Feedback regulation of the isoprenoid pathway by SsdTPS overexpression has the potential to enhance plant tolerance to drought stress.

In order to maintain the dynamic physiological balance, plants are compelled to adjust their energy metabolism and signal transduction to cope with the abiotic stresses caused by complex and changeable environments. The diterpenoid natural compound and secondary metabolites, sclareol, derived from Salvia sclarea, has gained significant attention owing to its economic value as a spice material and diverse physiological activities. Here, we focused on the roles and regulatory mechanisms of the sclareol diterpene synthase gene SsdTPS in the resistance of S. sclarea to abiotic stresses. Our results suggested that abiotic stresses could induce the response and upregulation of SsdTPS expression and isoprenoid pathway in S. sclarea. Ectopic expression of SsdTPS conferred drought tolerance in transgenic Arabidopsis, compared with wild-type. Overexpression of SsdTPS enhanced the transcription of ABA signal transduction synthetic regulators and induced the positive feedback upregulating key regulatory genes in the MEP pathway, thereby promoting the increase of ABA content and improving drought tolerance in transgenic plants. In addition, SsdTPS-overexpressed transgenic Arabidopsis improved the responses of stomatal regulatory genes and ROS scavenging enzyme activities and gene expression to drought stress. This promoted the stomatal closure and ROS reduction, thus enhancing water retention capacity and reducing oxidative stress damage. These findings unveil the potentially positive role of SsdTPS in orchestrating multiple regulatory mechanisms and maintaining homeostasis for improved abiotic stress resistance in S. sclarea, providing a novel insight into strategies for promoting drought resistance and cultivating highly tolerant plants.

Biotransformation of ginsenoside compound K using ß-glucosidase in deep eutectic solvents.

Ginsenoside compound K (CK) holds significant potential for application in the pharmaceutical industry, which exhibits numerous pharmacological activity such as cardioprotective and antidiabetic. However, the difficult separation technique and limited yield of CK hinder its widespread use. The study investigated the process of converting ginsenoside CK using ß-glucosidase. It aimed to determine the specific site where the enzyme binds and the most favorable arrangement of the enzyme. Molecular docking was also employed to determine the interaction between ß-glucosidase and ginsenosides, indicating a strong and spontaneous contact force between them. The effectiveness of the conversion process was further improved using a "green" deep eutectic solvent (DES). A univariate experimental design was used to determine the composition of DES and the optimal hydrolysis conditions for ß-glucosidase to convert ginsenoside Rb1 into ginsenoside CK. The employment of ß-glucosidase enzymatic hydrolysis in the synthesis of rare ginsenoside CK applying the environmentally friendly solvent DES is not only viable and effective but also appropriate for industrial use. The characterization methods confirmed that DES did not disrupt the structure and conformation of ß-glucosidase. In ChCl:EG = 2:1 (30%, v/v), pH 5.0 of DES buffer, reaction temperature 50 ℃, enzyme substrate mass ratio 1:1, after 36 h of reaction, the CK yield was 1.24 times that in acetate buffer, which can reach 86.2%. In this study, the process of using ß-glucosidase enzymatic hydrolysis and producing rare ginsenoside CK in green solvent DES is feasible, efficient and suitable for industrial production and application.

Mathematical analysis of robustness of oscillations in models of the mammalian circadian clock.

Circadian rhythms in a wide range of organisms are mediated by molecular mechanisms based on transcription-translation feedback. In this paper, we use bifurcation theory to explore mathematical models of genetic oscillators, based on Kim & Forger's interpretation of the circadian clock in mammals. At the core of their models is a negative feedback loop whereby PER proteins (PER1 and PER2) bind to and inhibit their transcriptional activator, BMAL1. For oscillations to occur, the dissociation constant of the PER:BMAL1 complex, [Formula: see text], must be ≤ 0.04 nM, which is orders of magnitude smaller than a reasonable expectation of 1-10 nM for this protein complex. We relax this constraint by two modifications to Kim & Forger's 'single negative feedback' (SNF) model: first, by introducing a multistep reaction chain for posttranscriptional modifications of Per mRNA and posttranslational phosphorylations of PER, and second, by replacing the first-order rate law for degradation of PER in the nucleus by a Michaelis-Menten rate law. These modifications increase the maximum allowable [Formula: see text] to ~2 nM. In a third modification, we consider an alternative rate law for gene transcription to resolve an unrealistically large rate of Per2 transcription at very low concentrations of BMAL1. Additionally, we studied extensions of the SNF model to include a second negative feedback loop (involving REV-ERB) and a supplementary positive feedback loop (involving ROR). Contrary to Kim & Forger's observations of these extended models, we find that, with our modifications, the supplementary positive feedback loop makes the oscillations more robust than observed in the models with one or two negative feedback loops. However, all three models are similarly robust when accounting for circadian rhythms (~24 h period) with [Formula: see text] ≥ 1 nM. Our results provide testable predictions for future experimental studies.

