Transcriptomic responses of Microcystis aeruginosa under electromagnetic radiation exposure

Electromagnetic radiation is an important environmental factor. It has a potential threat to public health and ecological environment. However, the mechanism by which electromagnetic radiation exerts these biological effects remains unclear. In this study, the effect of Microcystis aeruginosa under electromagnetic radiation (1.8 GHz, 40 V/m) was studied by using transcriptomics. A total of 306 differentially expressed genes, including 121 upregulated and 185 downregulated genes, were obtained in this study. The differentially expressed genes were significantly enriched in the ribosome, oxidative phosphorylation and carbon fixation pathways, indicating that electromagnetic radiation may inhibit protein synthesis and affect cyanobacterial energy metabolism and photosynthesis. The total ATP synthase activity and ATP content significantly increased, whereas H+K+-ATPase activity showed no significant changes. Our results suggest that the energy metabolism pathway may respond positively to electromagnetic radiation. In the future, systematic studies on the effects of electromagnetic radiation based on different intensities, frequencies, and exposure times are warranted; to deeply understand and reveal the target and mechanism of action of electromagnetic exposure on organisms.

Proteomic and transcriptomic methods have been successfully applied to cyanobacteria. These methods have been used to understand the mechanisms of environmental stress (such as antibiotics 17 , metal ions 18 , organic pollutants 19 , nitrogen limitation 20 ) on algae at the molecular level. However, the effects of electromagnetic radiation on the growth of cyanobacteria at the molecular level remain to be elucidated. In our previous study using proteomics, electromagnetic radiation altered the expression levels of photosynthesis-related proteins, and we speculated that the photoreaction system may be the target of electromagnetic radiation on cyanobacteria 21 . To further determine the effect of electromagnetic radiation on the growth of cyanobacteria, the present study investigated the effects of 1.8 GHz (the most common and widely used mobile communication frequency in mainland China is 1.8 GHz) and 40 V/m electromagnetic radiation on cyanobacteria cells through transcriptomic methods and measured the activity of ATP synthase. Analyze the influence mechanism of the electromagnetic environment on algae growth and explore the methods and means of physical to control algae growth rate, will be of great scientific significance and practical significance to evaluate the potential ecological risk of electromagnetic environment and solve the increasingly serious problem of water bloom.

Results
Differentially expressed genes analysis. Between the treatment and control groups, 306 differentially expressed genes were determined, 121 and 185 of which were upregulated and downregulated, respectively (Fig. 1). The full list of differentially expressed genes with the fold change and false discovery rate (FDR) detailed can be found as Supplementary Table S1.
Pathway enrichment analysis of differentially expressed genes. Exposure of M. aeruginosa to electromagnetic radiation caused significant differential gene expression and corresponding enrichment of the ribosome, oxidative phosphorylation and carbon fixation in photosynthetic pathways of organisms (Table 1).
Fourteen differentially expressed genes were present on the oxidative phosphorylation pathway of M. aeruginosa under electromagnetic stress. Among them, 13 were downregulated, and 1 was upregulated. The corresponding differentially expressed genes are listed in Table 2. The expression of genes regulating NAD(P)H dehydrogenase, subunit NdhF3 family protein, proton-translocating NADH-quinone oxidoreductase, chain M family protein, NADH-ubiquinone/plastoquinone oxidoreductase chain 6 family protein, and NAD(P)H-quinone oxidoreductase subunit 1, 2, I was downregulated. Moreover, the expression of genes regulating ATP synthase subunit alpha, b' , b, beta, delta, gamma, and epsilon chains was downregulated. By contrast, the expression of genes regulating succinate dehydrogenase/fumarate reductase and flavoprotein subunit was upregulated. Volcanic plot of C versus E (The control group mark was C and the electromagnetic radiation exposure group mark was E. The abscissa indicates the logarithm value of the difference multiple between E and C. The ordinate indicates the negative Log10 value of the FDR of the difference between the two groups. The red (E relative to C, gene expression is upregulated) and green (E relative to C, gene expression is downregulated) dots indicate the difference in gene expression (the criterion is FDR < 0.05, and the difference multiple is more than twice), and the black points are not different). www.nature.com/scientificreports/ Seven differentially expressed genes were related to carbon fixation. Among them, 6 were downregulated, and 1 was upregulated. As shown in Table 2, the expression of genes regulating glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase class 2, Calvin cycle subtype, d-fructose 1,6-bisphosphatase class 2/sedoheptulose 1,7-bisphosphatase, transketolase, phosphoribulokinase, and ribulose-phosphate 3-epimerase was downregulated. By contrast, the expression of genes regulating phosphoenolpyruvate carboxykinase was upregulated.
