were harvested 48 h after the third repetitive X-irradiation, and chlorophyll fluorescence was measured. Readings were taken from the wells after a 96-well black plate containing cell cultures was dark-incubated for 15 min at 25°C. Chlorophyll fluorescence parameters Fv/Fm, ETR, qP, and NPQ were calculated as described by Kim et al. (2005) and were used as representative parameters for photosynthetic activity. The maximum electron transport rate, ETRmax, was obtained from the relative ETR vs. PPFD curve, as reported previously (Bischof et al., 2000).

Chlorophyll and protein contents were determined using the methods described by Kim et al. (2009) with modifications for C. reinhardtii cells. Cells (1 × 10⁸) were harvested 48 h after the second repetitive X-irradiation. The cells were subjected to vigorous vortexing in 500 μL of 100% (v/v) ice-cold acetone for chlorophyll extraction, and cell debris was removed by centrifugation at 4°C and 21,000 × g for 5 min. Chlorophyll concentration was calculated using the equations of Lichtenthaler (1987) as follows: chlorophyll a = 11.24 × A_661.6 − 2.04 × A_644.8; chlorophyll b = 21.13 × A_644.8 − 4.19 × A_661.6; and total chlorophyll = 18.09 × A_644.8 + 7.05 × A_661.6.

For protein extraction, the harvested cells (1 × 10⁸) were resuspended and shaken in 300 μL of lysis buffer containing 60 mM DTT, 60 mM Na₂CO₃, 2% (w/v) SDS, and 12% (w/v) sucrose for 20 min at 25°C (Ferrante et al., 2011). The protein extracts were obtained by centrifugation at 4°C and 10,000 × g for 1 min and mixed with five volumes of 100% (v/v) ice-cold acetone. The mixture was kept at −20°C for 2 h to facilitate protein precipitation and centrifuged at 4°C and 5,000 × g for 15 min. The protein pellets were washed twice with 100% (v/v) ice-cold acetone and kept in a fume hood for the complete drying of acetone. The dried protein pellets were dissolved in distilled water and used to determine the protein concentration according to the manual of the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA), using the method of Bradford (1976).

Starch and lipid contents were determined using the methods of Xiao et al. (2006) and Tran et al. (2019) with some modifications. Mock and X-irradiated cells were harvested 48 h after the second repetitive X-irradiation. All experiments were performed in triplicate. The cells were incubated with 100 µL of iodine reagent (5 mM I₂ and 5 mM KI) at a ratio of 1:1 (v/v) in a 96-well plate to determine starch content. Following color development, the absorbance of the iodine-treated samples was measured at 580 nm. The relative starch content of the mock and X-irradiated samples was calculated from the absorbance before and after normalization against OD₇₅₀.

Neutral lipid content was quantified using a fluorescence-based microplate assay after staining mock and X-irradiated cells with Nile Red. The cells were incubated with 100 µL Nile Red (a stock solution of 2 µg mL^-1) at a ratio of 1:1 (v/v) in a 96-well plate for 30 min in the dark at 25°C. Nile Red fluorescence signals were measured at an excitation wavelength of 560 nm and an emission wavelength of 635 nm. The relative lipid content of the mock and X-irradiated samples was calculated from the fluorescence before and after normalization against OD₇₅₀, respectively.

Production and scavenging of ROS in C. reinhardtii cells after X-irradiation with 6 Gy was estimated by measuring the cellular H₂O₂ content in absence and presence of ascorbic acid (AA) as a ROS scavenger. The cells (6 × 10⁷) were harvested at 1 h post-irradiation without or with 1 mM AA treatment for 1 h at 25°C, and were incubated with 10 μM 2’,7’-dichlorofluorescin diacetate (H₂DCF-DA) for 25 min in darkness at 25°C. The cell-permeable fluorometric probe H₂DCFDA is cleaved to 2’,7’-dichlorofluorescin (H₂DCF) by endogenous esterases in cells and is oxidized to its fluorescent form DCF upon reaction with H₂O₂. DCF fluorescence signals were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The ROS level of the mock and X-irradiated samples was calculated relative to the mock without AA treatment by the DCF signal.