Modeling the temporal dynamics of master regulators and CtrA proteolysis in Caulobacter crescentus cell cycle.

The cell cycle of Caulobacter crescentus involves the polar morphogenesis and an asymmetric cell division driven by precise interactions and regulations of proteins, which makes Caulobacter an ideal model organism for investigating bacterial cell development and differentiation. The abundance of molecular data accumulated on Caulobacter motivates system biologists to analyze the complex regulatory network of cell cycle via quantitative modeling. In this paper, We propose a comprehensive model to accurately characterize the underlying mechanisms of cell cycle regulation based on the study of: a) chromosome replication and methylation; b) interactive pathways of five master regulatory proteins including DnaA, GcrA, CcrM, CtrA, and SciP, as well as novel consideration of their corresponding mRNAs; c) cell cycle-dependent proteolysis of CtrA through hierarchical protease complexes. The temporal dynamics of our simulation results are able to closely replicate an extensive set of experimental observations and capture the main phenotype of seven mutant strains of Caulobacter crescentus. Collectively, the proposed model can be used to predict phenotypes of other mutant cases, especially for nonviable strains which are hard to cultivate and observe. Moreover, the module of cyclic proteolysis is an efficient tool to study the metabolism of proteins with similar mechanisms.

A Robust and Efficient DNA Storage Architecture Based on Modulation Encoding and Decoding.

Synthetic DNA has been widely considered an attractive medium for digital data storage. However, the random insertion-deletion-substitution (IDS) errors in the sequenced reads still remain a critical challenge to reliable data recovery. Motivated by the modulation technique in the communication field, we propose a new DNA storage architecture to solve this problem. The main idea is that all binary data are modulated into DNA sequences with the same AT/GC patterns, which facilitate the detection of indels in noisy reads. The modulation signal could not only satisfy the encoding constraints but also serve as prior information to detect the potential positions of errors. Experiments on simulation and real data sets demonstrate that modulation encoding provides a simple way to comply with biological constraints for sequence encoding (i.e., balanced GC content and avoiding homopolymers). Furthermore, modulation decoding is highly efficient and extremely robust, which can correct up to ∼40% of errors. In addition, it is robust to imperfect clustering reconstruction, which is very common in practice. Although our method has a relatively low logical density of 1.0 bits/nt, its high robustness may provide a wide space for developing low-cost synthetic technologies. We believe this new architecture may boost the early coming of large-scale DNA storage applications in the future.

The E3 ubiquitin ligase MARCH1 regulates antimalaria immunity through interferon signaling and T cell activation.

Malaria infection induces complex and diverse immune responses. To elucidate the mechanisms underlying host-parasite interaction, we performed a genetic screen during early (24 h) Plasmodium yoelii infection in mice and identified a large number of interacting host and parasite genes/loci after transspecies expression quantitative trait locus (Ts-eQTL) analysis. We next investigated a host E3 ubiquitin ligase gene (March1) that was clustered with interferon (IFN)-stimulated genes (ISGs) based on the similarity of the genome-wide pattern of logarithm of the odds (LOD) scores (GPLS). March1 inhibits MAVS/STING/TRIF-induced type I IFN (IFN-I) signaling in vitro and in vivo. However, in malaria-infected hosts, deficiency of March1 reduces IFN-I production by activating inhibitors such as SOCS1, USP18, and TRIM24 and by altering immune cell populations. March1 deficiency increases CD86+DC (dendritic cell) populations and levels of IFN-γ and interleukin 10 (IL-10) at day 4 post infection, leading to improved host survival. T cell depletion reduces IFN-γ level and reverse the protective effects of March1 deficiency, which can also be achieved by antibody neutralization of IFN-γ. This study reveals functions of MARCH1 (membrane-associated ring-CH-type finger 1) in innate immune responses and provides potential avenues for activating antimalaria immunity and enhancing vaccine efficacy.