Genes and proteins with significant differences. Combined with the results of our previous proteomic study 21 , five genes or proteins showed simultaneous changes in gene and protein levels after electromagnetic radiation. The expression of the genes C789_RS04790, C789_RS04815 was downregulated, whereas that of iron stress-induced chlorophyll-binding protein was upregulated. The gene and protein levels of ATP synthase subunit b showed a downward trend. The expression of two unknown functional genes and proteins was upregulated. The corresponding differentially expressed genes and proteins are listed in Table 3.
Effect of electromagnetic radiation on ATP synthase. As shown in Fig. 2, 1.8 GHz electromagnetic radiation significantly increased total ATP synthase activity (p < 0.05) and ATP content (p < 0.05). Electromagnetic radiation reduced H + K + -ATP synthase activity, but no significance was observed.

Discussion
Ribosomes are the sites for proteins synthesis. We found that the expression of 16 ribosome-related genes in M. aeruginosa was downregulated after electromagnetic radiation, and the number of 50S ribosomal proteinregulated gene was 10, comprising 62.5% of the total ribosome-related differentially expressed genes. 50S ribosomal proteins were more sensitive to electromagnetic radiation than 30S ribosomal proteins. Significantly downregulated genes S10, L2, S19, L22, S3, L16, and L29 belonged to elongation factor thermo unstable (EF-Tu). EF-Tu is a guanosine nucleotide binding protein and plays a central role in protein synthesis, it is responsible for the extension of the peptide chain during protein synthesis 22,23 . EF-Tu forms a quaternary complex with the ribosome with aminoacyl-tRNA and guanosine triphosphate (GTP) and is coupled to GTP hydrolysis. As the aminoacyl-tRNA binds to the ribosome, EF-Tu forms a complex with guanosine diphosphate leaving the ribosome. This process continues to circulate so that the peptide chain continues to extend 24 . EF-Tu also functions as a molecular chaperone; to promote the renaturation of denatured rhodanese, the translation EF-Tu has chaperone-like capacity 25 When an organism is exposed to environmental stress, the protein denatures. The protein exposure residues aggregate due to interaction, causing irreversible inactivation of the protein. EF-Tu can prevent the aggregation of exposed hydrophilic groups and protect the structure, which returns to normal after the stress conditions disappear, avoiding permanent denaturation 26 . After electromagnetic exposure, the downregulation of genes related to EF-Tu may decrease the amount of EF-Tu synthesis, indicating that electromagnetic radiation may inhibit the peptide chain extension of M. aeruginosa cells, affecting protein synthesis, and weaken the function of EF-Tu in preventing protein aggregation.
Cyanobacteria NAD(P)H dehydrogenase is a multi-subunit photosynthetic membrane protein complex located on the thylakoid membrane and plays a vital role in energy metabolism. NAD(P)H-quinone Table 1. Differentially expressed genes enrichment KEGG Pathway. The second column is the pathways; the third column is the number of differentially expressed genes that are noted to a pathway and the percentage of differentially expressed genes to the total number of differentially expressed genes (number of headings); the fourth is the number of genes annotated to a pathway and the percentage of genes to the total number of genes (number of headings); the fifth column is the P value; the sixth column is the Q value after multiple checks; the seventh column is the pathway ID.  www.nature.com/scientificreports/ oxidoreductase is involved in many important energy reactions. It was responsible for many functions including respiration, cyclic electron flow around photosystem I (PSI) and CO 2 uptake [27][28][29] . Bernát et al. showed that active electron flow from metabolites to plastoquinone is suppressed upon deletion of ndhF1 and PSI-mediated cyclic electron transport is dependent on NdhF3/F4-type NDH-1 complexes 30 . Ogawa showed that the ndhB gene is required for C i transport, the inactivation of the ndhB gene also depressed dark respiration, and NADH dehydrogenase is essential to photoheterotrophic growth and inorganic carbon transport 31,32 . Stress conditions may alter the NAD(P)H dehydrogenase; for example, the contents of NAD(P)H-dehydrogenase are increased in cells grown in an environment with high salinity 33 , high light conditions also regulate NADH genes 34 . Our study shows that electromagnetic radiation also causes downregulation of NAD(P)H-quinone oxidoreductase regulatory gene expression, indicating that electromagnetic radiation may have a certain effect on the NAD(P) H complex of cyanobacteria, which in turn affects a series of physiological processes. ATP synthase is widely present in the mitochondria and chloroplasts of eukaryotic cells and cytoplasmic membrane of heterotrophic bacteria and photosynthetic bacteria. It participates