All experiments were repeated more than three times using biological replicates harvested after independent X- or γ-irradiation. The data were subjected to a two-sample independent t-test, one-way or multivariate analysis of variance followed by Tukey’s honest significance difference test, correlation or discriminant analysis using the statistical and graphical functions of R version 4.2.1 (R Core Team, 2022) and ggplot2 (Villanueva and Chen, 2019) in RStudio 2022.07.1 + 554 (Rstudio Team, 2022). A p-value less than 0.05 was considered significant.

The growth rates of C. reinhardtii cells were analyzed after X- or γ-irradiation with different doses of 3, 6, 12, 60, and 100 Gy to investigate the dose effect of ionizing radiation on algal cells. The two types of irradiation with the same absorbed doses but different dose rates, as described in the Materials and Methods section, revealed similar stimulatory and inhibitory effects on the growth curves of C. reinhardtii cells ( Supplementary Figures 1, 2 ). The similar dose-response curves following the two types of irradiation demonstrated that the growth response of C. reinhardtii cells is dependent on the absorbed dose rather than on the dose rate. In both X- and γ-irradiation, doses of 12 to 100 Gy inhibited cell growth in a dose-dependent manner, while doses of 3 and 6 Gy increased cell growth from 2 days after irradiation ( Figure 1 ). These results suggest that X- or γ-irradiation at a relatively low dose (< 10 Gy) can stimulate growth in C reinhardtii cells.

Subsequently, C. reinhardtii cells were subjected to repetitive X-irradiation at 3 or 6 Gy to substantiate the growth stimulatory effect of X- or γ-irradiation at a relatively low dose. We investigated whether repetitive X-irradiation with 3 or 6 Gy, with a final cumulative dose of 9 or 18 Gy, respectively, could further increase the growth of C. reinhardtii cells beyond the single X-irradiation ( Figure 2A ). The irradiation groups demonstrated 9.8−10.4% higher cell densities after the first X-irradiation, 19.2−19.6% after the second, and 20.5−21.7% after the third compared to the mock group, suggesting increasing growth stimulation at least up to the third ( Figure 2B ). In particular, repetitive X-irradiation with 6 Gy with a final cumulative dose of 18 Gy enhanced growth stimulation approximately two-fold, despite growth inhibition by single X- or γ-irradiation with doses above 12 Gy ( Figures 1 , 2 ). These results suggest that repetitive X-irradiation with a certain range of relatively low doses can enhance the growth stimulation of C. reinhardtii beyond single X-irradiation by preventing the growth inhibition of a high cumulative dose. No substantial difference in the radiation-induced growth stimulation between the 3 and 6 Gy groups after repetitive X-irradiation may imply that the two doses are within similar equilibrium between radiation-induced positive and negative effects.

Gamma irradiation at doses of 3–48 Gy induced the expression of DDR genes in a dose-dependent manner, following nuclear DNA damage in Arabidopsis and rice plants (Ryu et al., 2018; Kim et al., 2021). However, γ-H2AX induction and DDR gene expression were not statistically different within the dose range of 3–12 Gy and almost recovered to the control levels within 24 h. To evaluate the genotoxic stress of X- or γ-irradiation at two different doses proven to stimulate or inhibit cell growth, we compared the genome-wide transcriptomic profiles of C. reinhardtii by RNA-Seq analysis after X- or γ-irradiation at doses of 6 and 50 Gy. The overall transcriptome changes were discriminated by the radiation dose rather than the radiation type and manifested mainly by DDR genes ( Supplementary Figure 3 and Table 1 ). Transcription levels of the seven DDR genes selected from Table 1 were found to increase in a dose-dependent manner but were rarely affected by the radiation type in RT-qPCR analysis ( Figure 3 ). The concerted transcriptional activation of these DDR genes suggests that various types of DNA damage might be induced after X- or γ-irradiation, even at a dose of 6 Gy. Radiation-induced genotoxic stress needs to be further evaluated by physiological tests such as cell death and survival rate assays to explain the growth-stimulatory effect of X- or γ-irradiation at doses below 10 Gy.