Correction: Critical role of deadenylation in regulating poly(A) rhythms and circadian gene expression.

[This corrects the article DOI: 10.1371/journal.pcbi.1007842.].

Bioluminescence imaging of mouse monocyte chemoattractant protein-1 expression in inflammatory processes.

Monocyte chemoattractant protein-1 (MCP-1) plays a crucial role in various inflammatory diseases. To reveal the impact of MCP-1 during diseases and to develop anti-inflammatory agents, we establish a transgenic mouse line. The firefly luciferase gene is incorporated into the mouse genome and driven by the endogenous MCP-1 promoter. A bioluminescence photographing system is applied to monitor luciferase levels in live mice during inflammation, including lipopolysaccharide-induced sepsis, concanavalin A-induced T cell-dependent liver injury, CCl 4-induced acute hepatitis, and liver fibrosis. The results demonstrate that the luciferase signal induced in inflammatory processes is correlated with endogenous MCP-1 expression in mice. Furthermore, the expressions of MCP-1 and the luciferase gene are dramatically inhibited by administration of the anti-inflammatory drug dexamethasone in a septicemia model. Our results suggest that the transgenic MCP-1-Luc mouse is a useful model to study MCP-1 expression in inflammation and disease and to evaluate the efficiency of anti-inflammatory drugs in vivo.

Critical role of deadenylation in regulating poly(A) rhythms and circadian gene expression.

The mammalian circadian clock is deeply rooted in rhythmic regulation of gene expression. Rhythmic transcriptional control mediated by the circadian transcription factors is thought to be the main driver of mammalian circadian gene expression. However, mounting evidence has demonstrated the importance of rhythmic post-transcriptional controls, and it remains unclear how the transcriptional and post-transcriptional mechanisms collectively control rhythmic gene expression. In mouse liver, hundreds of genes were found to exhibit rhythmicity in poly(A) tail length, and the poly(A) rhythms are strongly correlated with the protein expression rhythms. To understand the role of rhythmic poly(A) regulation in circadian gene expression, we constructed a parsimonious model that depicts rhythmic control imposed upon basic mRNA expression and poly(A) regulation processes, including transcription, deadenylation, polyadenylation, and degradation. The model results reveal the rhythmicity in deadenylation as the strongest contributor to the rhythmicity in poly(A) tail length and the rhythmicity in the abundance of the mRNA subpopulation with long poly(A) tails (a rough proxy for mRNA translatability). In line with this finding, the model further shows that the experimentally observed distinct peak phases in the expression of deadenylases, regardless of other rhythmic controls, can robustly cluster the rhythmic mRNAs by their peak phases in poly(A) tail length and abundance of the long-tailed subpopulation. This provides a potential mechanism to synchronize the phases of target gene expression regulated by the same deadenylases. Our findings highlight the critical role of rhythmic deadenylation in regulating poly(A) rhythms and circadian gene expression.

EkFLS overexpression promotes flavonoid accumulation and abiotic stress tolerance in plant.

Flavonoids with great medicinal value play an important role in plant individual growth and stress resistance. Flavonol synthetase (FLS) is one of the key enzymes to synthesize flavonoids. However, the role of the FLS gene in flavonoid accumulation and tolerance to abiotic stresses, as well as its mechanism has not yet been investigated systematically in plants. The aim of this research is to evaluate the effect of FLS overexpression on the accumulation of active ingredients and stress resistance in Euphorbia kansui Liou. The results showed that when the EkFLS gene was overexpressed in Arabidopsis thaliana, the accumulation of flavonoids was improved. In addition, when the wild-type and EkFLS overexpressed Arabidopsis plants were treated with ABA and MeJA, compared with WT Arabidopsis, EkFLS overexpressed Arabidopsis promoted stomatal aperture to influence photosynthesis of the plants, which in turn can promote stress resistance. Meanwhile, under MeJA, NaCl, and PEG treatment, EkFLS overexpressed in Arabidopsis induced higher accumulation of flavonoids, which significantly enhanced peroxidase (POD) and superoxide dismutase (SOD) activities that can scavenge reactive oxygen species in cells to protect the plant. These results indicated that EkFLS overexpression is strongly correlated to the increase of flavonoid synthesis and therefore the tolerance to abiotic stresses in plants, providing a theoretical basis for further improving the quality of medicinal plants and their resistance to abiotic stresses simultaneously.

iTRAQ-Based Proteomics Analysis of Autophagy-Mediated Responses against MeJA in Laticifers of Euphorbia kansui L.

Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. Methyl jasmonate (MeJA), a plant hormone, mediates diverse developmental process and defense responses which induce a variety of metabolites. In plants, little is known about autophagy-mediated responses against MeJA. In this study, we used high-throughput comparative proteomics to identify proteins of latex in the laticifers. The isobaric tags for relative and absolute quantification (iTRAQ) MS/MS proteomics were performed, and 298 proteins among MeJA treated groups and the control group of Euphorbia kansui were identified. It is interesting to note that 29 significant differentially expressed proteins were identified and their associations with autophagy and ROS pathway were verified for several selected proteins as follows: α-L-fucosidase, ß-galactosidase, cysteine proteinase, and Cu/Zn superoxide dismutase. Quantitative real-time PCR analysis of the selected genes confirmed the fact that MeJA might enhance the expression of some genes related to autophagy. The western blotting and immunofluorescence results of ATG8 and ATG18a which are two important proteins for the formation of autophagosomes also demonstrated that MeJA could promote autophagy at the protein level. Using the electron microscope, we observed an increase in autophagosomes after MeJA treatment. These results indicated that MeJA might promote autophagy in E. kansui laticifers; and it was speculated that MeJA mediated autophagy through two possible ways: the increase of ROS induces ATG8 accumulation and then aotophagosome formation, and MeJA promotes ATG18 accumulation and then autophagosome formation. Taken together, our results provide several novel insights for understanding the mechanism between autophagy and MeJA treatment. However, the specific mechanism remains to be further studied in the future.

Increased CD40 Expression Enhances Early STING-Mediated Type I Interferon Response and Host Survival in a Rodent Malaria Model.

Both type I interferon (IFN-I) and CD40 play a significant role in various infectious diseases, including malaria and autoimmune disorders. CD40 is mostly known to function in adaptive immunity, but previous observations of elevated CD40 levels early after malaria infection of mice led us to investigate its roles in innate IFN-I responses and disease control. Using a Plasmodium yoelii nigeriensis N67 and C57BL/6 mouse model, we showed that infected CD40-/- mice had reduced STING and serum IFN-ß levels day-2 post infection, higher day-4 parasitemia, and earlier deaths. CD40 could greatly enhance STING-stimulated luciferase signals driven by the IFN-ß promoter in vitro, which was mediated by increased STING protein levels. The ability of CD40 to influence STING expression was confirmed in CD40-/- mice after malaria infection. Substitutions at CD40 TRAF binding domains significantly decreased the IFN-ß signals and STING protein level, which was likely mediated by changes in STING ubiquitination and degradation. Increased levels of CD40, STING, and ISRE driven luciferase signal in RAW Lucia were observed after phagocytosis of N67-infected red blood cells (iRBCs), stimulation with parasite DNA/RNA, or with selected TLR ligands [LPS, poly(I:C), and Pam3CSK4]. The results suggest stimulation of CD40 expression by parasite materials through TLR signaling pathways, which was further confirmed in bone marrow derived dendritic cells/macrophages (BMDCs/BMDMs) and splenic DCs from CD40-/-, TLR3-/- TLR4-/-, TRIF-/-, and MyD88-/- mice after iRBC stimulation or parasite infection. Our data connect several signaling pathways consisting of phagocytosis of iRBCs, recognition of parasite DNA/RNA (possibly GPI) by TLRs, elevated levels of CD40 and STING proteins, increased IFN-I production, and longer host survival time. This study reveals previously unrecognized CD40 function in innate IFN-I responses and protective pathways in infections with malaria strains that induce a strong IFN-I response, which may provide important information for better understanding and management of malaria.

A novel Pseudomonas aeruginosa strain with an oprD mutation in relation to a nosocomial respiratory infection outbreak in an intensive care unit.