in oxidative phosphorylation and photosynthetic phosphorylation and catalyzes ATP synthesis under the promotion of transmembrane proton potential. ATP synthase consists of F o unit and F 1 unit, also known as F o F 1 -ATPase, the F o unit acts as a proton channel, whereas F 1 unit catalyzes ATP synthesis 24 . The following factors affect the function of ATP synthase: ① ATP synthase function is affected through the subunit of F o ; such as oligomycin binding to the ATP synthase F o subunit, thereby inhibiting H + through F o ; dicyclohexylcarbodiimide also inhibits the action of protons through F o 24 . ② ATP synthase function is affected by the subunit of F 1 ; when 1 mol of IF 1 (the natural ATPase inhibitor from beef heart mitochondria) is bound to 1 mol of F 1 , the ATPase activity is fully inhibited 35 . ③ ATP synthase function is affected by the linkage; Subunit ε is a natural endogenous inhibitor of ATP synthase. It plays a dual role in F o F 1 from bacteria and chloroplasts. Subunit ε is indispensable for coupling between proton translocation Changes of the total ATP synthase activity, H + K + -ATP synthase activity and ATP content (The control group mark was C and the electromagnetic radiation exposure group mark was E. Bars represent the SD of the mean, "*" represents p < 0.05, E relative to C). www.nature.com/scientificreports/ though F o and ATP synthesis/hydrolysis in F 1 , and it has a regulatory role inhibiting the ATPase activity of the enzyme 36 . Stimulation of the external environment has a certain effect on ATPase activity, and ATPase is also part of a stress response mechanism of the organism against external stimuli. Plasma membrane H + ATPase mitigates of physiological disturbances imposed by salt press 37 . Our study showed that under the exposure of electromagnetic radiation, the expression of ATP synthase subunit gene was downregulated, it is indicated that electromagnetic radiation may have effects on ATP synthase function. The total ATP synthase activity and the ATP content of cyanobacteria increased significantly. H + K + -ATPase activity was decreased, but no significant change was observed. This may be because the total ATP synthase contains P-type ATPase, F-type ATPase, and many factors affecting ATPase activity. The specific reasons need further study. The Calvin cycle of photosynthesis consists mainly of three phases: fixation of CO 2 , reduction of 3-phosphoglycerate, and regeneration of ribulose-1,5-diphosphate. The external environment affects the photosynthetic carbon-fixing enzyme. Studies have shown that suboptimal temperature and low light intensity significantly decreased the growth, photosynthetic rate, activities and mRNA expressions of ribulose 1,5-biphosphate carboxylase/oxygenase, fructose-1,6-bisphosphatase (FBP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBA, transketolase 38 . Cucumber CsGAPDH gene was significantly induced at 2 h after waterlogging treatment, and the peak level of gene expression was observed at 12 h 39 . Adversity environment (high temperature stress, salt stress) will affect the expression of CpFBA gene, creating a "short-term inhibition" effect, where short-term stress will be expressed in large quantities and long-term stress will reduce gene expression levels [40][41][42] . Studies have also shown that CpFBA exhibits periodic variation under adverse conditions 43 . In our previous study, electromagnetic radiation significantly reduced the activity of FBA enzymes of M. aeruginosa 13 . The different response modes of cpFBPase mRNA levels to desiccation stress and high temperature indicated that cpFBPase played an important role in responsing to abiotic stress 44 . The expression of transketolase in rice leaves was upregulated in response to heat stress 45 , whereas the expression of transketolase in Manihot esculenta Crantz at 5 and 15 days of drought stress was downregulated 46 . SBPase overexpression enhances photosynthesis under high temperature or salt stress in transgenic rice plants, indicating SBPase plays an important role in plants in stress 47,48 . Studies have shown that the superior water-deficit tolerance in bermudagrass (Tifway) could be mainly associated with Phosphoribulokinase 49 . Phosphoribulokinase was downregulated by drought and abscisic acid in maize (Zea mays L.) 50 . A study has shown that phosphoenolpyruvate carboxylase activity was three times higher in infected plant leaves compared to healthy plants 51 . In summary, external stress can affect the gene or protein expression of photosynthetic carbon fixation enzymes or enzyme activity. Our study showed that under the exposure of electromagnetic radiation, seven differentially expressed genes were related to carbon fixation; among them, 6 (gap2, Fba, glpX, Tkt, PRK, Rpe) were downregulated, and 1 (pckA) was upregulated, it indicated that electromagnetic radiation may affect the related synthesis of photosynthetic carbon fixation enzymes through the regulation of gene expression.