Flow cytometric analysis has been used as a rapid ecotoxicological tool to evaluate the effects of toxicity in Chlamydomonas cells using appropriate probes (Jamers et al., 2009). Propidium iodide (PI) and 7-aminoactinomycin D (7-AAD) are popular probes for quantifying the proportion of dead cells (Seed and Tomkins, 2016). In this study, mock and X-irradiated C. reinhardtii cells were subjected to double staining with Annexin V and 7-AAD to analyze apoptotic cell death. The cell death rate was significantly higher at 6 h after X-irradiation with 50 Gy and then decreased with time until 48 h, whereas X-irradiation with 6 Gy induced no significant difference in cell death compared to the mock treatment ( Figures 4A, B ). In addition, when the mock and X-irradiated C. reinhardtii cells were cultivated on solid TAP medium for 7 days, the relative cell survival rate decreased to approximately 30% in the 50 Gy irradiated group but showed no significant difference in the 6 Gy group ( Figures 4C, D ). These results demonstrate that the DDR gene expression induced after X-irradiation with 6 Gy, as shown in Table 1 and Figure 3 , does not represent fatal genotoxic stress responses, such as increased cell death and decreased survival rates. Although radiation-induced genotoxic stress or mutagenesis is generally accepted, the physiological responses of C. reinhardtii cells after X- or γ-irradiation are differentially modulated, depending on the absorbed dose.

X-irradiation at 6 Gy stimulated the growth of C. reinhardtii without inducing cell death or reducing survival ( Figures 1 , 2 , 4 ). The gene ontology and KEGG pathway analyses of genome-wide transcriptomic changes after X- or γ-irradiation revealed substantial up-regulation in various metabolic pathways as well as DDR pathway ( Figure 5 ). Therefore, to understand the favorable growth stimulation of X-irradiation with 6 Gy, we compared the mock and X-irradiated groups in terms of various metabolic activities. When the photosynthetic activity was evaluated by chlorophyll fluorescence analysis, the maximal photochemical efficiency of photosystem II (Fv/Fm), as well as the parameter for photochemical quenching, qP, were not substantially different between the mock and X-irradiated groups ( Figures 6A, B ). However, the parameter for non-photochemical quenching of chlorophyll fluorescence, NPQ, and the maximal electron transport rate of photosynthesis, ETRmax, were significantly increased in the latter group ( Figures 6C, D ). The changes in Fv/Fm, qP, and NPQ after X-irradiation were similar to those previously reported in some γ-irradiated groups of C. reinhardtii (Gomes et al., 2017), but the increase in ETRmax was first observed in this study. The chlorophyll and protein contents were 19% and 16% higher in the X-irradiated group than in the mock group, respectively ( Figures 6E, F ). In addition, the major algal metabolites such as starch and lipid were significantly increased by up to 136% and 130% (or 113% and 109% after normalization against cell density) in the X-irradiated group compared to the mock group, respectively ( Figure 7 ). Correlation and discriminant analyses displayed differential correlations between the metabolic parameters including growth rate, photosynthetic activity, chlorophyll, protein, starch, and lipid contents with a distinct difference between the mock and X-irradiated groups of C. reinhardtii cells ( Supplementary Figure 4 ). These results suggest that the growth stimulation of C. reinhardtii cells by X- or γ-irradiation with 6 Gy or less can be associated with the modulation of photosynthetic activity and enhancement of metabolic activities such as chlorophyll, protein, starch, and lipid synthesis. Our data demonstrate that X- or γ-irradiation with a certain range of relatively low doses can contribute to the improvement of algal biomass and metabolite yields via the growth stimulation of algal cells, despite the potential genotoxicity and oxidative stress.