Seven imipenem-resistant Pseudomonas aeruginosa isolates were recovered from the sputum samples of pneumonia patients in southwestern China. They had identical antibiotic resistance patterns and indistinguishable pulsed-field gel electrophoresis profiles. Nucleotide sequence analysis revealed a 4-bp (AGTC) insertion in the oprD gene, resulting in a frameshift in the cognate open reading frame. These isolates became imipenem susceptible when the chromosomal oprD lesion was complemented, indicating that the 4-bp insertion in the oprD gene resulted in imipenem resistance.

Characterization of Staphylococcus aureus responses to spermine stress.

Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with ß-lactams against methicillin-resistant Staphylococcus aureus (MRSA). To explore the Spm-based antibacterial targets in S. aureus, time course-dependent transcriptome analysis was conducted on Mu50 (MRSA) in the absence and presence of Spm. Genes in the sigB regulon and most ATP-producing pathways were found down-regulated when exposure to high dose Spm. In contrast, a number of genes for iron acquisition and regulation showed significant induction, indicating a specific connection between Spm and iron-depletion. The tetM gene for tetracycline (Tc) resistance exhibited most significant fold change among the listed genes. It was specifically upregulated by Tc and Spm but not by other ribosome-targeted drugs or other polyamines; however, such induction of tetM cannot confer resistance to Spm. A set of genes for osmotic balance, including kdpABCDE for potassium ion uptake and regulation, was also induced by Spm stress. Addition of KCl or NaCl, but not high concentration sucrose, was found to increase Spm MIC over 30-fold. In summary, transcriptome analysis demonstrated a specific pattern of response upon Spm exposure, suggesting Spm may alter the intracellular iron status and suppress the SigB regulon to exert its toxicity.

Functional characterization of the potRABCD operon for spermine and spermidine uptake and regulation in Staphylococcus aureus.

Spermine, a potent bactericidal polyamine, exerts a strong synergistic effect with ß-lactams against methicillin-resistant Staphylococcus aureus. Transcriptome analysis revealed that the putative potRABCD operon for polyamine uptake and regulation exhibited significant fold change upon exposure to exogenous spermine. Properties of the PotABCD transporter in polyamine uptake were studied using wild-type and the pot deletion mutant. It was found that spermidine and spermine, but not putrescine, were the preferred substrates for the Pot system of high affinity. The PotR protein was purified from a recombinant strain of Escherichia coli, and binding of PotR to the pot regulatory region was demonstrated by electromobility shift assays. In summary, these results support the physiological function of PotR in regulation of the expression of PotABCD for spermidine and spermine uptake in S. aureus.

A Deniable Encryption Method for Modulation-Based DNA Storage.

Recent advancements in synthesis and sequencing techniques have made deoxyribonucleic acid (DNA) a promising alternative for next-generation digital storage. As it approaches practical application, ensuring the security of DNA-stored information has become a critical problem. Deniable encryption allows the decryption of different information from the same ciphertext, ensuring that the "plausible" fake information can be provided when users are coerced to reveal the real information. In this paper, we propose a deniable encryption method that uniquely leverages DNA noise channels. Specifically, true and fake messages are encrypted by two similar modulation carriers and subsequently obfuscated by inherent errors. Experiment results demonstrate that our method not only can conceal true information among fake ones indistinguishably, but also allow both the coercive adversary and the legitimate receiver to decrypt the intended information accurately. Further security analysis validates the resistance of our method against various typical attacks. Compared with conventional DNA cryptography methods based on complex biological operations, our method offers superior practicality and reliability, positioning it as an ideal solution for data encryption in future large-scale DNA storage applications.

A Novel Image Encryption Scheme for DNA Storage Systems Based on DNA Hybridization and Gene Mutation.

With the rapid development of DNA (deoxyribonucleic acid) storage technologies, storing digital images in DNA is feasible. Meanwhile, the information security in DNA storage system is still a problem to solve. Therefore, in this paper, we propose a DNA storage-oriented image encryption algorithm utilizing the information processing mechanisms in molecule biology. The basic idea is to perform pixel replacement by gene hybridization, and implement dual diffusion by pixel diffusion and gene mutation. The ciphertext DNA image can be synthesized and stored in DNA storage system after encryption. Experimental results demonstrate it can resist common attacks, and shows a strong robustness against sequence loss and base substitution errors in the DNA storage channel. A DNA storage-oriented image encryption algorithm based on gene hybridization and gene mutation, First, we scramble rows and columns of the plaintext image by dynamic Josephus traversing. Second, we replace the pixels by gene hybridization. Finally, we diffuse the image matrix in binary domain and encode pixels into 8-base strands which are later further diffused by gene mutation. The ciphertext image can be synthesized according to the mutant gene codes and stored in any DNA storage system.