After electromagnetic radiation, five genes or proteins showed simultaneous changes in gene level and protein levels. Iron stress-induced protein A (IsiA) is the main chlorophyll-binding protein in the thylakoid membrane, and significantly induced under iron deficiency conditions; dynamic changes in IsiA-containing complexes during long-term iron deficiency in cyanobacteria may represent an adaptation to iron limitation stress for flexible light energy distribution, which can balance electron transfer between PS I and PS II, thus minimizing photooxidative damage 52 . Havaux also showed that IsiA protects cyanobacteria from photooxidativestress 53 .
Our results indicate that electromagnetic radiation may affects the function of iron stress-induced chlorophyllbinding protein. In the study, IsiA gene expression is downregulated, whereas the corresponding protein levels are increased. We speculate that it may be a negative feedback regulation mechanism. CP43′ protein functions as a nonradiative dissipator of light energy, which is encoded by the IsiA gene 54 . Electromagnetic radiation is a kind of energy, which may promote the upregulation of CP43′ protein. The control and exposure groups were placed in the dark for 24 h. The lack of light may cause CP43′ to be downregulated. Electromagnetic radiation upregulated CP43′ protein. In order to keep CP43′ downregulated, the IsiA gene is downregulated, forming a negative feedback regulation mechanism. The influence of electromagnetic radiation on IsiA gene and related regulatory protein needs further study.
The gene and protein levels of ATP synthase subunit b showed a downward trend after eletcromagnetic exposure. ATP synthase acts on photosynthetic and oxidative phosphorylation to synthesize ATP. ATP synthase b subunit plays an important role in the growth, reproduction and adaptability of organisms. Chen 55 and Zheng 56 found that the ATP synthase b subunit gene is related to the testis development of drosophila, which will reduce the fecundity of male flies and cause male sterility. Li et al. 57 found that F o ATP synthase b-chain is involved in white spot syndrome virus infection. Feng et al. found that the ATP synthase b subunit gene is essential to the survival of rice brown planthopper (BPH), and the RNAi of ATP synthase b shows an effective inhibition effect of the BPH. ATP synthase b may serve as a potential target gene for BPH control 58 . ATP synthase b subunit is essential for normal function of ATP synthase. The gene and protein level of ATP synthase subunit b are downregulated by electromagnetic radiation. This finding indicated that electromagnetic radiation will affect the photosynthesis and energy metabolism of M. aeruginosa, which may reduce the ability of these biological processes.
This experiment is an exploratory experiment that found some response of cyanobacteria to electromagnetic radiation. Previous studies in our laboratory have found that electromagnetic waves have effects on the energy metabolism of Caenorhabditis elegans 59,60 and yeast 61 , which is similar to the results of this experiment. Next, we need to further study the mechanism of electromagnetic radiation's influence on energy metabolism pathway and changes in energy metabolism-related metabolites and so on. Further experiments investigating the altered pathways would be beneficial to reveal a mechanism by which electromagnetic radiation exerts these biological effects. In the future, it is necessary to base on different intensities (energy domain), frequencies (frequency domain), exposure times (time domain), from the level of cells lines, tissues, organs, individuals, colonies, and adopt different methods (appearance characteristics analysis, epigenetic analysis, genomic analysis, cytology www.nature.com/scientificreports/ atlas, etc.), systematically study the effects of electromagnetic radiation. It will help the scientific community to deeply understand and reveal the target sites, signaling pathway and mechanism of action of electromagnetic exposure on organisms. Future research outlook is shown in Fig. 3. From study the effects of electromagnetic radiation on organisms to electromagnetic dose effects, clarify the mechanism of electromagnetic radiation, and finally establish electromagnetic radiation protection programs, in order to ensure the safe development of the electromagnetic environment.