Carbon, nitrogen, and light are important factors in the growth and biomass yield of the green alga Chlamydomonas (Moon et al., 2013; Mortensen and Gislerod, 2015; Yang et al., 2018; Bialevich et al., 2022). Therefore, we investigated the combined effect of repetitive ‘low-dose’ X-irradiation and carbon source supplementation, as shown in Figure 8A . Although the standard TAP medium used in this study contained acetate as an organic carbon source, two concentrations (5 and 10 mM) of sodium bicarbonate (NaHCO₃) as an inorganic carbon source were added to the TAP medium for growth stimulation. The growth rates of C. reinhardtii cells in the NaHCO₃-supplemented TAP medium greatly increased in a concentration-dependent manner and significantly more increased when cultivated following X-irradiation at 6 Gy ( Figure 8B ). However, the enhanced growth stimulation after repetitive X-irradiation was not significant in TAP medium containing NaHCO₃. When cultivated in 5 or 10 mM NaHCO₃-supplemented TAP medium for 5 days after X-irradiation with 6 Gy, the growth rate of C. reinhardtii cells increased by 55% and 84% at 3 days after X-irradiation, respectively ( Supplementary Figure 5 ). This growth stimulation was much stronger than the 37% or 42% induced by a single treatment with 5 or 10 mM NaHCO₃ without X-irradiation. Taken together, the combination of X-irradiation at 6 Gy and NaHCO₃ supplementation at 5 or 10 mM induces a synergistic effect for growth stimulation in Chlamydomonas cultivation.

Even in the same species, general stress responses are expected to differ according to individual genotypes. As Chlamydomonas is a widely used algal platform for recombinant protein production (Kumar et al., 2013; Rasala and Mayfield, 2015; Doron et al., 2016; Ma et al., 2022), the applicability of radiation-induced growth stimulation is worth investigating in transgenic Chlamydomonas producing recombinant proteins. Therefore, two transgenic C. reinhardtii lines (AtTHI-OE1 and AtTHI-OE2), which overexpress AtTHI2.1 gene encoding Arabidopsis antibacterial proteins, thionins, were randomly selected and compared with wild type (WT) in the cell growth and survival rates after X-irradiation at a dose range of 6–50 Gy ( Supplementary Figure 6 and Figure 9 ). The cell growth rate after X-irradiation was distinctly different between WT and AtTHI-OE1 or AtTHI-OE2 lines, showing the growth stimulation in the 6 Gy group for WT and in the 12 Gy group for AtTHI-OE1 and AtTHI-OE2 ( Supplementary Figure 6B ). When the mock and X-irradiated C. reinhardtii cells were cultivated for 7 days on solid TAP medium, the relative cell survival rate was higher in the AtTHI-OE1 and AtTHI-OE2 lines than in the WT after X-irradiation with 25 or 50 Gy ( Figure 9 ). The survival rate started to decrease in the 6 Gy group for WT and the 12 Gy group for AtTHI-OE1 and AtTHI-OE2 in a dose- and genotype-dependent manner. In addition, the expression levels of two DDR genes CrRPA70A and CrRAD51 were generally lower in the AtTHI-OE1 and AtTHI-OE2 lines than in the WT after X-irradiation ( Supplementary Figure 6C ). These results suggest that the radiation sensitivity of C. reinhardtii cells differs among genotypes and may be closely associated with an optimal dose range of X-irradiation for growth stimulation.

Ionizing radiation generates various types of ROS such as hydrogen peroxide (H₂O₂) and hydroxyl radical (•OH) in a dose-dependent manner via water radiolysis (Lee et al., 2009). Especially, H₂O₂ is the main ROS to mediate both oxidative damage and signaling in plants and algae (Quan et al., 2008; Battah et al., 2015; Niu and Liao, 2016; Fal et al., 2022). X-irradiation at a dose range of 3–100 Gy induced a biphasic increase of ROS in a dose- and dose rate-dependent manner including the relatively low doses of 3 and 6 Gy ( Supplementary Figure 7 ). In contrast, the in vivo accumulation of radiation-induced ROS in C. reinhardtii cells after X-irradiation with 6 Gy decreased from 33.6% to 18.4% by a subsequent treatment of ascorbic acid (AA), a ROS scavenger ( Figure 10A ). Therefore, we investigated the effect of ROS accumulation on the radiation-induced growth stimulation of C. reinhardtii cells. The radiation-induced growth stimulation of C. reinhardtii cells after X-irradiation with 6 Gy significantly decreased from 9.6% to 3.3% or from 20.6% to 11.7% by AA treatment before heterotrophic or mixotrophic cultivation, respectively ( Figure 10B ). These results suggest that radiation-induced growth stimulation could be closely associated with ROS accumulation and signaling.