Limit and screen sequences with high degree of secondary structures in DNA storage by deep learning method.

BACKGROUND: In single-stranded DNAs/RNAs, secondary structures are very common especially in long sequences. It has been recognized that the high degree of secondary structures in DNA sequences could interfere with the correct writing and reading of information in DNA storage. However, how to circumvent its side-effect is seldom studied. METHOD: As the degree of secondary structures of DNA sequences is closely related to the magnitude of the free energy released in the complicated folding process, we first investigate the free-energy distribution at different encoding lengths based on randomly generated DNA sequences. Then, we construct a bidirectional long short-term (BiLSTM)-attention deep learning model to predict the free energy of sequences. RESULTS: Our simulation results indicate that the free energy of DNA sequences at a specific length follows a right skewed distribution and the mean increases as the length increases. Given a tolerable free energy threshold of 20 kcal/mol, we could control the ratio of serious secondary structures in the encoding sequences to within 1% of the significant level through selecting a feasible encoding length of 100 nt. Compared with traditional deep learning models, the proposed model could achieve a better prediction performance both in the mean relative error (MRE) and the coefficient of determination (R2). It achieved MRE = 0.109 and R2 = 0.918 respectively in the simulation experiment. The combination of the BiLSTM and attention module can handle the long-term dependencies and capture the feature of base pairing. Further, the prediction has a linear time complexity which is suitable for detecting sequences with severe secondary structures in future large-scale applications. Finally, 70 of 94 predicted free energy can be screened out on a real dataset. It demonstrates that the proposed model could screen out some highly suspicious sequences which are prone to produce more errors and low sequencing copies.

An image cryptography method by highly error-prone DNA storage channel.

Introduction: Rapid development in synthetic technologies has boosted DNA as a potential medium for large-scale data storage. Meanwhile, how to implement data security in the DNA storage system is still an unsolved problem. Methods: In this article, we propose an image encryption method based on the modulation-based storage architecture. The key idea is to take advantage of the unpredictable modulation signals to encrypt images in highly error-prone DNA storage channels. Results and Discussion: Numerical results have demonstrated that our image encryption method is feasible and effective with excellent security against various attacks (statistical, differential, noise, and data loss). When compared with other methods such as the hybridization reactions of DNA molecules, the proposed method is more reliable and feasible for large-scale applications.

Alleviation of cadmium-induced photoinhibition and oxidative stress by melatonin in Chlamydomonas reinhardtii.

As one of the most threatening challenges to the natural environment and human health, cadmium (Cd) pollution has seriously impacted natural organisms. Green algae, such as Chlamydomonas reinhardtii (C. reinhardtii), can provide a safer, lower cost, and more effective ecological approach to the treatment of heavy metal ions in wastewater due to their sorption properties. However, heavy metal ions affect C. reinhardtii when adsorbed. Melatonin is able to protect the plant body from damage when the plant is under biotic/abiotic stress. Therefore, we investigated the effects of melatonin on the cell morphology, chlorophyll content, chlorophyll fluorescence parameters, enzymatic activity of the antioxidant system, gene expression, and the ascorbic acid (AsA)-glutathione (GSH) cycle of C. reinhardtii under the stress of Cd (13 mg/L). Our results indicated that Cd significantly induced photoinhibition and overaccumulation of reactive oxygen species (ROS). By application with the concentration of 1.0 µM melatonin, the algal solute of C. reinhardtii under the Cd stress gradually regained its green color, the cell morphology became intact, and the photosynthetic electron transport function was retained. However, in the melatonin-silenced strain, there was a significant decrease in all of the above indicators. In addition, the use of exogenous melatonin or the expression of endogenous melatonin genes could enhance the intracellular enzyme activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR). It also upregulated the expression of active enzyme genes such as SOD1, CAT1, FSD1, GSH1, GPX5, and GSHR1. These results indicate that the presence of melatonin effectively protects the activity of photosynthetic system II in C. reinhardtii, enhances antioxidant activity, upregulates gene expression in the AsA-GSH cycle, and reduces the level of ROS, thereby alleviating the damage caused by Cd toxicity.