In conclusion, electromagnetic radiation affects the expression of ribosome-related regulatory genes, which may inhibit the protein synthesis of M. aeruginosa. Electromagnetic radiation affects the expression of Calvin cycle-related regulatory genes, and alters photoreaction system related protein expression levels, thereby disturbing the photosynthesis of M. aeruginosa. Moreover, it influences oxidative phosphorylation, thereby affecting cyanobacterial energy metabolism. Our finding suggests that energy metabolism pathway may respond positively to electromagnetic radiation.

Materials and methods
Experimental materials. The experiment involved species from M. aeruginosa (FACHB-905), and came from the Institute of Aquatic Biology, Chinese Academy of Sciences. The culture medium was BG 11 medium 21 .
Experimental treatment. M. aeruginosa were cultured in the normal light (Culture temperature was 25 ± 1 °C with 12 L: 12 D light-to-dark ratio, light intensity was 1000 ± 100 lx) for a period. Then, M. aeruginosa suspension were divided into two parts for exposure and control experiments. Based on our previous study result ( 13,21 ), a dark condition was chosen for the experiment. The exposure group M. aeruginosa was treated with 1.8 GHz and 40 V/m electromagnetic radiation in the dark for 24 h, temperature was 25 ± 1 °C, whereas the control group was not exposed to electromagnetic radiation and other conditions remained constant.
Electromagnetic radiation was generated using a vector signal generator (AgilentE8267DPSG) and a signal amplifier (AV38701E), and emitted from an antenna (ETS3180B). The antenna was placed at 24 cm above the sample area. The signal at the sample position was measured using an electromagnetic radiation analyzer (PMM8053B, Narta-STS, Italy) and a signal analyzer (AgilentN9030A). M. aeruginosa suspension were exposed to 1.8 GHz radio frequency electromagnetic radiation through a continuous sine wave, and at the position of the M. aeruginosa suspension, the radio frequency electromagnetic field strength was 40 V/m and the temperature was 25 °C. Experimental exposure device is the same as the device in the reference 21 .
Each experiment was repeated three times.
Transcriptomic analysis. Three samples each from the treatment and control groups were sent to GENE DENOVO Company to extract RNA and Transcriptome sequencing. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeqTM 4000 by Gene Denovo Biotechnology Co. (Guangzhou, China). To get high quality clean reads, reads were further filtered by fastp (version 0.18.0). The edgeR package (version 3.12.1) (http://www.r-proje ct.org/) was used to identify differentially expressed genes (DEGs) across samples or groups. We identified genes with a fold change ≥ 2 and a false discovery rate (FDR) < 0.05 in a comparison as significant DEGs. DEGs were then subjected to enrichment analysis of KEGG pathways (https ://www.kegg. jp/kegg/kegg1 .html). The calculated p-value has undergone FDR correction, taking FDR ≤ 0.05 as a threshold. Pathways meeting this condition were defined as significantly enriched pathways in DEGs. Determination of enzymes activity or content. Three samples each from the treatment and control groups were determinated total ATP synthase, H + K + -ATPase activity and ATP content.
Approximately 20 mL of M. aeruginosa suspension was taken, and algal cells were first collected by using a refrigerated centrifuge (Eppendorf 5810R). The cells were centrifuged at 10,000 r·min -1 and 4 °C for 3 min. Second, 1 mL of PBS or 0.9% saline solution and quartz sand were added to the suspension. Subsequently, a high-throughput tissue crusher (Shanghai Jingxin tiss-48) was used to crush the suspension for 6 min. Finally, the suspension was centrifuged at 10,000 r·min -1 for 5 min to obtain a test solution.
Total protein content, total ATP synthase activity, H + K + -ATPase activity, and ATP content were determined by kits (all kits used in the experiment were purchased from Nanjing Jiancheng Bioengineering Institute). Sample determination was performed in accordance with the kits' instructions. The experiment was repeated three times.
Statistical analysis of the data was performed using SPSS 18 software. The significance analysis was performed using an independent sample t-test, and the data were expressed as mean ± SD). When p < 0.05, a significant difference was present between the control group and the exposed group.

Data availability
All data generated or analysed during this study are included in this published article.